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Identifying and characterizing caveolin-1 derived peptides : a novel approach to promoting nitric oxide… Trane, Andy E. 2016

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     IDENTIFYING AND CHARACTERIZING CAVEOLIN-1 DERIVED PEPTIDES: A NOVEL APPROACH TO PROMOTING NITRIC OXIDE RELEASE FROM THE ENDOTHELIUM    by   Andy E Trane   B.SC. (Honours), The University of British Columbia, 2009  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY  in   THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES   (Pharmacology & Therapeutics)  THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)     July 2016   ©Andy E Trane, 2016ii  Abstract Cardiovascular diseases are one of the largest causes of mortality globally. One of the hallmarks of cardiovascular diseases is a reduction in systemic endothelial nitric oxide synthase (eNOS)-derived Nitric Oxide (NO), a critical regulator of vascular homeostasis. eNOS regulation is complex, involving phosphorylation and direct protein interactions. The main negative regulator is caveolin-1 (Cav-1), the homo-oligomeric coat protein of caveolae, which interacts with eNOS via its scaffolding domain (CAV). Studies have shown that alanine substitution of F92 in CAV can lead to abolishment of the inhibitory effect on eNOS; furthermore, CAV peptides with the F92A substitution can be used as an antagonist to promote basal eNOS-derived NO to reduce blood pressure and reduce cardiovascular disease progression. We hypothesized that identification of the eNOS binding motif in CAV could be used as the basis for a pharmacophore to develop antagonists aimed at increasing vascular NO.  We performed a protein interaction study to identify a 10 residue ‘binding site peptide’ (BSP) in CAV that could account for the majority of eNOS binding. Both BSP and its F92A counterpart (BSPF92A) bound eNOS with similar affinity as the full CAV sequence as confirmed by polarization assay, while computational modeling suggested that the peptides inserted themselves in to a hydrophobic pocket in eNOS. While substitution of F92 prevents inhibition of activated eNOS, we found that both BSP and BSPF92A could promote basal NO release from resting endothelial cells (ECs), independent of cell permeabilization sequence used. Furthermore, BSP and BSPF92A generated NO in an eNOS and lipid raft dependent manner. Subsequently, we found that neither BSP nor BSPF92A affected basic biochemical properties of eNOS and Cav-1, such as iii  oligomerization, subcellular targeting and co-localization. Instead, the presence of F92 was found to promote phosphorylation of eNOS, an important step in its activation. As a result of this finding, we have identified the basis for two different pharmacophores that increase NO in different manners. One that promotes activity indirectly (BSP) while the other one acts as an antagonist (BSPF92A). We hope to use this as the beginnings for a therapeutics development platform to promote cardioprotective NO.  iv  Preface  This thesis contains published and unpublished research. The data has been restructured for the thesis to provide a better flow.  Chapter 2 and a section of chapter 3 contains research published (Trane AE, Pavlov D, Sharma A, Saqib U, Lau K, van Petegem F, Minshall RD, Roman LJ, Bernatchez PN. 2014. Deciphering the binding of caveolin-1 to client protein endothelial nitric-oxide synthase (eNOS): scaffolding subdomain identification, interaction modeling, and biological significance. J Biol Chem, 289: 13273-83). Computer modeling of the eNOS/Cav-1 interaction was performed by Drs. Uzma Saqib and Richard Minshall. Polarization assays were performed by Dr. Dmitri Pavlov, but I was involved in the project design and data analysis and drafting of manuscript. Lastly, Dr. Linda Roman kindly provided the purified eNOS samples for polarization studies.  Parts of chapter 3 and chapter 4 are derived from a second research paper (Trane AE, Hiob M, Uy T, Pavlov D, Bernatchez PN. 2015. Caveolin-1 scaffolding domain residue phenylalanine 92 modulates Akt signaling. Eur J Pharmacol, 766: 46-55.). All work in chapters 3 and 4 were carried out by myself, as was drafting of the manuscript.  Lastly, the remainder of chapter 3 consists of unpublished data.   v  Table of Contents Abstract ................................................................................................................................... ii Preface .................................................................................................................................... iv Table of Contents .................................................................................................................... v List of Tables ......................................................................................................................... xii List of Figures ...................................................................................................................... xiii List of Abbreviations ........................................................................................................... xiv Acknowledgements .............................................................................................................. xvi Chapter 1. General Introduction........................................................................................... 1 1.1. Overview of Thesis ....................................................................................................... 2 1.2. Cardiovascular Disease ............................................................................................... 2 1.3. Endothelial Dysfunction .............................................................................................. 3 1.4. Link Between NO and Endothelial Dysfunction ....................................................... 5 1.5. General Overview of NO ............................................................................................. 6 1.6. Production of NO ......................................................................................................... 6 1.7. eNOS Polymorphism and Cardiovascular Disesases ............................................... 7 1.8. Origins of Vascular NO Research .............................................................................. 8 1.9. Role of NO in Vascular Health and Disease .............................................................. 9 1.9.1. NO and SMC .......................................................................................................... 9 1.9.2. NO and Inflammation ........................................................................................... 10 vi  1.9.3. NO and Platelet Activation ................................................................................... 10 1.9.4. NO and Neovascularization .................................................................................. 11 1.10. Linking NO loss to Cardiovascular Disease Symptoms ....................................... 11 1.11. Basis for Targeting eNOS-derived NO in Endothelial Dysfunction ................... 12 1.12. NO Availability Versus NO Signaling in Endothelial Dysfunction ..................... 13 1.13. Enzyme Expression in Endothelial Dysfunction ................................................... 13 1.14. Targeting Superoxide: A Natural NO Sink ........................................................... 14 1.15. Targeting eNOS for Better Vascular Health ......................................................... 14 1.16. eNOS Regulation ...................................................................................................... 16 1.16.1. Transcriptional Regulation of eNOS .................................................................. 16 1.16.2. Acylation and Subcellular Localization.............................................................. 17 1.16.3. Phosphorylation .................................................................................................. 19 1.16.4. S-Nitrosylation .................................................................................................... 21 1.16.5. Substrate Accessibility and the Arginine Paradox.............................................. 22 1.16.6. Co-Factor Insufficiency and eNOS Uncoupling................................................. 24 1.16.7. Protein-Protein Interactions ................................................................................ 25 1.17. Introduction to Caveolae and the Caveolin Family .............................................. 26 1.18. Cav-1: The Coat Protein of Vascular Caveolae .................................................... 27 1.19. Physiological Role of Caveolae and Cav-1 ............................................................ 29 1.19.1. Cholesterol Regulation ....................................................................................... 29 vii  1.19.2. Vesicular Trafficking .......................................................................................... 29 1.19.3. Signaling Regulation .......................................................................................... 30 1.20. Caveolae and Cav-1 in the Cardiovascular System .............................................. 31 1.21. A Closer Look at the eNOS-Cav-1 Relationship ................................................... 33 1.22. Experimental Results of Cav-1 Based Peptides in Cardiovascular Settings ...... 33 1.23. Summary .................................................................................................................. 34 1.24. Hypothesis ................................................................................................................ 36 1.25. Breakdown of Research Chapters .......................................................................... 36 Chapter 2. Identification of the eNOS Binding Motif within the Cav-1 Scaffolding Domain ................................................................................................................................... 38 2.1. Introduction ............................................................................................................... 39 2.2. Methods ...................................................................................................................... 40 2.2.1. Plasmids and Constructs ....................................................................................... 40 2.2.2. Plasmid Expression ............................................................................................... 40 2.2.3. GST-Fusion Protein Resin Preparation ................................................................ 43 2.2.4. Western Blotting ................................................................................................... 43 2.2.5. Fluorescent Polarization Assay............................................................................. 44 2.2.5. Molecular Modeling of Peptides .......................................................................... 45 2.2.6. Molecular Docking of Peptides into eNOS .......................................................... 45 2.2.7. Data Analysis ........................................................................................................ 46 viii  2.3. Results ......................................................................................................................... 46 2.3.1. eNOS Binding is F92 Independent, but CAV Dependent .................................... 46 2.3.2. CAV-BC Accounts for Almost All eNOS Binding .............................................. 48 2.3.3. Residues 90-99 of CAV Accounts for Bulk of eNOS Binding ............................ 51 2.3.4. Binding Site Motif Affinity for eNOS Unaltered by Truncation ......................... 55 2.3.5. Molecular Modeling Insights in to the eNOS/BSP Interaction ............................ 57 2.4. Discussion ................................................................................................................... 57 2.4.1. The Utilization of an Isolated System and Alanine Substitution .......................... 59 2.4.2. Comparison of Scaffolding Domains ................................................................... 60 2.4.3. Cav-1, the eNOS Binding Domain and the Cav-1 Binding Motif ........................ 62 2.5. Conclusion .................................................................................................................. 63 Chapter 3. Cav-1 Binding Site Derived Peptides and NO Release in Endothelial Cells 64 3.1. Introduction ............................................................................................................... 65 3.2. Methods ...................................................................................................................... 66 3.2.1. Cell Culture ........................................................................................................... 66 3.2.2. NO Measurements ................................................................................................ 66 3.2.3. Live Cell Imaging ................................................................................................. 67 3.2.4. siRNA Studies ...................................................................................................... 67 3.2.5. Western Blotting ................................................................................................... 68 3.2.6. Cyclodextrin Peptide Uptake Studies ................................................................... 68 ix  3.2.7. Data Analysis ........................................................................................................ 68 3.3. Results ......................................................................................................................... 69 3.3.1. Loss of F92 Prevents BSP-mediated Inhibition of Stimulated eNOS .................. 69 3.3.2. BSP and BSPF92A Increase Basal NO Release ...................................................... 69 3.3.3. F92A Substitution Does Not Affect Peptide Uptake ............................................ 71 3.3.4. NO Release is Blunted by eNOS, but not Cav-1, siRNA ..................................... 75 3.3.5. Peptide Stimulated NO Release is Lipid Raft Dependent .................................... 78 3.4. Discussion ................................................................................................................... 78 3.4.1. Cell Line Limitations ............................................................................................ 80 3.4.2. Carrier-based Uptake ............................................................................................ 81 3.4.3. eNOS and Cav-1 Dependence .............................................................................. 82 3.4.4. Interpreting the Importance of Lipid Rafts ........................................................... 84 3.5. Conclusion .................................................................................................................. 85 Chapter 4. Mechanistic Insight in to the Functional Significance of F92 on eNOS Regulation .............................................................................................................................. 87 4.1. Introduction ............................................................................................................... 88 4.2. Methods ...................................................................................................................... 89 4.2.1. Cell Culture ........................................................................................................... 89 4.2.2. Velocity Gradient Centrifugation ......................................................................... 89 4.2.3. NO Analysis.......................................................................................................... 89 x  4.2.4. Sucrose Fractionation ........................................................................................... 90 4.2.5. Immunofluorescence............................................................................................. 90 4.2.6. Western Blotting ................................................................................................... 91 4.2.7. Data Analysis ........................................................................................................ 91 4.3. Results ......................................................................................................................... 91 4.3.1. Peptide Treatment and the Biochemical Properties of Cav-1 ............................... 91 4.3.2. Subcellular Localization of eNOS and Cav-1 Not Affected by F92A ................. 92 4.3.3. F92A Substitution Does Not Affect eNOS and Cav-1 Co-localization ............... 97 4.3.4. F92 Inactivation Blunts Akt Signaling ................................................................. 97 4.4. Discussion ................................................................................................................... 99 4.4.1. Characterization of Critical Residue F92 ........................................................... 101 4.4.2. Peptide-induced Redistribution of Cav-1 ........................................................... 101 4.4.3. Akt and Regulation by CAV............................................................................... 102 4.5. Conclusion ................................................................................................................ 103 Chapter 5. General Discussion and Conclusion ............................................................... 104 5.1. Review of Findings ................................................................................................... 105 5.2. Significance of Findings .......................................................................................... 106 5.3. Applications of Research ......................................................................................... 108 5.4. Concerns with NO Upregulation ............................................................................ 110 5.5. NOS-related Pleiotropic Effects ............................................................................. 111 xi  5.6. F92 Versus F92A in Therapies Targeting Endothelial Dysfunction ................... 112 5.7.  Non-Cardiovascular Side Effects of CAV-derived Peptides ............................... 113 5.8. Future Studies .......................................................................................................... 113 5.9. Conclusion ................................................................................................................ 114 References ............................................................................................................................ 115    xii  List of Tables Table 1. Primer Sequences Used to Generate Library of Plasmids ................................. 41 Table 2. Primers Used for Sequencing ................................................................................ 42 Table 3. Comparison of the Scaffolding Domains of Cav-1, -2 and -3 ............................. 61     xiii  List of Figures Figure 1. Endothelial Dysfunction ........................................................................................ 4 Figure 2. Dynamic Post-translational Modifications Regulate eNOS Activity ............... 18 Figure 3. NO Production can be Regulated by Physical Interactions ............................. 23 Figure 4. Mutant Caveolin-1 Peptides for NO Release ..................................................... 35 Figure 5. F92A Substitution Does Not Affect eNOS Binding ........................................... 47 Figure 6. eNOS Binding is Cav-1 Scaffolding Domain Dependent .................................. 49 Figure 7. eNOS Binding Motif is Located in the B and C Sub-domains ......................... 50 Figure 8. Residues 90-95 of B Sub-Domain Contribute to eNOS Binding ...................... 52 Figure 9. Residues 95-99 of C Sub-domain Contribute to Bulk of eNOS Binding ......... 53 Figure 10. Residues 90-99 Together Account for Majority of eNOS Binding ................ 54 Figure 11. Wild Type and F92A eNOS Binding Site Peptide (BSP) Has Similar Affinity for eNOS as the Full-length Scaffolding Domain Peptide ................................................. 56 Figure 12. Computational Analysis of Binding Site Peptide (BSP) with eNOS .............. 58 Figure 13. Loss of F92 Prevents Inhibition of Stimulated NO ......................................... 70 Figure 14. Both BSP and BSPF92A-derived Peptides Increase NO Release from Endothelial Cells ................................................................................................................... 72 Figure 15. F92A Substitution Does Not Affect Peptide Uptake........................................ 74 Figure 16. Peptide-stimulated Release is eNOS Dependent .............................................. 76 Figure 17. Peptide-stimulated Release Not Affected by Cav-1 Knock-down .................. 77 Figure 18. Peptide-stimulated NO is Regulated by Lipid Rafts ....................................... 79 Figure 19. F92A Substitution Does Not Affect Cav-1 Oligomerization ........................... 93 Figure 20. F92A Substitution Does Not Affect Cav-1 Targeting to Cholesterol Enriched Fractions ................................................................................................................................ 95 Figure 21. F92A Substitution Does Not Affect eNOS Targeting to Cholesterol Enriched Fractions ................................................................................................................................ 96 Figure 22. F92A Substitution Does Not Affect eNOS and Cav-1 Co-localization .......... 98 Figure 23. F92 Promotes Akt-mediated NO Release ....................................................... 100 Figure 24. Hypothesized Sequence of Events Based on Mechanistic Findings ............. 107  xiv  List of Abbreviations a.a.   Amino acid(s) ADMA  Asymmetric dimethy-L-arginine Akt   Protein Kinase B ANOVA  Analysis of variance AP   Antennapedia peptide BAEC   Bovine aortic endothelial cells BH4   Tetrahydrobiopterin BSP   Binding site peptide BSPF92A  Binding site peptide (F92A substitution) CaM   Calmodulin Cav-   Caveolin (i.e. Cav-1, Cav-2 or Cav-3 isoforms) CAV   Caveolin-1 scaffolding domain CEM   Cholesterol-enriched membrane cGMP   Cyclic guanosine monophosphate CVD    Cardiovascular diseases DMEM  Dulbecco’s Modified Eagle Medium DTT   Dithiothreitol EDTA   Ethylenediaminetetraacetic acid eNOS   Endothelial nitric oxide synthase FAD   Flavin adenine dinucleotide FMN   Flavin mononucleotide GPCR   G-protein coupled receptor GST   Glutathione-S-transferase GTP   Guanosine triphosphate HSP-90  Heat shock protein 90 iNOS   Inducible nitric oxide synthase Kd   Dissociation Constant LB   Luria Broth  L-NAME  N-nitro-L-arginine methyl ester mCD   Methyl-β-cyclodextrin MBS   Mes buffered saline Myr   Myristoylated/Myristic acid PI3K   Phosphatidylinositol-4,5-bisphosphate 3-kinase xv  NADPH  Nicotinamide adenine dinucleotide phosphate NMMA  Monomethyl-L-arginine nNOS   Neuronal nitric oxide synthase NO   Nitric oxide nsRNA  Non-silencing ribonucleic acid ONOO¯  Peroxynitrite PBS   Phosphate-buffered saline PCR   Polymerase chain reaction PMSF   Phenylmethylsulfonyl fluoride RT   Room temperature SDS PAGE  Sodium dodecyl sulfate polyacrylamide gel electrophoresis S.E.M.   Standard error of the mean sGC   Soluble guanylyl cyclase siRNA   Silencing ribonucleic acid STE   Sodium-tris-EDTA buffer STET   STE with 1% triton-X TBS   Tris-buffered saline     xvi  Acknowledgements  I would like to first thank Dr. Pascal Bernatchez for mentoring me through the PhD process. In addition, I would like to thank my committee members Drs. Catherine Pang and Ivan Robert Nabi for their support and insights over the duration of my graduate studies.   This body of work would not have been possible without support from past and current members of the lab. In particular, there are three members I’d like to thank. Firstly, Arpeeta Sharma for the early phase of this project, and introduction to Dr. Bernatchez’s laboratory.  Secondly, Dr. Dmitri Pavlov for help, encouragement and support, from lab ordering to running experiments.  Lastly, I would like to thank Matti Hiob for his insights and company during the late hours of the evening when things got busy.  My research has been supported by the Canadian Institutes of Health Research which has provided me with a graduate award. Furthermore, I would like to extend my thanks to the Heart & Stroke Foundation, CIHR, and Michael Smith Foundation for Health research for providing research funding to carry out the experiments.    Last, but not least, I would like to thank my friends and family for their support and words of encouragement throughout the entire process.1          Chapter 1. General Introduction   2  1.1. Overview of Thesis Nitric oxide (NO) and its regulation is an essential component of vascular health. Endothelial dysfunction, marked by a reduction in systemic NO bioavailability, has been associated with a host of cardiovascular diseases. As such, there are many therapies utilized in modern medicine that target systemic NO signaling, perhaps best known of which are drugs like nitroglycerin or sildenafil, used in ischemic heart disease and erectile dysfunction respectively. Because of the inherent importance of the NO-signaling axis, there is a large amount of interest and research in the area to try and identify new therapies. Herein, we detail our approach to identify a novel avenue to promote NO signaling. Specifically, we study the endothelial NO synthase/caveolin-1 signaling interaction in order to characterize its properties and evaluate its potential in the field of cardiovascular therapies. 1.2. Cardiovascular Disease  Cardiovascular disease (CVD) is a general term referencing a family of cardiovascular conditions, which include coronary heart disease, cerebrovascular disease, hypertension and peripheral arterial disease, amongst others. According to the World Health Organization, in 2012, 31% of global deaths (17.5 million) were the result of CVD [1]. Of the vascular-related deaths, 7.4 million were the result of coronary heart disease, while stroke accounted for 6.7 million.  Given the large social and economic impact of CVD, there is a high level of interest in its prevention, diagnosis and treatment. One of the hallmarks of CVD is endothelial dysfunction, a clinical condition characterized by an impairment of the vascular endothelium, the inner most lining that is responsible for homeostasis of the vascular system.  3  1.3. Endothelial Dysfunction  The endothelium is responsible for regulating vasodilation, inhibiting smooth muscle cell (SMC) proliferation and migration, reducing leukocyte adhesion and migration and reducing platelet aggregation and adhesion, and other functions required for proper homeostasis. In the mid 1980’s, researchers observed blunted endothelium regulated relaxation of blood vessels from hypertensive rats [2,3] and hypercholesteremic rabbits [4] in comparison to control animals. These observations were further extended to human patients with coronary atherosclerosis [5]. Thus began the concept of “endothelial dysfunction”, a term that describes the transition of the vasculature into a misregulated, pro-inflammatory, pro-coagulatory and proliferative state [6,7], which has been found to be present in most CVD (Figure 1). Now, it has been shown that endothelial dysfunction can arise from a variety of causes, many of which can be considered classical risk factors for CVD, such as diabetes, smoking, aging, hypertension and dyslipidemia [7,8].  There is a strong interest in understanding and treating endothelial dysfunction because it possesses prognostic and diagnostic value within the realm of CVD. In fact, endothelial dysfunction can be considered the first step of CVD. For example, it was found that children and adults with risk factors for atherosclerosis, such as smoking and high cholesterol levels, exhibited signs of endothelial dysfunction without any evidence of atherosclerotic plaque [9]. Moreover, it was found that patients with cardiovascular disorders who showed greater levels of coronary artery endothelium-dependent relaxation in response  to vasodilating acetylcholine were less likely to experience acute vascular events, such as heart attacks and strokes [10]. Similarly, another study that assessed the endothelial function based on forearm blood flow of never-treated hypertensive patients found that those who  4  Endothelial CellLeukocytePlateletSmooth Muscle CellNormal Endothelial FunctionEndothelial DysfunctionCoagulationLeukocyte adhesion & infiltrationSMC proliferation & migration Figure 1. Endothelial Dysfunction  The shift of the vasculature from a normal into a dysfunctional state is associated with an increase in leukocyte adhesion and infiltration, platelet aggregation and vascular smooth muscle cell (SMC) proliferation and migration. This process has been associated with increased risk for cardiovascular events in patients.  5  exhibited poorer levels of dilation were also more likely to experience major adverse events, including coronary revascularization procedures, strokes and heart attacks during the follow-up period [11]. These examples are a small selection of research that indicates that endothelial dysfunction precedes and can predict cardiovascular outcomes. Hence, there is strong support for identifying and treating endothelial dysfunction to mitigate the severity of cardiovascular events. 1.4. Link Between NO and Endothelial Dysfunction One of the hallmarks of endothelial dysfunction is a reduced level of NO within the vasculature. This observation has been demonstrated in patients with endothelial dysfunction.  For example, a study in diabetic patients showed that NO regulatory proteins were misregulated [12]. Moreover, patients with peripheral arterial occlusive disease, a more general term describing the narrowing of arteries by atherosclerotic plaques outside of the heart and brain, were found to have reduced levels of urinary nitrate, a by-product of NO metabolism, in comparison to controls [13]. It was also shown that there is a positive correlation between serum levels of endogenous NO inhibitors and the degree of coronary vessel thickness in atherosclerotic patients [14]. Conversely, approaches that improved endothelial function were found to improve NO regulation. For instance, exercise, which has been demonstrated to improve health [15,16], has been associated with improved NO regulation in patients with coronary artery disease [17]. There is a plethora of additional evidence that link NO misregulation to endothelial dysfunction, which suggests that targeted treatment to correct the deficiency could prove beneficial. To ease in to the topic, herein, we will first give a general overview of NO before discussing its generation and physiological role, followed by possible means to promote NO release. 6  1.5. General Overview of NO Cell signaling processes are typically associated with proteins, peptides and chemical transmitters; however, it is becoming increasingly clear that gaseous chemicals, such as carbon dioxide, hydrogen sulphide and NO, play an important physiological role [18]. NO was recognized to be an important physiological mediator of blood vessel relaxation in the 1980s [19,20]. NO can be produced in response to a variety of stimuli including acetylcholine, histamine, thrombin, serotonin, and histamine [21]. Being a free radical, NO is extremely unstable, with a short half-life of 1.6 milliseconds [22]. Outside of vascular regulation, NO is involved in mediating the bactericidal activity of macrophages [23]; furthermore, NO is a neurotransmitter in the brain and gut [24–28]. In addition, NO interacts with a plethora of transcription factors and proteins via nitrosylation, leading to altered levels of enzymatic or signaling activity. For example, nitrosylation of hypoxia-inducible factor 1α, a transcription factor involved in the response to hypoxia, resulted in increased stability of the transcription factor [29]. Similarly, nitrosylation of G-protein coupled receptor (GPCR) kinase 2, which mark GPCRs for internalization, resulted in reduced activity of the kinase [30]. There are a plethora of processes in which NO play a crucial role and the above are just a small subset of NO’s known physiological properties, with more being identified on a regular basis.   1.6. Production of NO There are two main methods by which NO can be generated. The first, and better characterized, approach is via NO synthases (NOS). Enzymatically, NO is produced by one of three isoforms of synthases: inducible, neuronal or endothelial NOS (iNOS, nNOS and eNOS, respectively), which are named after the tissue from which they were first identified. 7  All NOS are homodimers which share many structural similarities [31]. Each monomer consists of a reductase and oxygenase domain bridged by a calmodulin binding motif. The reductase domain contains a nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) binding site, whereas the oxygenase domain contains the L-arginine, tetrahydrobiopterin (BH4) and heme binding site [32]. Aside from being structurally similar, all 3 isoforms are able to use L-arginine as a substrate to generate NO [33]. The mechanism has been well-studied, and reviewed extensively [32–34]. In brief, NADPH donates electrons to FAD, which transports it from the reductase to the heme moiety/BH4 complex in the oxygenase. It is at this active site that L-arginine and molecular oxygen are catalyzed to form L-citrulline and NO. Besides enzymatic synthesis via one of the three NOS isoforms, an alternate, though less understood, approach by which NO can be produced is through the conversion of exogenous inorganic nitrates and nitrites [35]. This source of NO is dependent on dietary habits and is reliant on a combination of bacterial reductases and ischemic conditions to generate NO. Our research focuses on eNOS, which generates NO to regulate vascular homeostasis and cardiovascular health. 1.7. eNOS Polymorphism and Cardiovascular Disesases Although there have been studies using eNOS knockout animals to demonstrate the significance of eNOS in the vasculature, there are doubts regarding the clinical significance due to the nature of translating animal results to humans. Instead, it may be more interesting to assess the impact associated with eNOS polymorphism in humans. Indeed, one such mutation is the glutamic acid 298 to asparagine (E298D) polymorphism in the eNOS gene. This eNOS protein resulting from the E298D substitution was suggested to be more 8  susceptible to cleavage versus its non-substituted counterpart [36]. Patients in the United Kingdom with an E298D polymorphism in their eNOS gene were found to have significantly greater odds of having coronary artery disease or experiencing a myocardial infarction [37] and to have reduced levels of collateral vessel development [38]. In addition, in subjects possessing the E298D substitution, elevated resting systolic blood pressure was observed when compared to subjects lacking the substitution [39]. Similarly, a coding polymorphism, thymine 789 to cytosine (T789C), in the promotor region is associated with decreased levels of eNOS mRNA and protein expression [40]. Similarly, this polymorphism has been shown to correlate with increased risk of coronary artery disease and cardiac problems [41,42]. In fact, artery ring preparations from patients with either, or both, E298D and T789C, had significantly blunted response to acetylcholine, compared to control biopsy samples [43]. There is a clear indication that eNOS, the main source of vascular NO, has a great influence on vascular health and the prognosis of CVD. To better understand the functional significance of eNOS in the vasculature, we need to understand the function of NO. 1.8. Origins of Vascular NO Research In 1980, the endothelium was shown to be  essential for inducing smooth muscle relaxation of arteries by Furchgott [44]; however, it was not until 1987 that the previously termed endothelium-derived relaxing factor was identified to be NO [19]. In 1989, an NADPH-dependent enzyme that used L-arginine as a substrate was identified [45]. Finally, in 1992, researchers cloned a 140 kDa protein which we have come to know as eNOS [46,47], leading to a burgeoning of research in an attempt to characterize its physiological role. 9  Since then, even though eNOS has been identified in a variety of tissues (including the placenta [48,49], adipose tissue [50], peritoneal mesothelial cells [51], testis (epithelium, Leydig cells and Sertoli cells [52]) and various regions of the brain, such as the olfactory bulb, cerebellum and hippocampus [53], it is still best known and recognized for being the main producer of vascular NO [54] and has a plethora of roles within the vasculature.  1.9. Role of NO in Vascular Health and Disease As mentioned, in order to understand the role of eNOS in health and disease, it is important to first understand the role of NO in the vasculature. To many, vascular NO is typically associated with arterial vasodilation. Typically, upon exposure to sheer stress, the endothelium releases NO [55,56] which then diffuses and activates soluble guanylyl cyclase in SMC, resulting in the conversion of cyclic guanosine triphosphate (GTP) to guanosine monophosphate, more commonly known as cyclic guanosine monophosphate (cGMP) [57,58]. The production of cGMP leads to both a reduction in the entry of Ca2+ into SMC and an increase in myosin light chain (MLC) phosphatase activity [59,60], resulting in a two-pronged approach to inducing relaxation. The reduction in intracellular Ca2+ by NO diminishes the activation of calmodulin, which is responsible for activating MLC kinase to phosphorylate the MLC to promote contraction. In contrast, the MLC phosphatase serves to dephosphorylate any existing phosphorylated MLC to induce relaxation.  1.9.1. NO and SMC NO has a much larger role in the vasculature than just regulation of vascular tone. For example, both NO gas and NO-donors, sodium nitroprusside (SNP) and nitroglycerin, were found to inhibit DNA synthesis in SMC, resulting in reduced proliferation of SMC [61,62]. Furthermore, NO donors S-nitroso-N-acetylpenicillamine and SNP were also shown to 10  reduce the rate of synthesis of both total protein and collagen, an extracellular matrix component involved in vascular remodeling (e.g. during atherosclerosis), in SMC [63]. Similarly, there is evidence suggesting that NO could contribute to SMC apoptosis [64–66]. In addition to proliferation and apoptosis, NO has also been suggested to reversibly inhibit SMC migration [67]. 1.9.2. NO and Inflammation NO also plays a large role in regulating inflammation and maintaining the integrity of the vascular endothelium. Similar to its effect on SMCs, NO can inhibit the proliferation of immune cells [68] and mediate cellular apoptosis [69,70]. In addition, NO has been shown to reduce leukocyte adhesion, migration and rolling in an in vivo preparation [70]. This is in part mediated by NO’s ability to reduce endothelial activation in response to noxious stimuli. In fact, flow-induced endothelium-derived NO can repress expression of vascular cell adhesion molecule-1 (VCAM-1), a cell surface protein involved in monocyte adhesion, following exposure to inflammatory stimuli such as cytokine interleukin-1 [71–73]. 1.9.3. NO and Platelet Activation NO also plays an important role in the regulation of platelet activity. Both exogenously applied and endothelium-stimulated NO have been shown to significantly reduce platelet adhesion to endothelial cells via a combination of upregulating cGMP, preventing influx of calcium in to the cytoplasm of platelets and blunting thrombogenic signaling pathways [74–78]. In addition, to prevention of platelet activation and platelet aggregation [79], NO also promotes platelet disaggregation [80]. Interestingly, platelets appear to be extremely sensitive to regulation by NO, as levels of NO sufficient to prevent 11  aggregation does not affect the blood pressure of patients undergoing coronary angioplasty [81].   1.9.4. NO and Neovascularization Another important role that NO plays is in the regulation of neovascularization, which includes the de novo formation of new vessels, formation of capillaries from a pre-existing bed, and the remodeling of pre-existing arterioles into arteries [82,83]. This is an important process for wound repair, as well as vascularization of ischemic tissue (e.g. following myocardial ischemia). Neovascularization is a complicated process that is still under intense investigation. Loss of vascular NO was shown to significantly reduce recruitment of endothelial progenitor/stem cells at the damaged tissue, and to decrease neovascularization [84]. Furthermore, NO plays a role in decreasing endothelial cell apoptosis [85,86] and increasing endothelial cell migration [87] . In addition, blockade of NO was found to elevate interstitial fluid expression of angiostatin, an endogenous inhibitor of angiogenesis, which resulted in reduced capillary density in an ischemia model of neovascularization [88]. Similarly, NO has been shown to be a critical mediator in signaling of vascular endothelial growth factor (VEGF), one of the most potent stimuli of angiogenesis [89]. 1.10. Linking NO loss to Cardiovascular Disease Symptoms Given that NO has such important vascular roles, researchers believe that endothelial dysfunction resulting in deficiency of NO production may lead to CVD. To provide some examples, patients with pulmonary hypertension (characterized by elevated pulmonary vascular resistance, medial hypertrophy, intimal proliferation and thrombosis [90]) were observed to have reduced levels of eNOS expression, which correlated with severity of the 12  disease [91]. Similarly, coronary artery disease is associated with increased expression of cellular adhesion molecules, macrophage penetration and risk of thrombus formation [44,92–95], which can be considered events inhibited by NO under normal situations. On a similar vein, reduced levels of basal NO was linked to elevated levels of leukocyte adhesion following reperfusion in a feline model of myocardial ischemia [96]. Lastly, Abaci et al speculated that patients with diabetes mellitus had poor development of coronary collateral blood vessel [97], a neovascularization event important for surviving myocardial infarction [98], due to the presence of endothelial dysfunction and impaired NO production [99–101]. While other factors may also be involved, many of the characteristic symptoms of CVD can be associated with impaired regulation of NO. 1.11. Basis for Targeting eNOS-derived NO in Endothelial Dysfunction Thus far, we have discussed the linkage between a decrease in NO and cardiovascular diseases; however, it is just as important to assess the other side of the coin: whether an increase in NO or relevant markers is associated with improved cardiovascular outcomes.  For example,  statins, which are widely used to reduce cholesterol levels, have been demonstrated to improve cardiovascular outcomes [102]. However, in recent years, clinical studies suggest that pleiotropic effects, those not associated with serum cholesterol reduction, may contribute to the improved cardiovascular outcomes [103,104]. Moreover, findings suggest that improved NO regulation could contribute to the observed positive outcomes. In patients with arterial disease, those who were taking lipid lowering medication, demonstrated improved dilation in the forearm in response to serotonin; furthermore, this improvement could not be improved by co-injection of L-arginine [105], which suggested that the initial improvement was NO-based. Similarly, the use of statins has been associated with increased 13  eNOS expression [106] and increased eNOS activity via elevation of relevant signaling cascades [107,108]. Since impaired NO production can lead to endothelial dysfunction, there may be merit in upregulating NO production to improve endothelial function and cardiovascular outcomes.  1.12. NO Availability Versus NO Signaling in Endothelial Dysfunction There is evidence to suggest that endothelial dysfunction is associated with an insufficiency in NO levels, but not impaired NO signaling. For example, it was found that systemic levels of NO in hypertensive patients were around half of the control levels up to 36 hrs following a bolus injection of labeled L-arginine, indicating an impairment in available NO [109]. Moreover, pre-treatment with NG-monomethyl-L-arginine (L-NMMA), an eNOS inhibitor, was found to reduce acetylcholine-induced vasodilation in healthy individuals, but not in hypertensive patients [110,111]. The lack of response to L-NMMA in hypertensive patients was associated with failure to increase NO availability and not NO signaling, as sodium nitroprusside, an NO donor, was found to have similar effects in both hypertensive and non-hypertensive patients [111]. Hence, the common believe is that there is an impairment with maintaining an appropriate level of NO, as opposed to impaired NO signaling responses. Furthermore, this suggests that it may be therapeutically beneficial to either target the cause behind the reduction in NO availability or focus on alternative approaches to improve NO production. 1.13. Enzyme Expression in Endothelial Dysfunction A direct reduction in the level of eNOS expression is perhaps the most straightforward reason for reduced bioavailability of NO; however, there is limited research regarding this issue, and often times there are conflicting reports. For example, one study 14  suggests that atherosclerotic arteries have reduced eNOS expression compared to normal mammary arteries from the same patient [112]. On the other hand, there are reports suggesting that eNOS is both up- [91] and down-regulated [113,114] in patients with pulmonary hypertension. There does not appear to be sufficient evidence to draw any conclusions on the status of eNOS expression in the presence of endothelial dysfunction; however, research suggests that other factors may play a role in reducing NO bioavailability. More specifically, either NO is being scavenged or there exists a problem with its production. 1.14. Targeting Superoxide: A Natural NO Sink In the presence of endothelial dysfunction, there is a tendency to have elevated levels of superoxide anions. Due to the high rate constant for the reaction between superoxide and NO [115], should NO and superoxide be in the same compartment, NO is likely to be rapidly scavenged to form peroxynitrite (ONOO-), thereby failing to reach its target site of action. The impact of superoxide on NO can be verified in experiments where the usage of superoxide dismutase, which breaks down superoxide, was shown to increase the level of NO produced. Furthermore, ONOO- may also destabilize the eNOS dimer and react with the heme moiety, leading to inactivation of the eNOS protein [116]. However, from a more clinical standpoint, the significance of NO scavenging and its significance in driving endothelial dysfunction is unclear, as clinical trials utilizing anti-oxidants have produced  mixed results [117]. The discrepancy may be due to confounding generalized oxidative stress with endothelial oxidative stress. 1.15. Targeting eNOS for Better Vascular Health Given 1) the inability to differentiate between different types of oxidative stress, and 2) the strong recognition that NO is a critical player in endothelial dysfunction, researchers 15  have been trying to identify approaches to increase NO. Classically, organic nitrate donors, such as sodium nitroprusside or nitroglycerin have been used as sources of NO; however, there are severe limitations associated with such an approach due to non-specificity and possible development of tolerance [118]. In order to counter this, there are various indirect and direct approaches that are being investigated. Indirectly, NO signaling could be potentiated via enhancing cGMP signaling. One such example is the use of sildenafil, an inhibitor of phosphodiesterase type 5, the enzyme responsible for breaking down cGMP [118], which has been shown to be beneficial in cardiovascular complications [119–121].  An alternative approach is to stimulate sGC directly with compounds such as riociguat, which has been found to be beneficial in patients with pulmonary arterial hypertension [122–125].  In contrast, recently, there have been more direct attempts at altering eNOS activity. For example, compounds such as AVE9488 and AVE3085, which enhance eNOS expression and activity [126], were found to have cardioprotective effects in different animal models of cardiovascular complications, such as diabetes [127] and atherosclerosis [126], and could improve endothelial function in spontaneously hypertensive rats [128]. Alternatively, gene transfer of the eNOS enzyme into rats that underwent balloon-injury to the carotid artery, was found to reduce restenosis [129], wherein SMCs hyperproliferate, resulting in re-occlusion of the vessel. Similar to these approaches, we are interested in enhancing NO bioavailability by directly targeting eNOS. To get a better understanding of our approach, we will first delve into more detail regarding eNOS and its regulation. 16  1.16. eNOS Regulation It is important to investigate how eNOS is regulated in order to develop targeted therapies. eNOS regulation can be subdivided into five categories: transcriptional, post-translational modifications, substrate accessibility, co-factor availability and direct protein-protein interactions. We will focus on some of the more well-defined aspects of these categories. 1.16.1. Transcriptional Regulation of eNOS Like many other proteins, eNOS mRNA and expression can modulated by physiological and pathophysiological stimuli or an external stimulus, such as statins, which was discussed in Section 1.11. For example, in vivo [130] and in vitro [131,132] studies have demonstrated that eNOS expression is upregulated following exposure to sheer stress, in part by increasing 3’-adenylation of the eNOS mRNA [132] and activation of the c-src signaling pathways [133]. In addition to sheer stress, another example of a physiological stimulus is hypoxia, which has been shown to be able to both up- and down-regulate eNOS mRNA, depending on conditions. In vitro studies have demonstrated that hypoxia can inhibit eNOS expression in endothelial cells by decreasing the rate of transcription and reducing mRNA stability [134], potentially by inducing expression of eNOS antisense sONE, which leads to breakdown of the mRNA [135]; however, some studies have suggested that hypoxia can upregulate eNOS RNA expression [136,137]. As mentioned, aside from physiological stimuli, pathophysiological settings can also alter transcriptional regulation. For example, oxidative stress has also been known to modulate the expression of eNOS. In vitro studies have demonstrated that oxidative stress such as hydrogen peroxide and oxidized low density lipoprotein (ox-LDL), a major player in 17  atherosclerosis progression and endothelial dysfunction, can increase eNOS mRNA in a concentration and time dependent manner [138,139]; however, it has also been shown that exposure to ox-LDL can reduce eNOS mRNA transcription and activity [140]. These results suggest the existence of a complex relationship between experimental conditions and mRNA stability. 1.16.2. Acylation and Subcellular Localization There are three major modifications by which eNOS can be regulated post-translation, the best known of which are acetylation and phosphorylation, whilst S-nitrosylation is a relatively new area of study (Figure 2). With respect to acetylation, eNOS can be myristoylated at glycine 2 (G2) or palmitoylated at cysteine 15 and 26 (C15 and C26) [141,142]. The covalent N-myristolyation of G2 on eNOS is necessary and sufficient for membrane and Golgi association, as confirmed in experiments where alanine substitution of the glycine residue resulted in almost all eNOS shifting from the membrane to the cytoplasm [143,144]; furthermore, myristoylation is required for full eNOS activation within the cellular environment [145]. In addition to myristoylation, eNOS can also undergo palmitoylation at C15  and C26 [146]; however, this process is dependent on the presence of myristoylation [147]. Palmitoylation, similar to myristoylation, helps eNOS target to the membrane fractions [146]. However, unlike with myristoylation, palmitoylation appears to be reversible and is thought to dynamically regulate eNOS localization in response to stimuli [147]. Subsequently, it was demonstrated that palmitoylation specifically promoted the targeting of almost all cellular eNOS to microdomains in the plasma membrane known as caveolae [148,149].  It should be noted that myristoylation and palmitoylation are required only for efficient response to stimuli, but not catalytic activity, as loss of acylation did not  18   Figure 2. Dynamic Post-translational Modifications Regulate eNOS Activity Endothelial NOS (eNOS) activity is highly regulated by a series of post-translational modifications. This includes phosphorylation, nitrosylation and palmitoylation. The effect of phosphorylation on eNOS activity is site-dependent, with serine 114 (S114), threonine 495 (T495) and tyrosine 657 (Y657) reducing eNOS activity. However, phosphorylation of Y81, S615, S633 and S1177 promote eNOS activity and are thought to play an important role in basal NO production. In contrast, S-nitrosylation at cysteines 94 (C94) and 99 (C99) are thought to reduce eNOS activity. Lastly, reversible palmitoylation at C15 and C26 helps promote targeting to the membrane for efficient NO synthesis.   eNOS Activity eNOS ActivityeNOSP P PPPPeNOSeNOSY657T495S114PY81S615S633S1177C94C99Phosphorylation:S-Nitrosylation:Phosphorylation:Palmitoylation:eNOSC15C2619  affect the catalysis kinetics of radiolabelled L-arginine into 3H-L-citrulline [143,150], which indicated that subcellular localization was an important regulator of the NO response. At this point, we’ve touched on the two major pools of eNOS: the plasma membrane and Golgi pools. Both of these pools behave differently in response to stimuli, which bears implications for their intended function. Zhang et al. found that plasma membrane eNOS was more responsive to stimulus versus their Golgi-counterpart, but was also more sensitive to plasma cholesterol changes [151]. Furthermore, even within the Golgi itself, localization plays an important role – trans-Golgi eNOS was markedly less active when compared to cis-Golgi eNOS in response to stimuli, as well as at rest [152]. The physiological implications of these findings are still an active area of investigation. However, it demonstrates the importance of acetylation on the regulation of eNOS localization and the subsequent impact on function. 1.16.3. Phosphorylation  Phosphorylation is an important regulator of eNOS activity and can be considered a major post-translational regulator of eNOS function. The human eNOS possesses two tyrosine (Y81 and Y657), four serine (S114, S615, S633 and S1177) and one threonine (T495) phosphorylation sites, for a total of seven [141]. It should be noted that different species may have an equivalent phosphorylation site at a similar position (e.g. S1179 in bovine); however, we will reference the human equivalent herein for simplicity. Of the seven phosphorylation sites, S1177 and T495 are probably the best understood, while tyrosine phosphorylation is the least understood. In part, this is due to the difficulty associated with researching tyrosine phosphorylation, as some groups could not find evidence of phosphotyrosine [153,154], while others only detected it as a minor species [55,155], making 20  it an elusive site to investigate. Regardless, tyrosine phosphorylation was known to occur since the mid 1990’s, but the tyrosine phosphorylation sites Y81 [156] and Y657 [157] have only been confirmed in the past decade, and appear to have opposing effects. Specifically, while phosphorylation of Y81 by v-src promotes NO [156], phosphorylation of Y657 by proline-rich tyrosine kinase 2 (PYK2) results in reduced eNOS activity.  In contrast, the serine sites on eNOS tend to favor the activation of eNOS; however, S114 can be considered an outcast in that it has been shown to be the only serine site capable of attenuating NO release by promoting increased interaction with caveolin-1 (Cav-1), a known inhibitor of eNOS activity, which will be explored at a greater length at a later point [158]. Of the remaining three serine sites, S1177 is the best characterized, as mentioned previously, likely due to its role in cardiovascular regulation. For example, infection of endothelial cells with a non-phosphorylatable S1177A eNOS mutant, where the serine is replaced with alanine, resulted in significantly lower levels of acetylcholine-induced vasodilation versus the S1177D mutant, which mimics phosphorylation of eNOS at site S1177 via an aspartic acid substitution [159]. Similarly, transgenic mice with the S1177D mutation show better cerebral blood flow and smaller stroke infarct sizes versus the S1177A mice [160]. Thus far, there have been several kinases that have been shown to phosphorylate S1177 including AMPK [161], protein kinase B (Akt) [162], protein kinase A [162] and calcium-calmodulin kinase kinase (CaMKK) II beta [163]. Mechanistically, phosphorylation of S1177 is thought to enhance electron flux through the reductase domain and allows eNOS to produce NO at resting levels of calcium by enhancing interaction of eNOS with the calcium sensing protein, calmodulin [164,165]. S633 is similar to S1177 in that phosphorylation of the site allows eNOS to remain active at low intracellular calcium levels 21  [166]. Temporally, S633 occurs more slowly than S1177 phosphorylation, suggesting that it may have a role in sustaining basal NO release [167]. Interestingly, the last serine site, S615, also appears to assist S1177. Dual phosphorylation of S615 and S1177 has been shown to result in greater levels of NO production than just phosphorylation of S1177 alone [168]. This is potentially due to the ability of S615 to further increase sensitivity of eNOS to intracellular calcium when compared to just phosphorylation of S1177 alone [169].  For this reason, both S633 and S615 are thought to play an important role in regulating basal NO. The final phosphorylation site, T495, can be phosphorylated by either protein kinase C (PKC) [163] or adenosine monophosphate (AMP)-activated protein kinase (AMPK) [161]. The phosphorylation of T495 results in decreased affinity of eNOS for calmodulin, leading to decreased sensitivity to Ca2+ and NO production [163]. As such, it has been considered by some to be the counterbalance of S1177. However, there is evidence suggesting that S1177 phosphorylated eNOS can maintain elevated levels of NO production even in the presence of T495 phosphorylation [170]. Conversely, it has also been observed that T495 can be dephosphorylated with no significant changes in S1177 status [171]. Lastly, T495 has also been suggested to help reduce eNOS derived superoxide at basal Ca2+ levels [172]. Unsurprisingly, the phosphorylation of multiple sites in eNOS is a dynamic process that has yet to be truly understood, and is still an area of active investigation. 1.16.4. S-Nitrosylation Similar to phosphorylation, there is a post-translational modification known as S-nitrosylation, wherein NO reacts with exposed cysteines on protein to generate SNO [173].  In recent years, it is being increasingly recognized as an important regulator of cellular function, and as recent as 2009, it was shown that over a hundred proteins were nitrosylated 22  under static conditions, and sheer stress could further promote eNOS-dependent nitrosylation of at least 12 different proteins [174]. With respect to eNOS itself, there are two nitrosylation sites – cysteine 94 and 99 [175]. Nitrosylation of the sites have been shown to inhibit eNOS activity [175], suggesting it may serve as a negative feedback mechanism to terminate signaling at the plasma membrane. The mechanism for this inhibition is still unclear. One report suggested that nitrosylation can lead to monomerization of eNOS [176],  although this has been disputed [175]. 1.16.5. Substrate Accessibility and the Arginine Paradox In addition to direct modification of eNOS, which are difficult to target for therapeutics, there exists pharmacologically targetable interactions that can govern eNOS activity (Figure 3). One such example comes from the arginine paradox – where exogenously applied L-arginine can increase NO production, even though extracellular levels of L-arginine exceed the Michaelis-Menton constant for eNOS by 15-30 fold [177]. One possibility put forth to address this strange observation is that the level of eNOS inhibition is elevated in endothelial dysfunction. The two hypothesized culprits are L-NMMA and NG,N’G-dimethy-L-arginine (ADMA), which are endogenous competitive inhibitors of  NO synthases; however, the majority of the focus has been on ADMA, as its plasma concentration is 10-fold that of  L-NMMA [178]. ADMA is formed following breakdown of protein containing methylated arginine [179] and is found elevated across a plethora of conditions where endothelial dysfunction is present including pulmonary hypertension [180], hypertension [181], atherosclerosis [182] and diabetes [183]. In fact, it is considered a predictor of cardiovascular events such as myocardial infarction, stroke and all-cause    23  Caveolin-1eNOSL-ArginineNOBH4L-ArginineROSL-ArginineNOL-ArginineNOADMACaMHSP90NOBH4BH4 Figure 3. NO Production can be Regulated by Physical Interactions Nitric oxide (NO) production is regulated by a series of pharmacologically targetable interactions. Depletion of tetrahydrobiopterin (BH4) leads to endothelial NO synthase (eNOS) uncoupling and the formation of reactive oxygen species (ROS). Binding of eNOS to caveolin-1 leads to inhibition of eNOS activity. Asymmetric dimethyl arginine (ADMA) can act as a competitive inhibitor to L-arginine, leading to reduced NO synthesis. While the above are associated with reduced NO synthesis, binding of heat shock protein 90 (HSP90) and calmodulin (CaM) is known to strongly promote NO generation.   24  mortality in diabetes and coronary artery disease [184–186]. While this may suggest that L-arginine supplementation may be useful in overcoming the competitive inhibition of eNOS by ADMA, clinical results have been mixed, with some reporting beneficial effects [187–190], and others reporting no effect [191–193]. While it may prove beneficial in the future, more studies are required.  1.16.6. Co-Factor Insufficiency and eNOS Uncoupling Under normal conditions, eNOS is an invaluable source of NO; however, under certain situations, it may instead produce superoxide/reactive oxygen radicals. This is known as eNOS uncoupling, wherein continued transfer of electrons from NADPH and reduction of O2 is not paired with reduction of L-arginine, and hence no NO is formed. While it is a normal process that may occur with aging, as shown in an aged mice model [194], it is also found to be exacerbated in disease states characterized by endothelial dysfunction, such as atherosclerosis, hypertension and diabetes [195]. Although the actual mechanism is unclear, eNOS uncoupling is associated with an increase in the monomeric form, as opposed to the homodimeric form, of eNOS [194,196]. Since BH4 has been shown to promote NOS dimer stability [31], it has been hypothesized that uncoupling is due to reductions in BH4 levels. The significance of reduced BH4 in endothelial dysfunction is supported by the observation that improvements in endothelial function were observed in smokers [197], hypercholesteremic subjects [198] and coronary disease patients [199] following administration of the co-factor, and that the improvements could be blocked by N-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS. However, there are limitations to the use of BH4, in part due to how easily it gets oxidized, resulting in a loss of effect [200]. 25  1.16.7. Protein-Protein Interactions  Another approach by which eNOS can be regulated is through the use of protein-protein interactions. It is still an active area of investigation with many unknowns. Three of the better documented direct interactions with eNOS include calmodulin, HSP90 and Cav-1. Unlike iNOS, eNOS, along with nNOS, were considered to be calcium dependent enzymes [201]; however, these enzymes do not react to Ca2+ directly. Instead eNOS senses changes in calcium indirectly by binding to CaM via the CaM binding motif. When intracellular calcium increases, Ca2+ occupy the four Ca2+ binding sites on CaM, causing CaM to undergo a conformation change [202–204]. This conformation change allows it to bind to the synthase and helps facilitate the transfer of electrons, and hence NO production, from NADPH to the heme group [205]. HSP90 is a ubiquitously expressed chaperone protein involved in protein folding, translocation and signaling [206]. In vitro studies have shown that HSP90 is recruited to and complexes with eNOS following exposure to external stimuli, such as VEGF and sheer stress, and may help in the stabilization of active eNOS complexes [207]. HSP90 has also been implicated in facilitation of Akt-mediated phosphorylation and promoting CaM-eNOS association, leading to calcium independent NO release [208].  Lastly, as mentioned above, eNOS is known to traffic to caveolae microdomains within the plasma membrane. Within caveolae, eNOS directly associates itself with Cav-1, which leads to the inhibition of eNOS activity [209]. This interaction is mediated by a specific 20 amino acid sequence (a.a. 81-101) within the Cav-1 protein known as the Cav-1 scaffolding domain (CAV) and a short aromatic sequence within the eNOS oxygenase domain known as the Cav-1 binding motif [210,211]. Like all other interactions mentioned 26  thus far, eNOS and Cav-1 have a dynamic relationship. Exposure of endothelial cells to external stimuli leads to the dissociation of eNOS from Cav-1, which is facilitated by HSP90 and calmodulin [212–214]. Subsequently, NO generated by eNOS induces Src activation, which can result in the binding of eNOS to Cav-1 [215]. The association with Cav-1 also helps facilitate the translocation of eNOS between the cytoplasm and plasma membrane [216]. Given that Cav-1 is essentially the only direct endogenous inhibitor of eNOS, it has received a fair amount of interest. The remainder of this chapter will be devoted to discussing caveolae/Cav-1 and their role in regulating cardiovascular health and eNOS. 1.17. Introduction to Caveolae and the Caveolin Family In the 1950s, analysis of cells led to the identification of flask-shaped invaginations in the plasma membrane, which came to be known as caveolae, or “little caves” [217,218], and could increase the area of the plasma membrane by as much as 50% [219]. Since then, we have come to learn that caveolae are specialized lipid microdomains within the plasma membrane that are rich in cholesterol and sphingolipids. Exposure of cells to cholesterol binding agents leads to the flattening of caveolae invaginations, which highlights the need for an adequate level of cholesterol content in the maintenance of caveolae [220,221]. However, the need for cholesterol is common to other vesicle formation processes, and cannot be considered a unique feature of caveolae. Instead, what defines caveolae are its resident proteins. In the past, the caveolar protein, caveolin, was thought to be necessary and sufficient to drive caveolae biogenesis. However, in recent years, an additional regulatory protein, the cavins, has also been identified [222]. While such proteins are important for caveolae formation and regulation, the focus of our discussion will be on the caveolin family, which is 27  still the main area of focus for many researchers. There are three known isoforms of caveolin: Caveolin-1, -2 and -3 (Cav-1, Cav-2 and Cav-3). Of the three isoforms, Cav-1 and Cav-3 are ~65% identical and ~85% similar [223]; in contrast, Cav-2 is ~40% identical and ~60% similar to both Cav-1 and Cav-3 [224,225]. With respect to caveolae biogenesis, the presence of Cav-1 or Cav-3 is essential – the loss of either results in the loss of caveolae invagination. In contrast, Cav-2 by itself is insufficient to promote caveolae biogenesis. Studies have shown that Cav-2 remains localized to the Golgi when expressed alone, and only trafficked to the membrane following co-expression with Cav-1 or Cav-3 to form hetero-oligomers [226–228].  With regards to expression, Cav-1 is fairly ubiquitously expressed and can be found in most major tissue types including, but not limited to, kidney, lung, intestines, heart, pancreas and adipose tissue; however, the challenge is in knowing which cells specifically express Cav-1, as it is expressed in both epithelium and endothelium, a component of many of these tissues [223] . In comparison, Cav-3 is known to be more muscle specific, and has been shown to be expressed in the heart and skeletal muscle [229,230]. Interestingly, SMC appear to express both Cav-1 and Cav-3 [229]. As Cav- 2 does not form caveolae on their own, their expression tends to coincide with either Cav-1 or -3 [224,228]. For the remainder of this introduction, we will be focusing on Cav-1, the major vascular isoform, and its role in cardiovascular physiology. 1.18. Cav-1: The Coat Protein of Vascular Caveolae Cav-1, as its name would suggest, was the first caveolin to be discovered and was predicted to be 178 amino acids in length [231,232]. However, it later became known that there were in fact two isoforms: Cav-1α, which consists of residues 1-178, and Cav-1β, 28  which contains only residues 32-178 [233]. Cav-1α has been shown to be more likely to generate deeper invaginations versus Cav-1β [234] ; in contrast, a report showed that only the beta isoform was phosphorylated following exposure to insulin , which suggests that the missing N-terminal confers some functional properties [235]. However, functional differences between these two isoforms are not typically investigated.  Structurally, Cav-1 can be broadly divided into three sections – a cytoplasmic N-terminal (a.a. 1-101) and C-terminal (a.a. 135-178), along with a hairpin loop (a.a. 102-134) that anchors Cav-1 to the membrane. Within the exposed N-terminal, there are two structural features that are of critical importance to caveolae biogenesis and proper functioning of Cav-1. The first is what is known as the oligomerization domain. Cav-1 is a 20-25 kDa protein; however, within cellular systems, they tend to form large homo-oligomeric structures that exceed 400 kDa [236]. This unique trait has been mapped to residues 61-101 of Cav-1; in fact, just these 40 residues alone could generate oligomeric activity [237]. The second feature is known as the Cav-1 scaffolding domain (CAV), which consists of residues 82-101 and serves two functions: interacting with client proteins [211] and facilitating membrane attachment [238]. Similarly, the C-terminus is also thought to help in assisting with membrane association [238], and may weakly assist with protein binding [210,239]. In addition, there are also three irreversible palmitoylation sites located at residues C133, 143 and 156 of the C-terminal [240]. Interestingly, palmitoylation does not appear to affect Cav-1’s ability to localize to caveolae [241], but instead affects its ability to interact with other proteins [242,243]. Lastly, the membrane spanning domain is thought to be in involved in interacting with other caveolins to generate oligomeric complexes [244].  29  1.19. Physiological Role of Caveolae and Cav-1 Caveolae have many physiological functions within cellular systems, many of which are mediated in part by Cav-1. The functions of caveolae can be broken down into three separate categories: cholesterol regulation, vesicular trafficking and signal transduction. 1.19.1. Cholesterol Regulation As we mentioned above, the formation of caveolae is highly dependent on the presence of cholesterol. Unsurprisingly, Cav-1 and cholesterol share an intricate relationship. Cav-1 itself has been shown to complex cholesterol in a 1:1 ratio [245]. This property allows Cav-1 to transport newly synthesized cholesterol from the endoplasmic reticulum to caveolae, where it can then be redistributed [246]. In the same study, the authors showed that presence of Cav-1 in cells could lead to a 4-fold increase in plasma membrane cholesterol levels. Others have also shown that caveolae are a major site for cholesterol efflux [247]. The ability of Cav-1 to target cholesterol to caveolae is thought to play a role in cholesterol removal, with overexpression of Cav-1 in cells being linked to increased levels of efflux [248]. Similarly, caveolae have been implicated in the uptake of extracellular cholesterol, with labeled cholesterol being disproportionately concentrated in caveolae [247]. It has been suggested that Cav-1/caveolae achieves these outcomes via regulation of proteins involved in the efflux process [249,250].  1.19.2. Vesicular Trafficking Following their discovery in the 1950s, the function of caveolae remained unknown. In the 1970’s, there was speculation that these “plasma membrane vesicles” were involved in the transcytosis of proteins across the endothelium [251]. Then in the 1980’s there was a report that caveolae could mediate endocytosis, providing a further foundation that caveolae 30  may be involved in vesicular trafficking [252]. To support their role in vesicular trafficking, caveolae have been shown to be rich in proteins involved in vesicle formation, docking and fusion [253]. However, the details surrounding cellular trafficking have proven difficult to elucidate, with conflicting studies being reported. For example, in 2001, the Helenius group initially described the existence of a novel organelle, the caveosome, being involved in caveolar endocytosis [254]; however, a decade later, the same group published a study indicating that the caveosome was an artifact associated with protein overexpression [255]. As a result, there are questions regarding the validity of conclusions drawn from studies that utilize overexpression of labeled Cav-1. However, in 2015, a group using genome-edited cell lines demonstrated that a small fraction of cellular Cav-1 has been shown to be highly mobile and capable of budding from the plasma membrane to participate in vesicular trafficking [256].  1.19.3. Signaling Regulation In the 1990’s, it was observed that glycosyl-phosphatidylinositol (GPI)-linked proteins could be isolated simultaneously with Cav-1, suggesting a linkage between the two [257]. Shortly afterwards, researchers came to realize it was more than just GPI-linked proteins that could be isolated in caveolin-rich membrane domains. This included receptors for cholesterol, cytoskeleton components and signaling proteins, such as G proteins and src kinases, amongst a host of other proteins [258]. Because of the hairpin structure of Cav-1, wherein both terminals were intracellular, it was hypothesized that Cav-1 could potentially act as a protein scaffold. This was confirmed by the identification of a 20 amino acid sequence within Cav-1 (a.a. 81-101) that came to be known as the scaffolding domain, which was shown to directly interact with many of the proteins isolated in Cav-1 enriched cell 31  fractions [259,260]. Thus, it was hypothesized that caveolae could serve as a method to spatially regulate signal transduction, with Cav-1 being responsible for regulating many of the relevant components, via its scaffolding domain. Subsequent investigations identified an aromatic residue rich sequence that was hypothesized to be found in all Cav-1 binding partners, which came to be known as the Cav-1 binding motif [211]; however, there is controversy regarding the validity of this and is still a point of debate. Regardless, the fact that many proteins target to caveolae and can be regulated by Cav-1 is not contested, which highlights the potential of caveolae and Cav-1 as a signal transduction hub. 1.20. Caveolae and Cav-1 in the Cardiovascular System Caveolae are abundantly expressed on the surface of endothelial cells and can cover up to 30% of the endothelial surface [261], which suggest that they may be physiologically important. Perhaps the quickest approach to assessing the functional importance of Cav-1 and caveolae in the vascular system is to study the vascular phenotype of a Cav-1 knock-out animal. Although viable, Cav-1-/- have been shown to have increased microvascular permeability, elevated blood triglycerides, impaired NO regulation, and reduced myogenic tone [262–264]. These abnormalities result in cardiovascular problems such as cardiomyopathy, pulmonary hypertension, and impaired angiogenesis [265,266]. Perhaps one of the most fascinating observations is that although Cav-1 knock out leads to the loss of all non-muscle caveolae (i.e. SMCs, adipocytes, fibroblasts, epithelium, endothelium, etc.), reconstitution of endothelium specific Cav-1 was sufficient to restore all previously studied vascular abnormalities such as cardiac hypertrophy, pulmonary hypertension, aortic ring contraction and relaxation, and microvascular permeability [267]. This highlights the 32  functional importance of endothelial caveolae and Cav-1 in the regulation of cardiovascular homeostasis.  One major area in which caveolae and Cav-1 play a role in is the response to sheer stress, which has important implications for endothelium gene regulation, cellular signaling and ultimately disease development [268,269], in addition to regulation of blood vessel diameter [270]. The conversion of mechanical forces into cellular signaling is known as mechanotransduction. Chronic exposure of endothelial cells to sheer stress increased the formation of caveolae on the surface of endothelial cells versus cells under static condition by increasing targeting of Cav-1 to the plasma membrane [271]. This suggested that caveolae acted as mediators in the mechanotransduction process. In support of the importance of caveolae, vessels isolated from Cav-1 knock out mice exhibited significantly reduced levels of dilation in response to sheer stress versus wild type; however, this deficiency was rescued by performing re-constitution of Cav-1 in the vascular endothelium [272]. But aside from regulating signal transduction, others have observed that caveolae flatten out in response to mechanical stretching, and suggested that it helps to ease raising membrane tension [273]. Beyond these processes, Cav-1 and caveolae have been implicated in angiogenesis, endothelial cell migration and proliferation, and SMC regulation [274]. There is still much to be elucidated regarding the role of caveolae and Cav-1 within the vasculature, given the complex nature of the organelle and its main coat protein. With everything in mind, a re-visit to the eNOS-Cav-1 interaction is warranted. 33  1.21. A Closer Look at the eNOS-Cav-1 Relationship Perhaps one of the best understood Cav-1/partner pairing is the Cav-1/eNOS interaction. Cav-1 plays a major role in the inhibition of basal eNOS activity, as well as the termination of stimulated NO production, as mentioned previously. In aortic rings prepared from Cav-1-/- mice, acetylcholine induced relaxation was significantly increased versus wild type; furthermore, basal NO release from Cav-1-/-  cells were roughly 31% higher [264]. True to its hypothesized role as a spatial regulator of proteins, loss of Cav-1 caused a shift of all eNOS to non-cholesterol rich subcellular fractions in addition to abolishing phosphorylation of S1177 in response to external stimuli [266]. These observations further highlight the intimate relationship between eNOS and Cav-1 in endothelial cells. While the precise mechanism by which Cav-1 regulates eNOS has not been elucidated, specific residues within the scaffolding domain are known to play essential roles. One amino acid in particular is phenylalanine 92 (F92). It was found that substitution of F92 with an alanine (F92A) was sufficient to almost entirely abolish Cav-1’s ability to inhibit eNOS activity, with threonine 90 and 91 playing a smaller role [275]. 1.22. Experimental Results of Cav-1 Based Peptides in Cardiovascular Settings The observation that substitution of F92 with alanine could lead to the loss of inhibition by Cav-1 led to the idea that maybe a Cav-1 scaffolding domain sequence with a substituted F92 residue could potentially act as an antagonist. Initial proof of concept was demonstrated by our lab in 2011, wherein we showed that a F92A scaffolding domain peptide could indeed promote NO release from endothelial cells [276]; furthermore, such a peptide could be used to reduce blood pressure in vivo in an eNOS dependent manner. As an extension of this study, our lab further demonstrated that the above peptide could reduce 34  atherosclerotic burden in diabetic mice via an eNOS dependent mechanism [277], further validating the idea that an F92A variant of the scaffolding domain can potentially act as an antagonist to increase therapeutically relevant NO in settings of endothelial dysfunction (Figure 4). 1.23. Summary Cardiovascular diseases are the largest cause of mortality on the global stage. One of the most salient features of CVD is a clinical condition known as endothelial dysfunction, which is characterized by increased vascular inflammation, impaired regulation of vascular tone and increased likelihood of coagulation. While there are several factors that have been suggested as drivers of endothelial dysfunction, one that has received a lot of attention is the reduction in systemic NO levels. Endothelial NOS-derived NO is an important regulator of vascular homeostasis and lack of it is associated with endothelial dysfunction. Furthermore, reductions in systemic NO levels has been associated with increased risk of cardiovascular events, while conversely, many treatments that show cardiovascular benefits are associated with improved NO regulation, thus providing validation for the role of NO. As such, improving eNOS-derived NO has been an active area of research in an attempt to develop novel therapies.  Endothelial NOS regulation is a complex process that involves a multitude of cellular events such as RNA regulation, subcellular targeting, phosphorylation and direct protein-protein interaction. One particularly notable protein-protein interaction is that with Cav-1, the main coat protein of caveolae, as it is the only known direct inhibitory interaction. The inhibitory interaction is mediated by a 20 a.a. region known as the scaffolding domain in Cav-1. Studies have shown that mutation of one particular residue, F92, within CAV can  35    Figure 4. Mutant Caveolin-1 Peptides for NO Release The Caveolin-1/endothelial nitric oxide synthase (Cav-1/eNOS) interaction is by default inhibitory; however, studies have shown that Cav-1 scaffolding domain (CAV)-derived peptides with an inactivated inhibitory domain (F92A; denoted by red ‘A’) can be used to promote basal NO release from endothelial cells. Moreover, the F92A peptide has been shown to induce eNOS-dependent decreases in blood pressure and atherosclerosis development in animal models. Hence, the use of inactivated CAV-based peptides may constitute a new approach to help in the treatment of endothelial dysfunction.  eNOSCAVL-ArgNOCaveolin-1eNOSL-ArgNOeNOSCAVL-ArgNOeNOSL-ArgNOA36  lead to loss of inhibitory ability. Furthermore, F92A variant peptides derived from CAV have been shown to increase NO from endothelial cells and can potentially be useful in NO regulation in settings of endothelial dysfunction and cardiovascular diseases. As such, this led to the idea that such peptides could act as eNOS/Cav-1 antagonists to promote NO release from endothelial cells.  1.24. Hypothesis We hypothesize that, within the Cav-1 scaffolding domain, there is an eNOS binding motif that could be used as the basis for a pharmacophore (i.e. set of structural and chemical properties required for interaction and functional response) for NO-increasing compounds; moreover, such compounds possess therapeutic benefits in the management of endothelial dysfunction. 1.25. Breakdown of Research Chapters Section 1: Initially, we postulated that the Cav-1 scaffolding domain contained the binding sequence for eNOS. As such, we sought to determine a sequence from the scaffolding domain of Cav-1 that can account for the bulk of eNOS binding. In this section, we: 1) Used a classical technique, the glutathione-S-transferase (GST) fusion protein pull-down assay, to identify the eNOS binding motif within the scaffolding domain 2) Verified the binding of the identified motif via a secondary assay 3) Used computer modeling to develop conjectures regarding the eNOS/Cav-1 interaction. Section 2: Following identification of the binding site motif, we hypothesized that a F92A variant of the peptide derived from the binding site motif could be used to promote NO 37  release in an eNOS and Cav-1 dependent manner. We compared a wild type binding site peptide (BSP), conjugated to two different cell permeabilization sequences, against its F92A counterpart (BSPF92A). In this section, we: 1) Assayed for peptide regulation of NO release from endothelial cells 2) Assessed dependence on eNOS and Cav-1 via protein knock-down experiments 3) Assessed for significance of cell permeabilization carriers and lipid rafts in mediating the effects of the peptides Section 3: Following the unusual observation that both BSP and BSPF92A peptides could promote NO release, we sought to identify the mechanism behind this. We conjectured that the peptides may have de-stabilized biochemical properties associated with the Cav-1/eNOS interaction. In this section, we: 1) Assessed for F92A dependent changes in the biochemical properties of Cav-1 and eNOS 2) Assessed for F92A dependent changes in co-localization of eNOS and Cav-1 3) Assessed for F92A dependent changes in eNOS-relevant signaling cascades    38         Chapter 2. Identification of the eNOS Binding Motif within the Cav-1 Scaffolding Domain    39  2.1. Introduction1 Endothelial NOS is a critical regulator of vascular homeostasis, and its reduced bioavailability is associated with a variety of vascular complications. Current therapies aiming to increase NO or signaling have limitations related to a lack of target specificity and the development of tolerance [118]. Hence, here is merit in identifying approaches that could produce localized increases in NO release. One approach would be to target the eNOS enzyme itself and reduce inhibitory influences that suppress its activity. To achieve this, we chose to target the Cav-1/eNOS interaction. Cav-1 is the main coat protein of caveolae, and is the main negative protein regulator of eNOS activity; more specifically, inhibition of eNOS is achieved through direct protein-protein interaction via a 20 amino acid sequence (a.a. 82-101) in Cav-1 known as the Cav-1 scaffolding domain (CAV) [210].  While the actual mechanism of inhibition is still unknown, we do know that F92 plays an important role, as the substitution of F92 with an alanine completely abolished Cav-1’s ability to inhibit eNOS activity [275]. This led to the hypothesis that a peptide bearing a F92A substitution could be used as an antagonist to promote NO release [278]. This was supported by a subsequent study from our lab that demonstrated that a full length scaffolding domain peptide lacking the critical inhibitory residue could be used to promote NO release and reduce blood pressure in vivo in an eNOS dependent manner [276]. However, the eNOS binding site within Cav-1 was still undefined. This question has more important implications for drug discovery, as a smaller, more optimized sequence would allow for better rational drug design and development of potential therapeutics. Herein, we detail our approach to identify a 10 amino acid sequence within the Cav-1 scaffolding domain that is                                                  1 A version of the text and figures in this chapter has been published in the J Biol Chem (2014), 289: 13273-13283; doi: 10.1074/jbc.M113.528695.  40  responsible for the bulk of the eNOS binding and to gain insight on the eNOS/Cav-1 interaction using fluorescent polarization assay and molecular modeling/docking. 2.2. Methods 2.2.1. Plasmids and Constructs Primers for the constructs (Table 1), containing a BAM HI and NOT I restriction site at the 5’- and 3’- ends respectively, were purchased from Sigma Aldrich. Polymerase chain reaction (PCR) was performed by using the primers on a plasmid encoding glutathione-S-transferase (GST) fused to residues 61-101 of the Cav-1 protein, a gift from Dr. Michael Lisanti and Jean-Francois Jasmin, as the template. The PCR product was isolated on a 1% agar gel, purified using an agar gel extraction kit (Qiagen), digested with BAM HI and NOT I and finally ligated into a pGEX-4T-3 GST vector (GE Healthcare) using a T4 ligase kit (Invitrogen). CaCl2 competent JM109 E. Coli were then heat shocked with the constructs and plated on to an ampicillin containing agar (100 µg/mL) plate for selection. Colonies were grown in Luria Broth (LB; 10g tryptone/L, 5g yeast extract/L and 5g NaCl/L) media and plasmids were purified using a miniprep kit (Promega). Purified constructs were verified by sequencing at UBC-NAPS using a custom forward primer targeted upstream of the insertion site and a stock M13R reverse primer (Table 2) before being used to transform CaCl2 competent BL21 E.Coli. 2.2.2. Plasmid Expression  BL21 E.Coli were transformed with the respective constructs and grown in liquid LB media at 37°C until an optical density, measured at 600 nm, between 0.3 and 0.5 was reached.    41  Table 1. Primer Sequences Used to Generate Library of Plasmids Base pair sequences for primers used in creating the modified scaffolding domains for glutathione-S-transferase pulldown assays.   Name Type Sequence CAVALA Reverse 5’-TCACGATGCGGCCGCCTAAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAAAACTGTGGGTACCTTCTGG-3’ CAV 61-81 Forward 5’-ATATGGATCCGATGACGTGGTCAAGATTGACTTTGAAGATGTGATTGCAGAACCAGAAGGTACCCACAGTTTTTAGGCGGCCGCATAT-3’ CAV 61-81 Reverse 5’-ATATGCGGCCGCCTAAAAACTGTGGGTACCTTCTGGTTCTGCAATCACATCTTCAAAGTCAATCTTGACCACGTCATCGGATCCATAT-3’ CAV-A Forward 5’-ATATGGATCCGATGACGTGGTCAAGATTGACTTTGAAGATGTGATTGCAGAACCAGAAGGTACCCACAGTTTTGACGGCATTTGGAAGGCCAGCTAGGCGGCCGCATAT-3’ CAV-A Reverse 5’-ATATGCGGCCGCCTAGCTGGCCTTCCAAATGCCGTCAAAACTGTGGGTACCTTCTGGTTCTGCAATCACATCTTCAAAGTCAATCTTGACCACGTCATCGGATCCATAT-3’ CAV-AB Reverse 5’-ATATATGCGGCCGCCTACGTCACAGTGAAGGTGGTGAAGCTGGCCTTCCAAATGCCGTCAAAACTGTGGGTACCTTCTGGTTCTG-3’ CAV-BC Reverse 5’-ATATATGCGGCCGCCTAGCGGTAAAACCAGTATTTCGTCACAGTGAAGGTGGTGAAAGCAGCAGCAGCAGCAGCAGCAAAACTGTGGGTACCTTCTGGTTCTG-3’ CAV-B Reverse 5’-TCACGATGCGGCCGCCTAAGCAGCAGCAGCAGCAGCCGTCACAGTGAAGGTGGTGAAAGCAGCAGCAGCAGCAGCAGCAAAACTGTGGGTACCTTCTGG-3’ CAV-C Reverse 5’-TCACGATGCGGCCGCCTAGCGGTAAAACCAGTATTTAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAAAACTGTGGGTACCTTCTGG-3’ CAV-C + 93-95 Reverse 5’-TCACGATGCGGCCGCCTAGCGGTAAAACCAGTATTTCGTCACAGTAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAAAACTGTGGGTACCTTCTGG-3’ CAV-C + 91-95 Reverse 5’-ATATATATGCGGCCGCCTAGCGGTAAAACCAGTATTTCGTCACAGTGAAGGTAGCAGCAGCAGCAGCAGCAGCAGCAGCAAAACTGTGGGTACCTTCTGGTTCTGCAATCACA-3’ CAV-C + 90-95 Reverse 5’-ATATATATGCGGCCGCCTAGCGGTAAAACCAGTATTTCGTCACAGTGAAGGTGGTAGCAGCAGCAGCAGCAGCAGCAGCAAAACTGTGGGTACCTTCTGGTTCTGCAATCACA-3’ CAV-B + 96-97 Reverse 5’-ATATATATGCGGCCGCCTAAGCAGCAGCAGCGTATTTCGTCACAGTGAAGGTGGTGAAAGCAGCAGCAGCAGCAGCAGCAAAACTGTGGGTACCTTCTGGTTCTGCAATCACA-3’ CAV-B + 96-99 Reverse 5’-ATATATATGCGGCCGCCTAAGCAGCAAACCAGTATTTCGTCACAGTGAAGGTGGTGAAAGCAGCAGCAGCAGCAGCAGCAAAACTGTGGGTACCTTCTGGTTCTGCAATCACA-3’ CAV 90-99[Ala] Reverse 5’-ATATATATGCGGCCGCCTAAGCAGCAAACCAGTATTTCGTCACAGTGAAGGTGGTAGCAGCAGCAGCAGCAGCAGCAGCAAAACTGTGGGTACCTTCTGGTTCTGCAATCACA-3’ CAV 90-100[Ala] Reverse 5’-ATATATATGCGGCCGCCTAAGCGTAAAACCAGTATTTCGTCACAGTGAAGGTGGTAGCAGCAGCAGCAGCAGCAGCAGCAAAACTGTGGGTACCTTCTGGTTCTGCAATCACA-3’ GST-Fwd Forward 5’-ATATATGGATCCGATGACGTGGTCAAGATTGACTTTGAAGATGTGATTGCAGAACCAGAAGGTACCCACAG-3’  42  Table 2. Primers Used for Sequencing Sequence for forward and reverse primers used in sequencing of plasmid library.  Name Type Sequence GST-CAV Forward 5’-GTTTTATACATGGACCCAATGTGCCTG-3’ M13R Reverse 5’-CAGGAAACAGCTATGACC-3’   43  Subsequently, the bacteria were stimulated with 1mM of isopropyl β-D-1-thiogalactopyranoside for 60 mins at 34.5°C. The BL21 was then centrifuged at 3000g for 10 mins at 4°C, and the pellet was stored in a -80°C freezer until required.  The pellet was rinsed with STE (150 mM NaCl, 50 mM Tris, 5 mM EDTA, ph 8.0) and centrifuged (3000g, 10 mins, 4°C) before being re-suspended in STE a second time. The suspension was then incubated with lysozyme (100 µg/mL) and phenylmethylsulfonyl fluoride (PMSF; 1 mM) for 10 mins on ice, following which dithiothreitol (DTT; 5 mM) and N-laurylsarcosyl (1.5% final concentration) was added. The solution was then homogenized on ice for 2 mins before triton-x (2% final concentration) was added. The suspension was then centrifuged (30 mins, 4°C, 16,000g) and the supernatant was collected and stored at       -80°C until needed. 2.2.3. GST-Fusion Protein Resin Preparation Glutathione-coated resins (Pierce) were rinsed with STET (STE with 1% triton-X). The resin was then pelleted and incubated with the protein solution described in section 2.2.2 for 60 mins at 4°C on a rotator. The resins were then rinsed 3 times with STET before being re-suspended in STET and stored at 4°C. During the washes, resins were pelleted by centrifugation at 1,300g for 1 min to allow for removal of wash solution. 2.2.4. Western Blotting To ensure comparable levels of GST-fused proteins between conditions, a small, but equal, volume of resins from each construct was treated with sample loading buffer and resolved using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). The gel was then treated with SimplyBlue Commassie stain (Bio-RaD) to assess relative levels of protein in each sample. Samples were then loaded accordingly and discrepancies in resin volume 44  were resolved by addition of blank resins. Recombinant eNOS (Cayman Chemical), diluted to 200 µg/mL in a binding buffer (50 mM Tris, 20% glycerol, pH 7.4), was the added to the resins and allowed to incubate for 2 hrs at 4°C on a rotator. Subsequently, the resins were rinsed with a wash buffer (150 nM NaCl, 50 mM Tris, 1 mM EDTA, pH 7.7) 5 times in succession. After the final wash, the resins were incubated with sample loading buffer and resolved using SDS PAGE, followed by a transfer to a nitrocellulose membrane (Bio-Rad). Membranes were blocked with 1% casein solution and probed with rabbit anti-GST (Santa Cruz) and mouse anti-eNOS (BD Transduction) primary antibodies. Goat anti-mouse 790 (Invitrogen) and goat anti-rabbit 680 (Invitrogen) was used to detect the primary antibodies, and the results were imaged with our LICOR scanner. GST-protein and eNOS were then quantified using the Odyssey software; furthermore, eNOS levels were normalized against GST levels. 2.2.5. Fluorescent Polarization Assay Purified eNOS was provided for this study by Dr. Linda Roman (University of Texas). Custom peptides conjugated to fluorescein were purchased from Elim Biopharm. Peptide concentrations were assessed using spectrophotometry under the assumption of one fluorescent moiety per molecule and that the extinction coefficient was ɛ492nm = 79,000 M-1 cm-1. Subsequently, the peptides (100 nM) were added to varying concentrations of eNOS diluted in a phosphate buffer (HyClone) containing 50 mM KCl and 5 mM DTT (pH 7.5), and allowed to incubate for 60 mins in the dark at room temperature (RT). Samples were performed in triplicates. After 60 mins, sample wells were read from the top using a Tecan Infinite M1000 Pro in fluorescent polarization mode, using an excitation and emission wavelength of 470 nm and 515 nm respectively. Results were averaged and normalized 45  against the maximum and minimum response and fitted with a one site binding model on GraphPad Prism to determine the dissociation constant and Hill’s coefficient.   2.2.5. Molecular Modeling of Peptides The sequence from the wild type and mutant peptides were ran through NCBI protein blast to search for proteins having similar sequences. The Crystal structure of NmrA-like family domain containing protein 1 (PDB: 2WMD), showing a very high (75%) sequence identity was used for modeling the three dimensional structure of the peptides. The modeled structure of the peptides were energy minimized using CharMM forcefiled ( in Accelerys Discovery studio (, to relax any of the disordered regions in the peptides.  2.2.6. Molecular Docking of Peptides into eNOS The crystal structure of heme-containing human eNOS (PDB: 1M9M) was chosen as the receptor for molecular docking studies. The chain A of the full protein was used for the docking studies, followed by addition of hydrogens and further energy minimization in Discovery studio. Molecular docking of the modeled peptides was performed individually into the eNOS structure, using the GRAMM-X server ( sources/gramm/grammx). Further, top ranking model from each of the docking run was chosen as the final docked pose of the peptides. The eNOS-peptide binding was further confirmed by KFC2 server (, which predicts binding "hot spots" within protein-protein interfaces by recognizing structural features indicative of important binding contacts. Since peptides are highly flexible as ligands, we further validated the docked peptide poses by running an iterative molecular dynamics. A 10 ns Molecular dynamics (MD) simulation was carried out for the top ranking lowest energy structures given 46  by GRAMM-X server. The resulting conformations were compared with the original starting conformations and based on the superimposition and comparison of the docked conformations of each, wild type and mutant peptides, before and after the molecular dynamics study, we concluded that there were little to no deviation among them (less than 0.5Å), further validating our docking studies [279–282]. 2.2.7. Data Analysis Immunoblot images were quantified with the Odyssey system. Values were expressed as a ratio of the density of the eNOS band over the GST band, and expressed as a percentage of the positive control binding, as specified in the text. Normalized data for each construct was pooled together and was analyzed using one-tailed t-test to see if values where lower than 75% of control binding, as we were interested in identifying a motif that accounted for the majority of eNOS binding, and deemed 25 – 30% a reduction as being reasonable. One-way ANOVA analysis with a Dunnett’s post-hoc test was used to compare for differences between experimental groups in the polarization studies. Significance was defined as P < 0.05 in all cases. 2.3. Results 2.3.1. eNOS Binding is F92 Independent, but CAV Dependent As mentioned prior, the substitution of F92 with alanine within CAV prevented eNOS inhibition. To determine whether this was due to a reduction in eNOS binding or a failure to inhibit, we directly compared the wild type sequence (CAVWT) against the mutant CAVF92A and a negative control, an unmodified GST vector (Figure 5A). We observed that there was no difference in eNOS binding between CAVWT and CAVF92A (Figure 5B,C),    47   Figure 5. F92A Substitution Does Not Affect eNOS Binding A) Diagrammatic representation of the glutathione-S-transferase (GST)-fusion protein constructs utilized and their respective names. GST constructs were incubated with 200 µg/mL of  eNOS for 2 hrs to assay for binding. B) Representative immunoblot of the level of GST and the relative level of endothelial nitric oxide synthase (eNOS) pulled down by each construct. C) Densitometric quantification of eNOS binding (n = 3; mean ± S.E.M.), normalized to the level of GST (eNOS/GST), and expressed as a percentage of binding versus caveolin-1 scaffolding domain (CAVWT). Phenylalanine F92 substitution did not affect eNOS binding.  GST61-81eNOSGSTGST (-)CAV F92AA.B.C.61-81 CAVWT (+)DGIWKASFTTFTVTKYWFYRDGIWKASFTTATVTKYWFYR48  which suggested that F92 was not involved in eNOS binding, thus confirming our initial hypothesis that eNOS binding and eNOS inhibition by Cav-1 were two distinct events. As we intended to perform a series of alanine substitutions, we first had to verify that eNOS could not bind to alanine as a result of hydrophobic interactions, hence we compared a series of negative controls (Figure 6A). We found that GST alone, GST plus residues 61-81 of Cav-1 and GST plus 61-101 with a poly-alanine scaffolding domain (CAVALA) could not bind appreciable levels of eNOS (5.9 ± 5.4%, 9.8 ± 11.3%, 10.5 ± 7.0%, respectively; Figure 6B, C), indicating that there should be minimal levels of interaction. 2.3.2. CAV-BC Accounts for Almost All eNOS Binding To facilitate analysis of CAV, we arbitrarily segmented the sequence into 3 sub-regions named A (a.a. 82-88), B (a.a. 89-95) and C (a.a. 96-101). We first determined which sub-domain eNOS bound to by using a series of deletion mutants (Figure 7A) wherein we had sub-domain A alone (CAV-A), A and B (CAV-AB), or B and C, with a poly-alanine sequence in lieu of CAV-A (CAV-BC). We found that little to no eNOS bound to CAV-A; in contrast, CAV-AB had approximately 50% of maximal binding and CAV-BC had almost full binding (91.8 ± 12.9%), indicating that the latter two-thirds of CAV was the most essential for eNOS binding (Figure 7B, C). We then performed pull-down experiments with either regions B or C alone to determine which was more important for eNOS binding. Interestingly, neither the B nor the C subdomains could account for more than 50% of the eNOS binding (26.9 ± 6.3 and 38.8 ± 10.3%, respectively; Figure 7D, E), which suggested that the eNOS binding motif exists between these two subdomains.   49   Figure 6. eNOS Binding is Cav-1 Scaffolding Domain Dependent A) Diagrammatic representation of the glutathione-S-transferase (GST)-fusion protein constructs utilized and their respective names. GST constructs were incubated with 200 µg/mL of  eNOS for 2 hrs to assay for binding. B) Representative immunoblot of the level of GST and the relative level of endothelial nitric oxide synthase (eNOS) pulled down by each construct. C) Densitometric quantification of eNOS binding (n = 3-5, mean ± S.E.M.), normalized to the level of GST (eNOS/GST), and expressed as a percentage of binding versus caveolin-1 (Cav-1) scaffolding domain (CAVWT). Loss of the scaffolding domain, in the form of truncation or alanine (Ala) substitution, resulted in almost complete loss of eNOS binding. * = p < 0.05 value is lower than 75% of positive control.  GST61-8161-8161-81eNOSGSTGST (-)61-81 (-)CAVALA (-)CAVWT (+)A.B.C.DGIWKASFTTFTVTKYWFYRAAAAAAAAAAAAAAAAAAAA50   Figure 7. eNOS Binding Motif is Located in the B and C Sub-domains A) Diagrammatic representation of the glutathione-S-transferase (GST)-fusion protein constructs utilized and their respective names. GST constructs were incubated with 200 µg/mL of  eNOS for 2 hrs to assay for binding. B) Representative immunoblot of the level of GST and the relative level of endothelial nitric oxide synthase (eNOS) pulled down by each construct. C) Densitometric quantification of eNOS binding (n = 3-5, mean ± S.E.M.), normalized by the level of GST (eNOS/GST), and expressed as a percentage of  binding versus full-length caveolin-1 scaffolding domain (CAVWT). * = p < 0.05 value is lower than 75% of positive control. Residues 90-95 contribute to the bulk of CAV-B associated eNOS binding.  GST + 61-81CAV-ACAV-ABDGIWKASDGIWKASFTTFTVTKYWFYR CAVWT (+)DGIWKAS FTTFTVTKYWFYRAAAAAAA FTTFTVT CAV-BCAGST (-)CAV-BAAAAAAAAAAAAA FTTFTVTKYWFYRAAAAAAA AAAAAAA CAV-CAAAAAAAAAAAAA AAAAAAA CAVAla (-)CAV-A (82-88) CAV-B (89-95) CAV-C (96-101)eNOSGST GSTeNOSB DC E51  2.3.3. Residues 90-99 of CAV Accounts for Bulk of eNOS Binding To identify the binding motif, we decided to reconstitute residues with either CAV-B or CAV-C as a starting point. We first started with the CAV-C sequence and re-introduced CAV-B residues sequentially (Figure 8A). As expected, as residues 93-95, 91-95 and 90-95 were reconstituted, we recovered greater and greater levels of eNOS binding (57.3 ± 8.6%, 61.5 ± 8.7%, and 82 ± 15.2% respectively; Figure 8B, C), indicating that a.a. F89 was the least essential for eNOS binding out of the residues in CAV-B. We then performed the complementary experiment using CAV-B as the starting point (Figure 9A). Addition of a.a. lysine K96 and tyrosine Y97 did not change eNOS binding significantly; however, a large increase was observed following addition of residues tryptophan W98 and F99 (from 31.6 ± 3.4% to 68.0 ± 12.7%; Figure 9). Similarly, an additional, but small increase, in binding could be observed following re-introduction of a.a. tyrosine Y100 and arginine R101, indicating that these could be potential contributors to eNOS binding.  As mentioned, we are interested in identifying the sequence in CAV that could account for the majority of eNOS binding, and use that as a starting point for future novel therapeutics. Since a.a. F89 was not important for eNOS binding, we decided to focus our next phase on investigating a.a. 90-101, with a specific focus on the trailing a.a. 99-101 (Figure 10A). Also, as our previous studies have found that CAV-BC had comparable levels of eNOS binding as the full-length sequence, we decided to use that as our positive control. We found that the absence or presence of a.a. Y100 and R101 did not affect eNOS binding (Figure 10B, C), which was in line with our previous observation that addition of the two residues produced only a small effect on eNOS binding. Furthermore, residues 90-99 could    52   Figure 8. Residues 90-95 of B Sub-Domain Contribute to eNOS Binding A) Diagrammatic representation of the glutathione-S-transferase (GST)-fusion protein constructs utilized and their respective names. GST constructs were incubated with 200 µg/mL of  eNOS for 2 hrs to assay for binding. B) Representative immunoblot of the level of GST and the relative level of endothelial nitric oxide synthase (eNOS) pulled down by each construct. C) Densitometric quantification of eNOS binding (n = 3-5, mean ± S.E.M.), normalized to the level of GST (eNOS/GST), and expressed as a percentage of binding to versus caveolin-1 scaffolding domain (CAVWT). * = p < 0.05 value is lower than 75% of positive control. Residues 90-95 contribute to the bulk of CAV-B associated eNOS binding.  eNOSGST + 61-81KYWFYR CAVWT (+)DGIWKAS FTTFTVTKYWFYRAAAAAAA FTTFTVTCAV-C +90-95AAAAAAAAAAAAA AAAAAAAKYWFYRAAAAAAA AAAATVTCAV-CCAV-BC (+)KYWFYRAAAAAAA AATFTVTCAV-C +93-95KYWFYRAAAAAAA ATTFTVTCAV-C +91-95A.B.KYWFYRAAAAAAA AAAAAAACAVALA(-)C.Direction of ReconstitutionCAV-A (82-88) CAV-B (89-95) CAV-C (96-101)GST53   Figure 9. Residues 95-99 of C Sub-domain Contribute to Bulk of eNOS Binding  A) Diagrammatic representation of the glutathione-S-transferase (GST)-fusion protein constructs utilized and their respective names. GST constructs were incubated with 200 µg/mL of  eNOS for 2 hrs to assay for binding. B) Representative immunoblot of the level of GST and the relative level of endothelial nitric oxide synthase (eNOS) pulled down by each construct. C) Densitometric quantification of eNOS binding (n = 3-5, mean ± S.E.M.), normalized to the level of GST (eNOS/GST), and expressed as a percentage of binding versus caveolin-1 scaffolding domain (CAVWT). * = p < 0.05 value is lower than 75% of positive control. Residues tryptophan 98 and phenylalanine 99 contribute to eNOS binding, with potentially less importance on tyrosine 100 and arginine 101.  GST + 61-81KYWFYRAAAAAAA FTTFTVTAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAA FTTFTVTKYAAAAAAAAAAA FTTFTVTKYWFAAAAAAAAA FTTFTVTA.B.C.CAV-BCAVALA (-)CAV-B +96-97CAV-B +96-99CAV-BC (+)GSTeNOSCAV-A (82-88) CAV-B (89-95) CAV-C (96-101)Direction of ReconstitutionKYWFYR CAVWT (+)DGIWKAS FTTFTVT54   Figure 10. Residues 90-99 Together Account for Majority of eNOS Binding A) Diagrammatic representation of the glutathione-S-transferase (GST)-fusion protein constructs utilized and their respective names. GST constructs were incubated with 200 µg/mL of  eNOS for 2 hrs to assay for binding.B) Representative immunoblot of the level of GST and the relative level of endothelial nitric oxide synthase (eNOS) pulled down by each construct. C) Densitometric quantification of eNOS binding (n = 6, mean ± S.E.M.), normalized to the level of GST (eNOS/GST), and expressed as a percentage of binding versus residues 89-101 of the scaffolding domain (CAV-BC). * = p < 0.05 value is lower than 75% of positive control. Residues tyrosine 100 and arginine 101 did not appear to provide any additional benefits to eNOS binding.  GST + 61-81AAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAA ATTFTVTKYAAAAAAAAAAA ATTFTVTKYWFAAAAAAAAA ATTFTVTA.CAV 90-99-[Ala]CAVAla (-)CAV 90-100-[Ala]CAV 90-101GSTeNOSB.C.CAV-A (82-88) CAV-B (89-95) CAV-C (96-101)KYWFYRAAAAAAA FTTFTVT CAV-BC (+)55  account for the bulk of the observed eNOS binding (73.2 ± 16.2 %) associated with the CAV-BC sub-region. 2.3.4. Binding Site Motif Affinity for eNOS Unaltered by Truncation  We recognized that while GST pulldowns and western blotting may indicate whether two proteins are interacting, they are less ideal for matters involving sensitivity, such as relative binding. As such, upon identification of the eNOS binding motif, we next set out to verify that the identified sequence interacts with eNOS with a similar binding affinity as the full length sequence by performing a fluorescent polarization study. We verified our assay by comparing polarization produced by eNOS incubated with the fluorescein-linker alone or a fluorescein probe conjugated to the wild type 20 a.a. CAV sequence. As would be expected, as the concentration of eNOS increased, an increased level of interaction, or polarization, was observed with the CAV sequence (Figure 11A). In contrast, the negative control demonstrated very little change in polarization as the eNOS concentration increased. It was also interesting to note that the basal polarization levels of the negative control were noticeably higher than that of CAV; however, this did not affect analysis of results. After verifying our assay, we directly compared the full length CAV against the identified binding site peptide (BSP) and its F92 substituted counterpart (BSPF92A), and normalized the responses against the maximal response observed to allow for better comparison (Figure 11B). Subsequently, we fitted the plot with a one site binding model to determine the dissociation constant (Kd) for the interaction. Qualitatively, no major differences could be identified between the Kd values for CAV, BSP and BSPF92A, which was further verified through a direct comparison of Kd values (45 ± 7, 59 ± 9, 34 ± 7 nM,  56   Figure 11. Wild Type and F92A eNOS Binding Site Peptide (BSP) Has Similar Affinity for eNOS as the Full-length Scaffolding Domain Peptide Fluorescein conjugated peptides (100 nM) were incubated for 60 mins with recombinant eNOS to assay for interaction. A) Arbitrary fluorescent units (A.F.U.) versus endothelial nitric oxide synthase (eNOS) concentration (µM) for a negative control linker sequence (Neg Ctrl; ) and a linker plus Cav-1 scaffolding domain peptide (CAV; ) conjugated to a fluorescein probe. eNOS bound CAV in a concentration dependent manner, which was not observed for the negative control. B) A representative comparison of normalized AFU (versus maximum) against eNOS concentration for CAV (blue), BSP (black) and BSPF92A (red). C) The derived dissociation (Kd; µM) constant for the three peptides were highly similar (n = 5; mean ± S.E.M.).  A.B.C.57  respectively; Figure 11C). This confirmed our pulldown data, which suggested that F92A was not involved in binding and that a.a. 90-99 could account for eNOS binding. 2.3.5. Molecular Modeling Insights in to the eNOS/BSP Interaction  As no crystal structure currently exists for Cav-1, we decided to undertake computer modeling to better understand the interaction between eNOS and the BSP and BSPF92A sequences. The CAV-derived peptides were modeled after the crystal structure of NmrA-like family domain-containing protein 1 due to high amino acid homology (75%). Little change in structure, hydrophobicity (F92 and valine V94) and hydrophilicity (K96) distribution was observed between BSP and BSPF92A (Figure 12A), which may explain the similarity in eNOS binding affinity. Upon docking the BSP (green) and BSPF92A (magenta) sequences to the crystal structure of human eNOS, we found that both docked with a similar orientation and position (Figure 12B) and in close proximity to the heme moiety (orange; Figure 12B inset), which was verified by a second simulation program. Viewed from an alternate angle (Figure 12C), it is possible to note that the F92 on BSP allows for deeper penetration in to the hydrophobic binding pocket on eNOS. Careful analysis of the BSP-eNOS interaction shows that F92 allows for the formation of π-π interactions with tryptophan 447 and 445, tyrosine 475 and cysteine 184; in contrast, these interactions were lost following alanine substitution (Figure 12D). 2.4. Discussion Cav-1 is one of the most critical regulators of eNOS activity, and a better understanding of the interaction between the two proteins could potentially help identify novel therapeutics for diseases characterized by NO deficiencies. We have thus far shown that F92, although thought to be critical for eNOS inhibition, is not a major player in eNOS  58   Figure 12. Computational Analysis of Binding Site Peptide (BSP) with eNOS A) Homology-modeled structure of BSP and BSPF92A, showing high hydrophobicity (red color) for F92 and a lower hydrophobicity (pink color) for F92A. Blue color shows high hydrophilicity of the lysine residue. B) Molecular docking of BSP (green) and BSPF92A (purple) to eNOS (blue) hydrophobic pocket in close proximity to heme and iron (red) in the endothelial nitric oxide synthase (eNOS) groove (inset is zoomed in on heme group). C) Docked conformations of BSP (green) and BSPF92A (purple) in the eNOS binding site (surface representation). Yellow arrow shows how the wild type F92 residue is deeply buried in the hydrophobic patch (red color) in the eNOS binding site, whereas the F92A mutant stays far off the eNOS pocket. D) Detailed view of docked conformations of wild type F92 (green) and mutant F92A (magenta) in eNOS binding site (cyan ribbons). F92 makes hydrophobic and π-π interactions in the hydrophobic core of eNOS via tryptophan 447 and 445, and cysteine 184 whereas F92A (purple) is devoid of such interactions.   F92 A92A.C.B.D.59  binding to Cav-1. Furthermore, using a series of alanine substitution mutants, we have clearly identified a 10 a.a. protein sequence that could account for the bulk of the eNOS binding. Subsequently, peptides created from this sequence, with or without a F92A substitution, were shown to have a similar dissociation constant as that of the full-length sequence, which supported our GST-pull down results. Finally, molecular modeling was utilized to shed some light on the interaction between the Cav-derived peptides and eNOS. 2.4.1. The Utilization of an Isolated System and Alanine Substitution Regulation of interaction between eNOS and its partners is a highly complex event. For example, binding of calmodulin to eNOS is thought to induce conformational changes to eNOS that prevent it from interacting with Cav-1. Similarly, other proteins, such as HSP90 can serve to modulate the interaction between eNOS and Cav-1. For this reason, we choose to use an isolated system consisting of just recombinant eNOS and GST-CAV, which allows us to specifically study the interaction between eNOS and Cav-1 in the absence of other regulatory factors and cellular events. Furthermore, GST pull-down assays, along with techniques such as yeast-two-hybrid, are highly validated and have been used extensively to study a plethora of protein-protein interactions, not only for eNOS, but a host of other proteins as well [209,283].  In addition to initial queries regarding an intact cellular system versus an isolated system, there were also concerns regarding how alanine substitution could impact the eNOS/Cav-1 interaction. Stretches of alanine have been known to take on an alpha helix configuration; however, experimental results have suggested that the Cav-1 scaffolding domain similarly is likely to adapt an alpha helical structure [284,285]. In such a case, alanine substitution would have relatively less of an impact on the overall shape adapted by 60  CAV and, overall, may provide a better platform than expected. However, there is a very real possibility, regardless, of there being an impact on eNOS binding, and may also contribute to an explanation as to why we could not recover full eNOS binding with some of the smaller sequences (e.g. when more than half the CAV sequence was substituted).   2.4.2. Comparison of Scaffolding Domains It has been demonstrated that both Cav-1 and Cav-3, a skeletal muscle specific form of caveolin, can interact with eNOS [286]; in addition, introduction of a peptide encoding the Cav-1 CAV sequence, or the analogous sequence from Cav-3, was sufficient to inhibit eNOS activity, whereas the Cav-2 CAV peptide could not produce this effect [210]. This suggested that there should be homologous regions between Cav-1 and Cav-3 that are essential for eNOS binding and inhibition that would not be present in Cav-2. Interestingly, when comparing the CAV sequences of Cav-1, Cav-2 and Cav-3 [210,287] , it was found that there was a high degree of similarity between the putative eNOS-binding site in Cav-1 and the analogous region in Cav-3, with only a one amino acid residue difference (Table 3); in contrast, the analogous region in Cav-2 differed from Cav-1 by 5 residues – chiefly the replacement of T90/91/93 with alanine, leucine and glutamic acid respectively, in addition to W97 with valine and F98 with methionine. Furthermore, the Cav-2 and Cav-3 scaffolding domains possess a phenylalanine group in an analogous position to F92 of Cav-1, indicating that the failure of Cav-2 to inhibit eNOS activity could not be due to a simple lack of an “inhibitory” domain. This is in agreement with the idea that if Cav-1 and Cav-3 can interact with and inhibit eNOS, they would be sharing a highly similar eNOS binding motif, and should significantly differ from the analogous region in Cav-2, which has been demonstrated    61  Table 3. Comparison of the Scaffolding Domains of Cav-1, -2 and -3 Amino acids for the scaffolding domains of Caveolin (Cav)-1, -2 and -3 are shown. The starting position of the initial residue has been noted with a superscript. Different (i.e. non-identical) residues are bolded in black. The inhibitory F92 and its equivalent in the other caveolin isoforms are shown in red.   Cav-1 82D G I W K A S F T T F T V T K Y W F Y R Cav-2 54D K V W I C S H A L F E I S K Y V M Y R Cav-3 55D G V W K V S Y T T F T V S K Y W C Y R 62  to be unable to inhibit eNOS activity [210]. This observation may explain the differences in binding capabilities between the different members of the caveolin family. 2.4.3. Cav-1, the eNOS Binding Domain and the Cav-1 Binding Motif Previously, a Cav-1 binding motif, ΦXΦXXXXΦXXΦ (where Φ can be tryptophan, phenylalanine or tyrosine) had been identified using phage library [211]. It was conjectured that this aromatic residue rich motif was necessary for client protein binding to Cav-1. In line with this, eNOS also contains such a sequence. Furthermore, alanine substitution of the aromatic residues (in bold) in the Cav-1 binding motif found in eNOS, F350SAAPFSGW358, prevented the inhibition of eNOS activity by Cav-1 [210]. In similar studies with a different protein, it was demonstrated that removal of the aromatic residues from the caveolin binding motif of a Gi2-derived peptide prevented the peptide from associating with Cav-1 [211]. This highlighted the significance of aromatic rings in the interaction between caveolin-interacting proteins and the caveolin protein. Furthermore, the eNOS-binding motif identified herein (T90TFTVTKYWF99) also happens to significantly overlap with the Cav-1 binding motif found in Cav-1 itself (F92TVTKYWFY100), which may have implications for protein interactions. For example, other researchers, using a combination of biophysical techniques and computer modeling, have hypothesized that the scaffolding domain formed an alpha helical structure, where one face consisted of interdigitating aromatic residues [285]; hence, there is a possibility that the aromatic rings could serve as a form of interlocking mechanism that is further stabilized by surrounding amino acid residues. But beyond protein interaction, the “caveolin-1 binding motif” may have broader applications, such as mediating the inhibition of client proteins (e.g. F92 inhibition of eNOS), and could be an interesting subject to explore with other client proteins. 63  However, there has been some recent controversy regarding the existence, and significance of such a binding motif. In the opinion piece, Collins argued, with particular emphasis on eNOS, that such sequences are typically buried and are not accessible, short of detrimental or drastic conformational changes to the eNOS protein [288]. However, this is unlikely to be the case, as demonstrated in a study mentioned prior [210]. In the study, cells that did not endogenously express eNOS were transfected with a mutant eNOS bearing alanine substitution in lieu of the aromatic residues within the eNOS Cav-1 binding motif, F350SAAPFSGW358. This substitution study, and the subsequent observation that eNOS failed to interact with Cav-1, suggests that the sequence is indeed accessible. Furthermore, there is a known eNOS antagonist that has been shown to insert itself in to this “inaccessible” binding pocket, as was demonstrated using crystallography [289]. Hence, there is sufficient evidence to indicate that “buried” binding sequence is in fact accessible and is likely to be involved in the regulation of the Cav-1 interaction. 2.5. Conclusion In summary, we have demonstrated that F92’s role is chiefly regulatory and it does not participate to a significant extent in eNOS-Cav-1 binding. We have successfully generated a library of binding constructs to identify CAV binding partners and motifs, and have subsequently identified a core motif within the CAV that appears to be critical to eNOS binding. This finding contributes not only to our understanding of eNOS regulation, but may also help us to understand the defining characteristics within the caveolin-1 protein that allows it to interact with and regulate a plethora of molecules and signaling cascade.   64        Chapter 3. Cav-1 Binding Site Derived Peptides and NO Release in Endothelial Cells   65  3.1. Introduction2 To date, essentially all published literature, with respect to the usage of CAV-derived peptides, have been based on the full length sequence. However, as we are interested in developing new therapies, it would be much more feasible to optimize the sequence, which would facilitate any additional analysis. As demonstrated in the previous chapter, we have found that inhibition of eNOS activity (i.e. residue F92) is independent of binding, which is predominantly attributable to a 10 residue sequence that could account for the majority of the binding. Given that we have identified the binding sequence, we are now interested in determining whether or not this sequence could in fact be used to promote basal NO release from resting endothelial cells. Previous studies have suggested that the F92A derivative could act as an antagonist and promote NO release [290]; in contrast, we hypothesized that the wild type sequence would not affect basal NO release, given its association with eNOS inhibiton. Typically, peptides do not readily permeate into cells. As such there is a need for a cell permeabilization sequence. Antennapedia peptide (AP), a cell permeabilization sequence derived from Drosophila [291], is one of the most commonly used cell uptake sequence for Cav-1 peptides – essentially all literature published on Cav-1 peptides have been using this sequence. However, we were curious if this was in fact the most suitable choice, and have chosen to explore other options of cell permeabilization. We investigated the feasibility of peptide myristoylation (Myr) as a method of delivery, given the role of myristoylation in protein targeting and trafficking to the plasma membrane [292], where our targets of interest, eNOS and Cav-1, reside.                                                  2 A version of figures 14 thru 16 and parts of the text in this chapter can be found published in the J Biol Chem (2014), 289: 13273-13283; doi: 10.1074/jbc.M113.528695 and Eur J Pharmacol (2015): 766: 46-55; doi:10.1016/j.ejphar.2015.09.033. 66  We hypothesized that a BSPF92A peptide could be used to promote NO release from endothelial cells. Herein, we assayed the ability of BSP and BSPF92A to increase NO release from endothelial cells. Subsequently, we characterized eNOS and Cav-1 dependence through silencing ribonucleic acid (siRNA) knock-down and caveolae/lipid raft dependence through cyclodextrin pre-treatment. We also gained insight into the effect of F92A substitution on cellular uptake of BSP and BSPF92A peptides and how different permeabilization sequences can affect peptide uptake. 3.2. Methods 3.2.1. Cell Culture Bovine aortic endothelial cells (BAECs) were isolated from bovine aortas. The BAECs were utilized between passages 3 and 10 and were grown in Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen) supplemented with 5% fetal bovine serum, 100 units of penicillin and 0.1 mg streptomycin in a 37°C incubator with 7% CO2. 3.2.2. NO Measurements For inhibition of NO release studies, BAECs were incubated with the stated peptide (10 µM) for 6 hrs in serum free DMEM. Subsequently, the media was replaced with one containing 1 nM vascular endothelial growth factor (VEGF), and NO was allowed to accumulate for 30 mins. For basal NO release studies, BAECs were incubated with the stated peptide and after 6 hrs, the media was collected. In the case of cyclodextrin, cells were pre-treated with the compound (1 mM) for 60 min before replacing the media with serum-free media containing the stated peptide. If there was a need to determine the total amount of NO produced (NOx), we performed a nitrate conversion assay according to the manufacturer’s instructions. Sample NO3¯ was 67  converted into nitrite by incubating samples with 0.1 units of nitrate reductase, 50 µM NADPH, and 5 µM FAD for 30 min at 37 ºC in the dark. For nitrite analysis, samples were centrifuged for 2 min at 2000g and the supernatant was harvested in order to remove any pelleted cells. Following which, the supernatent was analyzed for nitrite using our Sievers NO analyzer according to manufacturer’s instructions. The molar amount of nitrite present in the sample was determined by using a sodium nitrite standard curve. 3.2.3. Live Cell Imaging BAECs were grown in 35 mm glass-bottom dishes (Mattek) and transfected with Cav-1 YFP, a kind gift from Dr. Richard Minshall, using Lipofectamine 2000 (Invitrogen). Following 2 days of expression, cells were blocked with 10 µM BSP peptides for 60 min before being incubated with either AP-BSP or Myr-BSP (10 µM) conjugated to a Cy-5 fluorophore. Cells were then imaged using an inverted Leica microscope with a confocal scanner and results were analyzed using the Volocity software (PerkinElmer).  The identity of the fluorophore was confirmed by performing a λ-scan and comparing the emission pattern against the known signature of the fluorophore. 3.2.4. siRNA Studies All siRNA sequences were ordered from Santa Cruz Biotechnology. eNOS and Cav-1 siRNA (or a matching amount of non-silencing siRNA) was prepared with oligofectamine (Invitrogen) in Opti-MEM. BAECs were treated with the resulting working solution for 6 hrs before the media was replaced with DMEM containing 10% FBS. The cells were then allowed to rest for 48 hours before they were utilized for NO experiments.  68  3.2.5. Western Blotting Cells were lysed in a buffer containing: 50mM Tris-HCl, 1% NP-40, 0.1% SDS, 0.1% deoxycholic acid, 0.1mM EDTA, 0.1mM EGTA, 1mM PMSF and Sigma protease inhibitor cocktail. The lysate was allowed to rotate at 4ºC for an hour before being centrifuged at 20,000g for 30 mins at 4°C, which was followed by collection of the supernatant. Protein concentrations were determined using a Bio-Rad DC assay and 20 µg of each condition was loaded on to a polyacrylamide gel for resolving. Gels were transferred on to a nitrocellulose membrane and probed with mouse anti-Hsp90 (BD Transduction), rabbit anti-Cav-1 (Santa Cruz Biotechnology) and mouse anti-eNOS (BD Transduction). Primary antibodies were detected using goat anti-mouse 790 or goat anti-rabbit 680 IR Dye (Invitrogen) respectively and imaged on our LICOR scanner. 3.2.6. Cyclodextrin Peptide Uptake Studies BAECs were plated in black 96-well plates with clear flat bottoms (Fischer Scientific) and grown to confluency. Following cyclodextrin and Cy-5 conjugated peptide treatment as outlined in Section 3.2.2, plates were read with a Tecan Infinite M1000 Pro plate reader with an excitation wavelength of 630 nm and an emission capture set to 645-670 nm. Results were performed in triplicate and averaged. 3.2.7. Data Analysis NO release data was analyzed using one-way ANOVA followed by a Dunnet’s test post-hoc to determine if was any differences within groups. For siRNA, cyclodextrin and peptide uptake studies, data was analyzed using two-way ANOVA. Significance was defined as p < 0.05. 69  3.3. Results 3.3.1. Loss of F92 Prevents BSP-mediated Inhibition of Stimulated eNOS From our findings in chapter 2, we know that the majority of eNOS binding is associated with a 10 a.a. sequence, 90TTFTVTKYW99F, furthermore, within the identified motif, residue F92 is known to play a critical role in inhibition of activated eNOS (Figure 13A). We first sought to confirm whether this fundamental property of the peptide was changed by the shortening of CAV sequence. To do this, we first had the BSP and BSPF92A sequences conjugated to AP, which is a commonly used cellular uptake sequence used for the vast majority of CAV peptide studies currently published.  Following AP pre-treatment, VEGF induced a large increase in measured nitrite from bovine aortic endothelial cells (BAECs) versus baseline (686.4 ± 78.9 versus 2114.0 ± 307.1 pmol NO/mL).  In contrast, 6 hour pre-treatment of BAECs with either the full length AP-CAVWT or AP-BSP resulted in a significant reduction in VEGF stimulated NO production, which was not observed following F92A substitution (1309 ±177.3, 1336 ± 204.1 and 1981 ± 210.3 pmol NO/mL, respectively; Figure 13C). This confirmed that truncation of the CAVWT sequence did not affect eNOS inhibition, but rather that the inhibitory activity was linked to residue F92 of CAV. 3.3.2. BSP and BSPF92A Increase Basal NO Release We next wanted to determine if the BSPF92A peptide could increase basal NO production. For this we measured both nitrite accumulation and total NO release (nitrite and nitrate), after stimulating the cells for 6 hrs with AP, AP-BSP or AP-BSPF92A. As would have been expected, AP had little effect on nitrite release; in contrast, we noticed elevated levels of NO production following treatment with the AP-BSPF92A peptide, which would align with  70   Figure 13. Loss of F92 Prevents Inhibition of Stimulated NO A) Summary graphic outlining the binding contribution of different areas of the Cav-1 scaffolding domain (CAVWT). F92 does not affect binding, but is responsible for inhibition of nitric oxide (NO) synthesis. B) Amino acid representation of the peptides used, with the F92 residue substitution bolded in red. C)  Endothelial cells were pretreated with either antennepedia peptide (AP) vehicle or a treatment sequence (CAVWT, binding site peptide (BSP) or BSPF92A; All peptides were used at 10 µM) before being stimulated with 1 nM vascular endothelial growth factor (VEGF; AP (V) indicates AP pre-treatment followed by VEGF stimulation). Loss of F92 residue resulted in the failure of the peptide sequence to inhibit NO release (mean ± S.E.M.; n = 6; * = 0.05 < p versus AP (V)).   KYWFYRDGIWKAS FTTFTVT~ 8% ~ 92%K     Y     W     F     Y     R65%68%F     T     T    F T     V     TA. CAV-A (82-88) CAV-B (89-95) CAV-C (96-101)0%; inhibitoryCAVWT: DGIWKASFTTFTVTKYWFYRBSP: TTFTVTKYWFBSPF92A: TTATVTKYWFB. C.** *71  our hypothesis that these peptides could be used to promote NO release (Figure 14A). However, we also noted an increase in NO release following treatment of BAECs with AP-BSP, which was rather unexpected, given the inhibitory nature of the sequence. To confirm whether this was an effect of the AP cellular uptake sequence, we tested conjugation to myristic acid (Myr), a 14-carbon lipid chain typically associated with post-translational modification, as discussed in Chapter 1. Interestingly, Myr-BSP and Myr-BSPF92A incubation also produced strong nitrite and nitrate release (Figure 14B), while Myr had no effect on both measures, indicating that our previous observation with AP was unlikely to be a carrier associated artifact. Lastly, in all instances, nitrite appeared to serve as a viable marker for total NO release, so subsequent studies will assess for nitrite solely and all reference to NO will refer to thus.   Another point of interest was that AP-BSP induced higher levels of NO compared to AP-BSPF92A (1200.0 ± 137.6 and 388.4 ± 110.7 pmol NO/mL, respectively); in contrast, Myr-BSPF92A stimulation resulted in higher levels of NO compared to Myr-BSP (2167 ± 290.1 and 794.6 ± 88.9 pmol NO/mL). This suggests either 1) the F92A mutation has an effect on uptake, resulting in different levels of activity or 2) that the F92A and wild type sequence induces NO uptake via different mechanisms, which is affected by carrier sequence utilized. 3.3.3. F92A Substitution Does Not Affect Peptide Uptake We first decided to investigate whether uptake of peptides was affected by carrier (AP vs Myr) or peptide sequence (BSP vs BSPF92A). For this, we performed live cell imaging of BAECs using Cy5-fluorophore labelled AP and Myr peptides to determine total uptake at 1 and 4 hrs. We selected four hours for this because in initial pilot studies, we found that NO  72   Figure 14. Both BSP and BSPF92A-derived Peptides Increase NO Release from Endothelial Cells Endothelial cells were incubated for 6 hrs with 10 µM of different of peptides before the media was collected for NO analysis. A) The amount (pmol NO/mL) of nitrite (white) and total NO (nitrite and nitrate; black) stimulated by antennapedia peptide (AP), AP-binding site peptide (BSP) and AP-BSPF92A from endothelial cells. Both BSP and BSPF92A-conjugated peptides promoted NO release (mean ± S.E.M.; n = 6). B) Further testing for nitrite (white) and total NO (nitrite and nitrate; black) stimulated by myristic acid (Myr), Myr-BSP and Myr -BSPF92A from bovine aortic endothelial cells (BAECs), showed the peptides behaved similar to their AP counterparts (mean ± S.E.M.; n = 6), regardless of F92A substitution.   B.A.73  was already promoted by this point in time. However, the pilot studies were performed in 6-well plates, whereas subsequent studies were performed in 96-well plates, where the plate surface to media volume was much lower. As such, we compensated for the decreased ratio by increasing incubation time, resulting in incubation times of 6 hrs for NO release studies. However, for mechanistic insights, we deemed it was of greater value to capture the events at an earlier time. For the uptake study, results were normalized to the maximum observed value at 4 hrs. In the study, we found that the rate of uptake for Myr-conjugated peptides to be slower than that of AP-peptides. At 1 hr, Myr-conjugated peptides were on average 5-6 fold lower in intensity versus their AP-counterparts (Figure 15A). This difference in relative total uptake was less than 2-fold by 4 hrs, but still significantly different. This suggested that AP-conjugated peptides were taken up quickly, but that the uptake plateaus rapidly as well in comparison to Myr-conjugated peptides. Interestingly, at 1 hr, we can see that the AP-peptides are spread out across the entire cell in a diffused pattern; however, Myr-peptides had a much more focused staining along the cell membrane (Figure 15B). This suggested that the Myr-peptides may indeed be able to target membrane interactions much more efficiently than the AP-conjugated peptides. Similarly, it is worthwhile to note that while cell permeabilization affected rate and targeting of peptide, the F92A substitution did not affect either property. Given that F92 does not affect uptake nor distribution, the fact that BSP and BSPF92A elicited different amounts of NO production (Figure 14A, C), suggest that BSP and BSPF92A most likely induced NO release via different mechanisms of action.   74  A.1 hour:4 hour:AP-BSP AP-BSPF92A Myr-BSP Myr-BSPF92AB.**** Figure 15. F92A Substitution Does Not Affect Peptide Uptake Endothelial cells were incubated with 10 µM of Cy-5 conjugated peptides and imaged with an inverted confocal microscope. A) Relative uptake, normalized against the maximum, of Cy-5 labeled, antennapedia peptide (AP)-conjugated binding site peptide (BSP; black) and BSPF92A (red) and myristic acid(Myr)-conjugated BSP (green) and BSPF92A (orange) at 1 and 4 hrs by endothelial cells. Each data point is expressed as mean ± S.E.M. (n = 5) B) Representative sample images from the uptake study showing cellular distribution of the stated peptides at 1 and 4 hrs. Myr-conjugated peptide uptake was slower and more localized along the cell membrane versus AP-peptides. * p < 0.05 versus their time matched counterpart.  75  3.3.4. NO Release is Blunted by eNOS, but not Cav-1, siRNA As all our peptides were able to promote NO release, we next determined whether these peptides functioned in an eNOS and Cav-1 dependent manner. We first gauged eNOS dependence by use of an eNOS siRNA (Figure 16A). In the absence of siRNA, we observed fairly robust responses triggered by AP-BSP and BSPF92A (1098 ± 101.2 and 926.5 ± 64.3 pmol NO/mL, respectively) versus AP-baseline (328.6 ± 49.8 pmol NO/mL; Figure 16B). Similarly, Myr-BSP and Myr-BSPF92A (1124.0 ± 85.14 and 802.4 ± 74.39 pmol NO/mL) could induce robust responses versus vehicle (7.263 ± 32.19 pmol NO/mL; Figure 16C). In contrast, following eNOS knock-down, the NO response was essentially reduced to baseline levels for all peptides. This demonstrated that both BSP and BSPF92A, regardless of carrier sequence, were able to promote NO release in an eNOS dependent manner.   We then tried to knock down Cav-1 siRNA to gauge Cav-1/caveolae dependence (Figure 17A). In the absence of Cav-1 siRNA, the BSP- and BSPF92A-peptides (AP-BSP siRNA: 2122 ± 832.2, AP-BSPF92A: 867.8 ± 292.3, Myr-BSP: 1206 ± 356.6, Myr-BSPF92A:2217.0 ± 335.0 pmol NO/mL), regardless of the uptake sequence, elicited fairly robust levels of NO production from the BAECs. Unexpectedly, however, we did not observe any reduction in NO levels following Cav-1 siRNA treatment in AP-peptide treated cells. Instead, we saw comparable levels of NO production compared to that of non-silencing siRNA treated samples (AP-BSP siRNA: 2857 ± 1080; AP-BSPF92A siRNA: 940.7 ± 229.7 pmol NO/mL; Figure 17B). Similarly, Myr-treated Cav-1 silenced cells displayed comparable levels of NO release to non-silenced cells (Myr-BSP siRNA: 1489 ± 406.3; Myr-BSPF92A siRNA: 3026 ± 559.0 pmol NO/mL; Figure 17C). However, this doesn’t necessarily    76   Figure 16. Peptide-stimulated Release is eNOS Dependent Endothelial cells were treated with silencing ribonucleic acid (siRNA) treatment or non-silencing (NS) siRNA for 6 hrs, then allowed to rest for 48 hrs before being stimulated with 10 µM of peptide to gauge for eNOS dependence. A) Western Blot showing knock-down of endothelial nitric oxide synthase (eNOS) in endothelial cells by siRNA treatment versus NS siRNA, with coatomer-protein I beta (β-COP) used as a loading control. B) NO release (pmol/mL) following 6 hrs of treatment with antennapedia peptide (AP), AP-binding site peptide (BSP) or AP-BSPF92A in eNOS silenced cells decreased to baseline versus non-silenced endothelial cells. C) Similarly, NO released following treatment with vehicle myristic acid (Myr), Myr-BSP or Myr-BSPF92A in eNOS silenced cells was at baseline levels versus non-silenced endothelial cells. Both experiments expressed as mean ± S.E.M. (n = 6; * p < 0.05 vs NS).  eNOSβ-COPL 15 75 15 75NS siRNA eNOS siRNAA.B. C.****77   Figure 17. Peptide-stimulated Release Not Affected by Cav-1 Knock-down Endothelial cells were treated with silencing ribonucleic acid (siRNA) treatment or non-silencing (NS) siRNA for 6 hrs, then allowed to rest for 48 hrs before being stimulated with 10 µM of peptide to gauge for caveolin-1 (Cav-1) dependence. A) Western Blot showing knock-down of endogenous Cav-1 in endothelial cells by Cav-1 siRNA versus NS siRNA treatment, with heat shock protein (HSP) 90 used as a loading control. B) Nitric oxide (NO) release (pmol/mL) following 6 hrs of treatment with antennapedia peptide (AP), AP-binding site peptide (BSP) or AP-BSPF92A in Cav-1 silenced cells was similar to that of non-silenced endothelial cells (mean ± S.E.M.; n = 4). C) NO release (pmol/mL) released following treatment with myristic acid (Myr), Myr-BSP or Myr-BSPF92A in Cav-1 silenced cells was similar to NO release from non-silenced endothelial cells (mean ± S.E.M.; n = 4).  Cav-1HSP 90NS siRNA Cav-1 siRNAA.B. C.78  eliminate Cav-1 as an important player in the regulation of BSP/BSPF92A-induced NO release, as will be discussed later.  3.3.5. Peptide Stimulated NO Release is Lipid Raft Dependent As Cav-1 knock-down did not produce the expected response, we examined if methyl-β-cyclodextrin (mCD), which is known to disrupt lipid rafts, including caveolae, could affect NO release. We first tested the effect of 1hr mCD pre-treatment (1mM) on AP-peptide induced NO release. Following mCD treatment, NO levels induced by the AP-peptides were increased compared to their control counterparts (AP-BSP: 1237 ± 314.3 versus 380.4 ± 113.2 and AP-BSPF92A: 408.9 ± 41.29 versus 201.8 ± 85.96 pmol NO/mL; Figure 18A). In contrast, following mCD pre-treatment, NO levels induced by Myr-conjugated peptides were reduced compared to control counterparts (Myr-BSP: 271.4 ± 57.2 versus 784.7 ± 47.46 and Myr-BSPF92A: 252.4 ± 76.48 versus 1236 ± 143.1 pmol NO/mL; Figure 18B). Once again, we determined if the uptake of any of the NO-inducing peptides (AP- and Myr-BSP/BSPF92A) were affected by the cyclodextrin treatment, which may explain the observed results. Interestingly, we observed that mCD treatment increased the amount of peptide taken up by BAECs by 15 – 50% compared to control conditions (Figure 18C), indicating that any NO decrease was not associated with a drop in peptide uptake.  3.4. Discussion Having identified the eNOS binding motif in Chapter 2, we were now concerned about the practical implications of the identified sequence. We demonstrated that the sequence retained its inhibitory properties when used in the presence of VEGF stimulated eNOS. However, both F92A and wild type BSP peptides were capable of promoting basal NO release from resting BAECs; however, the levels of NO produced appeared to be both  79   Figure 18. Peptide-stimulated NO is Regulated by Lipid Rafts Endothelial cells were pre-treated with 1 mM cyclodextrin (mCD), before being stimulated with 10 µM of peptides for 6 hrs. Cell media was then collected for nitric oxide (NO) analysis. A) Antennepedia peptide (AP), AP-binding site peptide (BSP) and AP-BSPF92A induced NO release (pmol/mL) from endothelial cells was increased in endothelial cells pre-treated with mCD versus normal media. B) Equivalent study performed using myristic acid (Myr), Myr-BSP and Myr-BSPF92A; however, a decline in NO release was observed instead. Both data sets are expressed as mean ± S.E.M. (n = 5; * p < 0.05). C) Uptake of Cy-5-conjugated peptides AP-BSP, AP-BSPF92A, Myr-BSP and Myr-BSPF92A following mCD pre-treatment, normalized against uptake from untreated cells and expressed as a percentage (mean ± S.E.M.; n = 5).    C.A. B.***80  sequence and carrier dependent, but uptake independent. More specifically, uptake of AP-conjugated peptides was more rapid compared to Myr-peptides, although Myr-peptides targeted the membrane much better than their AP counterparts. However, given the same carrier, there was essentially no discernable difference in uptake between BSP and BSPF92A sequences. Furthermore, initial evidence suggest that BSP and BSPF92A sequences promote NO via different mechanisms. Subsequently, we confirmed that NO release was eNOS dependent. However, confirmation of Cav-1 dependence proved difficult. To work around this conundrum, we utilized mCD and found that the results suggested that the peptides acted in a lipid raft dependent manner. 3.4.1. Cell Line Limitations  For our studies, we choose to use bovine aortic endothelial cell. They are a widely used cell line for studying endothelial function, readily available and easily accessible. However, there are several shortcomings that should be considered. The first consideration is that BAECs are a non-human cell line. This may result in species-dependent effects, even though we are studying a very fundamental property of endothelial cells, chiefly eNOS regulation by Cav-1. Hence, there may be value in validating key experiments in an equivalent primary human cell line, such as human aortic endothelial cells. In addition, we do not know the age and health status of the bovine from which the cells were derived, which could potentially lead to unexpected outcomes. For example, one study showed that age and atherosclerosis was associated with a higher number of multinuclear aortic endothelial cells in humans [293], which may have functional consequences.   Moreover, we are solely studying an aortic, or macrovascular, cell line. This has two important implications. The first consideration is that different vascular beds are known to 81  behave differently under the same context. The second is that the macrovascular system is different from the microvasculature. There have been many reviews that discuss the difference between veins and arteries, as well as between arteries, arterioles and capillaries, and how endothelium structure, function and phenotype differ depending on the vascular bed studied [294,295]. For example, one group showed that thromboxane inhibitors could enhance acetylcholine-induced relaxation in the aorta, but not superior mesenteric artery, of aged rats [296]. Similarly, another study suggested that expression of endothelial von Willebrand factor was dependent on tissue microenvironment [297]. Hence, it is essential that we are careful in how we interpret and extrapolate our findings, particularly when it comes to microvascular (e.g. diabetic retinopathy) and macrovascular (e.g. atherosclerosis) diseases. Given our cell line of choice, it may be more appropriate to extrapolate in the direction of macrovascular diseases. 3.4.2. Carrier-based Uptake  Cav-1-derived peptides have been typically conjugated to cell permeable AP, a cellular internalization sequence derived from the third helix of the DNA binding domain of antennapedia [291]. However, the uptake sequence can have a large effect on uptake and distribution. We addressed potential uptake and sub-cellular trafficking-related issues by comparing AP-conjugated peptides against another carrier. Of interest is myristic acid (Myr), a fourteen carbon lipid chain, which has been used to deliver agents such as PKC-α pseudosubstrate peptides [298] and gadolinium-DOTA [299], a magnetic resonance imaging contrast agent, in to cells. In addition, myristoylation is a naturally occurring form of post-translational modification for proteins such as eNOS, which helps target it to the plasma membrane [149]. Furthermore, myristoylation of peptides is thought to promote increased 82  association with the plasma membrane [300], making it an appealing comparator to AP, given the site of eNOS/Cav-1 interaction. To some extent, we observed this in the early stages of our live cell imaging process, where cells treated with Myr-conjugated exhibited a more membrane-centric staining pattern. Beyond uptake, different cell lines have been known to exhibit preference for specific uptake carriers, as shown in a study comparing Myr to TAT (Trans-activator of Transcription; a cell-penetrating peptide sequence derived from HIV-1 [301]) which found that lymphocytes more readily took up the Myr-conjugated peptide sequence [302]. In contrast, our study demonstrated that uptake of AP-based peptides was greater than Myr-based peptides. This may be the result of the uptake mechanisms, as protein based uptake sequences, such as AP and TAT, have been conjectured to be uptaken by lipid raft dependent, clathrin-independent endocytosis [303] and macropinocytosis [304], whilst lipidated peptides (e.g. palmitoylated and myristoylated peptides) are thought to associate with the membrane and enter the cell via flip-flop exchange [302,305]. The membrane targeted entry method of lapidated peptides may allow it to better target membrane specific processes (i.e. Cav-1/eNOS interactions), which may explain why lower levels of Myr-based peptide uptake could generate such robust responses and contribute to some of the carrier dependent effects observed, which could lead to substantial differences in therapeutic outcomes. This suggests that Myr-conjugation may be a better alternative than the traditionally utilized AP-conjugation for investigating Cav-1 based regulation of cellular mechanisms and therapies. 3.4.3. eNOS and Cav-1 Dependence As an initial proof of mechanism of action, we wanted to demonstrate that the peptides were both eNOS and Cav-1 dependent. With respect to eNOS, following siRNA 83  knock-down, the NO response was essentially abolished, suggesting that, indeed, the NO release was mediated by eNOS. In contrast, Cav-1 knock-down did not produce the expected response. We initially hypothesized that Cav-1 knock-down would reduce NO release versus control, as the level of eNOS/Cav-1 interaction would be reduced, resulting in reduced efficacy of the peptides. However, counterintuitively, we do not observe a decrease. There are two probable explanations for the above observation. The first explanation could be that the knock-down of Cav-1 was insufficient, with the remaining cellular Cav-1 being able to compensate for the reduction in Cav-1 expression, thereby resulting in a lack of effect. There could be two approaches to address this problem. One method would be to use multiple types of Cav-1 siRNA to produce a greater knock-down and hoping that it would be sufficient to alter the response. Another approach would be to use cells that naturally lack Cav-1 and eNOS, such as LNCaP cells [306], and transiently express Cav-1, eNOS, or both proteins in the cell in order to gauge for Cav-1 dependence. Aside from an insufficient knock-down of Cav-1, a second explanation could be that it was due to a unique property of CAV. The CAV sequence is part of a larger sequence known as the Cav-1 oligomerization domain (residue 61-101), which is responsible for direct interaction between Cav-1 proteins. In a study, the Weiss group showed that full length CAV-derived peptides could interact with phage-displayed CAV sequences and increase binding of client proteins such as protein kinase  A [307] and eNOS [308]. As such, there is a possibility that CAV-derived peptides could interact with the remaining Cav-1 and promote increased eNOS/Cav-1 interaction to counter the effects of the knock-down. Beyond knock-down experiments, this suggests that under basal conditions, these peptides could interact with endogenous Cav-1 protein, via their scaffolding domain, to increase Cav-1/client 84  protein interaction, which would have implications for other signaling processes, in addition to eNOS regulation. As such, this oligmeric tendency would present a challenge in elucidating the significance of Cav-1 in the NO-response within an intact system. Interestingly, Levin et al. [308] utilized an in vitro assay to demonstrate that this oligomeric/recruitment effect of the Cav-1 scaffolding sequence can be overcome at high concentrations of synthetic peptides; however, such concentrations are unfeasible for cellular studies.  3.4.4. Interpreting the Importance of Lipid Rafts  Cyclodextrins are cyclic oligosaccharides capable of extracting cholesterol from the plasma membrane by forming inclusion complexes with cholesterol to enhance the solubility of cholesterol in aqueous solution [309,310]. Depletion of membrane cholesterol results in the loss of cell surface caveolae [220]. Researchers have utilized this unique trait of mCDs to study mechanisms of actions associated with lipid rafts and caveolae.  While it is difficult to discern whether the effects are mediated by lipid rafts or caveolae due to the non-specific effects of mCD, some light can be shed on this issue by a previous study performed by Sowa et al [311]. In the study, they demonstrated that LNCaP cells, which do not express Cav-1, do not form caveolae following infection with a Cav-1 adenovirus. What was striking was that, while Cav-1 distributed to cholesterol rich membrane fractions, it failed to regulate eNOS in the absence of caveolae. This demonstrated that it was caveolae Cav-1 that was involved in eNOS regulation and not lipid rafts. Hence, changes observed in peptide function are most likely the outcome of disturbed caveolar regulation, as opposed to disruption of lipid rafts. This would help towards explaining the following observation. 85  With regards to our results, following mCD treatment, we observed that AP-BSP and BSPF92A elicited similar, if not higher levels of NO relative to their untreated counterparts. In contrast, Myr-BSP and BSPF92A-induced NO levels were noticeably reduced. Interestingly, in all instances, total peptide uptake was not decreased in comparison to their control counterpart. This outcome would make sense when taken in line with our previous findings. More specifically, in Figure 15 we observed that AP-based peptides were uptaken faster and possessed a more diffused cellular staining pattern in comparison to Myr-conjugated peptides, which suggested that cellular localization of these peptides are most likely carrier dependent. Following the dissolution of caveolae by cholesterol depletion, the AP-conjugated peptides would have greater access to eNOS and Cav-1 that were previously trapped in caveolae. In contrast, Myr-based peptides most likely failed to be effectively concentrated at the relevant sites of interaction following mCD treatment, resulting in lower level of induced NO.  3.5. Conclusion In summary, we have confirmed that the identified BSP and BSPF92A motifs can be used to promote NO in endothelial cells, upon conjugation to a suitable uptake sequence, for example Myr or AP. Both Myr and AP have their own unique characteristics that affect the level of NO released by resting BAECs. Furthermore, NO release was eNOS dependent, whereas Cav-1 knock-down showed no effect on NO release. This could either be due to insufficiency of the knock down or the oligomeric nature of Cav-1. Regardless, it is difficult to discern the extent of contribution from Cav-1 to NO release. However, our cyclodextrin results, in conjunction with findings from the literature, suggest that effects of the peptides are linked to caveolae. Lastly, the discrepancies in NO release between the BSPF92A- and 86  BSP-based peptides provide indirect evidence that the two motifs act via different mechanisms of action, which warrants further investigation to validate.   87          Chapter 4. Mechanistic Insight in to the Functional Significance of F92 on eNOS Regulation   88  4.1. Introduction3 As discussed, the wild type CAV sequence has been associated with eNOS inhibition. Indeed, we have observed that even the shorter BSP sequence can be used to reduce VEGF-stimulated NO release from BAECs. However, as demonstrated in the previous section, it was unexpected that both BSP and BSPF92A were able to stimulate basal NO production. While initial evidence appears to suggest that BSP and BSPF92A induce NO via different mechanisms of actions, preliminary investigations in to uptake of these peptides and eNOS/Cav-1 dependence did not reveal anything worthwhile. This has led to postulation of two possible causes behind this unexpected observation.  The first potential cause is that the peptides, being as they are part of the oligomerization domain [237], could interact with and destabilize endogenous Cav-1 oligomeric complexes or endogenous interactions, resulting in redistribution of eNOS and Cav-1, leading to some anomalous results. Alternatively, given the promiscuous nature of the scaffolding domain, it is also conceivable that other relevant signaling cascades may be disrupted by the substitution of F92 with alanine. For example, an F92A mutation-containing peptide was previously shown to be better at inducing ERK phosphorylation in fibroblasts versus its wild type counterpart [312]. As eNOS regulation is highly complex and dynamic, involving events such as subcellular targeting, phosphorylation and other mechanisms, either approach could foreseeably alter NO production. As such, we sought to investigate whether the mutation of F92 into alanine could unexpectedly alter eNOS activation indirectly, via changing biochemical properties of either eNOS or Cav-1, or directly, such as changing relevant signaling cascade.                                                  3 A version of the text and figures in this chapter has been published in the Eur J Pharmacol (2015), 766: 46-55; doi:10.1016/j.ejphar.2015.09.033. 89  Herein, we demonstrate that neither BSP, nor BSPF92A causes destabilization of Cav-1 oligomeric complexes. Similarly, neither peptide was found to cause a shift in Cav-1 or eNOS subcellular localization, as determined by subcellular fractionation studies. Furthermore, F92A substitution did not affect eNOS and Cav-1 co-localization. However, the substitution of F92 with an alanine was found to prevent Akt-based activation of eNOS, thus suggesting that F92 could play an essential role in cellular signaling.  4.2. Methods 4.2.1. Cell Culture Refer to section 3.2.1. 4.2.2. Velocity Gradient Centrifugation Cells were lysed in a MES-buffered saline solution (MBS; 25mM Mes, 0.15M NaCl, pH 6.5) containing 60mM octyl-β-glucoside (Sigma Aldrich). The lysate was briefly sonicated to disrupt visible debris before being loaded on to a linear 5% - 45% sucrose gradient. Samples were centrifuged using a SW 41 Ti at 140, 000g for 18 hours before being collected in 1 mL fractions in a top-down manner for western blot analysis.  4.2.3. NO Analysis For inhibitor studies, cells were pre-treated with the inhibitor for 60 mins at the stated concentration. Following which, the BAECs were incubated with fresh serum free media with the stated peptides at 10 µM for 6 hrs, before the media was collected and analyzed as described in section 3.2.2. 90  4.2.4. Sucrose Fractionation BAECs were grown to a post-confluent stage in C100 plates (BD Falcon) and were treated with peptides (10 µM). Cells were lysed in a detergent free buffer consisting of 500mM NaCO3 with general protease inhibitor cocktail (Sigma Aldrich). Samples were loaded on to discontinuous sucrose gradient (5 and 45% sucrose dissolved in MBS) and centrifuged at 140,000g for 18 hrs at 4°C using a SW 41 Ti rotor. Fractions were collected in 1 mL aliquots in a top-down manner for Western blot analysis. 4.2.5. Immunofluorescence BAECs were grown in 4-chamber slides and stimulated with peptides (10 µM) after confluence was reached. Four hours post-stimulation, the cells were washed with phosphate buffered saline (PBS) and fixed with formalin for 10 min at RT. The cells were washed with PBS and incubated with 0.1% triton-X for 10 mins at RT, following which 3 PBS washes were performed. The fixed samples were then blocked with PBS containing 0.1% BSA (Millipore) and 2.5% normal goat serum for at least 60 min. Samples were then incubated with rabbit anti-Cav-1 (Santa Cruz Biotech) and mouse anti-eNOS (BD transduction) in blocking solution overnight at 4°C. The chambers were then washed 3 times with PBS and incubated for 45 min with goat anti-mouse Alexa Fluor 488 and goat anti-rabbit Alexa Fluor 594 (Molecular Probes, Invitrogen) in the dark at RT. Samples were washed with PBS and coverslips were mounted using Vectashield mounting media with DAPI (Vector Laboratories). Slides were allowed to set overnight in the dark at RT before being imaged on an inverted Leica microscope with a confocal scanner. Results were analyzed on the Volocity software (PerkinElmer). Specificity of signal was confirmed via comparison against the 91  respective immunoglobulin G controls. Three to five cells were averaged per slide to generate an average for each n-value. 4.2.6. Western Blotting Cells were treated for the stated duration and lysed in a buffer containing: 50mM Tris-HCl, 1% NP-40, 0.1% SDS, 0.1% deoxycholic acid, 0.1mM EDTA, 0.1mM EGTA, 5mM NaF, 2mM sodium pyrophosphate, 2mM sodium orthovanadate, 1mM PMSF and Sigma protease inhibitor cocktail. The lysate was centrifuged at 20,000g for 30 mins at 4°C and the supernatant was collected. Protein concentrations were determined using a Bio-Rad DC assay and 20 µg of each condition was loaded on to a polyacrylamide. Gels were transferred on to a nitrocellulose membrane and probed with rabbit anti-phospho-Akt-473 (Cell Signaling), rabbit anti-Akt (Cell Signaling), mouse anti-Hsp90 (BD Transduction), rabbit anti-phospho-eNOS 1179 (Cell Signaling) and mouse anti-eNOS (BD Transduction). Primary antibodies were detected using goat anti-mouse 790 or goat anti-rabbit 680 secondaries respectively and imaged on our LICOR scanner. 4.2.7. Data Analysis Co-localization studies were analyzed using one-way ANOVA. In contrast, NO release studies were analyzed using two-way ANOVA. A post-hoc Dunnett’s test was used to find significant difference between samples. Significance was defined as a p-value < 0.05. 4.3. Results 4.3.1. Peptide Treatment and the Biochemical Properties of Cav-1 As discussed in Chapter 3, these CAV-derived peptides can interact with the oligomerization domain of Cav-1, which could potentially destabilize Cav-1 oligomers, and 92  indirectly affect caveolae function, which may explain why both BSP and BSPF92A could promote NO release in BAECs. To determine if oligomerization was affected, we used a technique called velocity gradient centrifugation, which allows us to sort proteins based on molecular weights. This had been validated in our lab previously, using a series of molecular weight markers including carbonate anhydrase, BSA, catalase, and thyroglobulin [276] and has been further confirmed by directly comparing the distribution of Cav-1 to eNOS (140 kDa) and Akt (60 kDa). Akt starts to appear in fraction 2 whilst eNOS shows up in fraction 4 and above (Figure 19A). Cav-1, being a 25 kDa protein, would be expected to show up in fractions 2 or earlier; however, due to its oligomeric nature, it shows up in fractions 4 and above. Qualitatively, treatment of BAECs with BSP and BSPF92A peptides, regardless of carrier sequence, did not produce any noticeable shift in the distribution of Cav-1 oligomers compared to their respective vehicular control (Figure 19B, C). This was verified by direct quantification of the Western Blot bands, which demonstrated that BSP and BSPF92A treatment produced almost exactly the same distribution profile, with the bulk of the Cav-1 oligomers found in fractions 4 to 7, while the remainder was spread out between fractions 8 through 12 (Figure 19D, E).  This indicated that disruption of Cav-1 oligomers, and therefore caveolae function, was unlikely to be a major contributor to BSP and BSPF92A stimulated NO release. 4.3.2. Subcellular Localization of eNOS and Cav-1 Not Affected by F92A We next determined if the BSP and BSPF92A peptides altered sub-cellular distribution of Cav-1 and eNOS. A sub-cellular fractionation technique was used to determine if proteins are associated with cholesterol-enriched membranes (CEM). We chose to investigate this  93   Figure 19. F92A Substitution Does Not Affect Cav-1 Oligomerization Endothelial cells were treated with 10 µM of peptides for 6 hrs before lysis and placed on top of a 5 – 45% sucrose gradient for centrifugation at 140, 000 x g. Fractions were collected top-down. A) Representative immunoblot indicating the distribution of endothelial nitric oxide synthase (eNOS), Akt and caveolin-1 (Cav-1) following velocity gradient centrifugation. Molecular weight increases from left to right (indicated by gradient bar above figure). B and C) Representative immunoblots demonstrating the distribution of Cav-1 oligomers following antennapedia peptide (AP)- or myristic acid (Myr)-conjugated peptide treatment, respectively. Cargo sequences were binding site peptide (BSP) or BSPF92A. D and E) Graphical representation of the percent of total Cav-1 in each fraction following treatment by peptides conjugated to either AP or Myr respectively (mean ± S.E.M.; n = 3). The oligomer distribution of Cav-1 was not altered by phenylalanine F92 substitution.   A.eNOSAktCav-11 12Fraction:B.1 12Fraction:APAP-BSPAP-BSPF92AMyrMyr-BSPMyr-BSPF92AC.1 12Fraction:Increasing MWD.E.94  since it is well known that the function of proteins, including eNOS, can be affected by cellular targeting. Under normal conditions, HSP 90, a bulk cytosolic protein, can be found in fractions 7 through 12 following subcellular fractionation; in contrast, Cav-1, CEM associated protein, is targeted to fractions 2 through 4. Lastly, eNOS, could be found in both CEM and non-CEM fractions (Figure 20A). We initially assayed for changes in Cav-1 distribution, as redistribution of Cav-1 could affect caveolae function and signaling. However, we found that BSP and BSPF92A peptides, regardless of uptake sequence, did not affect the CEM distribution of Cav-1 relative to vehicle treated cells. More specifically, the bulk of the Cav-1 protein was found in fractions 1 through 5, whilst a small portion (~20%) could be found in the non-CEM fractions (Figure 20B-E). This indicated that the peptides did not affect the CEM distribution of Cav-1. Similarly, we sought to confirm whether eNOS distribution was altered by peptide treatment, particularly with respect to the F92A substitution. Both the wild type BSP and the F92A variant had a similar effect on eNOS targeting to cholesterol-rich microdomains (Figure 21A-D), regardless of whether it was conjugated to AP or Myr. On the other hand, when compared to control AP or Myr stimulation, the CAV-derived peptides triggered a slight shift of eNOS from the buoyant fractions to the non-lipid fractions (Figure 21E), a known outcome of eNOS activation [154,313], regardless of F92 inactivation. This simply re-affirms the finding that the BSP sequence can change underlying mechanisms of eNOS regulation to promote NO release and that this triggers a shift of eNOS outside of caveolae/lipid rafts, but does not provide insight into the actual identity of the mechanism itself. 95   Figure 20. F92A Substitution Does Not Affect Cav-1 Targeting to Cholesterol Enriched Fractions Endothelial cells were treated with 10 µM of peptides for 6 hrs before lysis and placed on top of a discontinuous sucrose gradient for centrifugation. Fractions were collected top-down. A) Representative immunoblot demonstrating distribution of endothelial nitric oxide synthase (eNOS), heat shock protein (HSP) 90 and caveolin-1 (Cav-1) to cholesterol enriched lipid rafts (1-7) and cytosolic fractions (8-12) following sucrose fractionation. B and C) Representative immunoblots demonstrating the distribution of Cav-1 following antennapedia peptide (AP)- or myristic acid (Myr)-conjugated peptide treatment, respectively. Cargo sequences were binding site peptide (BSP) or BSPF92A. D and E) Graphical representation of the percent of total Cav-1 associated with each individual fraction following treatment with peptides conjugated to either AP (D) or Myr (E) respectively (mean ± S.E.M.; n = 3). The distribution of Cav-1 was not affected by phenylananine F92 substitution.  APAP-BSPAP-BSPF92AMyrMyr-BSPMyr-BSPF92A1 12Fraction:A.C.Fraction:WB: Cav-1WB: Cav-1eNOSHSP 90Cav-1Fraction: 12B.Cholesterol-rich Bulk proteinsCholesterol-rich Bulk proteins D.E.112Cholesterol-rich Bulk proteins196   Figure 21. F92A Substitution Does Not Affect eNOS Targeting to Cholesterol Enriched Fractions Endothelial cells were treated with 10 µM of peptides for 6 hrs before lysis and placed on top of a discontinuous sucrose gradient for centrifugation. Fractions were collected top-down. A and B) Representative immunoblots demonstrating the distribution of endothelial nitric oxide synthase (eNOS) following antennapedia peptide (AP)- or myristic acid (Myr)-conjugated peptide treatment, respectively. Cargo sequences were binding site peptide (BSP) or BSPF92A. C and D) Graphical representation of the percent of total eNOS associated with each individual fraction following treatment by peptides conjugated to either AP (C) or Myr (D) respectively. E) Total Percentage of eNOS found in the buoyant fractions following treatment w either AP- or Myr-conjugated peptides (mean ± S.E.M.; n = 3). Substitution of F92 did not alter distribution of eNOS between cholesterol-enriched and cytosolic fractions.  E.B.MyrMyr-BSPMyr-BSPF92AWB: eNOSFraction: 1 12Cholesterol-rich Bulk proteinsA.APAP-BSPAP-BSPF92AWB: eNOSFraction: 1 12Cholesterol-rich Bulk proteinsD.C.97  4.3.3. F92A Substitution Does Not Affect eNOS and Cav-1 Co-localization As eNOS was mobilized from cholesterol-enriched to bulk protein fractions following peptide treatment, we investigated if F92A affected co-localization of eNOS and Cav-1, which may explain how these scaffolding domain-derived peptides could promote NO. For this, we performed a fixed-cell immunofluorescence study on BAECs following peptide treatment. Qualitatively, all treatments affected eNOS distribution similarly, leading to a heavy perinuclear concentration and cytoplasmic distribution. In contrast, following AP-BSP and AP-BSPF92A treatment, Cav-1 appears to aggregate at the plasma membrane (Figure 22A); in comparison, following Myr-BSP and Myr-BSPF92A treatment, Cav-1 tends to have a more cytoplasmic distribution (Figure 22B). This was also reflected in the Pearson’s correlation, which indicates the extent of co-localization between eNOS and Cav-1, wherein AP-treated samples (0.210 ± 0.027, 0.224 ± 0.043 and 0.205 ± 0.049 for AP, AP-BSP, and AP-BSPF92A respectively) tended to have slightly lower levels of correlation than Myr-treated samples (0.281 ± 0.048, 0.307 ± 0.048 and 0.332 ± 0.029 for Myr, Myr-BSP and Myr-BSPF92A respectively). However, analysis of co-localization within the same treatment group showed no significant difference. 4.3.4. F92 Inactivation Blunts Akt Signaling  As we could find no changes in the basic biochemical profile of either Cav-1 or eNOS, we decided to investigate whether there were any alterations in cell signaling cascades. Endothelial NOS is a protein whose function is heavily regulated. One of the most important regulators of eNOS is Akt. Activation of Akt leads to phosphorylation of eNOS at S1177, resulting in prolonged and increased levels of NO synthesis.   98   Figure 22. F92A Substitution Does Not Affect eNOS and Cav-1 Co-localization A) Representative confocal images of formaldehyde fixed endothelial cells demonstrating endothelial nitric oxide synthase (eNOS; green) and caveolin-1 (Cav-1; red) co-localization following treatment with antennapedia peptide (AP) or AP-conjugated peptides for 4 hrs. Cargo peptides were either binding site peptide (BSP) or BSPF92A. Plasma membrane staining was observed for Cav-1 (white arrow); however, all treatment conditions (AP, AP-BSP and AP-BSPF92A) had similar levels of eNOS/Cav-1 co-localization. B) Equivalent images for endothelial cells treated with myristic acid (Myr)-conjugated peptides, showing a more cytoplasmic staining pattern for Cav-1. Similarly, Pearson’s correlation indicated that the degree of co-localization between Cav-1 and eNOS following treatment with Myr-conjugated peptide treatment was similar for all conditions. n = 5-6 (mean ± S.E.M) for both experiments.  APAP-BSPAP-BSPF92AeNOS Cav-1 MergeA.eNOS Cav-1 MergeMyrMyr-BSPMyr-BSPF92AB.99  We first assessed for the presence of phospho-Akt (p-Akt) following peptide stimulation and found that, interestingly, p-Akt was upregulated by both AP- and Myr-BSP (Figure 23A, B). In contrast, AP- and Myr- BSPF92A had little effect on p-Akt levels. To confirm this, we used a well-known inhibitor of Akt phosphorylation, wormtannin. Wortmannin is an irreversible inhibitor of phosphoinositide 3-kinase, which is responsible for the phosphrylation of Akt. Following 60 mins pre-treatment of BAECs with wortmannin, we found that both AP- and Myr-BSP mediated NO release was significantly blunted by approximately 46% and 53% respectively (Figure 23C and D). In comparison, wortmannin caused a very mild, and non-significant reduction, in AP- and Myr-BSP mediated NO release, which is in line with our western blot observations.    4.4. Discussion Cav-1, via its scaffolding domain, is a critical regulator of a plethora of proteins including eNOS, which is essential for cardiovascular homeostasis. Due to its significant vascular role in health and disease, there is merit in exploring novel approaches to promoting eNOS-mediated NO release. We propose that peptides derived from Cav-1 could be used to promote eNOS-mediated NO release for better management of cardiovascular outcomes. Interestingly, we found that, in the context of unstimulated settings, both BSP and BSPF92A could promote NO release, with initial evidence pointing at differing mechanisms. Since the peptide sequences used originated from the oligomerization domain, defined as residues 61-101 of the Cav-1 [237], there were initial concerns about disrupting Cav-1 oligomers. However, as Cav-1 complex sizes were unchanged, it suggests that, within the time frame of assessment, changes in oligomerization status was not a major contributor to NO production, as would be expected, since Cav-1 oligomeric complexes are known to be stable [314].  100   Figure 23. F92 Promotes Akt-mediated NO Release Endothelial cells were stimulated with 10 µM of peptides for 4 hrs before being lysed for protein analysis. A and B) Representative blots comparing changes in Akt and endothelial nitric oxide synthase (eNOS) phosphorylation states (denoted by ‘p-‘) in antennpaedia peptide (AP)- or myristoylated (Myr)-peptide treated endothelial cells. Cargo peptides were binding site peptide (BSP) or its F92A variant (BSPF92A). Heat shock protein (HSP) 90 was utilized as a loading control. The peptides were able to promote phosphorylation of Akt (n = 3). C) AP-, AP-BSP- and AP-BSPF92A-induced nitrite (pmol/mL) in endothelial cells in the absence (white) or presence (black) of wortmannin (1μM), an inhibitor of Akt activation (mean ± S.E.M.; n = 4; *: P < 0.05 versus ). D) Equivalent study using Myr-conjugated peptides (mean ± S.E.M.; n = 4; *: P < 0.05) in the absence (white) or presence (black) of wortmannin. A reduction in sensitivity to wortmannin was observed following F92A substitution.  B.p-eNOSeNOSHSP 90p-AKTAKTA.p-eNOSeNOSHSP 90p-AKTAKTC. D.101  Furthermore, sub-cellular targeting of Cav-1 and eNOS did not seem to be affected by F92A substitution. In contrast, F92A substitution played a unique role in uncoupling NO release from Akt activation. Taken together, our results indicate that that the F92 residue has a significant impact on basal NO regulation via Akt signaling, independent of the uptake sequence utilized.  4.4.1. Characterization of Critical Residue F92 There have been many studies investigating CAV to date; however, such studies tend to study the impact of several residues simultaneously, as part of what is known as the Cav-1 binding motif [211]. Another common approach is to perform a F92A/V94A substitution, which has been used for a range of studies including insulin-mediated Cav-1 phosphorylation [315], negative regulation of raft-dependent endocytosis [316], Elk-1 signaling in adipose cells [317] and epidermal growth factor-induced p-ERK signaling [318]. However, such studies would not be able to attribute the observed effects to specific residues. Furthermore, as shown by our previous investigations [278,290,319], and also in this study, a one point mutation could have large implications for eNOS regulation. Similarly, another group studying monocytes and fibroblasts observed that a single point mutation, F92A, could have a profound effect on cellular events [312]. While F92 has been highlighted, other residues may be similarly important; hence, there is a need to focus on individual residues to develop a true understanding of how the Cav-1 scaffolding domain is regulating cellular biology. 4.4.2. Peptide-induced Redistribution of Cav-1 Protein distribution is well known to alter function. As such, we hypothesized that there could be a change in localization of either eNOS or Cav-1 induced by the F92A substitution. We first investigated eNOS, which is differentially regulated based on its 102  localization in the cytoplasm or plasma membrane [320]; however, our sucrose fractionation results suggested that this was not a major contributor, which was qualitatively supported by our immunofluorescence study. In contrast, a noticeable shift was observed in the Cav-1 distribution in the imaging studies. Interestingly, AP-peptide treatment induced the re-distribution of Cav-1 to the plasma membrane; in comparison, Myr-peptides produced a diffused staining pattern. As Cav-1 at the plasma membrane is known to drive caveolae formation, redistribution of Cav-1 could have profound consequences on downstream events. Even if caveolae formation was not affected, Cav-1 is a regulator of a plethora of proteins; hence, it is likely that other aspects of cell signaling could be perturbed. This suggests that Myr- and AP-based peptides may be utilized to study the functional significance of specific mutations on cargo sequences in different regions of the cell, which could help with the development of targeted therapies. However, in the context of our study, by combining observations from both carriers, we better understand the role of F92 in cellular signaling processes. 4.4.3. Akt and Regulation by CAV As the basic biochemical properties of both eNOS and Cav-1 were not affected by F92A substitution, we decided to investigate endothelial cell signaling instead. Cav-1 has been shown to promote PI3k/Akt activity, which is known to drive eNOS activity by promoting phosphorylation of S1177 [321,322], in events such as type I procollagen expression [323], cancer cell migration [324], cell survival  and vascular mechanotransduction [325] through its scaffolding domain [326]. Similarly, in our study, we observed that the wild type BSP sequence was able to increase pAkt and drive NO production. This was further confirmed by the significant decrease in NO release following 103  treatment with wortmannin, a well-known irreversible inhibitor of PI3k. In contrast, the substitution of F92 in BSP reduced sensitivity to wortmannin, suggesting that this residue plays an important role in PI3k/Akt signaling regulation. This is supported by findings from another group that suggested F92A substitution in cavtratin, the full length scaffolding domain peptide, led to increased pERK and pMEK [312], indicating that a single residue could have profound implications on scaffolding domain peptide-mediated signaling. Lastly, a small, but insignificant, decrease in NO production was observed in Myr-BSPF92A treated cells following wortmannin incubation, which may be due to the nature of Myr having the potential to promote Akt phosphorylation [327], or residual regulatory effects of the cargo sequence.  4.5. Conclusion We have observed that the basic biochemical properties of both eNOS and Cav-1, such as oligomerization, trafficking to cholesterol rich membrane and co-localization are not altered by the F92A substitution in the BSP sequence. However, we demonstrated that the F92 residue is important for eNOS phosphorylation and Akt signaling, suggesting that there is more to the role of F92 than what is currently known. This suggests the need for more studies that focus on specific amino acid substitutions over the current norm of performing multiple substitutions, where it becomes difficult to isolate the significance of the residues. Lastly, it is interesting to know that the carrier sequence utilized may do more than just alter trafficking patterns, but may also produce unexpected downstream events such as redistribution of proteins of interest.   104         Chapter 5. General Discussion and Conclusion   105  5.1. Review of Findings Cardiovascular diseases are the largest cause of global mortality. One of the hallmarks of cardiovascular disease is a marked reduction in vascular NO, an important gaseous molecule with cardiovascular protective properties. Vascular NO formation is predominantly governed by eNOS, which is regulated by a variety of mechanisms, including phosphorylation and direct protein-protein interactions. Cav-1, the main coat protein of caveolae, is an important protein regulator of eNOS. Interaction with a 20 a.a. region of Cav-1, known as the scaffolding domain (CAV), leads to inhibition of eNOS activity. Our lab had previously demonstrated that F92 in CAV is responsible for the inhibition of eNOS and that inactivation of the F92 residue with alanine could lead to loss of inhibitory action. This finding could have potential therapeutical relevance, as CAV peptides with an F92A substitution can increase NO release from endothelial cells to regulate blood pressure and reduce atherosclerotic plaque load. As an extension of our previous studies, we showed that 10 residues within CAV can account for the majority of eNOS binding. As expected, the wild type variant of the sequence, BSP, could inhibit stimulated NO release, which was lost in the F92A variant, BSPF92A. However, both BSP and BSPF92A could promote NO release following conjugation to either Myr or AP. In both instances, NO release was eNOS and most likely caveolae dependent. As we have demonstrated that F92A substitution does not change uptake, in a further attempt to understand if there is a mechanistic difference in NO release between BSP and BSPF92A peptides, we further investigated the biochemical properties of Cav-1 and eNOS. We found that F92A substitution did not affect Cav-1 oligomerization, eNOS and Cav-1 subcellular localization, and eNOS/Cav-1 co-localization. Instead, F92 played a role in promoting Akt 106  activity. Based on our results, we propose the following two mechanisms of action for BSP and BSPF9A: BSP peptides promote the activation of PI3K, leading to phosphorylation of Akt and downstream activation of eNOS. However, due to the presence of the F92A residue, there is likely a fine equilibrium between activation and inhibition of NO production. In contrast, F92A substitution serves as a non-inhibitory sequence to help maintain eNOS in an active state (Figure 24). 5.2. Significance of Findings  The above research provides insight into Cav-1/protein interactions, Cav-1 signaling and cell penetration sequence selection. First, we confirmed that 10 residues (a.a. 89-99) of CAV is sufficient to account for majority of eNOS binding. This confirms a similar observation where 16 residues (a.a. 86-101) were found to be required for CAV interaction with Gi2α, a G-protein subunit [211]. However, the difference in required number of residues for binding suggests that specificity of binding proteins can be imparted by residues surrounding the Cav-1 binding motif. This knowledge provides support for the possibility of optimizing the selectivity of BSP and BSPF92A for eNOS, or for other proteins of interest. Moreover, our research confirms that different properties (i.e. binding vs regulation) can be independently identified from within CAV, which may be applicable to other proteins. Second, we provide support for the multi-functional significance of F92. This is the first time a CAV-derived sequence has been shown to be able to promote NO release via Akt upregulation. Furthermore, upregulation of NO via Akt can be significantly affected by the presence or absence of residue F92. This suggests that F92 is not only important for regulation of eNOS, but also for Akt. Moreover, there is a possibility that F92 may similarly regulate other proteins that localize to caveolae. In addition, our findings support the value of  107   Figure 24. Hypothesized Sequence of Events Based on Mechanistic Findings A) Wild type binding site peptides (BSP) regulates eNOS activity via two different paths. First, it can stimulate phosphoinositide 3-kinase (PI3K), which ultimately results in phosphorylation and activation of eNOS. Secondly, it can bind to activated eNOS to inhibit activity. These two counterbalancing effects create a dynamic equilibrium. B) In contrast, the substitution of the F92 residue in BSPF92A (denoted by red ‘A’), prevents activation of PI3K signaling. Instead, it is thought that BSPF92A can act as an antagonist, preventing eNOS from binding to endogenous Cav-1 and maintain it in an active state.  A.B.BSPPI3kAKTAKTPeNOSPNONOAAeNOSNOBSPeNOSPI3kAKTAKTBSP108  focusing efforts on identifying functional significance of individual residues within CAV. Lastly, there have been no exhaustive comparisons of uptake sequences for Cav-1 peptides, with AP being essentially ubiquitously used. While different uptake sequences can affect cargo trafficking, we clearly showed that both AP- and Myr-conjugation can also promote unique, permeabilization sequence-dependent changes in Cav-1 distribution. This has significant implications for studies that have relied on AP-conjugated Cav-1 peptides to study cellular mechanisms, where redistribution of the Cav-1 protein can influence cellular events and subsequent interpretation of the results.   5.3. Applications of Research Peptide based therapies have been gaining interest in recent years, with 6 of 39 FDA approved therapies being peptide-based in 2012 and over 128 different additional therapies being investigated in Phase I, II and III studies in diverse areas such as pain, endocrinology, immunology, oncology and dermatology [328]. Though peptide therapies are generally thought to be efficacious, tolerable, and selective, peptides tend to be quickly metabolized and difficult to administer [329]. Given their quirks, this leads to two possible routes regarding application – find a route of administration that is less degradation prone or use the identified sequence as a pharmacophore.  To the first point, one disease to target could be pulmonary hypertension. The reason for selecting pulmonary hypertension is two-fold. The first is that NO-based therapies have been shown to be effective in pulmonary hypertension. In pulmonary hypertension, the phosphodiesterase 5 inhibitor sildenafil, which prevents the breakdown of cGMP, and thereby potentiates NO-mediated sGC signaling, has been shown to reduce pulmonary 109  pressure, reverse a pathological shift of the cardiac septum, and increase performance in the 6-minute walk test [330,331]. Although the studies did not assess for pulmonary hypertension associated mortality, the 6-minute walk test is positively associated with survival [332]. In support of the value of increased NO in pulmonary hypertension, animal studies have shown that inhaled nitrite, which can be converted into NO in biological systems, could be used to mitigate pulmonary hypertension-induced pathological remodeling of the heart and lung [333]. The second reason for delving in to pulmonary hypertension is that the pulmonary system is an end target, which allows for bypassing of systemic absorption and metabolism inefficiencies and greater flexibility in the manipulation of doses delivered. However, an investigation into the usage of BSP and BSPF92A peptides in pulmonary hypertension would be little more than a proof of concept, with subsequent optimization studies still required. The second application for our findings is to use it as a basis for a pharmacophore to create a compound that is more stable and readily administrable, opening up treatment opportunities for endothelial dysfunction in a greater number of conditions such as atherosclerosis, systemic hypertension and diabetes. The simplest approach would be peptidomimetics, a relatively recent development. This generally involves performing chemical modifications to improve distribution, bioavailability, protease resistance, stability and other pharmacologically desirable properties of identified peptide sequences [334,335]. To improve performance, there is also an additional possibility to generate a library of sequences based on the identified sequence, potentially in conjunction with a computation modeling study, before performing modifications, as evidenced by others [336,337]. 110  5.4. Concerns with NO Upregulation When attempting to promote NO release, there are potentially two concerns – shifting eNOS into uncoupled reactions and overproduction of NO release. There have been no studies performed with the peptides on uncoupling. However, a study showed that eNOS/Cav-1 interaction increased under cellular conditions that favored eNOS  uncoupling, resulting in reduced superoxide formation [338]. Similarly, our lab demonstrated that cells infected with the full-length F92A Cav-1 protein could lead to inhibition of superoxide formation [290].  This would indirectly suggest that both BSP and BSPF92A could potentially be able to reduce superoxide formation.  Similar to increased uncoupling and oxidative stress, high levels of NO are also a concern. Indeed, NO has been implicated in both cytotoxicity and cellular proliferation, as seen when immune cells release NO as a means to destroy bacteria, which highlights the dangers of NO. Whether NO presents a threat or not is linked to the concentration – the greater the concentration, the higher the likelihood of being cytotoxic or detrimental to surrounding cells. The question then becomes “Can increased NO from eNOS become detrimental?” While we can assess for effects following application of an exogenous source of NO, such as nitroglyercin, there is a degree of non-specificity due to the diffused nature of drug distribution within physiological and cellular systems. Instead, it may be better to use eNOS overexpression in models of cardiovascular disease as a surrogate.  While there have not been many studies performed, current studies have shown both benefits and detriments associated with elevated NO. In a diabetic ischemic reperfusion injury model, both overexpression of eNOS and pharmacological inhibition of eNOS proved detrimental [339]. Likewise, in an atherosclerotic model, one group showed that eNOS 111  overexpression led to reduced cholesterol, blood pressure and atherosclerotic lesions [340]; however, another group demonstrated that eNOS overexpression had the undesirable effect of accelerating the development of atherosclerosis [341]. As with all therapies, there will come a point wherein it becomes necessary to perform in vivo titration to better understand the relationship between NO, the pathophysiological context and the selected therapy. 5.5. NOS-related Pleiotropic Effects Having discussed the importance of NO levels, this brings up an interesting point regarding pleiotropic NO regulation of the other two NOS isoforms. As the active site for the NOS family is relatively conserved, it is unsurprising that CAV-derived peptides have been shown to be able to inhibit both iNOS and nNOS activity [210]. Of particular interest is iNOS, which is perhaps best known for its role in inflammation and generating oxidative stress. iNOS has been shown to be upregulated in animal models where endothelial dysfunction is observed [342–345], suggesting it may be beneficial to block it. Similarly, others have shown that pharmacological inhibition of iNOS or iNOS knockout could attenuate pathological remodeling in murine models [346,347]. On a similar vein, in vivo overexpression of myocardial iNOS was shown to promote immune cell infiltration, cardiac hypertrophy and sudden cardiac death [348]. In addition, iNOS has been shown to be upregulated in the aging endothelium, leading to increased arginase activity and endothelial dysfunction [349]. The majority of these above highlighted studies have suggested nitrositive stress as a potential concern. This may suggest that there could be certain advantages to BSP derived peptides over BSPF92A-based peptides, which could serve to reduce the level of inflammatory NO in the surrounding environment. 112  5.6. F92 Versus F92A in Therapies Targeting Endothelial Dysfunction Herein, we have demonstrated that both BSP and BSPF92A can promote NO release; however, it is difficult to say which is more viable in endothelial dysfunction. While many researchers study Cav-1 in cardiovascular diseases, not as many utilize Cav-1 derived peptides. Similarly, as the F92A peptide is a relatively novel development, there is little available information on extraneous effects. However, we can draw some conclusions regarding the benefits of both by taking a look at a few studies. With respect to the wild type sequence, Rodriguez-Feo et al. found that Cav-1 was reduced in the atherosclerotic lesions of patients, and that the reductions were positively correlated with increased risk of vascular events [350]. In the same study, the authors showed that Cavtratin, the full length CAV peptide, could be used to prevent expansive remodeling, which is associated with vulnerable plaque phenotypes, in an in vivo setting. In addition, in our results presented herein, we show that BSP sequences could stimulate NO via Akt signaling, which has been shown to be important in vascular protection [351]. This would suggest that the wild type could prove beneficial in atherosclerosis.  On the other hand, our lab has just similarly demonstrated that the F92A variant of the CAV-peptide can reduce leukocyte adhesion, oxidative stress and atherosclerotic plaque load in an eNOS dependent manner in an animal model of atherosclerosis [277]. Taking all these studies together, there may be value in both the wild type and mutant peptides in different disease contexts, although it would be difficult to predict the outcomes, owing to the complex nature of the systems and the potential mechanisms involved.   113  5.7. Non-Cardiovascular Side Effects of CAV-derived Peptides We showed that BSP can upregulate Akt signaling, while another group showed that substitution of F92 in CAV peptides can promote phospho-MEK and ERK signaling [312]. This raises concerns about promoting cancer, as activation of MEK, ERK and Akt pathways have been suggested to contribute to development and progression of tumors [352,353], with some even suggesting that these could be potential anti-cancer therapeutic targets. However, there is limited information on the tumorigenic potential of both BSPF92A- and BSP-based peptides. Studies have shown that the relationship between Cav-1, CAV peptides and cancer is highly complex. While Cav-1 has been shown to be down-regulated in fibroblasts that undergo oncogenic transformation [354], a study in prostate cancer cells showed that Cav-1 deletion reduced cell viability [326]. There are many more additional studies that show Cav-1 can be up- or down-regulated in cancer [355], showing that endogenous Cav-1 has a complex relationship with cancer. In addition, an in vivo study showed that CAV peptides can reduce microvascular permeability in tumors by inhibiting NO production, resulting in reduced tumor burden [356]. Given the above, the role of Cav-1 in cancer appears to be complex and multi-faceted; hence, it is plausible that CAV-derived peptides, such as BSP and BSPF92A, could also share a complex relationship with cancer, making their tumorigenic implications difficult to predict.   5.8. Future Studies We have identified optimized motifs for targeting eNOS binding, and validated that they can be used to promote NO from endothelial cells. The next step should focus more so on translational research, as opposed to more molecular studies. Since our lab has already demonstrated the value of the parent peptides in an atherosclerotic model of endothelial 114  dysfunction, it may be of greater use to start an organic synthesis and computational biology project to generate compounds for screening. Lead candidates could then be selected for typical pharmacokinetic studies, such as adsorption, distribution, metabolism and excretion, as well as toxicology studies. Selected compounds can then be tested in animal models of endothelial dysfunction to determine efficacy and potency.  5.9. Conclusion Endothelial dysfunction is associated with a pronounced decrease in system NO bioavailability and is a hallmark of many cardiovascular diseases, which as a group, is the largest contributor to global mortality. As such, it is an active area of research, with many trying to devise approaches by which to increase vascular NO. We attempted to directly increase eNOS-derived NO by studying its regulation. Herein, we have identified a binding sequence from the main protein inhibitor of eNOS, Cav-1, and derived two peptide sequences, BSP and BSPF92A. Both peptides show promise, as they can increase eNOS derived NO, albeit via different mechanisms. Given the complex role of Cav-1, it is difficult to conclude which of the two peptides would be more promising. Furthermore, in the future, we hope to potentially use these peptides as the basis for pharmacophores to develop more targeted approaches to promoting vascular NO release.  115  References  1.  World Health Organization, Cardiovascular diseases (CVDs), Last updated 2015, Accessed on 2015.  2.  Lockette W, Otsuka Y, Carretero O (1986) The loss of endothelium-dependent vascular relaxation in hypertension. Hypertension 8: II61–I66.  3.  Winquist RJ, Bunting PB, Baskin EP, Wallace AA (1984) Decreased endothelium-dependent relaxation in New Zealand genetic hypertensive rats. J Hypertens 2: 541–545.  4.  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