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Domains in islet amyloid polypeptide important in fibril formation and toxicity Park, Kirily 2003

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DOMAINS IN ISLET A M Y L O I D POLYPEPTIDE IMPORTANT IN FIBRIL FORMATION A N D TOXICITY by KIRILY P A R K B.Sc, The University of British Columbia, 1993 Hons., The University of Queensland, 1997 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF GRADUATE STUDIES (Department of Pathology and Laboratory Medicine) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July 2003 © Kirily Park, 2003 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of VznUC^ The University of British Columbia Vancouver, Canada DE-6 (2/88) Abstract: Islet amyloid deposits are a characteristic pathologic lesion of the pancreas in type 2 diabetes and are composed primarily o f the islet beta cell peptide islet amyloid polypeptide (IAPP or amylin). Islet amyloid fibrils are toxic to insulin-producing beta cells. Heparan sulfate proteoglycans (HSPGs) are also a component of islet amyloid in vivo and accelerate amyloid fibril formation in vitro. Although the cause of amyloid formation in pancreatic islets is unknown, HSPGs have been proposed to play an initiating role. Impaired processing o f proIAPP, the I A P P precursor, has also been implicated in the mechanism of islet amyloid formation. The N - and C-terminal cleavage sites at which proIAPP is processed by prohormone convertases contain series of basic amino acid residues which we hypothesized may interact with heparan sulfate proteoglycans. We found that a monomeric fragment o f proIAPP (residues 1-30) bound both heparin and heparan sulfate affinity columns. Substitution of alanine residues for two basic residues in the N-terminal cleavage site abolished heparin and heparan sulfate binding activity. These data suggest that monomeric N-terminal human proIAPP contains a heparin-binding domain that is lost during normal processing o f proIAPP. Although islet amyloid fibrils bind to heparin and heparan sulfate, it is unknown which residues in IAPP may be responsible for this interaction or whether nonfibrillar (monomeric) human I A P P also binds to heparin. Three basic residues in human I A P P (Lys 1 , A r g 1 1 , and His 1 8 ) could potentially contribute to this binding by interacting with negatively charged sulfate groups in heparin. We found heparin significantly enhanced fibril formation by IAPP 1-37 and 11-37, but not IAPP 12-37, suggesting that A r g 1 1 may be important in the ability o f heparin to enhance IAPP fibril formation. Fibrils generated from I A P P 20-29 (which contains no basic residues) did not bind heparin, supporting the possibility that specific basic residues within I A P P may be important in the formation of heparin binding domains within amyloid fibrils. These findings suggest that basic amino acids may play important roles in I A P P and amyloid fibril interaction with heparin/heparan sulfate and may contribute to islet amyloid formation. A point mutation (serine to glycine at position 20) in IAPP has been identified in a subpopulation of Japanese patients with a severe, early onset form of type 2 diabetes. We find S20G IAPP , when applied to INS-1 beta cells in culture, is cytotoxic at lower concentrations than wild-type IAPP. Using electron microscopy and thioflavin T assays, we also found that the S20G mutant fibrils form much more rapidly. Increased fibrillogenicity o f S20G I A P P could cause the increased beta cell toxicity of this peptide and may contribute to early beta cell death and onset of diabetes in patients carrying this mutation. In conclusion, we speculate that i f N-terminally extended proIAPP is secreted in excessive amounts from the beta cell in type 2 diabetes, this peptide may be retained by heparan sulfate i i i proteoglycans in the extracellular matrix and form a nidus for subsequent islet amyloid formation. The possible importance o f the three basic residues in mature IAPP in amyloid fibril formation suggests potential therapeutic targets in preventing islet amyloid formation and beta cell death in type 2 diabetes. Finally, IAPP toxicity is increased under conditions that hasten the onset of fibrillogenesis, such as a substitution of glycine for serine at position 20 of the molecule. Domains in IAPP and proIAPP involved in H S P G binding, fibrillogenesis, and toxicity may make novel therapeutic targets for inhibition o f islet amyloid formation and the progressive loss of beta cells in type 2 diabetes. iv Table of Contents: Abstract: i i Table of Contents: v List o f Tables: v i i List o f Figures: v i i i Abbreviations: x Acknowledgements: x i Dedication: x i i 1. Introduction 1 1.1 Diabetes Mellitus 1 1.1.1 Background 1 1.1.2 Genetic Component 2 1.1.3 Environmental Component 5 1.1.4 Insulin Requirement Versus Insulin Sensitivity 6 1.1.5 Beta Cel l Mass in Type 2 Diabetes 8 1.1.6 Potential Causes o f Beta Cel l Death in Type 2 Diabetes 11 1.1.7 Islet A m y l o i d 13 1.1.8 Islet A m y l o i d Polypeptide 14 1.1.9 Processing of Proinsulin in Health and Type 2 Diabetes 15 1.1.10 Processing of ProIAPP 17 1.1.11 Progression of Type 2 Diabetes 19 1.2 A m y l o i d 20 1.2.1 General Characteristics 20 1.2.2 Common Amyloids 21 1.2.3 Common Components of Amyloids 23 1.3 Islet A m y l o i d 24 1.3.1 Proteoglycans in Islet Amylo id 25 1.3.2 Effect of G A G s on IAPP Fibr i l Formation 27 1.3.3. Domains in IAPP Important in Amylo id Formation 29 1.3.4 Mechanisms of IAPP Fibrillogenesis 34 1.3.5 A n Unfolded Intermediate as a Precursor to Amylo id Fibrils 36 1.3.6 Mechanisms of IAPP Toxicity 38 1.3.7 Inhibition of Fibrillogenesis In Vitro 41 1.3.8 Inhibition of Amylo id Formation In Vivo 43 1.4 Overall Rationale and Specific Aims 46 1.4.1 A i m 1: To determine whether proIAPP binds to heparin 47 1.4.2 A i m 2: To determine which residues in IAPP are important in heparin binding to fibrils, and in heparin stimulation of fibril formation 48 v 1.4.3 A i m 3: To determine whether the S20G mutant form of I A P P is more cytotoxic to beta cells than wild-type I A P P 49 2. Methods 51 2.1 Materials 51 2.1.1 Affinity Chromatography 51 2.1.2 Fluorometry 51 2.1.3 Ce l l Death Studies 52 2.1.4 3 H-Heparin Binding Assays 52 2.1.5 Electron Microscopy 52 2.2 Methods 53 2.2.1 Affinity Chromatography 53 2.2.2 Coupling of Glycosaminoglycans to Sepharose® C L - 4 B Beads 53 2.2.3 Toluidine Blue Assay 54 2.2.4 Preparation of Peptides 55 2.2.5 Fluorometry 56 2.2.6 Cel l Death Studies 57 2.2.7 3 H-Heparin Binding Assays 58 2.2.8 Electron Microscopy 58 2.2.9 Statistical Analysis 59 3. Results 60 3.1 Affinity o f Human ProIAPP Fragments for Heparin 60 3.2 Affinity of Human ProIAPP Fragments for Heparan Sulfate 65 3.3 Affinity of Human ProIAPP Fragments for Chondroitin Sulfate 68 3.4 Human N-and C-terminal proIAPP Fragments are Non-Fibrillogenic 69 3.5 N-terminal ProIAPP Fragment Does Not Inhibit Fibri l Formation 71 3.6 Involvement of Histidine Residues in Heparin Binding at Ac id i c p H 75 3.7 Residues Involved in Heparin Stimulation of IAPP Fibr i l Formation 77 3.8 Heparin Does Not Augment Cytotoxicity of Human IAPP 11-37 or 12-37 81 3.9 Residues Important in Binding of Human IAPP to Heparin 87 3.10 Rifampicin Does Not Inhibit Fibr i l Formation 94 3.11 Lag Time for Fibr i l Formation of Wild-Type and S20G hIAPP 96 3.12 Toxicity of S20G hIAPP 102 4. Discussion 110 4.1 N-terminally Extended ProIAPP Binds to Heparan Sulfate I l l 4.2 Residues in IAPP that Mediate Heparin Binding and Heparin Stimulation o f Fibr i l Formation 119 4.3 Increased Fibrillogenicity and Beta Cel l Toxicity of S20G Mutant I A P P 123 4.4 Future Studies 128 Bibliography: 131 v i List of Tables: Table I: Major protein component o f several amyloids List of Figures: Figure 1: Proinsulin and proIAPP processing 16 Figure 2: The sequence of IAPP is well-conserved across species 30 Figure 3: Amino acid sequence of (A) full-length human proIAPP and (B) the synthetic isl-and C-terminal proIAPP peptides used in these studies 34 Figure 4: Amino acid sequence of wild-type human IAPP 1-37, and S20G mutant 50 Figure 5: Affinity of proIAPP fragments for heparin 62 Figure 6: Affinity of N-terminal proIAPP fragment for heparin-agarose in the presence o f J3-mercaptoethanol 64 Figure 7: Affinity of N-terminal proIAPP fragment for heparin Af f i -Ge l 65 Figure 8: Affinity of proIAPP fragments for heparan sulfate 67 Figure 9: Affinity o f proIAPP fragments for chondroitin sulfate 69 Figure 10: N-terminal and C-terminal proIAPP fragments are not fibrillogenic 71 Figure 11: N-terminal proIAPP fragments does not inhibit fibril formation by human I A P P in vitro 73 Figure 12: N-terminal proIAPP fragment does not protect INS-1 cells from I A P P fibril toxicity 74 Figure 13: Affinity o f N-terminal proIAPP fragment for heparin and heparan sulfate is increased at p H 5.5 76 Figure 14: Heparin augments fibrillogenesis of human IAPP 78 Figure 15: Addit ion of heparin to the culture medium increases the cytotoxicity of human I A P P 79 Figure 16: Sequence of synthetic IAPP peptides 81 Figure 17: Heparin stimulates the initial rate of fibrillogenesis of human I A P P 11-37 and not human I A P P 12-37 82 Figure 18: Addit ion of heparin to the culture medium does not increase the cytotoxicity o f human IAPP 11-37 or 12-37 83 Figure 19: Human I A P P 20-29 does not bind to heparin 86 Figure 20: Addit ion of unlabeled heparin displaces 3H-heparin from I A P P fibrils 87 Figure 21: Affinity of rat IAPP for heparin and heparan sulfate 89 Figure 22: K 1 0 A / R 1 1 A mutant proIAPP fragment shares basic residues with human I A P P 1-37 but does not bind to heparin 90 Figure 23: Binding of 3H-heparin to IAPP at p H 7.5 and p H 5.5 90 Figure 24: Human IAPP 20-29 is not toxic to INS-1 beta cells 92 Figure 25: Treatment with heparinase I or III, or 13-D-xyloside does not protect INS-1 cells from I A P P cytotoxicity 93 Figure 26: Summary of residues proposed to be important in IAPP fibril formation, toxicity, and heparin stimulation of fibril formation 94 Figure 27: Effect o f rifampicin on thioflavin T binding to IAPP in the presence o f heparin. 95 v i i i Figure 28: Binding of 3H-heparin to IAPP fibrils in the presence o f rifampicin 96 Figure 29: S20G mutant IAPP undergoes seeding event for fibrillogenesis earlier than wi ld-type 98 Figure 30: Lag time to seeding event of amyloidogenesis for wild-type human I A P P and S20G at different concentrations 99 Figure 31: S20G mutant fibrils form faster than those of wild-type I A P P 101 Figure 32: S20G mutant human IAPP is more cytotoxic to beta cells than wild-type human I A P P 103 Figure 33: Rat IAPP is not cytotoxic to INS-1 cells 105 Figure 34: Addit ion of heparin to the culture medium increases the cytotoxicity o f wild-type human IAPP 106 Figure 35: Comparable amount of 3H-heparin bound by wild-type IAPP and S20G mutant 109 Figure 36: Proposed role of proIAPP in the formation of islet amyloid 112 IX Abbreviations: A A inflammation associated A p o E apolipoprotein E A P P amyloid precursor protein A B amyloid beta B S A bovine serum albumin C N B r cyanogen bromide D M S O dimethylsulfoxide E M electron microscopy G A G glycosaminoglycan HFIP hexafluoroisopropanol H S heparan sulfate H S P G heparan sulfate proteoglycan hIAPP human islet amyloid polypeptide I A P P islet amyloid polypeptide M O D Y maturity-onset diabetes of the young P B S phosphate-buffered saline P C prohormone convertase ProIAPP pro-islet amyloid polypeptide PrP prion protein S A P serum amyloid P component S A A serum amyloid A S20G serine to glycine substitution at position Acknowledgements: I would like to thank Dr. Bruce Verchere for allowing me the opportunity to study in his lab, for his guidance, his support, and his sense of humour. I also thank the members of my supervisory committee, Dr. John H i l l , Dr. Mladen Korbelik, and Dr. Christopher Mcintosh, for guidance and thought-provoking discussion. A heartfelt thanks goes to all the members of the Verchere Lab. Y o u have been a wonderful group o f people to work with. In particular, I am grateful to Jennifer Finnerty, Amara Garcia, M i k e Kennedy, Lucy Marzban, Annette Plesner, and Daniel Bruch. I am also grateful to M s . Penny Woo, who has made grad studies that much easier by taking care of so many o f my administrative responsibilities. I would like to thank Dr. Bob Kisi levsky and Dr. John Ancsin at Queen's University, for much advice on affinity chromatography. I am also grateful to Dr. Anne Clark, Dr. Emma Jaikaran, and Cathryn Sloan at Oxford University for their technical assistance, and for the opportunity to perform electron microscopy in their lab. Thank you Carolyn Kavanagh for your encouragement, your friendship, and for your humorous impersonations of me panicking about PhD coursework. A very big thanks to Dr. Trina Mcllhargey, for your cheerful companionship, especially during one very difficult year, and for your shared love of Dairy Queen. I am extremely grateful to my parents, and all o f my family, for their unconditional love, support, enthusiasm and encouragement, and for all the things they have taught me over the years. Brad, thanks for being such a great friend. N o one could ask for a better big brother. Finally, to my husband, Denis Hughes, thank you from the bottom of my heart for your incredible generosity, kindness, and support, your boundless love, and for the way you can always make me laugh. Thanks also for encouraging me to go outdoors once in a while during the production of this thesis. Je t'aime. In loving memory of Douglas James Reginald Park August 19 t h, 1933 - M a y 29 t h , 2002 x i i 1. Introduction 1.1 Diabetes Mellitus 1.1.1 Background Diabetes mellitus encompasses a heterogeneous group o f disorders that have hyperglycemia as a common feature (Crawford and Cotran 1999). The most common forms of diabetes can be subdivided into two major groups: type 1 and type 2. Type 1 diabetes is an autoimmune disorder in which the cellular immune system targets and destroys the insulin-producing beta cells of the pancreas. This disease is marked by insulitis and subsequent severe insulin deficiency. It is also called 'juvenile-onset' diabetes, or insulin-dependent diabetes mellitus, as the onset occurs typically at less than 20 years o f age. Type 2 diabetes, also called adult-onset diabetes, and formerly called non-insulin dependent diabetes mellitus, is a progressive disease also characterized by fasting hyperglycemia. Its onset occurs typically in persons over 30 years of age. Similar to type 1 diabetes, the chronic elevation of blood sugar can arise through defects in the production o f the glucoregulatory hormone insulin (DeFronzo 1988; Kahn 1994). In type 2 diabetes, however, defects in the action o f insulin, termed 'insulin resistance' are also present in various tissues. Over many years, hyperglycemia leads to complications such as nephropathy, neuropathy, retinopathy, and cardiovascular disease. Type 2 diabetes is widespread in the western world, accounting for 90-95% of all cases o f diabetes. Approximately two mil l ion Canadians and sixteen mil l ion Americans suffer from type 2 diabetes, and the incidence is increasing. In 2000, the World Health Organization estimated that 177 mil l ion people worldwide were afflicted with diabetes ( W H O 2003). This number is expected to climb to 370 mil l ion by the year 2030. The resulting costs to healthcare are enormous. In 1998 in Canada, approximately $5 bi l l ion ($US) was spent on treating people with diabetes and its complications (Dawson et al. 2002). Previously thought o f as a disease unique to adults, type 2 diabetes is now being diagnosed in young adults and children as young as 8 years of age (Brosnan et al. 2001; Kaufman 2002). Both genetic and environmental influences interact to contribute to the development of type 2 diabetes, although the exact nature of these interactions has not been fully elucidated. 1.1.2 Genetic Component A genetic basis for type 2 diabetes is indicated by the fact that first degree relatives of persons with this disease have an increased chance of developing the disease (Gottlieb 1980; Kahn 1994) and that monozygotic twins have a 50-90% concordance rate (Newman et al. 1987; L o et al. 1991; Medic i et al. 1999; Poulsen et al. 1999). In addition, certain ethnic groups show a much higher incidence of this disease than others (Zimmet et al. 1978; Abate and Chandalia 2001). For example, persons of African American, Hispanic, Native American, or Asian descent in the United States have a prevalence o f type 2 diabetes that is 2 two to six times greater than those of Caucasian descent (Carter et al. 1996; Hosey et al. 1998). In a small percentage of cases, the cause of type 2 diabetes can be linked to one specific gene. For example, obesity and diabetes in one human subject are associated with mutations in the gene encoding prohormone convertase 1 (PCI) , an enzyme involved in the conversion of proinsulin to insulin (see Section 1.1.9) (Jackson et al. 1997). In rodents, mutations in the gene encoding leptin, a hormone produced by adipose tissue that is involved in satiety signaling, and the leptin receptor (Coleman 1978; Chua et al. 1996) are associated with diabetes and obesity. Maturity Onset Diabetes of the Young ( M O D Y ) is a relatively rare type o f diabetes, which accounts for 1-5% of all cases of diabetes in the U S A (Fajans et al. 2001). Although M O D Y is a disorder distinct from type 2 diabetes, it shares certain common features. A s the name suggests, M O D Y patients develop a non-autoimmune diabetes at a young age (usually before the age of 25) (Fajans et al. 2001). M O D Y is inherited in an autosomal dominant pattern, and is classified into 6 subtypes, resulting from mutations in different genes (Fajans et al. 2001), such as glucokinase and the beta cell transcription factors hepatocyte nuclear factor-la and - IB. Searches for genes that segregate with the more common 'garden variety' type 2 diabetes in human populations have been largely unsuccessful. Certain gene polymorphisms have been 3 established as conferring an increased risk of developing the disease in specific populations. For example, two diabetes susceptibility loci, termed ' N I D D M 1 ' and ' N I D D M 2 ' have been identified, the former in Mexican-Americans (Hanis et al. 1996) and the latter in Finnish families (Mahtani et al. 1996). Although the exact gene responsible has yet to be identified conclusively, certain polymorphisms in the calpain 10 gene, found in the N I D D M 1 locus (Horikawa et al. 2000), are associated with metabolic differences in Pima Indians which may predispose to enhanced carbohydrate stores, and insulin resistance (Permutt et al. 2000). Another gene in which polymorphisms may be associated with increased risk of developing type 2 diabetes is the peroxisome proliferator-activated receptor gamma, a nuclear receptor which regulates l ipid and glucose metabolism, and adipocyte differentiation (Auwerx 1999). The A l a l 2 allele of this gene has been associated with increased risk of developing diabetes in subjects with impaired glucose tolerance (Lindi et al. 2002). Other studies have, however, associated the same allele with a protective effect against the development o f type 2 diabetes (Altshuler et al. 2000; M o r i et al. 2001). A mutation in the hepatocyte nuclear factor-la gene is predictive of diabetes susceptibility in the Oji-Cree (Hegele 2001), a First Nations people l iving in northern Ontario. In a small percentage of Japanese diabetic patients, a mutation in the gene encoding islet amyloid polypeptide (IAPP), a thirty seven amino acid peptide which is co-secreted with insulin (see Section 1.1.8) has been identified (Sakagashira et al. 1996). This point mutation has been associated with an earlier onset and more severe form of type 2 diabetes, although not all patients who harbour this mutation develop diabetes (Chuang et al. 1998; Yamadae ta l . 1998). Thus far, mutations that have been associated with a predisposition to type 2 diabetes are confined to small populations. The major predisposing genes for type 2 diabetes remain to be elucidated, and these genes are unlikely to cause disease on their own. Instead, type 2 diabetes l ikely arises from the presence of a complex set of predisposing genes in the appropriate environment (Gerich 1998; Kahn 1994). 1.1.3 Environmental Component It is evident that genetics are not the sole factor underlying the development o f type 2 diabetes. Environmental influences such as sedentary lifestyle (Gerich 1998; Hegele 2001), poor physical fitness (Eriksson and Lindgarde 1996; Wang et al. 2002), and cigarette smoking (Wannamethee et al. 2001; W i l l et al. 2001) are risk factors for the development of type 2 diabetes. The consumption of a high fat diet (Mann 2002; Summers et al. 2002) is also a risk factor for the development of type 2 diabetes in humans. This is mirrored in several animal models o f type 2 diabetes (Kalderon et al. 1986; Verchere et al. 1996; Westermark et al. 2000). One such model is the gerbil Psammomys obesus, native to Israel. In its natural habitat P. obesus eats a very low calorie diet, feeding strictly on a plant called the salt bush. When placed on a higher calorie diet, this animal very rapidly develops 5 hyperglycemia associated with obesity, insulin resistance, and beta cell dysfunction (Kalderon et al. 1986; Barnett et al. 1994; Nesher et al. 2001). 1.1.4 Insulin Requirement Versus Insulin Sensitivity Although the interplay between genetic and environmental factors remains to be unraveled, it is known that once type 2 diabetes develops, fasting hyperglycemia is the result of a combination of insulin resistance in peripheral tissues and beta cell dysfunction. In type 2 diabetes, insulin resistance occurs in skeletal muscle, liver, and adipose tissue (DeFronzo 1988), and there is recent evidence that it may also occur at the level o f the pancreatic beta cell (Kulkarni et al. 1999). Insulin resistance can be detected prior to the onset o f diabetes and is also present in many persons who never develop the disease. For example, obese persons are usually insulin resistant, yet many are able to compensate for their diminished peripheral insulin sensitivity by secreting elevated levels of insulin (DeFronzo 1988; Polonsky et al. 1988) and do not necessarily develop diabetes. In fact, as long as the relationship between insulin sensitivity and insulin resistance is constant, normoglycemia is maintained (Bergman et al. 2002). The term "disposition index" refers to the product of insulin sensitivity and insulin secretion (Bergman et al. 2002), which describes the relationship between peripheral insulin sensitivity and beta cell function. There is evidence that groups at high risk for developing type 2 diabetes have abnormal disposition 6 indices. For example, first degree relatives of patients with type 2 diabetes, women with a history o f gestational diabetes, and women with polycystic ovary syndrome, as well as older subjects (Bergman et al. 2002) have been shown to have disposition indices that fall outside the normal range. When beta cells fail to secrete adequate amounts of insulin to compensate for insulin resistance, fasting hyperglycemia indicative of type 2 diabetes develops (DeFronzo 1988; Bergman et al. 2002). Insulin resistance tends not to worsen markedly as diabetes progresses (DeFronzo 1988). In contrast, beta cell function progressively declines over time, resulting in the secretion of less and less insulin in response to glucose and a progressive worsening o f hyperglycemia. Beta cell dysfunction in type 2 diabetes manifests in several ways. People with this disease demonstrate a markedly reduced or absent early ("first phase") insulin response to glucose (Perley and Kipnis 1967; Pfeifer et al. 1981). Beta cells also secrete elevated amounts of proinsulin in type 2 diabetes (Duckworth et al. 1972; Ward et al. 1987; Kahn and Halban 1997), and have reduced ability to entrain insulin secretion in response to oscillations of blood glucose levels (O'Meara et al. 1993). The beta cell dysfunction of type 2 diabetes is apparent prior to the onset of disease and therefore cannot be solely a consequence of a toxic effect of glucose on the beta cell, since it is detectable before hyperglycemia develops. For example, a study o f persons with impaired glucose tolerance revealed impaired first phase insulin secretion in response to glucose compared to age, sex, and weight-matched controls with no difference in insulin sensitivity (Fritsche et al. 2000). The subjects with impaired glucose tolerance also showed a decreased insulin secretory response to glucagon-like-peptide-1 (Fritsche et al. 2000). Subjects with impaired glucose tolerance also show beta cell defects in entrainment of insulin secretion in response to oscillations of blood glucose levels (O'Meara et al. 1993; Ehrmann et al. 2002). A defect in processing of proinsulin by the beta cells is also detectable prior to the onset o f overt disease (Kahn et al. 1996). 1.1.5 Beta Cell Mass in Type 2 Diabetes The mechanism underlying beta cell compensation for insulin resistance, or failure to compensate, is the subject of intense study. It has been proposed that the pancreatic beta cell mass may expand to meet demand in insulin resistant states (Bonner-Weir 2000). N e w beta cells may arise through replication, neogenesis, and transgenesis (Finegood et al. 1995). A deficiency in these processes, or an increase in the rate of cell death could lead to a failure o f the beta cell mass to expand (Finegood et al. 1995). Inadequate expansion of beta cell mass 8 could therefore play a role in progression from insulin resistance to diabetes (Pick et al. 1998). It is very difficult to study the human pancreas in vivo, and most studies o f human beta cell mass are based on samples taken at autopsy. The dynamics of beta cell mass in vivo have therefore been studied most extensively in animal models. Several models o f insulin resistance in animals indicate that beta cell mass can indeed expand to compensate for insulin resistance. For example, mice lacking the insulin receptor substrate-1 are insulin resistant, and exhibit extreme islet hyperplasia (Araki et al. 1994). Obese (ob/ob) mice lacking leptin are also insulin resistant and demonstrate increased beta cell mass compared to control mice (Flier et al. 2001). Mouse islets transplanted into insulin resistant recipient mice show an increase in surface area and beta cell mitosis compared with controls (Flier et al. 2001). Rats have also been shown to increase beta cell mass in response to hyperglycemia (Lipsett and Finegood 2002) or following a partial pancreatectomy (L iu et al. 2000). One rat model in particular that may yield important clues to the mechanism of failure o f beta cell mass expansion is the Zucker Diabetic Fatty rat. These rats have a mutation in the leptin receptor, become obese and develop a type 2 diabetes-like syndrome. Their beta cell mass does not expand sufficiently in the face of insulin resistance to prevent the onset of diabetes (Pick et al. 1998). In contrast, the Zucker Fatty rat, a partially outbred strain, demonstrates a much more robust beta cell mass expansion and does not develop diabetes (Pick et al. 1998). A n increased rate of beta cell apoptosis rather than a decrease in replication accounted for the difference in beta cell mass between these two strains. Insufficient beta cell mass expansion due to beta cell apoptosis in this rat model can therefore lead to hyperglycemia. Studies in humans indicate that similar processes may contribute to the development of type 2 diabetes. There is evidence that humans, like rodents, increase their beta cell mass in response to increased demand for insulin. In 1985, Kloppel found that beta cell mass is increased in the obese non-diabetics compared to lean controls (Kloppel et al. 1985). However, a decrease in beta cell mass was found in humans who have type 2 diabetes compared to weight-matched controls (Kloppel et al. 1985). Other studies of humans at autopsy also report a decrease in islet mass in type 2 diabetes (Maclean and Ogilvie 1955; Westermark and Wilander 1978; Clark et al. 1988). A recent study in human autopsy material found that obese non-diabetics had approximately a 50% increase in relative beta cell volume (Butler et al. 2003). However, the relative beta cell volume in humans with type 2 diabetes was decreased, and this correlated with an increase in beta cell apoptosis (Butler et al. 2003). Therefore, as in the Zucker Diabetic Fatty rat, insufficient beta cell mass leading to hyperglycemia correlates with an increased rate of apoptosis rather than a decrease in replication. There are several factors that may contribute to the death o f beta cells in type 2 diabetes. 10 1.1.6 Potential Causes of Beta Cell Death in Type 2 Diabetes A t least three different causes of beta cell death in type 2 diabetes have been identified: hyperlipidemia, hyperglycemia, and islet amyloid. i. Hyperlipidemia: There are elevated circulating levels of lipids in the blood o f most persons with type 2 diabetes, a condition which has been shown to have detrimental effects on beta cell function in vitro. For example, prolonged exposure to fatty acids compromises the ability o f the rat islets to respond to high glucose concentrations (Sako and G r i l l 1990; Zhou and G r i l l 1994; Bollheimer et al. 1998). Prolonged exposure of rat beta cells to fatty acids in vitro leads to defective processing of proinsulin, and secretion of unprocessed forms of the molecule (Furukawa et al. 1999). High fat feeding leads to impaired glucose-induced insulin secretion from islets of hyperglycemic Goto-Kakizaki rats but not islets from normoglycemic Wistar rats (Briaud et al. 2002). Exposure to free fatty acids for a prolonged period decreases glucose-induced insulin secretion in rat islets (Sako and G r i l l 1990) and in healthy non-obese humans (Carpentier et al. 1999). These conditions also lead to delayed processing o f proinsulin, as well as of the enzymes that process proinsulin (Furukawa et al. 1999). Free fatty acids may also contribute to beta cell death. Exposure to elevated levels of free fatty acids leads to apoptosis of beta cells in culture (Cnop et al. 2001). It has been proposed that the increased rate of apoptosis in the Zucker Diabetic Fatty rat is related to its 11 increased intraislet l ipid content in a hyperglycemic environment (Shimabukuro et al. 1998; Unger and Zhou 2001). ii. Hyperglycemia: The hyperglycemia experienced by persons with impaired glucose tolerance or type 2 diabetes may contribute to dysfunction and death o f beta cells (Kloppel et al. 1985). In animal models of type 2 diabetes, beta cell mass maintenance is attained by improvement of glycemic control (Donath et al. 1999; Tanaka et al. 1999). Studies in vitro also indicate that glucose has a toxic effect on beta cells. Exposure to elevated glucose in culture induces dysfunction (Eizirik et al. 1992) and apoptosis in human islets (Federici et al. 2001; Maedler et al. 2001) and rat islets (Efanova et al. 1998; Piro et al. 2002). Prolonged exposure of rat beta cells to high glucose leads to increased insulin secretion and synthesis during subsequent exposure to low glucose levels (Leahy et al. 1992). In addition to its detrimental effects on beta cell function and viability, it has also been suggested that hyperglycemia provides a permissive environment for the detrimental effects o f fatty acids (Cruz et al. 2001; Prentki et al. 2001; Poitout and Robertson 2002). Hyperglycemia and hyperlipidemia both may cause beta cell death and help lead to a failure to compensate for insulin resistance with an appropriate increase in beta cell mass in humans. Another important factor that may be a major culprit in inhibiting beta cell mass expansion and increasing beta cell death, is islet amyloid. 1.1.7 Islet Amyloid The onset o f impaired insulin secretion in type 2 diabetes is associated with the deposition of islet amyloid (Hansen and Bodkin 1986; Rocken et al. 1992). Islet amyloid is a fibrillar protein deposit that accumulates adjacent to beta cells, largely around islet capillaries. Islet amyloid develops in up to 90% of patients with type 2 diabetes (Westermark 1972; Clark et al. 1990). Interestingly, both a high fat diet (Hull et al. 2003) and hyperglycemia (Verchere et al. 1996) have been associated with the development o f islet amyloid in transgenic mice. Islet amyloid is composed primarily of islet amyloid polypeptide (IAPP; amylin), a normal product o f the beta cell (Cooper et al. 1987; Westermark et al. 1987; Lukinius et al. 1989). IAPP is co-secreted along with insulin in response to beta cell stimuli, although at approximately one hundred fold lower concentrations (Kanatsuka et al. 1989; Kahn et al. 1990). Normally I A P P passes into the bloodstream in monomelic form; for unknown reasons in type 2 diabetes it aggregates adjacent to beta cells and forms islet amyloid. A m y l o i d fibrils cause beta cell apoptosis (Lorenzo et al. 1994) (see Section 1.3.6). Islet amyloid may therefore be a major culprit in the loss of beta cell mass in type 2 diabetes, and therefore play a role in development of relative insulin sufficiency. In addition, it has been shown that islet amyloid develops in animal recipients of transplanted human islets 13 (Westermark et al. 1995; 1999), raising the possibility that rapid deposition o f amyloid in transplanted islets may contribute to graft failure. The likelihood that islet amyloid plays an important role in the progression of type 2 diabetes is supported by a number of findings. The degree of islet amyloid deposition correlates with the severity o f hyperglycemia at the time of death in a post-mortem study (Rocken et al. 1992). The degree o f islet amyloid correlated with a reduction in beta cell mass in a mouse model (MacArthur et al. 1999) and in monkeys (de Koning et al. 1993). 1.1.8 Islet Amyloid Polypeptide I A P P is a 37 amino acid polypeptide that bears significant homology to calcitonin gene-related peptide (Roberts et al. 1989). Although it is produced primarily by beta cells, small amounts are present in parts of the gut (Mulder et al. 1994) and in sensory neurons (Mulder et al. 1995). It is present in the plasma in low picomolar concentrations. The physiological function of IAPP is still the subject of much debate. Proposed functions include inhibition of gastric emptying (Young et al. 1995), satiety signaling (Morley and Flood 1991; Rushing et al. 2000), inhibition of glucose-stimulated insulin release (Silvestre et al. 1990; Wang et al. 1993), lowering of plasma calcium levels (Kassir et al. 1991; Datta et al. 1989), promotion of post-prandial calcium absorption from the intestines (Datta et al. 1989), and mi ld vasodilation (Chin et al. 1994; DeWitt et al. 1994). However, many of 14 these effects are only observed at non-physiological concentrations of the peptide, and mice lacking I A P P have no discernable phenotype except a mi ld increase in glucose -stimulated insulin secretion (Gebre-Medhin et al. 1996; 1998). Thus, although its co-release with insulin suggests a role in glucose metabolism, and its homology to calcitonin gene-related peptide suggests a role in calcium metabolism (Westermark et al. 1992), the physiological function of IAPP remains unknown. In addition, no IAPP receptor has been identified (Christopoulos et al. 1999) although IAPP binding sites are created (Muff et al. 1999) when the calcitonin receptor is co-expressed with receptor activity modifying proteins. 1.1.9 Processing of Proinsulin in Health and Type 2 Diabetes Proinsulin, the precursor to insulin, is processed in the secretory granules o f the pancreatic beta cell. This processing is carried out by two prohormone convertase (PC) enzymes, PC2 and PC3 (also called P C I ) , which cleave at the C-terminal side o f dibasic residues (see Fig . 1), liberating insulin and C-peptide. Insulin-containing secretory granules are exocytosed in response to metabolism of glucose by the beta cell, or in response to other beta cell secretagogues such as arginine, or the gut hormone glucagon-like peptide-1 (Nauck etal . 1993). 15 Proinsulin C-peptide PC2 PC3 ProIAPP P C 2 PC3 OCOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOC3000# rlFf N-Term Pro Region IAPP 1-37 C-Term Pro Region Figure 1: Proinsulin and proIAPP processing. Both proinsulin and proIAPP are cleaved by prohormone convertase enzymes at the C-terminal side of dibasic residues to liberate insulin and C-peptide, and IAPP 1-37. Proinsulin processing in beta cell granules is known to be impaired in type 2 diabetes, leading to hyperproinsulinemia (Porte and Kahn 1989; Leahy 1990; Kahn and Halban 1997). The cause o f this defective processing is not known. It has been proposed that the elevated ratio o f proinsulin to insulin in the blood of persons with type 2 diabetes and in rat models o f type 2 diabetes is the result of high secretory demand causing the release of beta cell secretory granules before proinsulin processing is complete (Mako et al. 1977; Leahy 1993). 16 However, experimental insulin resistance induced by nicotinic acid does not lead to an increase in proinsulin in the blood of human subjects, suggesting that increased demand per se can not lead to the elevated release of proinsulin in persons with type 2 diabetes (Porte and Kahn 1989). A second proposed cause of hyperproinsulinemia is the existence o f a genetic or acquired impairment o f proinsulin processing in the granule (Porte and Kahn 1989) or by missorting of proinsulin to the constitutive secretory pathway (Porte and Kahn 1989). The constitutive secretory pathway does not contain PC2 or PC3 (Steiner 1998). The endopeptidase furin is present, but it is not capable of fully processing human proinsulin in vitro (Vollenweider et al. 1995). Unprocessed proinsulin is also secreted by insulinomas (beta cell tumours), which have a high degree of constitutive secretion (Halban 1994). 1.1.10 Processing of ProIAPP Similar to proinsulin, proIAPP is processed in the secretory granules of the beta cell. It too is cleaved at the C-terminal side of dibasic residues, to liberate I A P P 1-37. It is thought that the same prohormone convertases process proIAPP as process proinsulin. P C 2 has been shown to be necessary for cleavage at N-terminal cleavage site of proIAPP (Wang et al. 2001). M i c e lacking PC2 do not completely process proIAPP, and accumulate an N -terminally extended intermediate form of the molecule. PC3 is also important although not 17 essential for complete processing of proIAPP. Islets from mice lacking PC3 show reduced levels of fully processed IAPP 1-37, and an increase in C-terminally extended proIAPP (Marzban et al. 2002). These data indicate that PC3 is important for efficient processing o f proIAPP at the C-terminal cleavage site, but that it is not essential; PC2 can process proIAPP completely (Marzban et al. 2003). There are several models of compromised beta cell function which exhibit altered sorting and processing of proinsulin and proIAPP (Kahn et al. 1999). For example, neonatal rat beta cells exhibit decreased glucose-induced insulin secretion in addition to releasing a significant proportion of I A P P through the constitutive pathway (Verchere et al. 2000). The immortalized PTC-3 beta cell line exhibits similar alterations to neonatal rat beta cells both in glucose-induced insulin secretion (Nagamatsu and Steiner 1992) and in sorting o f newly synthesized proinsulin and proIAPP (Nagamatsu et al. 1991). Although such phenomena could arise from hyperglycemia-induced release of immature granules, it is also possible that prolonged exposure to high levels of glucose impairs normal functioning of beta cells. Failure to process proinsulin or proIAPP could be secondary to this impaired function. Recent studies with human islets have demonstrated increased synthesis of I A P P correlated with release of proIAPP via the constitutive pathway (Gasa et al. 2001). It is therefore possible that sorting of proIAPP and proinsulin is impaired in situations where beta cell function is compromised, such as type 2 diabetes. Immunoreactivity for the N-terminal 18 flanking peptide of proIAPP has been detected in human islet amyloid deposits (Westermark et al. 1989), suggesting that unprocessed forms of proIAPP may indeed be secreted from the beta cell in type 2 diabetes and may contribute to islet amyloid formation. 1.1.11 Progression of Type 2 Diabetes Several longitudinal studies have examined the progression of type 2 diabetes in humans. The Diabetes Prevention Program, which addressed the progression from impaired glucose tolerance to type 2 diabetes (1999), showed that it is possible to delay the progression from impaired glucose tolerance to overt diabetes by intensive lifestyle modification (diet and exercise) or by administration of a drug that enhances insulin action and decreases blood glucose levels. Once diabetes is diagnosed, however, it appears that the progression towards beta cell failure cannot be prevented. The United Kingdom Prospective Diabetes Study followed patients with type 2 diabetes over the course of a decade. This study showed that regardless of the type of treatment, once type 2 diabetes has developed, there is a progressive increase in blood glucose levels (1998; Turner et al. 1999). Since the appearance of islet amyloid is correlated with a decline in insulin secretion in isolated islets (MacArthur et al. 1999), in the onset o f hyperglycemia in mice transgenic for human I A P P (Verchere et al. 1996; Hoppener et al. 1999), a reduction in beta cell mass in mice (MacArthur et al. 1999) and monkeys (de Koning et al. 1993), and with severity of hyperglycemia at the time o f death in humans (Rocken et al. 1992), the development of islet amyloid may play a role in the 19 progression of diabetes to increasing hyperglycemia. The occurrence o f amyloid is not confined to type 2 diabetes; it is also present in and implicated in the progression o f a number o f other disease states. 1.2 Amyloid 1.2.1 General Characteristics A m y l o i d is a generic term referring to fibrillar protein deposits o f similar ultrastructural and tinctorial characteristics. Amylo id fibrils can be formed by a variety o f proteins: at least 20 different proteins and polypeptides form amyloid deposits in vivo (Kisilevsky 2000). N o obvious sequence homology or consistent similarities in the three dimensional structure under normal physiological conditions o f these proteins has been found (Kallijarvi et al. 2001). There are, however, a number of common features that amyloid fibrils share, no matter what the protein source (Sipe 1992; Kisi levsky and Fraser 1997). These include: 1) a high degree o f B-sheet in a "cross-B" pattern; 2) fibrillar morphology when viewed with an electron microscope, with fibrils being straight and unbranched, and on average 10 nm in diameter (Sunde and Blake 1997); and 3) the ability to bind the dyes Congo Red and thioflavin T. 20 1.2.2 Common Amyloids In humans, amyloid may occur either systemically or locally. Systemic amyloid is distributed throughout the body. Localized amyloid is confined to one part of the body, for example, the brain, the skin, or the pancreas. Each different disease state in which amyloid occurs has a specific pattern o f distribution. For example, in Alzheimer's disease, amyloid forms in the brain, particularly in the hippocampus, amygdala, and neocortex, and in the walls o f cerebral blood vessels (De Girolami et al. 1999). In type 2 diabetes, amyloid is deposited in the pancreatic islets, adjacent to beta cells and islet capillaries. Additionally, each different type of amyloid contains a unique major protein component (see Table I for examples). U p to 20 different proteins have been found to form amyloid (Kisilevsky and Fraser 1997); for the purpose of this discussion, six w i l l be introduced. The first four occur in the central nervous system; the latter two deposit systemically. One of the hallmarks of Parkinson's disease is Lewy bodies, fibrillar intracellular inclusions in neuronal cells in the brain (Lotharius and Brundin 2002). Alpha synuclein is the main protein component of these deposits. A-beta (AJ3) forms the basis of senile plaques in the brain in Alzheimer 's disease. It is derived from a larger transmembrane protein, the amyloid precursor protein (APP) , by proteolytic processing (Kang et al. 1987; Haass et al. 1992; Shoji et al. 1992). Huntingtin is a protein of unknown function that forms intraneuronal aggregates in the central nervous system in Huntington's Disease (Di Prospero and Tagle 2000). Prion 21 protein is an extracellular membrane protein of unknown function, expressed in neurons as well as a number of other tissues (Bendheim et al. 1992). It aggregates in the brain during Cruetzfeldt-Jakob Disease, Gerstmann-Straussler-Scheinker syndrome, and Kuru in humans, in the sheep and goat disease scrapie, and in bovine spongiform encephalopathy (Prusiner 1991). Transthyretin is a plasma protein produced mainly in the liver which forms systemic amyloid in familial amyloid polyneuropathy. Serum amyloid A ( A p o S A A ) is the main component of inflammation-associated amyloid (Uhlar and Whitehead 1999; Urieli-Shoval et al. 2000), which deposits systemically. A p o S A A is an acute phase protein that increases 500-1000 fold in concentration in the plasma during inflammation. Its function is unknown (Uhlar and Whitehead 1999; Urieli-Shoval et al. 2000). 22 Table I. Major protein component of several amyloids. Disease Major Protein Component of Amyloid Type 2 diabetes, Insulinomas Islet Amylo id Polypeptide ( IAPP; Amyl in) Alzheimer 's Disease A-beta Chronic Inflammation Serum A m y l o i d A Huntington's Disease Huntingtin Cruetzfeldt-Jakob Disease Prion protein Familial A m y l o i d Polyneuropathy Transthyretin Parkinson's Disease Alpha-Synuclein 1.2.3 Common Components of Amyloids Besides the major protein constituent, a set of additional components is also consistently present in amyloid deposits. These include glycosaminoglycans (GAGs) , the heparan sulfate proteoglycan perlecan, laminin, serum amyloid P, collagen I V , and apolipoprotein E (apo E) (Kisilevsky 1997). A p o E is an apolipoprotein associated with very-low density lipoprotein. It is important in cholesterol transport and lipoprotein metabolism (Weisgraber and Mahley 1996; Fazio et al. 2000). A p o E is also synthesized in the brain (Williams et al. 1985; Nakai et al. 1996), where it is believed to play a role in 23 synaptic plasticity and membrane repair after neuronal injury (Boyles et al. 1989; Guillaume et al. 1996). Serum amyloid P component (SAP) is a glycoprotein that is normally found circulating in the plasma (Pepys et al. 1997). Its physiological function is unknown (Pepys et al. 1997), but it may play a role in bacterial survival during infection (Noursadeghi et al. 2000) and in preventing the development of autoimmunity to chromatin (Bickerstaff et al. 1999). Laminins are the main noncollagenous component of basement membranes. They are glycoproteins, and together with collagen I V and several other proteins, provide structural organization to the basement membrane (Colognato and Yurchenco 2000). Heparan sulfate proteoglycans and G A G s are also components of the basement membrane. These molecules w i l l be discussed in more detail in section 1.3.1. 1.3 Islet Amyloid A s discussed in section 1.1.7, islet amyloid accumulates in type 2 diabetes, and likely contributes to beta cell death. Heparan sulfate proteoglycans are associated with islet amyloid deposits (Young et al. 1992) and may play a role in their formation (Kisilevsky and Fraser 1997; Park and Verchere 2001). 24 1.3.1 Proteoglycans in Islet Amyloid Proteoglycans are large molecules consisting of a protein core with long, negatively charged polysaccharide side chains. Proteoglycans are found on the plasma membrane o f all cells and in the extracellular matrix. These important molecules have a multitude o f diverse functions, including cell adhesion, growth factor sequestration, cell signaling, viral entry into cells (Shieh et al. 1992; Compton et al. 1993; Shukla et al. 1999), and binding to interleukin, lipoprotein lipase, interferon gamma, and platelet factor 4 (Rosenberg et al. 1997; Lindahl et al. 1998; Shukla etal . 1999). The polysaccharide side chains o f proteoglycans consist of repeating disaccharide subunits, and are termed glycosaminoglycan ( G A G ) chains. G A G s are subdivided into four classes, depending on the individual sugars that constitute the polymer: chondroitin sulfate, dermatan sulfate, keratan sulfate and heparan sulfate. The heparan sulfate proteoglycan perlecan is found in islet amyloid (Young et al. 1992) in association with IAPP fibrils rather than simply distributed diffusely throughout the deposit. The presence of perlecan has been proposed to have a role in the initiation of islet amyloid (Young et al. 1992; Castillo et al. 1998). It has been suggested that the perlecan that accumulates in islet amyloid is l ikely synthesized locally, since immunoreactivity for perlecan is present not just in islet amyloid but also in the islet cells (Kahn et al. 1999). In 25 further support o f this possibility, beta cells have been shown to secrete heparan sulfate proteoglycans which bind to human IAPP (Potter-Perigo et al. 2003). Other animal models of amyloidosis further support a role for perlecan in the initiation o f islet amyloid deposition. For example, in a murine model of A A amyloidosis, perlecan m R N A becomes upregulated prior to the histological detection of amyloid deposition (Ailles et al. 1993), suggesting that, at least in one other type of amyloid, perlecan may be involved in the generation of the deposit rather than simply accumulating subsequent to amyloid formation. Snow et al. (1991) found that HSPGs and amyloid were deposited at the same time in the spleen and liver in this model of A A amyloidosis, and the H S P G s were closely associated with amyloid fibrils when viewed with electron microscopy ( E M ) . In a mouse model of Alzheimer 's Disease, perlecan enhances fibrillar A B amyloid deposition and persistence in brain when coinfused with A B into rodent hippocampus (Snow et al. 1994). The inability of the body to remove amyloid may be one factor that contributes to its persistence in vivo (Kisilevsky 1998; 2000). Amylo id fibrils share a relative resistance to the action o f proteases, compared to their constituent protein in its monomelic form. For example, the prion protein P r P c becomes relatively resistant to protease degradation when in the fibrillar, P rP S c conformation (Baldwin et al. 1995). Similarly, A B possesses a degree o f protease resistance when in fibrillar form (Soto and Castano 1996). Proteoglycans have been 26 shown to have a role in contributing to this protease resistance in vitro. Heparan sulfate and chondroitin proteoglycans prevent protease degradation of A B fibrils in vitro (Gupta-Bansal et al. 1995), a property which could aid their persistence in vivo (Snow and Wight 1989; Snow et al. 1994). Rat neuronal cells are able to internalize and degrade fibrillar A B from culture medium (Shaffer et al. 1995), but addition of C S P G to A B prior to addition o f microglia resulted in a decreased ability to do so. H S P G association with basic fibroblast growth factor, a non-amyloidogenic protein, also confers a degree o f resistance to proteolytic degradation (Saksela et al. 1988). 1.3.2 Effect of GAGs on IAPP Fibril Formation Sulfated G A G s have been found in every amyloid deposit examined to date (Kisilevsky and Fraser 1997). Heparan sulfate moieties in the extracellular matrix have been proposed to play a particularly important role in the initiation of amyloid formation (Kisilevsky and Fraser 1997). They have the potential to accelerate islet amyloid deposition as they bind to I A P P fibrils and other amyloids in vitro (Watson et al. 1997; Ancs in and Kisi levsky 1999; Cohlberg et al. 2002), and enhance fibril formation. Heparan sulfate increases the beta sheet content of monomeric IAPP and other amyloid precursors, facilitating their stacking into fibrils (Castillo et al. 1997; Kisi levsky and Fraser 1997; Castillo et al. 1998; McLaur in et al. 1999). Heparan sulfate, and its analogue heparin, 27 stimulate not only the polymerization o f A B fibrils but also their nucleation or seeding (Fraser et al. 1992; McLaur in , Franklin et al. 1999). Heparan sulfate accelerates the rate o f A B fibril formation, and also leads to more fibrils being formed (Castillo et al. 1997). Similarly, both the rate and extent of fibril formation of alpha synuclein are increased in the presence o f heparin and heparan sulfate (Cohlberg et al. 2002). Sulfated glycans such as heparan sulfate stimulate formation of the protease resistant form o f prion protein from the protease sensitive form (Wong et al. 2001). In order to exert this stimulatory effect it has been proposed that G A G s act as a scaffold for the assembly of fibrils (McLaurin et al. 1999), potentially by serving as an anchor for fibril organization or by stabilizing the tertiary structure of the fibril. This ability o f G A G s to accelerate amyloid fibril formation seems to be dependent on the sulfate groups (Fraser et al. 1992). Desulfation of heparin leads to a significant decrease in its augmentation o f I A P P fibril formation (Castillo et al. 1998), and a complete loss o f its ability to enhance A B , . 4 0 fibril formation (Castillo et al. 1999). Sulfation patterns of heparan sulfate in vivo may be important, as there is evidence that they are altered in amyloidosis. Studies have indicated that the heparan sulfate proteoglycans in the A D brain are more heavily N-sulfated than in normal aged brains (Lindahl et al. 1995) or that N-sulfated G A G s were highly enriched (Snow et al. 1990). There is also a different pattern of sulfation of these molecules in amyloid laden spleens and livers (Lindahl and Lindahl 1997). In fact, Kisi levsky (Kisilevsky 1992) has suggested that a disturbance in basement membrane metabolism may be present during amyloid formation in vivo. One way in which this disturbance could occur has been proposed to be the interaction o f amyloidogenic precursors interacting with basement membrane precursors (Narindrasorasak et al. 1995). This may interfere with the formation of a structured basement membrane, which in turn could take part in the nucleation of amyloid deposits (Narindrasorasak et al. 1995). 1.3.3. Domains in IAPP Important in Amyloid Formation Several regions in IAPP 1-37 have been identified as being important in the ability o f the molecule to form fibrils. i. Residues 20-29: The 20-29 region (Betsholtz et al. 1989; Westermark et al. 1990) in I A P P (Fig. 2) is a critical beta sheet region which largely confers on the molecule the propensity to rapidly form amyloid fibrils in vitro, and to form amyloid in vivo. The minimal unit for fibril formation is amino acids 23-27 (Tenidis et al. 2000). Rat IAPP has three proline substitutions in this region and does not form fibrils in vitro (Westermark et al. 1990). Rodents also never develop islet amyloid in type 2 diabetes-like syndromes. Notable 29 exceptions are some transgenic mice which express human IAPP (Verchere et al. 1996; Hoppener et al. 1999). Substitution of a proline residue for amino acids at positions 20, 21, or 29 still allowed for amyloid fibril formation by residues 20-29 (Moriarty and Raleigh 1999), whereas substitution o f a proline at positions 22,24, or 26-28 inhibits the ability o f the peptide to form amyloid fibrils. 1 10 20 30 Human KCNTATCATQRLANFLVHSSMNFGAILSSTNVGSNTY NH2 Monkey R T D-- NH2 Cat IR L P NH2 Rat R L-PV-PP NH2 Figure 2: The sequence of IAPP is well-conserved across species. Human I A P P 20-29, although representing a part of the molecule that is essential for fibril formation, clearly does not possess all properties of full length IAPP . For example, human I A P P 20-29 is 1000-fold less toxic (on a molar basis) than full length human I A P P (Tenidis et al. 2000). Another study found human IAPP 20-29 non-toxic to neuronal cells in culture (May et al. 1993; Pike et al. 1993) but full length human IAPP to be toxic. 30 ii. Residues 30-37 and 8-20: Two other regions of IAPP have recently been identified as beta sheet regions capable of forming amyloid fibrils in vitro. High concentrations o f synthetic peptides corresponding to I A P P 30-37 (Nilsson and Raleigh 1999) or 8-20 (Jaikaran et al. 2001) were both shown to form amyloid fibrils. Interestingly, both rat and human IAPP 8-20 form amyloid fibrils. Since rat IAPP does not form amyloid deposits in vivo, even in the most severe diabetic states, it seems that this domain is not sufficient for amyloid formation in vivo. Two other pentapeptide sequences in human I A P P have been found to self-assemble: h IAPP 14-18 ( N F L V H ) and hIAPP 15-19 ( F L V H S ) (Mazor et al. 2002). iii. S20G mutant: A subpopulation of Japanese patients with type 2 diabetes possess a mutation in the I A P P gene such that serine at position 20 in the mature human I A P P is substituted with a glycine. These patients may experience a more severe, earlier onset form o f type 2 diabetes (Sakagashira et al. 1996; Seino 2001). This mutant form o f I A P P has been shown to be more toxic when expressed intracellularly in C O S cells (Sakagashira et al. 2000) and forms more fibrils for a given concentration of peptide, as examined by E M and thioflavin T assay (Ma et al. 2001). Interestingly, a mutant form of alpha synuclein exhibits accelerated fibril formation in vitro (Conway et al. 1998), and this mutant form of the protein is expressed in some patients with an early-onset form of Parkinson disease. A mutant form o f A P P (the "Swedish" mutation) leads to increased A P P cleavage and increased production of A B , associated with an earlier onset form of Alzheimer's Disease (Citron et al. 1995; Haass etal . 1995). iv. Influences of other domains: ProIAPP forms amyloid fibrils, but is less amyloidogenic, and less cytotoxic to RIN5fm beta cells (Krampert et al. 2000), suggesting that the propeptides' effect on fibril formation influences toxicity. The propeptides, particularly the N-terminal, may have a role in the initiation of islet amyloid formation (see section 1.4.1). Other residues within IAPP which have an effect are residues 1-7, which Goldsbury et al. (Goldsbury et al. 2000) have found to influence the speed of I A P P fibril formation. v. Inconsistencies: Although the 20-29 region appears to be very important for amyloidogenicity, there are two animals which produce an amyloidogenic form o f I A P P , and yet do not develop islet amyloid in type 2 diabetes-like conditions. Dogs produce an amyloidogenic form of IAPP , and develop islet amyloid in insulinomas (O'Brien et al. 1990), but to date islet amyloid has not been reported in any dog with a type 2 diabetes-like syndrome (Jordan et al. 1990). Similarly, the European hare produces I A P P with a sequence that is amyloidogenic in vitro (Christmanson et al. 1993) but islet amyloid has not been documented in this animal in type 2 diabetes. In contrast, cats have an identical I A P P sequence to dogs and do develop islet amyloid (Jordan et al. 1990). 32 vi. Domains in AB that interact with heparan sulfate proteoglycans: Since heparan sulfate in the extracellular matrix has been implicated in the generation of amyloid deposits, interactions of amyloidogenic proteins with components o f the basement membrane have been investigated. Interactions between A P P and A B , and heparan sulfate proteoglycans have a number o f parallels to putative interactions between IAPP with HSPGs . A B binds to heparan sulfate (Narindrasorasak et al. 1991; Brunden et al. 1993), but the domains involved seem dependent on the aggregation state. It has been proposed that fibrillar A B binds to heparan sulfate via clusters of lysine residues aligned on adjacent A B monomers (Gupta-Bansal et al. 1995). A t p H 7.4, fibrillar, but not monomeric A B binds H S P G (Gupta-Bansal et al. 1995; Brunden et al. 1993). Binding of nonfibrillar A B to proteoglycans is thought to occur via a different mechanism, since it does not occur at p H 7.4 and therefore seems in part governed by the protonation of histidines (Gupta-Bansal et al. 1995; Brunden et al. 1993). A P P also binds to heparan sulfate proteoglycans (Narindrasorasak et al. 1991) through two domains in addition to that thought to mediate A B binding (Clarris et al. 1997; M o k et al. 1997). I A P P has been proposed to interact with heparan sulfate through clusters of positive charge created through alignment of basic residues on adjacent monomers in a fibril (Watson et al. 1997), similar to the proposed alignment of lysine residues in fibrillar A B (Gupta-Bansal et al. 1995). IAPP contains histidine residues (see Fig.2), but to date the role o f these in heparin 33 binding has not been established. ProIAPP also contains basic residues (see F ig . 3) which have the potential to interact with HSPGs. A. +++ + + ++ ++ TPIESHQVEK^KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTYGKR^NAVEVLKREPLNYLPL B. N-terminal fragment: K10A mutant: R11A mutant: K10A/R11A mutant: C-terminal fragment: TPIESHQVEKR KCNTATCATQRLANFLVHS TPIESHQVEAR^KCNTATCATQRLANFLVHS TPIESHQVEKAiKCNTATCATQRLANFLVHS T PI ESHQVEAAiKCNT ATC ATQRL ANFLVH S TNVGSNTYGKRJTSTAVEVLKREPLNYLPL Figure 3: Amino acid sequence of (A) full-length human proIAPP and (B) the synthetic N- and C-terminal proIAPP peptides used in these studies. Arrow denotes sites o f proIAPP cleavage during normal processing to produce mature I A P P (in bold). Underlined amino acids in (A) represent sequence of synthetic peptides shown in (B). "+" designates basic amino acid. 1.3.4 Mechanisms of IAPP Fibrillogenesis It has long been known that in vitro, the formation of amyloid fibrils from monomeric amyloid precursors follows a lag phase of varying interval, followed by a rapid onset accompanied by a transition to a conformation enriched in beta sheet. The lag phase and sudden onset of this transition resemble the nucleation of a crystallization process (Kisilevsky and Fraser 1997). In fact, amyloid formation by many amyloidogenic precursors, 34 including IAPP , has been demonstrated to be nucleation dependent (Scherzinger et al. 1999; Wood et al. 1999; Kayed et al. 1999; Conway et al. 1998). With the advent of atomic force microscopy, which allows far greater resolution than E M , processes that occur at a molecular level during fibril formation have been examined more closely. A m y l o i d fibrils begin as oligomeric assemblies, which in turn form an intermediate assembly state called the protofibril. These assemblies later form mature fibrils, and the protofibrils, which represent a small population of the total molecules present, disappear (Anguiano et al. 2002). A m y l o i d fibril assembly has been suggested to occur in four stages (Harper et al. 1999): 1) protofibril formation: a process that may be nucleation-dependent, and may require as few as 20 molecules to form one protofibril; 2) protofibril elongation: the coming together of smaller protofibrils; 3) protofibril-to-fibril transition: at this stage protofibrils begin to disappear, as they associate and wind together to form mature fibrils; and 4) fibril elongation. Protofibrils have been identified in a number of amyloidogenic peptides, including A B (Harper et al. 1999), in IAPP (Goldsbury et al. 1999; Anguiano et al. 2002), in transthyretin (Lashuel et al. 1998) and a-synuclein (Conway et al. 2000). Protofibrils are susceptible to disassembly (Voiles et al. 2001; Harper et al. 1999). It is the protofibril-to-fibril transition that does not appear to be easily reversible. Once protofibrils disappear during fibril 35 formation, they do not reappear, even upon dilution of fibrils (Harper et al. 1999; Kayed et al. 1999). Factors that cause nucleation may be an aggregate of protein or an exogenous seed. In the case o f A B , conversion of protofibrils to fibrils can be seeded by preformed A B fibrils but not protofibrils (Harper et al. 1997). Amylo id fibril formation can also be seeded with exogenous fibrillar molecules, such as silk (Kisilevsky et al. 1999). 1.3.5 An Unfolded Intermediate as a Precursor to Amyloid Fibrils It has recently been suggested that the ability to form amyloid fibrils is not restricted to a small number of proteins, as previously thought (Guijarro et al. 1998; Chit i et al. 1999). Instead, the ability to form amyloid may be an inherent property o f polypeptide chains, when exposed to the appropriate conditions (Chiti et al. 1999; Dobson 1999). Studies have indicated that rather than arising directly from proteins in their native conformation, amyloid fibrils form through a partially folded state of the precursor protein (Wood et al. 1996; Prusiner 1997; Guijarro et al. 1998; Chit i et al. 1999). Chit i et al. propose that partially denaturing conditions that destabilize the native fold of a protein but permit noncovalent interactions between various groups (Chiti et al. 1999) may encourage amyloid fibril formation in a wide variety of proteins. For example, Guijarro et al. (1998) found that a 36 small globular protein, SH3, which normally folds without intermediates and is not identified with any amyloid-associated disease, forms amyloid fibrils when kept in a partially-folded state for several days in vitro. In the case of IAPP, Kayed et al. have indicated that fibril formation indeed occurs via a partially folded state (Kayed et al. 1999). Studies with transthyretin support the hypothesis that an unfolded intermediate is the precursor to amyloid fibrils. Transthyretin normally exists as a tetramer in the plasma. In vitro, keeping the transthyretin tetramer associated prevents amyloid formation (Redondo et al. 2000). Similarly, Lashuel et al. (1998) found that the tetramer was incapable o f forming amyloid fibrils but, using acid dissociation, the monomer folded in an alternate conformation and ultimately formed amyloid fibrils. Hammarstrom et al. (2002) examined a series of common transthyretin mutants, and found that those associated with increased rate o f tetramer dissociation favoured amyloid formation, and correlated with a more serious and earlier onset form of amyloid in vivo. A m y l o i d formation likely is influenced in vivo by many factors that influence the physiological folding of proteins (Kel ly 1998). For example, certain values of p H or ionic strength may be more permissive for amyloid fibril formation than others. Prusiner (1996) and K e l l y (1996; 1998) have proposed that partial denaturing of amyloid precursors could 37 occur in compartments in the cell such as lysosomes. In the case of islet amyloid, however, much evidence suggests that the deposits form extracellularly. 1.3.6 Mechanisms of IAPP Toxicity The mechanism through which IAPP fibrils exert their toxic effect remains debated, although it seems to be dependent on the peptide adopting a beta sheet conformation and forming fibrils, as monomeric amyloid precursors are not toxic. For example, monomeric A B is not cytotoxic to neuronal cells, while fibrillar A B is (Pike et al. 1993; Lorenzo and Yankner 1994). Similarly, rat IAPP , which remains monomeric, is not cytotoxic to beta cells, while human I A P P is (Lorenzo and Yankner 1994). There is evidence that human IAPP and a number of other amyloidogenic peptides insert into phospholipid bilayer membranes and form ion-permeable channels (Mirzabekov et al. 1996; Kawahara et al. 2000). This observed ability to form channels seems dependent on fibril formation. Non-toxic rat IAPP does not form channels in l ipid bilayers. Similarly, fibrillar A B induces changes in membrane fluidity in liposomes (Kremer et al. 2000), while monomeric A B does not have this effect. A fragment of the prion protein, P r P 1 0 6 . 1 2 6 forms fi-sheets in water and ion channels across planar l ipid bilayer membranes (L in et al. 1997; Kawahara et al. 2000). 38 These channels have been proposed to form in the plasma membrane o f cells exposed to amyloidogenic proteins, and to allow passage of ions such as calcium. Evidence supporting this idea includes the finding that exposure of immortalized neuronal cells to fibrillogenic I A P P caused increases in intracellular calcium. The amyloidogenic peptides A B M 0 and P r P l 0 6 . 1 2 6 (Kawahara et al. 2000) had a similar effect. Other studies have failed to show a critical role for intracellular calcium in IAPP-induced cell death (Lorenzo et al. 1994; Ba i et al. 1999). The ability of I A P P to form channels appears to be dependent on l ipid membrane composition (Mirzabekov et al. 1996; Kawahara et al. 2000), as it is unable to form channels in cholesterol-rich membranes (Mirzabekov et al. 1996). The addition o f soluble cholesterol to the medium prior to addition of ARU40 increases the latency of the intracellular calcium increase and markedly decreases its magnitude (Kawahara et al. 2000). In a subsequent study, bilayer cholesterol content in total brain l ipid extracts correlated inversely with the ability o f A B M 0 to insert into the membrane (Yip et al. 2001). L ip id bilayers may in fact aid in the formation o f amyloid fibrils, and hence may aid channel formation by an amyloidogenic protein. For example, IAPP monomers are thought to form protofibrils faster in the presence of synthetic vesicle phospholipid membranes (Anguiano et al. 2002). Only membrane lipids bearing a negative charge were found to foster fibrillogenesis o f A B in another study (Yip and McLaur in 2001). 39 The fact that cell membrane composition is important for toxicity and fibrillogenesis of amyloid supports the hypothesis that moieties on the plasma membrane, such as heparan sulfate, have the potential to mediate toxicity. Others have suggested that proteoglycans are a potential candidate for a cell surface mediator of amyloid toxicity (Pollack et al. 1995; McLaur in et al. 1999). Although amyloid precursors are only toxic when they are in the form of fibrils, the toxicity o f the fibril itself is thought to be dependent on the stage of assembly. The mature amyloid fibril may be inert, and it may be the protofibril that is cytotoxic (Lansbury 1999; Goldberg and Lansbury 2000). Human I A P P toxicity to freshly dispersed human islet cells correlates with the presence of "intermediate sized" amyloid particles (Janson et al. 1999). Preferential protofibril interaction with synthetic membranes is supportive of protofibrils being the toxic species, since membrane interaction is necessary for IAPP fibril-induced death (Lorenzo et al. 1994). Pore formation by IAPP in synthetic vesicles temporally correlated with the disappearance of the protofibrils and the appearance of mature fibrils (Anguiano et al. 2002). Protofibrillar oc-synuclein has a higher affinity for the synthetic vesicles than does the monomeric form of this protein (Voiles et al. 2001). Aged A B fibrils lose their ability to cause changed in liposome membrane fluidity (Kremer et al. 2000). 40 In addition to changes in membrane ion flux, other mechanisms of cytotoxicity have been proposed for I A P P and other amyloidogenic proteins. For example, Schubert et al. (1995) found that IAPP , A B , and several other amyloidogenic peptides were toxic to and generated free radicals in rat neuronal cells. Several other studies have indicated that A B generates free radicals in cells (Butterfield et al. 1994; Hensley et al. 1994) whereas a more recent study found that it does not (Dikalov et al. 1999). Interestingly, antioxidants do not prevent the A B -induced change in liposome membrane fluidity (Kremer et al. 2000), indicating that perhaps membrane pore formation may occur independently of free radical generation. 1.3.7 Inhibition of Fibrillogenesis In Vitro Since amyloid fibrils are cytotoxic and are suspected to contribute to the pathogenesis of a number o f degenerative diseases, there has been much interest in the discovery of molecules that inhibit fibrillogenesis. A number of molecules have been used successfully to inhibit amyloid fibril formation in vitro and in vivo. i. Fragments of amyloidogenic precursors: One approach to inhibiting amyloid fibril formation in vitro has been the creation of fragments of amyloidogenic precursors that interfere with the interaction of full-length monomers. For example, a five amino acid peptide with homology to the N-terminal hydrophobic region of A B inhibits A B fibril 41 formation in vitro, and even causes disassembly of preformed fibrils (Soto et al. 1998). This fragment also prevents A B , . 4 2 neurotoxicity to neuroblastoma cells. Another approach to the design of inhibitors for A B toxicity has been to synthesize similar peptides attached to a disrupting element (oligolysine, which possesses polar hydrophilic groups), which interferes with A B self-assembly (Ghanta et al. 1996). This inhibitor, when preincubated with A B , . 3 9 , inhibited toxicity to PC-12 cells. Short peptides homologous to the central hydrophobic region o f A B , but bearing proline substitutions (so-called "beta sheet breakers") inhibit A B fibril formation and attenuate toxicity (Soto et al. 1996). Similar approaches have been successful in inhibiting IAPP fibril formation. Hexapeptides homologous to certain areas o f the 20-29 beta sheet region in IAPP strongly inhibit fibril formation o f the full length I A P P (Scrocchi et al. 2002). ii. Rifampicin and Congo Red: Rifampicin is an antibiotic used in the treatment of leprosy. It was reported in 1992 that elderly leprosy patients showed a reduced frequency o f senile plaques in their brains compared to age-matched controls (Namba et al. 1992), and subsequently rifampicin was shown to inhibit fibril formation by A B , and toxicity to P C 12 cells (Tomiyama et al. 1996). Rifampicin was proposed to inhibit toxicity by inhibiting binding o f fibrils to the cell surface (Tomiyama et al. 1997). Anionic sulfonates or sulfates, such as the cotton dye Congo Red, have been shown to reduce or abolish the toxicity of several forms of amyloid fibrils in vitro (Caughey and Raymond 1993; Lorenzo and Yankner 42 1994; Pollack et al. 1995; Heiser et al. 2000). Rifampicin and Congo Red were shown to significantly reduce the increase in membrane conductance induced by I A P P in synthetic bilayers (Harroun et al. 2001) and toxicity (Lorenzo and Yankner 1994) without inhibiting fibril formation. Treatment of C O S cells transiently transfected with exon 1 o f the huntingtin protein with Congo Red reduced the formation of huntingtin aggregates in the cells (Heiser et al. 2000). 1.3.8 Inhibition of Amyloid Formation In Vivo i. Immunization: In a mouse model of Alzheimer's Disease which overexpresses human amyloid precursor protein, immunization with A B M 2 led to almost complete prevention of amyloid-B deposition in the brain (Schenk et al. 1999), i f immunization was initiated before the onset of amyloid deposition, and >90% reduction in detectable amyloid i f immunization was initiated at an age when amyloid plaques normally have developed in the brains o f the mice. The same group later showed similar results with immunization with an antibody to A B ^ (Bard et al. 2000). A similar study found an approximately 50% reduction in amyloid plaque deposition in addition to a reduction in the behavioural impairment observed in the mice (Janus et al. 2000). The investigators concluded that it may be because the antibody recognized beta sheet forms of A B , as it has been shown that monoclonal antibodies against regions of A B that initiate fibril formation can inhibit fibril formation in vitro (Solomon et al. 1996). A recent study (Dodart et al. 2002) indicated that passive 43 immunization with an antibody to A f i M 2 can also reverse cognitive deficits, but did not reduce the amount o f amyloid in the brain. When incubated with pre-formed A B M 2 fibrils, sera from mice immunized with A B M 2 caused the fibrils to disassemble (McLaurin et al. 2002). Similarly, antibodies directed at the fibrillogenic exon 1 of the huntingtin protein inhibit aggregation in vitro in a dose-dependent manner (Heiser et al. 2000). ii. Other in vivo therapies: Oral administration of low molecular weight anionic sulfonates or sulfates attenuated the deposition o f splenic amyloid in a murine model of inflammation-associated amyloid (Kisilevsky et al. 1995). Intraperitoneal injection with porphyrin and phthalocyanine significantly delayed disease onset in a mouse model of scrapie (Priola et al. 2000). Recently, the administration of Congo Red intraperitoneally or directly into the brain was shown to lead to extensive clearance of huntingtin aggregates in a mouse model of Huntington's disease (Sanchez et al. 2003). iii. Potential endogenous inhibitors of amyloid formation in vivo: Several non-amyloidogenic proteins produced endogenously may play a role in preventing or predisposing to amyloid formation. For example, the e4 allele of apoE is associated with increased risk o f developing Alzheimer's disease (Strittmatter et al. 1993; Weisgraber and Mahley 1996). M i c e lacking apoE do not develop amyloid in a transgenic mouse model o f Alzheimer's disease (Bales et al. 1999). A p o E knockout mice can, however, develop islet 44 amyloid (Vidal et al. 2003), and are not protected from inflammation-associated amyloidosis (Elliott-Bryant and Cathcart 1997). Similarly conflicting results are found in vitro: apoE has been found to inhibit A B nucleation and fibrillogenesis (Wood et al. 1996) or to enhance fibrillogenesis of A B (Ma et al. 1994). M i c e lacking S A P , which is consistently found associated with amyloid deposits in vivo (Pepys et al. 1994) have a delayed onset of inflammation-associated amyloid (Botto et al. 1997). A subsequent study found that the rate o f regression o f the amyloid was not significantly different from control animals expressing S A P , however (Usui et al. 2001). In vitro, S A P also inhibits fibril formation by A B , ^ (Janciauskiene et al. 1995). Proteins found in the secretory granules of beta cells have been shown to have differential effects on I A P P fibrillogenesis (Westermark et al. 1996; Kudva et al. 1998). Insulin and proinsulin both inhibit fibril formation, although the latter much less potently. C-peptide, in combination with zinc ions, also has an inhibitory effect on IAPP fibril formation. Mouse I A P P inhibits fibril formation by human IAPP in vitro in a dose-dependent manner (Westermark et al. 2000). Mice that do not express endogenous IAPP but are transgenic for human I A P P develop islet amyloid earlier than their littermates which express both mouse I A P P and human IAPP , data which support the idea that mouse IAPP inhibits islet amyloid formation by human IAPP in vivo (Westermark et al. 2000). iv. Qualifications: In designing inhibitors of amyloid, care must be taken not to actually cause amyloid deposition or exacerbate existing amyloid deposits. Lundmark et al. have shown that a predisposition to develop A A amyloid can be transmitted by the addition o f preformed amyloid fibrils to the drinking water (Lundmark et al. 2002). This active immunization with A B fibrils might conceivably lead to seeding of further plaque formation. Certain monosaccharides derived from glycosaminoglycans have the ability to retain A B in the form o f protofibrils (McLaurin et al. 1999). I f protofibrils are indeed the toxic species, the administration o f a molecule with this effect would exacerbate rather than ameliorate the damaging effect of amyloid fibrils (Harper et al. 1999). 1.4 Overall Rationale and Specific Aims Although regions such as residues 20-29 in human IAPP are known to be important for fibrillogenesis, we hypothesized that residues outside of this region and in proIAPP are important for fibrillogenesis and cytotoxicity. Three specific aims were identified for the purposes of this investigation. 46 1.4.1 Aim 1: To determine whether proIAPP binds to heparin In order for islet amyloid to form in vivo, it is necessary that a fibrillogenic form o f I A P P be present (Westermark et al. 1990). Production, or even overproduction, of a fibrillogenic form o f IAPP , however, is not sufficient for islet amyloid formation to occur. Other factors must therefore be necessary for islet amyloid to form. One candidate is impaired processing of proIAPP, the IAPP precursor, in type 2 diabetes. In type 2 diabetes, processing of proinsulin is known to be impaired, leading to hypersecretion of proinsulin, and hyperproinsulinemia (Ward et al. 1987). ProIAPP processing is therefore likely to be similarly impaired in type 2 diabetes, resulting in the release o f unprocessed or partially processed forms of proIAPP from the beta cell ( M a et al. 1997; Kahn et al. 1999). Supporting this hypothesis is the finding that immunoreactivity for the N-terminal propeptide of proIAPP has been detected in amyloid deposits from human patients (Westermark et al. 1989), suggesting that at least an N-terminally extended form of proIAPP contributes to islet amyloid deposits. A n additional study suggests that hyperglycemic environment in type 2 diabetes could lead to the release o f incompletely processed proIAPP - in particular N-terminally extended proIAPP - from beta cells (Hou et al. 1999; Gasaeta l . 2001). 47 Proteins interact with heparin and heparan sulfate via clusters of positive charge. The cleavage sites for processing of proIAPP are dibasic residues, and at the N-terminus this site is adjacent to a third basic residue (see Figure 3). We hypothesized that the cluster of basic residues in the N-terminal region of proIAPP mediates binding to heparan sulfate, and that normal processing of proIAPP would be predicted to destroy this heparin-binding domain. I f proIAPP processing by beta cells is defective in type 2 diabetes, incompletely processed forms of proIAPP might bind to H S P G s following their secretion from the beta cell, forming a nidus for initiation of islet amyloid. 1.4.2 Aim 2: To determine which residues in IAPP are important in heparin binding to fibrils, and in heparin stimulation of fibril formation Although the sequence o f I A P P is wel l conserved across species (Figure 2), key differences between rodent I A P P and that o f other mammals shed light on domains essential for fibri l formation. I A P P from humans, monkeys, and cats form fibrils readily in vitro, and all o f these species develop islet amyloid in type 2 diabetes. In contrast, rat I A P P does not form fibrils, and rats never develop islet amyloid, even in the most severe diabetic states. A critical beta sheet region (amino acids 24-29; the ' G A I L S ' region) is largely responsible for the propensity of IAPP 48 to form fibrils (Westermark et al. 1990). Rat I A P P contains three proline substitutions in the G A I L S region, which are thought to inhibit fibril formation. One factor l ikely influencing islet amyloid formation in vivo is the rate of recruitment o f new I A P P fibrils into existing plaques. We therefore studied factors affecting fibrillogenesis of I A P P . In particular, we investigated the effect of heparin stimulation of this process. The ability o f human I A P P to bind to heparin is thought to be dependent on its aggregation state (Watson et al. 1997). Basic resides on adjacent strands in the beta sheet may align, forming heparin-binding "patches". We hypothesized that basic residues in IAPP including lysine at position 1, arginine at position 11, and histidine at position 18 are responsible for this interaction. 1.4.3 Aim 3: To determine whether the S20G mutant form of IAPP is more cytotoxic to beta cells than wild-type IAPP A small subpopulation of Japanese patients with type 2 diabetes bear a point mutation in the I A P P gene that leads to a serine to glycine substitution at position 20 in I A P P (Fig. 4). These patients may experience a more severe, early onset form of type 2 diabetes (Sakagashira et al. 1996; Seino 2001). It has recently been shown that the S20G mutant IAPP forms fibrils more quickly than wild-type in vitro (Sakagashira et al. 2000). When expressed in COS-1 cells, S20G 49 human I A P P is more cytotoxic than wild-type IAPP. We hypothesized that S20G mutant human IAPP is more cytotoxic to beta cells, and that this increased cytotoxicity may be related to increased heparin binding. 1 1 0 2 0 3 0 H u m a n K C N T A T C A T Q R L A N F L V H S S N N F G A I L S S T N V G S N T Y N H 2 S 2 0 G G N H 2 Figure 4: Amino acid sequence of wild-type human IAPP 1-37, and S20G mutant. 50 2. Methods 2.1 Materials 2.1.1 Affinity Chromatography Heparin, heparan sulfate, chondroitin sulfate A , bovine serum albumin ( B S A ) , Sepharose C L - 4 B , heparin-agarose, heparin-Affi-Gel, fi-mercaptoethanol, and Sigma Cote™ were purchased from Sigma (St.Louis, M O ) . Cyanogen bromide (CNBr)-activated Sepharose 4B was purchased from Pharmacia Biotech (Baie D'Urfe , Canada). A l l human proIAPP fragments were synthesized at the Nucleic A c i d Protein Synthesis unit at the University o f British Columbia, except human proIAPP 1-11, which was synthesized by Dr. Melanie Nilsson at the State University of New York at Stony Brook, New York. A l l synthetic peptides were H P L C purified, and the N-terminal peptides containing cysteine residues at positions 6 and 9 of I A P P were cyclized. Rat IAPP was purchased from Bachem (Torrance, C A ) . 2.1.2 Fluorometry Thioflavin T, heparin, dimethylsulfoxide ( D M S O ) , hexafluoroisopropanol (HFIP), T r i s - H C l , N a C l , and Triton X-100 were purchased from Sigma (St.Louis, M O ) . Synthetic rat I A P P 1-37 and human IAPP 1-37, 8-37, and 20-29 were purchased from Bachem. S20G human I A P P 1-37 and wild-type human IAPP 1-37 synthesized in parallel were obtained 51 from Multiple Peptide Systems (San Diego, C A ) . Syringe top filters (0.20 um) were obtained from Mil l ipore (Billerica, M A ) . 2.1.3 Cell Death Studies WST-1 reagent was purchased from Boehringer-Mannheim (Mannheim, Germany). Congo Red, heparinases I and III, and B-D-xyloside were purchased from Sigma Chemical Company (St. Louis, M O ) . INS-1 beta cells were a gift from Dr. Philippe Halban, University of Geneva. 2.1.4 3H-Heparin Binding Assays 3H-heparin was purchased from American Radiolabeled Peptides. Ninety-six well plates with 0.45 jam Durapore filters were purchased from Mill ipore. 2.1.5 Electron Microscopy Uranyl acetate was purchased from Sigma Chemical Company, U K . 52 2.2 Methods 2.2.1 Affinity Chromatography Heparin-agarose or heparin-Affi-Gel affinity chromatography was performed as per Ancs in and Kisi levsky (Ancsin and Kisi levsky 1999). In brief, an 8 m l column o f heparin-agarose or heparin-Affi-Gel was equilibrated with 20 m M Tr i s -HCl at either p H 7.5 or 5.5, as indicated. Peptide (300 jag) was dissolved in buffer and loaded onto the column, washed with four column volumes of buffer, then developed at a flow rate of 0.5 ml/min with a 0-1 M N a C l gradient over five column volumes using a G M - 1 gradient mixer (Pharmacia Biotech). Fractions (0.5 ml) were collected and absorbance measured at 214 nm. Salt concentrations in fractions were directly measured using a hand-held conductivity meter. Heparan sulfate and chondroitin sulfate affinity chromatography were performed in a similar manner, except the columns were made by coupling free heparan sulfate or chondroitin sulfate to C N B r -activated Sepharose 4B beads. 2.2.2 Coupling of Glycosaminoglycans to Sepharose® CL-4B Beads Coupling of glycosaminoglycan ( G A G ) to CNBr-activated Sepharose 4B beads was carried out following the protocol provided by the manufacturer. Chondroitin sulfate (600 mg) was dissolved in 15 m l coupling buffer (0.1 M N a H C 0 3 , p H 8.3, containing 0 .5M NaCl ) . CNBr-activated Sepharose C L - 4 B beads (4g) were swollen in 1 m M HC1, then 53 washed for 15 min with 1 m M HC1 on a sintered glass filter pre-coated with Sigma Cote. The chondroitin sulfate solution was mixed with the gel suspension in a 50 m l stoppered vessel also pre-coated with Sigma Cote. The mixture was rocked overnight at 4°C. The next day, excess chondroitin sulfate was washed from the gel on a sintered glass filter using five gel volumes of coupling buffer. The gel was then placed in 0.1 M Tr i s -HCl p H 8.0 and rocked at room temperature for 2h in a stoppered vessel to block any remaining active groups. The gel was subsequently subjected to three alternating washes of 0.1 M sodium acetate buffer (pH 4.0) containing 0.5 M N a C l , and 0 .1M Tr i s -HCl (pH 8.0) containing 0 .5M N a C l . To minimize cost for preparation of heparan-sulfate Sepharose, the coupling protocol was scaled down and a 3 ml column used. Glycosaminoglycan coupling efficiency to the Sepharose beads, assessed by the toluidine blue method (Smith et al. 1980; Ancsin and Kisi levsky 2001) was found to be 0.25 mg/ml gel for chondroitin sulfate and 0.1 mg/ml gel for heparan sulfate. 2.2.3 Toluidine Blue Assay The toluidine blue assay was carried out as per Ancsin and Kisi levsky (Ancsin and Kisi levsky 2001). The glycosaminoglycan solution (0-35 ul, in increments of 5 ul; 2 mg/ml) was added to tubes containing 0.75 m l 0.2% N a C l , as standards. Sepharose C1-4B beads coupled to G A G were also diluted 1:4 with 0.2% N a C l to yield a total volume o f 0.75 ml . A control tube, which contained uncoupled Sepharose C1-4B in 0.2% N a C l , was also prepared. Toluidine blue solution (0.75 ml ; 5 mg/lOOml in 0.01N HC1, 0.2% NaCl ) was added to all tubes, and the tubes were vortexed for 30s. Hexane (1 ml) was then added to each tube, and tubes were vortexed for a further 30s. The tubes were then centrifuged at 3000 rpm for 5 min. The hexane and the top half of the water layers were aspirated and discarded. A sample of the remaining water layer was diluted 1:10 with absolute ethanol, mixed, and absorbance read at 631 nm. Values from standards were graphed to create a standard curve, and the amount o f G A G linked to the matrix was calculated using this curve. 2.2.4 Preparation of Peptides For the E M analysis, and for fluorometric experiments with the S20G and wild-type peptides from Multiple Peptide Systems, the peptides were prepared in the following manner. To ensure no aggregated forms were present, peptides were first dissolved in hexafluoroisopropanol at 1 mg/ml and allowed to incubate at room temperature for l h (Higham et al. 2000). The solution was then filtered through a 0.22 um syringe-top filter (Millipore). The filtrate was aliquoted into microfuge tubes in 100 ul aliquots, and placed at - 2 0 ° C for 2h, then at -80°C overnight. The following day, the samples were placed on dry ice and then lyophilized. The lyophilized peptides were stored in an airtight container until use. For cell death assays, the peptides were prepared in a similar manner but were not filtered. In parallel cell death experiments, filtered peptide was found to yield comparable results to those observed with unfiltered peptide (data not shown). 55 For all other fluorometric assays and for heparin binding assays, I A P P peptides were dissolved directly in D M S O without prior lyophilization. These experiments were performed prior to the study by Higham et al. (Higham et al. 2000), which established that the protocol described above leads to optimal maintenance of IAPP in a monomeric state. 2.2.5 Fluorometry Measurement of amyloid fibril formation by thioflavin T fluorescence was performed using an assay adapted from Na ik i et al. (1989) and Kudva et al. (1998) for a 96 well plate format. Peptides were dissolved in 100% D M S O to 400 u M immediately prior to use. Peptide was added to wells containing 10 u M thioflavin T in 10 m M T r i s - H C l (pH 7.4), 100 raM N a C l , and 0.1 % Triton X-100 to final peptide concentrations o f 20 u M or 40 u M in 5% D M S O . In some experiments, peptides at 20 u M were incubated with 50ug/ml heparin added to the buffer. The plate was sealed with parafilm and fluorescence measured at 37°C using a Fluoroskan (Labsystems, Vista C A ) fluorometer with filters set at 444 (excitation) and 485 (emission) nm and bandwidth slits of 12 and 14 nm, respectively. When bound to amyloid fibrils, thioflavin T fluoresces with excitation and emission maxima of 450 and 482 nm, respectively (Naiki et al. 1989) and fluorescence under these conditions correlates with the degree of amyloid fibril formation as assessed by E M (Kudva et al. 1998). Triplicate measurements for each peptide were made every minute from 0-100 min, every 10 minutes 56 from 100-150 min, and finally every 60 minutes from 150 min to 15 h, allowing kinetic assessment of IAPP fibrillogenesis. The mean data were plotted and analyzed using Kaleidagraph™ software (Synergy Software, Reading P A ) . 2.2.6 Cell Death Studies INS-1 cells were grown in R P M I medium (Gibco-BRL) supplemented with 11 m M glucose, 10% fetal bovine serum, 100 U / m l penicillin (Gibco-BRL) , 100 U / m l streptomycin (Gibco-BRL) , and 50 u M 13-mercaptoethanol. Cells were maintained at 37°C in a humidified atmosphere supplemented with 5% C 0 2 . For the viability assay, cells were seeded into 96 well plates at a density of 3x10 4 cells per well . After 48 hours of culture, the medium was removed and replaced with medium containing 20 u M or 40 u M IAPP , or 20 u M I A P P plus 50 ng/ml heparin. After a further 48 hours, cell viability was assessed using the WST-1 assay. Cells were incubated with the WST-1 reagent for 4 hours, after which absorbance (450 nm) was recorded on a Bio-Rad 3550 microplate reader. Triplicate determinations were made for each condition and each experiment was replicated at least two times. Ce l l viability was also assessed qualitatively by morphological changes observed using a phase-contrast inverted microscope (Nikon). Morphological changes indicative of apoptosis include more prominent nuclei, and cell shrinkage, rounding, and lifting off of the bottom of the culture plate. 57 2.2.7 3H-Heparin Binding Assays Peptides were kept in I m M stock solutions in D M S O and frozen in aliquots at -20°C until use. On the day of each experiment, human or rat IAPP was diluted to 25 u M in 1 m l phosphate-buffered saline (PBS) and allowed to incubate at room temperature overnight. For the spin assay, tubes were spun at 10,000 rpm for 3 minutes. The supernatant was carefully removed with a pipette, except for the final 20 ul of solution, which was designated the "pellet" and contained fibrillar material. Non-fibrillar IAPP remained in the supernatant. Radioactivity ( 3H-beta emission) in both supernatant and pellet was assessed on a beta counter (Wallac, Turku, Finland), following addition of 300 ^1 scintillant (Fisher Scientific). For the filtration assay, following overnight incubation, the sample was gently resuspended using a pipette, and passed through a 0.45 um Durapore® membrane using a Mil l ipore Mult iscreen® 96-well filtration manifold. Fibrillar IAPP is expected to remain on the filter (Scherzinger et al. 1999). The filter was washed with P B S (200 ul). Scintillant was added to each wel l and 3 H radioactivity retained on the filters determined on a beta counter. 2.2.8 Electron Microscopy Peptides were dissolved at 1 mg/ml in P B S , and incubated without shaking. Aliquots (2 ul) were removed at 30 min, 2, 4, 24, 48, and 72 h, and 6 days. A t each time point, the aliquot was placed on a formvar-coated nickel grid for 5 minutes, then the buffer removed with filter paper. Uranyl acetate (2%) was immediately applied and allowed to incubate for a 58 further 2 minutes. Finally the uranyl acetate was removed, and fibrils on the grid detected using a Jeol 1010 Transmission Electron Microscope operated at 80 k V . 2.2.9 Statistical Analysis Lag time for fibrillogenesis in the fluorometric assays was deemed to be when the level o f fluorescence increased to two standard deviations above baseline fluorescence. Ce l l viability and 3H-heparin binding experiments were performed in triplicate and repeated on at least two occasions. Data represent mean ± standard deviation from representative experiments. Statistical significance in differences in cell viability and in 3H-heparin binding were determined using either a one-way Analysis of Variance and Tukey's Multiple Comparison Test, or Mann-Whitney Test. 59 3. Results 3.1 Affinity of Human ProIAPP Fragments for Heparin To test whether the cluster o f basic amino acids in the regions o f the N - and C -terminal cleavage sites in proIAPP might constitute heparin-binding domains, we synthesized peptides corresponding to the 30 N-terminal or 27 C-terminal amino acids o f human proIAPP (see F ig . 3) containing these domains, and applied these peptides to a heparin-agarose column. Since fibril formation might enhance heparin binding (Watson et al. 1997), a critical amyloidogenic region in IAPP (amino acids 31-40 of proIAPP) was omitted from these synthetic peptides to minimize the likelihood of protein aggregation. The N-terminal proIAPP fragment was retained by the heparin column, eluting at 0 .18M N a C l on a 0 - 1 M N a C l gradient developed over 80 minutes (Fig. 5a). In contrast, B S A , a protein known to not bind to heparin (Ancsin and Kisi levsky 1999) eluted in the void volume. When applied to a column containing uncoupled Sepharose, the N-terminal proIAPP fragment eluted in the void volume (Fig. 5a), indicating that its retention on heparin-agarose was not due to a nonspecific interaction between the peptide and the column matrix. The C-terminal region of proIAPP contains two pairs of basic residues, including one at the C-terminal cleavage site involved in processing of proIAPP to IAPP 1-37. Unlike the N-terminal proIAPP fragment, the C -terminal proIAPP peptide interacted only weakly with heparin, eluting in wash fractions prior to commencement of the N a C l gradient (Fig. 5b). 60 A. 0 50 100 150 Fraction # 0 50 100 150 200 250 Fraction # 61 0 50 100 150 Fraction # Figure 5: Affinity of proIAPP fragments for heparin. Synthetic peptides corresponding to (A) human proIAPP amino acids 1-30 (N-terminal), (B) human proIAPP 41-67 (C-terminal), or (C) human proIAPP 1-30 with alanines substituted for lysine 1 0 -arginine n (K10A;R11 A ) , were applied to a heparin-agarose or uncoupled Sepharose column, as indicated in (A) . Bovine serum albumin (BSA) was used as a control in (A). The column was washed with four column volumes of buffer (20 m M Tr i s -HCl , p H 7.5), developed with a 0-1 M N a C l gradient, fractions (0.5 ml) collected and absorbance determined at 214 nm. To determine whether the K 1 0 R H K 1 2 sequence in the N-terminal proIAPP cleavage site is essential for heparin binding, we synthesized a peptide in which alanine residues were substituted for the two basic residues (lysine and arginine) that comprise the cleavage site recognized by prohormone convertases during normal proIAPP processing (see F ig . 3). The K 1 0 A / R 1 1 A mutant N-terminal proIAPP peptide fragment had no affinity for heparin (Fig. 5c), eluting in the void volume when applied to the heparin-agarose column. The complete 62 loss of heparin binding in the K 1 0 A / R 1 1 A mutant peptide indicates that one or both o f the basic residues in the N-terminal cleavage site of proIAPP is essential for heparin binding. The peptide eluted earlier in the salt gradient (0.09M NaCl) in the presence of 5 m M B-mercaptoethanol (Fig. 6), indicating that the interaction may be partially dependent on intramolecular disulfide bond formation between cysteine residues at positions 13 and 18 (Fig. 3). The N-terminal proIAPP fragment was also tested for its ability to bind to heparin-Aff i -Ge l , a matrix in which heparin is coupled via carboxyl groups rather than by amino and hydroxyl groups as in heparin-agarose. The peptide bound the heparin-Affi-Gel (Fig. 7) but eluted at a lower concentration of N a C l , suggesting that carboxyl groups in heparin participate in the interaction with the N-terminal proIAPP fragment. 63 0 50 100 150 Fraction # Figure 6: Affinity of N-terminal proIAPP fragment for heparin-agarose in the presence of C-mercaptoethanol. A synthetic peptide corresponding to human proIAPP amino acids 1-30 was applied to a heparin agarose column. The column was washed with four column volumes of buffer (20 m M Tr i s -HCl , 5 m M B-mercaptoethanol, p H 7.5), developed with a 0-0.5 M N a C l gradient, fractions (0.5 ml) collected, and absorbance determined at 214 nm. 64 Fraction # Figure 7: Affinity of N-terminal proIAPP fragment for heparin Affi-Gel. A synthetic peptide corresponding to human proIAPP amino acids 1-30 was applied to a heparin-Affi-Gel column. The column was washed with four column volumes of buffer (20 m M Tris-HC1, p H 7.5), developed with a 0-0.5 M N a C l gradient, fractions (0.5 ml) collected, and absorbance determined at 214 nm. 3.2 Affinity of Human ProIAPP Fragments for Heparan Sulfate To determine whether the N-terminal region of human proIAPP has the ability to bind to the heparan sulfate chains o f heparan sulfate proteoglycans, as it does heparin, we applied the synthetic N-terminal proIAPP fragment to a column in which heparan sulfate was coupled to Sepharose. A s shown in Figure 8a, the N-terminal proIAPP peptide bound to the heparan sulfate, eluting at a N a C l concentration (0.17 M ) almost identical to that 65 observed when the peptide was applied to heparin-agarose. The C-terminal proIAPP fragment did not, by contrast, bind to the heparan sulfate, eluting in wash fractions (Fig. 8a). Similarly, the K 1 0 A / R 1 1 A mutant N-terminal proIAPP fragment eluted in the void volume when applied to the heparan sulfate-Sepharose column (Fig. 8a). Thus, the complete cluster of basic residues in the N-terminal cleavage site of proIAPP appears to confer affinity o f this molecule for both heparin and heparan sulfate. 66 A. E ^1-1.5 r -K10A/R11A .\ N-terminal fragment CM 1 r CD o c CO .a o (/) _Q < B. E CD O c CO _Q O 0) < o.5 r 1.5 r 0.5 0 50 100 Fraction # R11A N-terminal fragment K10A N-terminal fragment 0 50 100 Fraction # Figure 8: Affinity of proIAPP fragments for heparan sulfate. Synthetic peptides corresponding to (A) human proIAPP amino acids 1-30 (N-terminal), 41-67 (C-terminal), or 1-30 with two alanine substitutions (K10A;R11 A ) , or (B) human proIAPP 1-30 with single alanine substitutions ( K 1 0 A or R l 1 A ) were applied to a heparan sulfate-Sepharose column. The column was washed with four column volumes of buffer (20 m M T r i s - H C l , p H 7.5), developed with a 0-1 M N a C l gradient, fractions (0.5 ml) collected, and absorbance determined at 214 nm. 67 To elucidate which of the basic residues in the N-terminal cleavage site o f proIAPP may be critical for heparan sulfate binding, we next synthesized two mutant peptides, in which an alanine residue was substituted for either the lysine at position 10 (K10A) or the arginine at position 11 ( R l 1 A ) of proIAPP. When applied to the heparan sulfate column, both of these mutant peptides eluted in the void volume (Fig. 8b), indicating that both o f these basic residues are critical for interaction of N-terminal proIAPP with heparan sulfate. 3.3 Affinity of Human ProIAPP Fragments for Chondroitin Sulfate When applied to a chondroitin sulfate-Sepharose column, both the N-terminal and C -terminal proIAPP peptide fragments appeared to have weak interaction with this glycosaminoglycan, eluting after the void volume but prior to commencement of the salt gradient (Fig. 9). Thus, the affinity of the N-terminal proIAPP peptide appears to be much stronger for heparan sulfate than for chondroitin sulfate. Nonetheless, the weak binding of the N-terminal proIAPP peptide to chondroitin sulfate does seem to be dependent on the presence of the basic residues in the N-terminal cleavage site, since the K 1 0 A / R 1 1 A mutant N-terminal proIAPP peptide did not bind to chondroitin sulfate, eluting in the void volume (Fig. 9). 68 0 50 100 150 Fraction # Figure 9: Affinity of proIAPP fragments for chondroitin sulfate. Synthetic peptides corresponding to human proIAPP amino acids 1-30 (N-terminal), 41-67 (C-terminal), or 1-30 with alanine substitutions (K10A;R11 A ) were applied to a chondroitin sulfate-Sepharose column. The column was washed with four column volumes of buffer (20 m M T r i s - H C l , p H 7.5), developed with a 0-1 M N a C l gradient, fractions (0.5 ml) collected, and absorbance determined at 214 nm. 3.4 Human N-and C-terminal proIAPP Fragments are Non-Fibrillogenic Amylo id fibrils formed by mature human IAPP (proIAPP 12-49) and other amyloidogenic peptides including the amyloid-B (AB) protein o f Alzheimer 's disease, are known to bind to heparin (Watson et al. 1997). This affinity is thought to be dependent on the aggregation state of the peptide, since human but not non-fibrillogenic rodent I A P P has 69 been found to bind to both heparin (Watson et al. 1997) and perlecan (Castillo et al. 1998). To rule out the possibility that the heparin binding activity of our synthetic N-terminal proIAPP peptide was simply due to its aggregation into fibrils that subsequently bound heparin, we measured fibril formation using thioflavin T fluorescence and transmission E M . A s expected, thioflavin T fluorescence rapidly increased in the presence of human I A P P but was unchanged in the presence o f rat IAPP, demonstrating the known fibrillogenic properties o f human but not rat I A P P (Fig. 10). Neither the N - nor the C-terminal proIAPP fragments formed fibrils as measured by thioflavin T fluorescence (Fig. 10), producing identical traces to non-fibrillar rat IAPP . Over the seven day time course examined using transmission E M , no fibrillar aggregates were observed (data not shown). These data indicate that neither N -nor C-terminal proIAPP fragments were fibrillar over the time course of these experiments and therefore that the affinity for heparin and heparan sulfate demonstrated by N-terminal proIAPP was not due to its prior aggregation. 70 CD O c 0 12 10 hIAPP 1-37 N-term 8 — " • C-term rat IAPP 1-37 6 4 2 h 0 0 20 40 60 Time (minutes) 80 100 Figure 10: N-terminal and C-terminal proIAPP fragments are not fibrillogenic. Synthetic peptides (12.5 uM) corresponding to human proIAPP amino acids 1-30 ( N -terminal), human proIAPP 41-67 (C-terminal), mature human IAPP (proIAPP 12-49) or rat I A P P were incubated in 10 m M Tr i s -HCl (pH 7.4), 100 m M N a C l , 0.1% Triton X-100, and 10 u M thioflavin T. Amylo id fibril formation was assessed as thioflavin T fluorescence measured at 444 (excitation) and 485 (emission) ran. Values are expressed as mean ± S.D. 3.5 N-terminal ProIAPP Fragment Does Not Inhibit Fibril Formation It has been hypothesized that monomelic rat IAPP is an inhibitor of islet amyloid formation in vivo (Westermark et al. 2000). L ike rat IAPP, the N-terminal proIAPP fragment we created is monomelic, and the two molecules share substantial sequence homology. We tested the ability of the N-terminal proIAPP fragment to inhibit I A P P fibril formation in vitro (n=3). 71 (Fig. 11). Under the conditions tested, equimolar or tenfold excess of our monomeric N -terminal proIAPP fragment had no effect on IAPP fibril formation. A t equimolar or tenfold excess concentrations, the N-terminal fragment was similarly unable to protect INS-1 beta cells from IAPP fibril toxicity, as measured by the WST-1 assay (Fig. 12), in which a salt added to the culture medium forms a water soluble dye when reduced by mitochondrial superoxide anions. Samples containing greater numbers of metabolically active cells can then be detected by E L I S A , as described in Methods. 72 ¥ 3 r 2 r 1 h 0 0 hIAPP hIAPP + Nterm pro 1x •X- hIAPP + Nterm pro 10x 50 100 150 Time (minutes) 200 250 Figure 11: N-terminal proIAPP fragments does not inhibit fibril formation by human IAPP in vitro. Mature human IAPP (proIAPP 12-49) (12.5 uM) was incubated in 10 m M T r i s - H C l (pH 7.4), 100 m M N a C l , 0.1% Triton X-100, and 10 u M thioflavin T alone or in the presence of N-terminal proIAPP fragment (proIAPP 1-30) at equimolar (hIAPP + Nterm pro l x ) or ten times (hIAPP + Nterm pro lOx) the molar concentration of mature IAPP. A m y l o i d fibril formation was assessed as thioflavin T fluorescence measured at 444 (excitation) and 485 (emission) nm. Values are expressed as mean ± S.D. (n=3). 73 Figure 12: N-terminal proIAPP fragment does not protect INS-1 cells from IAPP fibril toxicity. Forty-eight hours following seeding, medium was removed and replaced with fresh medium alone or containing 40 u M wild-type h i A P P , with or without the addition o f a 50 (50x Nterm pro) or 100 (lOOx Nterm pro) fold molar excess o f the N -terminal proIAPP fragment. Cel l viability was assessed after a further 48h using the WST-1 assay. Values are expressed as mean ± S.D. (n = 3). Asterisk indicates significant decrease from medium alone, determined using a one-way Analysis of Variance and Tukey's Multiple Comparison Test (P < 0.001). Cel l viability in the presence of hIAPP with N-terminal proIAPP was not significantly different from that in the presence of hIAPP alone. The N-terminal proIAPP fragment alone had no effect on cell viability (data not shown). 74 3.6 Involvement of Histidine Residues in Heparin Binding at Acidic p H Having determined that the lysine and arginine residues at positions 10 and 11 o f N -terminal proIAPP are crucial for its ability to bind heparin and heparan sulfate at p H 7.5 (Figs. 5a and 8a), we next investigated the involvement of histidine residues, which would be protonated in the acidic milieu of beta cell secretory granules. There are two histidine residues present in human proIAPP, one in the N-terminal flanking region (position 6 o f proIAPP) and one at amino acid 18 o f mature IAPP (position 29 of proIAPP) (see F ig . 3). A t p H 5.5, the heparin affinity of the N-terminal human proIAPP fragment was increased, with the peptide eluting at 0 .28M N a C l (versus 0.18M at p H 7.5) (Fig. 13a). The affinity of the peptide for heparan sulfate was similarly increased at acidic p H (Fig. 13b). However, the K 1 0 A / R 1 1 A mutant had little affinity for heparin (Fig. 13a) or heparan sulfate (Fig. 13b) under these conditions. Thus, although either or both o f the two histidine residues present in proIAPP may increase its affinity for heparin/heparan sulfate at acidic p H , they cannot substitute for the K , 0 R n K 1 2 sequence in the N-terminal cleavage site, which is still essential for heparin binding at either acidic or neutral p H . 75 A. 0 50 100 150 Fraction # 0 50 100 150 Fraction # Figure 13: Affinity of N-terminal proIAPP fragment for heparin and heparan sulfate is increased at pH 5.5. Synthetic peptides corresponding to human proIAPP amino acids 1-30 (N-terminal) or 1-30 with alanine substitutions (K10A;R11 A ) were applied to a (A) heparin-agarose or (B) heparan sulfate-Sepharose column and affinity chromatography performed as in Figure 2, except the Tr i s -HCl buffer was p H 5.5, as indicated. Arrows denote elution time of N-terminal proIAPP peptide in T r i s - H C l at p H 7.5. 76 3.7 Residues Involved in Heparin Stimulation of IAPP Fibril Formation It is well documented that heparin augments the fibrillogenesis of I A P P and other amyloid precursors (McCubbin et al. 1988; Fraser et al. 1992; Castillo et al. 1998; McLaur in et al. 1999); (Fig. 14). Using a cell culture model of amyloid toxicity, we found the presence of heparin also augmented the toxicity of 20 u M human IAPP (Fig. 15) (P < 0.001). Since proteins are known to interact with heparin via basic residues, we synthesized two abbreviated fragments of human IAPP in order to elucidate which basic residues in human I A P P are responsible for interaction with heparin such that fibril formation is stimulated: I A P P 11-37 (Fig. 16), which lacks the first ten residues of IAPP (notably lysine 1), and I A P P 12-37, which lacks the first eleven residues (notably lysine 1 and arginine 1 1). In the presence of heparin, the initial rate o f fibril formation by IAPP 11-37 was stimulated compared to I A P P 11-37 alone (Fig. 17a), however the final steady state amount o f fibrils formed was not different from the control. N o difference in amount o f fibril formation at any point in the time course was observed in the presence of heparin for IAPP 12-37 (Fig. 17b). These data indicate that lysine 1 may be important for heparin increasing the final amount of fibrils formed by human I A P P , and that arginine 1 1 is important for heparin stimulating the initial rate of fibril formation. 77 Time (minutes) Figure 14: Heparin augments fibrillogenesis of human IAPP. Human IAPP was incubated in 10 m M Tr i s -HCl (pH 7.4), 100 m M N a C l , 0.1% Triton X-100, and 10 u M thioflavin T, in the presence or absence of heparin (50 (ig/ml). A m y l o i d fibril formation was assessed as thioflavin T fluorescence measured at 444 (excitation) and 485 (emission) nm. Values are expressed as mean ± S.D. (n=3). 78 A. Figure 15: Addition of heparin to the culture medium increases the cytotoxicity of human IAPP. Forty-eight hours following seeding, medium on INS-1 cells was replaced with fresh medium containing 20 u M wild-type hIAPP, with or without the addition of heparin (50 ng/ml). Ce l l viability was assessed after a further 48 hours. A ) WST-1 assay for cell viability. Values are expressed as mean ± S.D. (n = 3). Asterisk indicates significant decrease from control (medium alone), determined by one-way Analysis o f Variance and Tukey's Multiple Comparison Test (P < 0.001). B) Cells viewed by phase-contrast microscopy. Note rounded appearance and prominent nuclei of cells treated with 20 u M hIAPP in the absence, or, more markedly, in the presence of heparin. Arrow indicates cell with morphology suggestive of apoptosis. Each photograph is representative of an assay performed in triplicate. 79 B. Heparin only Wild-type human IAPP 20 pM 20 wild-type hIAPP + heparin 80 + + + IAPP 1-37 IAPP 11-37 IAPP 12-37 KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY RLANFLVHSSNNFGAILSSTNVGSNTY LANFLVHSSNNFGAILSSTNVGSNTY Figure 16: Sequence of synthetic IAPP peptides. "+" designates basic amino acid. 3.8 Heparin Does Not Augment Cytotoxicity of Human IAPP 11-37 or 12-37 In contrast to its effect on the cytotoxicity o f h i A P P 1-37 (Fig. 15), heparin failed to augment cytotoxicity of hIAPP 11-37 or hIAPP 12-37 (Fig. 18a). N o obvious morphological difference in heparin-treated cells was apparent using phase-contrast microscopy (Fig. 18b,c). To determine whether there were differences in the heparin binding o f fibrils formed from each o f these peptides, we assessed the amount of 3H-heparin bound to fibrils in solution after overnight incubation, as described in Methods. Fibrils formed by I A P P 11-37 and 12-37 peptides bound amounts of 3H-heparin that were significantly greater than the negative control, rat I A P P (P < 0.001)(Fig. 19), but not significantly different than the positive control, human IAPP . We confirmed that IAPP was interacting with the 3H-heparin, as we were able to displace radioactivity from the pellet by the addition of unlabeled heparin into the incubation (Fig.20). Residues 1-10 in I A P P appear therefore to be important in both heparin stimulating fibril formation and in heparin augmenting toxicity. 81 A CD 8 n c CD 2 ? 6 - > , 5 LL . CO 4 , E 5 3 O 1 i— o o 20 40 60 80 100 Time (minutes) rin (50 |jg/ml) 120 hIAPP 12-37 • — hIAPP 12-37 + heparin (50 ug/ml) 20 40 60 80 100 120 Time (minutes) Figure 17: Heparin stimulates the initial rate of fibrillogenesis of human IAPP 11-37 and not human IAPP 12-37. Human IAPP 11-37 (A) or 12-37 (B) (12.5 uM) was incubated in 10 m M Tr i s -HCl (pH 7.4), 100 m M N a C l , 0.1% Triton X-100, and 10 u M thioflavin T alone or in the presence heparin (50 ug/ml). Amylo id fibril formation was assessed as thioflavin T fluorescence measured at 444 (excitation) and 485 (emission) nm. Values are expressed as mean ± S.D. (n=3). 82 A. Figure 18: Addition of heparin to the culture medium does not increase the cytotoxicity of human IAPP 11-37 or 12-37. Forty-eight hours following seeding, medium on INS-1 cells was replaced with fresh medium containing 20 u M hIAPP 11-37 or 12-37, with or without the addition of heparin (50 ng/ml). Ce l l viability was assessed after a further 48 hours. A ) WST-1 assay for cell viability. Values are expressed as mean ± S.D. (n = 3). B ,C) Cells viewed by phase-contrast microscopy. Arrow indicates cell with morphology suggestive of apoptosis. Each photograph is representative of an assay performed in triplicate. 83 84 c . I hIAPP 12-37 20IJM < hIAPP 12-37 20 jvM + heparin 85 Figure 19: Human IAPP 20-29 does not bind to heparin. I A P P was diluted to 25 u M in 1 m l P B S containing 3H-heparin. Samples were treated in one of two ways: A ) Following overnight incubation, the sample was gently resuspended and passed through a 0.45 um Durapore® membrane using a Mill ipore Multiscreen® 96-well filtration manifold. 200 ul o f P B S was then passed through the filter, as a washing step. Scintillant (Fisher Scientific) was added to each well and 3H-radioactivity retained on the filters read. Values are expressed as mean ± S.D. (n = 3). Asterisk indicates significant increase from rat I A P P control as determined by one-way Analysis of Variance and Tukey's Mult iple Comparison Test (P < 0.001); B) Following incubation at room temperature for 90 min, samples were spun at 10,000 rpm for 3 min, and the supernatant carefully removed with a pipette, except for the final final 20 u l of solution, which was designated the "pellet". 3 H radioactivity in pellets was assessed on a beta counter (Wallac). Values are expressed as mean ± S.D. (n = 3). Asterisk indicates significant decrease from 3 H radioactivity retained in 1-37 pellet as determined by an unpaired t-test (P < 0.0001). (Undiluted sample of 3H-heparin contained 4500 C P M ) . 86 5000 40001 SN pellet SN pellet (+cold (+cold heparin) heparin) Figure 20 : Addition of unlabeled heparin displaces 3H-heparin from IAPP fibrils. Human IAPP 1-37 was diluted to 25 u M in 1 ml P B S containing 3H-heparin, in the presence or absence of an equivalent amount of unlabeled heparin (+ cold heparin), and allowed to incubate at room temperature overnight. The next day, samples were spun at 10,000 rpm for 3 minutes, and the supernatant (SN) carefully removed with a pipette, except for the final 20 ul of solution, which was designated the "pellet". 3 H counts in both supernatant and pellet were assessed on a beta counter (Wallac). Values are expressed as mean ± S.D. (n = 3). (Undiluted sample of 3H-heparin contained 5000 C P M ) . 3.9 Residues Important in Binding of Human IAPP to Heparin To establish which residues in IAPP are important for the binding of heparin to fibrils, we performed a series of assays with different IAPP fragments. Human I A P P 20-29 87 did not bind to heparin (Fig. 19), indicating that residues outside o f this region are responsible for the interaction of I A P P fibrils with heparin. B y affinity chromatography, rat I A P P was shown to bind to both heparin and heparan sulfate, eluting at approximately 0 .19M N a C l (Fig. 21). The double mutant N-terminal proIAPP fragment (K10A/R11 A ) used in other affinity column assays (Fig. 3) shares basic residues at the same positions as rat I A P P (Fig. 22). One difference is that human N-terminal proIAPP fragment possesses a histidine residue where rat I A P P possesses arginine (position 18 of rat IAPP) . The N-terminal human proIAPP fragment (K10A/R11 A ) did not bind to either heparin (Fig. 5c) or heparan sulfate (Fig. 8), eluting in the void volume. From these data, we would predict that monomelic human I A P P does not bind to heparin, and that the arginine substitution for the histidine confers affinity for heparin in monomelic rat IAPP. Protonation of this residue, however, did not lead to binding of the double mutant peptide to heparin or heparan sulfate (Fig. 13), nor did it lead to an increase in the amount of 3H-heparin bound by any fibrillar human I A P P peptide containing this residue (Fig. 23). 88 A. 0 50 100 150 200 Fraction # Fraction # Figure 21:Affinity of rat IAPP for heparin and heparan sulfate. Rat IAPP was applied to a (A) heparin-agarose column, or (B) heparan sulfate-Sepharose column. The column was washed with four column volumes of buffer (20 m M T r i s - H C l , p H 7.5), developed with a 0-1 M N a C l gradient, fractions (0.5 ml) collected, and absorbance determined at 214 nm. 89 Human: KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY R a t : R L - P V - P P P r o I A P P f r a g m e n t : TPIESHQVEAA H -Figure 22: K10A/R11A mutant proIAPP fragment shares basic residues with human IAPP 1-37 but does not bind to heparin. Figure 23: Binding of 3H-heparin to IAPP at pH 7.5 and pH 5.5. I A P P was diluted to 25 u M in 1 m l P B S , p H 7.4 or 5.5. Following overnight incubation, the sample was gently resuspended and passed through a 0.45 um Durapore® membrane using a Mil l ipore Multiscreen® 96-well filtration manifold. 200 ul of P B S was then passed through the filter, as a washing step. Scintillant (Fisher Scientific) was added to each well and 3 H radioactivity retained on the filters read. Values are expressed as mean ± S.D. (n = 3). 90 Interestingly, human IAPP 20-29 was not significantly cytotoxic to beta cells (Fig. 24), confirming findings in previous studies (Pike et al. 1993; Tenidis et al. 2000). Because IAPP 20-29 does not bind to heparin (Fig. 19) and is not cytotoxic, we hypothesized that there is a relationship between the heparin binding ability of IAPP fibrils, and fibril toxicity. Culture in the presence of 1 unit /ml heparinase I or III, which degrade cell surface heparan sulfate moieties, or 1 m M 13-D-xyloside, which prevents proteoglycan assembly, did not protect INS-1 cells from I A P P amyloid-induced cell death (Fig. 25) (P < 0.001). A summary o f putative residues important in fibril formation, toxicity, and heparin stimulation of fibril formation is outlined in Figure 26. 91 Figure 24: Human IAPP 20-29 is not toxic to INS-1 beta cells. Forty-eight hours following seeding, medium on INS-1 cells was replaced with fresh medium containing 20 u M hIAPP 1-37 or 20-29, or rat IAPP1-37. Ce l l viability was assessed after a further 48 hours using the WST-1 assay. Values are expressed as mean ± S.D. (n = 3). Rat I A P P was not significantly different from vehicle alone (data not shown). Treatments were not significantly different using a one-way Analysis o f Variance, followed by Tukey's Multiple Comparison test. 92 1.6 r 1.4 1.2 1 CD c 0.8 o 0.6 C/) < 0.4 0.2 0 o Q_ Q_ < 2 CJ) ~o "co o ST q Q_ Q_ < JZ + = Q l co _ i • i + CO CL CD CD CO co CO Q. CD Q_ Q. < i f ••5 ° CD Figure 25: Treatment with heparinase I or III, or B-D-xyloside does not protect INS-1 cells from IAPP cytotoxicity. Forty-eight hours following seeding, medium on INS-1 cells was replaced with fresh medium containing l U / m l heparinase I, l U / m l heparinase III, or I m M B-D-xyloside, with or without the addition of human I A P P (40 u M ) . Ce l l viability was assessed after a further 48 hours using the WST-1 assay. Values are expressed as mean ± S.D. (n = 3). Asterisk indicates significant increase from 40 u M human I A P P ( P < 0.001), determined using a one-way Analysis o f Variance and Tukey's Multiple Comparison Test. The three enzymes alone had no effect on cell viability (data not shown). 93 Regions other than 20-29 important in flbril-hcparin interactions Arg 1 ^Initial rate or" fibril formation KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY Lys 1: Final amount of fibrils formed Arg 1 8 : Rat IAPP binding to heparin Figure 26: Summary of residues proposed to be important in IAPP fibril formation, toxicity, and heparin stimulation of fibril formation. 3.10 Rifampicin Does Not Inhibit Fibril Formation Rifampicin, an antibiotic used in the treatment of leprosy, has been postulated to inhibit amyloid fibril formation by A13 (Tomiyama et al. 1996) but not I A P P (Tomiyama et al. 1997). Using our fluorometric assay of IAPP fibril formation, rifampicin appeared to completely inhibit fibril formation (Fig. 27). However, in our spin assay of 3H-heparin binding, we found that rifampicin did not inhibit a significant amount of 3H-heparin counts being present in the pellet (Fig. 28), suggesting that fibrils still formed regardless of the presence o f rifampicin, and that this drug merely inhibits thioflavin T binding to I A P P fibrils. Other studies have shown that rifampicin does not inhibit fibril formation in I A P P or exon 1 of the huntingtin protein (Heiser et al. 2000). 0 O C 0 00 j / f o 13 20.0 1 15.0 1 C g 10-0 O 5.0 hIAPP 1-37 + heparin "*— hIAPP 1 -37 + heparin + rifampicin o.o 0 25 50 75 100 Time (minutes) Figure 27: Effect of rifampicin on thioflavin T binding to IAPP in the presence of heparin. Human I A P P 1-37 (12.5 uM) was incubated in 10 m M T r i s - H C l (pH 7.4), 100 m M N a C l , 0.1% Triton X-100 ,10 u M thioflavin T, and heparin (50 ug/ml) in the presence or absence of rifampicin (100 uM). Amylo id fibril formation was assessed as thioflavin T fluorescence measured at 444 (excitation) and 485 (emission) nm. Values are expressed as mean ± S.D. (n = 3). 95 6000 5000 4000 CL ° 3000 2000 1000 0 1-37 1-37 + rifampicin Figure 28: Binding of 3H-heparin to IAPP fibrils in the presence of rifampicin. IAPP was diluted to 25 u M in 1 ml P B S containing 3H-heparin, in the presence or absence o f rifampicin (100 jaM) and allowed to incubate at room temperature for 90 minutes. Samples were then spun at 10,000 rpm for 3 minutes, and the supernatant carefully removed with a pipette, except for the final 20 ul of solution, designated the "pellet". 3 H radioactivity in the pellet was assessed on a beta counter (Wallac). Values are expressed as mean ± S.D. (n = 3). 3H-heparin control contained 5000 C P M (data not shown). The treatment with rifampicin was not significantly different from h i A P P 1-37 alone, determined using a one-tailed Mann-Whitney test. 3.11 Lag Time for Fibril Formation of Wild-Type and S20G hIAPP The S20G mutant form of IAPP (Fig. 4) is found in a small number of Japanese patients with type 2 diabetes. This mutation has been hypothesized to accompany a more severe, early onset form of diabetes. A t high concentrations of peptide (50 | i M ) , S20G IAPP 96 began to show increased thioflavin T fluorescence 40 minutes before wild-type h IAPP (Fig. 29). In contrast, at 40 u M and lower concentrations (Fig. 30), thioflavin T fluorescence increased simultaneously with both wild-type and S20G hIAPP, although the S20G mutant peptide bound more thioflavin T at all time points, indicative o f a greater amount o f fibrils formed. In the presence of heparin, 20 u M concentrations of the peptides followed the same trend. Over the 1200 minute time course of the experiment, very little signal was observed with a concentration of 20 u M peptide of either the mutant or wild-type species (data not shown). 97 0 50 100 150 200 250 Time (minutes) Figure 29: S20G mutant IAPP undergoes seeding event for fibrillogenesis earlier than wild-type. Synthetic human IAPP (wild-type or S20G mutant) was incubated in 10 m M T r i s - H C l (pH 7.4), 100 m M N a C l , 0.1% Triton X-100, and 10 u M thioflavin T. A m y l o i d fibril formation was assessed as thioflavin T fluorescence measured at 444 (excitation) and 485 (emission) nm. Values are expressed as mean ± S.D. (n = 3). 140 120 100 • Wild-type • S20G 20 | J M 40 | J M 50 |jM Peptide Concentration 20 M M + heparin Figure 30: Lag time to seeding event of amyloidogenesis for wild-type human IAPP and S20G at different concentrations. Synthetic human IAPP (wild-type or S20G mutant) was incubated in 10 m M Tr i s -HCl (pH 7.4), 100 m M N a C l , 0.1% Triton X-100, and 10 u M thioflavin T. Amylo id fibril formation was assessed as thioflavin T fluorescence measured at 444 (excitation) and 485 (emission) nm. Peptide concentrations were either 20, 40, or 50 |o,M, with or without the presence of 50 ug/ml heparin. Values are expressed as mean ± S.D. (n = 3). To attain higher resolution of differences in rates of fibril formation between wild-type and S20G h i A P P , aliquots from incubated samples were examined using transmission E M (Fig. 31). Fibrils derived from the S20G mutant peptide were detected at earlier time points than those o f wild-type h i A P P . B y 30 minutes, both peptides had formed amorphous nonfibrillar 99 aggregates; however, small protofilaments were visible in the S20G preparation, which progressed to fibril accumulations by 2 hours. In contrast, at 24 hours wild-type peptide was still at the stage o f short protofibrillar assemblies (small non-fibrillar oligomers) attached to amorphous aggregates, with no classical fibrils present. B y 48 hours, the wild-type h IAPP preparation contained fibrils similar to the S20G fibril assemblies, although displaying markedly more twisted morphology. A t 7 days no further change in fibril complexity or morphology was observed. 100 30 min Wild-type IAPP S20G Mutant 2h 24h m 48h 72h 6 days , 1 Figure 31: S20G mutant fibrils form faster than those of wild-type IAPP. Synthetic human I A P P (wild-type or S20G mutant) was incubated in phosphate-buffered saline (pH 7.2) for 7 days. Samples were taken from the solution at 30 minutes, 2 ,4 , 24,48, 72 hours, and 7 days, and the fibrils negatively stained with 2% uranyl acetate as described in experimental procedures. Fibrils were viewed with a Jeol 1010 transmission electron microscope operated at 80 k V . Bar = 200 nm. 101 3.12 Toxicity of S20G hIAPP The addition of 20 u M wild-type hIAPP to the culture medium of INS-1 beta cells did not lead to a significant decrease in cell viability after 48 hours compared to untreated controls (Fig. 32). However, the addition of 20 u M S20G mutant h IAPP to the culture medium led to a substantial reduction o f cell viability (58.1±4.0% viability compared to untreated control; P < 0.001). A t a higher concentration (40 uM) both forms o f h IAPP were similarly cytotoxic (cell viability 34.6±0.4% for wild-type treated cells; 30 .8±2.4% for S20G-treated cells; P < 0.001). Rat IAPP slightly increased cell viability at both 2 0 u M (P < 0.01) and 40 u M (P < 0.001) (Fig. 33), possibly by stimulating proliferation (Karlsson and Sandler 2001) thus it was not used as a negative control in the statistical analysis. 102 Figure 32: S20G mutant human IAPP is more cytotoxic to beta cells than wild-type human IAPP. Forty-eight hours following seeding, medium on INS-1 cells was replaced with fresh medium containing 20 u M or 40 u M wild-type or S20G mutant hIAPP. Ce l l viability was assessed after a further 48 hours. A ) WST-1 assay for cell viability .Values are expressed as mean ± S.D. (n = 3). Statistical differences were determined using one- way A N O V A and Tukey's Multiple Comparison Test. B ) Cells viewed by phase-contrast microscopy. Each photograph is representative o f an assay performed in triplicate. Note rounded appearance of INS-1 cells treated with 20 u M S20G h i A P P . Arrow indicates cell with morphology suggestive of apoptosis. 103 104 E c o IT) CD O c CO \— O CV) _Q < E CD c Q- 5 Q-5 Q-5 9-^  pan c 0- = L par 0_ =i. pan o < o < o < o par < o CD ^ CM CO CD I co co DC DC + Figure 33: Rat IAPP is not cytotoxic to INS-1 cells. Forty-eight hours following seeding, medium on INS-1 cells was replaced with fresh medium containing 20 u M rat I A P P 1-37 with or without the addition of heparin (50 ug/ml). Ce l l viability was assessed after a further 48 hours using the WST-1 assay. Values are expressed as mean ± S.D. (n = 3). Asterisks indicate significant increase from medium alone, as determined by one-way A N O V A and Tukey's Multiple Comparison Test (* P < 0.01; ** P < 0.001). $ indicates significant decrease from medium alone (P < 0.001). Addit ion of 50 ug/ml heparin (to stimulate fibrillogenesis) to the medium containing the 20 u M peptide led to an increase in toxicity for wild-type I A P P compared to the same concentration in the absence of heparin (P < 0.001) (Fig. 34). There was a trend towards an increase in toxicity of S20G IAPP in the presence of heparin, but this was not statistically 105 significant (Fig. 34). The addition of heparin alone to the culture medium had no effect on cell viability. A. o o CM CM Figure 34: Addition of heparin to the culture medium increases the cytotoxicity of wild-type human IAPP. Forty-eight hours following seeding, medium on INS-1 cells was replaced with fresh medium containing 20 u M S20G mutant hIAPP, with or without the addition of heparin (50 ug/ml). Cel l viability was assessed after a further 48 hours. A ) WST-1 assay for cell viability. Values are expressed as mean ± S.D. (n = 3). Statistically significant differences were determined by one-way A N O V A and Tukey's Mult iple Comparison Test. B) S20G-treated cells viewed by phase-contrast microscopy. (See also Fig . 15b). Each photograph is representative of an assay performed in triplicate.Note rounded appearance of cells treated with S20G IAPP . Arrow indicates cell with morphology suggestive of apoptosis. 106 20 j;M S20G hIAPP + heparin L 107 To confirm the increased cytotoxicity of S20G hIAPP using an independent approach, cells incubated with peptides for 48h were viewed by phase-contrast microscopy. Cells with reduced viability detected by the WST-1 assay also exhibited changes in cellular morphology associated with apoptosis (Fig. 32b; F ig 34b). Individual nuclei cell borders were visible, and cells appeared rounded, in contrast to viable cells, in which individual cell borders were not evident and nuclei were not visible. In accordance with WST-1 cytotoxicity data, apoptotic morphology was observed in cells treated with 20 u M S20G mutant, and in cells treated with 40 u M hIAPP or 20 u M hIAPP plus heparin (Fig. 32b; F i g 15b). N o sign o f apoptosis was observed in untreated cells upon visual inspection. Apoptotic morphology was observed in cells treated with 20 u M wild-type hIAPP but to a lesser degree than in those treated with 20 u M S20G h i A P P . The viability of these cells did not differ significantly from control when measured using the WST-1 assay. A correlation between the amount of heparin bound by S20G fibrils and their increased toxicity was not observed in preliminary heparin binding assays. S20G fibrils bound an amount of 3H-heparin that was not significantly different from the amount bound by wi ld -type human I A P P (Fig. 35). Both wild-type IAPP and S20G I A P P bound significantly more 3H-heparin than I A P P 20-29 (P < 0.001). Rat IAPP was used as a control as it remains 108 monomelic and would be expected to pass through the filter, leaving a minimal amount of 3H-heparin on the filter. Figure 35: Comparable amount of 3H-heparin bound by wild-type IAPP and S20G mutant. I A P P peptides were diluted to 25 u M in 1 m l P B S , p H 7.4. Fol lowing overnight incubation, the samples were gently resuspended and passed through a 0.45 urn Durapore® membrane using a Mil l ipore Multiscreen® 96-well filtration manifold. 200 ul of P B S was then passed through the filter, as a washing step. Scintillant (Fisher Scientific) was added to each well and 3H-counts retained on the filters read. Values are expressed as mean ± S.D. (n = 3). Asterisks indicate significant decrease from 1-37, as determined by one-way Analysis of Variance and Tukey's Multiple Comparison Test (P < 0.001). 109 4. Discussion Islet amyloid develops in up to 90% of persons suffering from type 2 diabetes. This fibrillar deposit is implicated in the decreased beta cell mass observed in these patients, as I A P P fibrils are toxic to beta cells in vitro. Islet amyloid is therefore thought to be an important factor in the progression of type 2 diabetes. In order for IAPP to form amyloid fibrils, a critical beta sheet region spanning amino acids 20-29 must be present. Therefore a beta sheet region must also be present in this part of the molecule for IAPP to exert the toxic effect characteristic of amyloid fibrils. IAPP is a 37 amino acid molecule, however, and domains outside of the 20-29 region are also likely important for fibrillogenesis and toxicity. Similar to other amyloid precursors, it is thought that interactions between I A P P and basement membrane components are important in the initiation and persistence o f amyloid deposits. Heparan sulfate proteoglycans may be particularly important as they are present in the extracellular matrix, are intimately associated with amyloid fibrils in vivo, and accelerate fibril formation in vitro. In this thesis, we have provided evidence that domains in I A P P and its precursor, proIAPP, are important in heparin binding and toxicity of I A P P fibrils. First, we have shown that the N-terminal cleavage site of proIAPP constitutes a heparin- and heparan sulfate-binding domain. This domain is predicted to be lost following normal processing of proIAPP. Second, basic residues in the region N-terminal to amino acid 20 of human I A P P 1-37 also mediate binding to heparin, and heparin stimulation of fibril 110 formation. The presence of heparin augments the cytotoxicity of human I A P P in vitro. Third, the S20G mutation observed in some Japanese diabetic patients leads to a species o f I A P P with increased fibrillogenicity and beta cell toxicity. 4.1 N-terminally Extended ProIAPP Binds to Heparan Sulfate In addition to IAPP , islet amyloid deposits have been shown to contain immunoreactivity for the N-terminal region of proIAPP (Westermark et al. 1989) and for basement membrane heparan sulfate proteoglycan (Young et al. 1992). The presence o f these molecules in islet amyloid deposits suggests their involvement in the mechanism of islet amyloid deposition, yet it is unknown what role they might play. In this thesis, we demonstrated that a synthetic fragment of the N-terminal region of proIAPP binds to heparin and heparan sulfate. We further demonstrated that the heparin-binding domain in N -terminally extended proIAPP requires the presence of basic residues in the N-terminal cleavage site at which one step in proIAPP processing occurs, and therefore that normal processing of proIAPP would be predicted to destroy this heparin-binding domain. In contrast, the C-terminal region of proIAPP showed no affinity for heparin or heparan sulfate, despite the presence o f two pairs of basic residues in this region. Our findings raise the possibility that i f secretion o f unprocessed proIAPP (or partially processed, N-terminally extended proIAPP) from the beta cell is increased in type 2 diabetes, it might bind to the sulfated glycosaminoglycan side chains of heparan sulfate proteoglycans, creating a nidus for 111 amyloidogenesis within the pancreatic islet (see Fig . 36). We speculate that this mechanism may be an important initiating step in islet amyloid formation in type 2 diabetes. Similar seeding mechanisms have been proposed for Alzheimer amyloid precursors (Narindrasorasak et al. 1992). Figure 36: Proposed role of proIAPP in the formation of islet amyloid. Insulin and IAPP are synthesized and secreted by beta cells. In type 2 diabetes, proIAPP may be secreted from beta cells in increased amounts. B y virtue of the heparan sulfate binding domain at the N-terminal cleavage site, proIAPP may have the ability to bind to heparan sulfate proteoglycans in the beta cell extracellular matrix, forming a seed for subsequent amyloid formation. In this model IAPP secreted from the beta cell can bind to proIAPP in the extracellular matrix instead of passing into the bloodstream as it normally would, thereby contributing to growth of an amyloid deposit. 112 ProIAPP is thought to be processed to mature IAPP in beta cell secretory granules by the action o f PC2 and/or PC3 (PCI) cleaving on the C-terminal side o f pairs o f basic residues (in both cases lysine-arginine), followed by trimming of these basic residues by carboxypeptidase E . A s a result, normal proIAPP processing results in the removal o f the basic residues that we have shown are critical for heparin binding o f the N-terminal region o f proIAPP. Interestingly, the K 1 0 R U K 1 2 cluster of basic residues in the N-terminal proIAPP fragment does not represent a classic linear heparin-binding domain as proposed by Cardin and Weintraub (1989); however, many proteins that bind heparin do not possess these sequences (Cardin et al. 1986; Baird et al. 1988). One model has suggested that a spacing o f ~20 A between two basic amino acids is a critical determinant of heparin binding ability (Margalit et al. 1993). Such spacing can be achieved by a peptide in alpha helical conformation by basic amino acids spaced 13 residues apart or, in beta-strand conformation, 7 residues apart. Although the basic amino acids close to the N-terminal K 1 0 R n cleavage site ( H 6 , R 2 2 , and H 2 9 ) are not appropriately spaced according to this model, it is still conceivable that they contribute to the binding o f N-terminal proIAPP to heparin/heparan sulfate. Indeed, the increased affinity for heparin and heparan sulfate of the N-terminal proIAPP peptide that we observed at p H 5.5 implies a possible role for the histidine residues at positions 6 and 29. Also , the disulfide bond between the cysteines at positions 13 and 18 of proIAPP might be 113 predicted to bring the arginine at position 22 in closer proximity to the K 1 0 R n K 1 2 sequence. The interaction appeared weaker in the presence of B-mercaptoethanol, indicating that the heparin binding activity of the N-terminal proIAPP peptide may depend upon a conformational change induced by formation of the disulfide bond. While the K 1 0 R n K 1 2 sequence at the N-terminal cleavage site of proIAPP is clearly essential for heparin binding, whether this sequence is in itself sufficient or whether the basic residues outside o f this sequence are also critical w i l l require further study using additional mutant peptides. Heparin binding domains may also be created by protein aggregation, as illustrated by the binding to heparin (Watson et al. 1997) and perlecan (Castillo et al. 1998) of aggregated human I A P P . Since our synthetic N - and C-terminal proIAPP fragments remained soluble and did not form fibrils as assessed by thioflavin T fluorescence, our findings cannot be explained by prior aggregation of the N-terminal proIAPP peptide to form a heparin-binding domain. It is possible, however, that binding of soluble proIAPP to heparin/heparan sulfate might induce conformational changes in the protein that would enhance fibrillogenesis. Indeed, binding of soluble A B to glycosaminoglycans is known to stimulate beta-sheet conformation and aggregation (McLaurin et al. 1999) and perlecan has been shown to stimulate fibril formation from mature human IAPP (Castillo et al. 1998). 114 Our finding that heparin/heparan sulfate binding activity o f the N-terminal proIAPP peptide is increased at p H 5.5 raises the possibility that alterations in the local p H , for example in acidic intracellular compartments, might impact proIAPP binding to heparan sulfate proteoglycans and subsequently amyloidogenesis. Alternatively, it cannot be ruled out that such interactions might play a normal physiological role, for example in (pro)IAPP trafficking and/or processing, although it is unknown whether heparan sulfate proteoglycans are a component o f the acidic beta cell secretory granules in which (pro)IAPP resides. Heparan sulfate proteoglycans have been shown to be secreted via the constitutive pathway in other secretory cells (Passafaro et al. 1996) and it was recently found that immature (neonatal) rat beta cells secrete a significant proportion of (pro)IAPP immunoreactivity by the constitutive secretory pathway (Verchere et al. 2000). The possible significance o f the increased heparin binding of proIAPP at acidic p H in both amyloid pathogenesis and in normal physiology may therefore be worthy of further investigation. The affinity of the N-terminal proIAPP peptide for another highly sulfated glycosaminoglycan, chondroitin sulfate, was much less than that observed for heparan sulfate. The weak interaction between chondroitin sulfate and the N-terminal proIAPP peptide likely does, however, involve the K 1 0 R H K 1 2 sequence in the N-terminal cleavage site, since substitution of two of these basic residues with alanines resulted in total loss o f binding. Chondroitin sulfate has not been shown to be a component of islet amyloid although it may 115 be a component of amyloid deposits in experimental murine A A amyloidosis (Inoue and Kisi levsky 1996) and Alzheimer's disease (DeWitt et al. 1993). This glycosaminoglycan has also been shown to bind to human IAPP-derived fibrils in vitro, albeit less strongly than does heparan sulfate (Castillo et al. 1998). We speculate that the spacing o f the sulfate groups in heparan sulfate and heparin are more appropriate than that o f chondroitin sulfate for their interaction with proIAPP, as has been recently suggested for interaction o f S A A , another amyloidogenic precursor, with glycosaminoglycans (Ancsin and Kisi levsky 1999). Altered proteolytic processing of precursors to produce a more amyloidogenic molecule may be a general mechanism underlying amyloid deposition in several different amyloidoses. This idea was first suggested by the work of Glenner et al. (1971) on A L amyloidosis, but has since been implicated in the mechanism of amyloid formation in Alzheimer's disease (Sisodia et al. 1990; Kisi levsky and Fraser 1997; Gervais et al. 1999) and recently in familial British dementia ( K i m et al. 1999). In the case of islet amyloid, we propose that impaired proteolytic processing o f proIAPP by beta cells may result in disproportionate secretion of forms of (pro)IAPP with high affinity for heparan sulfate proteoglycans present on islet cell basement membranes. Once exocytosed from the beta cell, these molecules may bind to perlecan on the basement membranes of beta cells or islet vascular endothelial cells, preventing their entry into islet capillaries. Indeed, ultrastructural evidence from a transgenic mouse model of islet amyloid formation suggests that amyloid fibrils first accumulate 116 extracellularly between islet beta cells and blood vessels (Verchere et al. 1996). Interaction with the sulfated glycosaminoglycan side chains might then induce conformational changes in proIAPP that favor beta sheet formation, enhancing its tendency to aggregate. The local accumulation of proIAPP bound to perlecan might form a nidus for amyloid formation to which other amyloidogenic forms of IAPP including the major secreted form, I A P P , could be incorporated following their secretion from neighboring beta cells. In non-diabetic patients (in which islet amyloid is not usually observed) (Clark et al. 1990), proIAPP processing would be expected to be nearly complete (based on proinsulin; (Kahn and Halban 1997)), resulting in loss of the proIAPP heparin-binding domain prior to exocytosis from the beta cell. Interestingly, the heparin binding activity of proIAPP that we identified was observed only in the N-terminal and not the C-terminal region of the peptide. Immunoreactivity for the N-terminal flanking region of proIAPP, but not the C-terminal region, is present in islet amyloid in type 2 diabetic human pancreas (Westermark et al. 1989), raising the possibility that a partially processed, N-terminally extended proIAPP conversion intermediate may be an important molecule in islet amyloid formation. Whether partially processed proIAPP is secreted in excessive amounts in type 2 diabetes is unknown, however beta cells o f patients with type 2 diabetes have disproportionately elevated secretion of both proinsulin and a partially processed proinsulin conversion intermediate, des-31,32 proinsulin (Kahn and Halban 1997). This intermediate is the result of the cleavage by PC3 (see F i g . l ) (Bailyes et al. 1992; Neerman-Arbez et al. 1993), after which PC2 acts to produce insulin and C-peptide 117 (Bennett et al. 1992). Prolonged culture in high glucose causes marked accumulation o f the N-terminally extended proIAPP conversion intermediate in human islets (Hou et al. 1999) as well as rapid islet amyloid formation in islets from transgenic mice expressing amyloidogenic human IAPP (de Koning et al. 1994). It is believed that proIAPP may be cleaved by prohormone convertases in a similar order to proinsulin: PC3 may act first at the C-terminal cleavage site (Marzban et al. 2002), followed by cleavage by PC2 at the N -terminal cleavage site (Wang et al. 2001). Accumulation of the first conversion intermediate of proIAPP in type 2 diabetes (N-terminally extended proIAPP) would therefore be similar to the observed accumulation of des-31,32 proinsulin. If indeed hyperglycemia in type 2 diabetes is associated with excessive secretion o f the N -terminal proIAPP conversion intermediate and its subsequent deposition as islet amyloid, we hypothesize that binding of N-terminal proIAPP to basement membrane heparan sulfate proteoglycans may be an important pathogenic event in this pathway. Interference with this interaction could be a potential target for therapeutics to inhibit the formation of islet amyloid. 118 4.2 Residues in IAPP that Mediate Heparin Binding and Heparin Stimulation of Fibril Formation Proteins are known to interact with heparan sulfate by virtue of clusters o f basic residues. I A P P fibrils bind to heparin and heparan sulfate, but I A P P monomers do not contain a classic heparin binding domain as proposed by Cardin and Weintraub (Cardin and Weintraub 1989). It has been proposed that heparin binding domains are formed from alignment o f basic residues on adjacent IAPP molecules within a fibril (Watson et al. 1997). In order to effectively target therapeutics to specific areas of the IAPP molecule, it is essential to know which residues are involved in binding to heparan sulfate and, and those which mediate their stimulatory effect on fibril formation. In this thesis, we demonstrate that lysine 1 and arginine 1 1 are both important for the stimulatory effect of heparin on IAPP fibril formation. In addition, we determined overall that residues outside of 20-29 are important in binding to heparin. Heparin augments the toxicity o f human IAPP to beta cells in culture, but has no effect on the toxicity o f truncated peptides lacking the first 10 or 11 residues. The basic residues found to be important for heparin stimulation of I A P P fibrillogenesis are arginine at position 11 (for heparin stimulation of the initial rate of fibril formation) and lysine at position 1 (for heparin augmentation of the final amount of fibrils formed). 119 Protonation o f the basic residue at position 18 may be critical to confer the ability of monomeric IAPP to bind heparin. Rat IAPP, which possesses arginine at this position, binds to heparin, but a synthetic peptide used in our affinity column assays with similar distribution of basic amino acids, but with a histidine at position 18, did not bind to either heparin or heparan sulfate. Others have demonstrated a role for histidine residues in the interaction o f G A G s and amyloidogenic proteins. For example, histidine residues are also thought to be important in the interaction of A B with heparin and heparan sulfate, as A B binds significantly greater amounts o f heparan sulfate at p H 5 than it does at p H 7.5 (Gupta-Bansal et al. 1995) (Brunden et al. 1993). These residues would bear a positive charge at p H 5.5, the p H o f the beta cell secretory granule. A n interaction contingent on protonation of histidines might conceivably occur in the beta cell secretory granule i f heparan sulfate proteoglycans are present. Beta cells have been shown to secrete proteoglycans which bind to I A P P , indicating that both are present in secretory granules and may interact (Potter-Perigo et al. 2003). We found that heparin stimulates not only the fibrillogenesis but also the toxicity of human I A P P to INS-1 beta cells. These findings are in accordance with those of McLaur in et al (1999) who found that the presence of a marine sponge heparan sulfate proteoglycan enhanced A B toxicity to neuronal cells, although not as marked as we observed. These authors suggested that the marine sponge proteoglycan may increase the number o f small aggregates or oligomers which may be responsible for the toxic effect o f A B . The authors 120 used E M to examine the morphology of the fibrils in the presence or absence o f the marine sponge proteoglycan, which showed the increased population of small fibers and aggregates. Pollack et al. found an opposite effect o f heparin and heparan sulfate on A B toxicity, in that the presence of these G A G s had an inhibitory effect on A B toxicity (Pollack et al. 1995). McLaur in et al. (1999) postulate that the effect of heparan sulfate is dependent on the manner in which it stimulates fibril formation. For example, i f heparin increases the lateral aggregation of the peptide, the toxicity is decreased. Another possible mechanism through which heparin has the potential to reduce toxicity is to decrease the amount o f time in which the amyloid precursor spends in the form of protofibrils. Future studies employing E M to observe the morphology of IAPP fibrils in our system would reveal whether the lifespan or population o f protofibrils is increased in our model. This possibility has been raised with respect to the design of inhibitors and therapeutics for amyloidosis, since compounds that inhibit amyloid fibril formation may have the unwanted effect o f prolonging the life of protofibrils thereby increasing toxicity (Lansbury 1999; Conway et al. 2000; Goldberg and Lansbury 2000). Heparin might also prevent toxicity by binding to fibrils, i f heparan sulfate moieties on the cell surface mediate toxicity, as has been hypothesized (Pollack et al. 1995; Castillo et al. 1998; McLaur in et al. 1999). Our data that human I A P P 20-29 is not cytotoxic are in agreement with those of others (May et al. 1993; Pike et al. 1993; Tenidis et al. 2000) that the 20-29 fragment has much reduced 121 toxicity compared to IAPP 1-37. Thioflavin T did not bind to human I A P P 20-29 (data not shown), eliminating our ability to use this dye to test the stimulatory activity o f heparin on fibril formation by this peptide. Since we found human IAPP 20-29 did not bind to heparin, however, we conclude that heparin most probably does not have a stimulatory effect on fibril formation by this peptide. Residues outside o f the 20-29 region, in particular lysine 1 and arginine 1 1, are important for heparin stimulation of fibril formation. In addition, the residues 30-37 (Nilsson and Raleigh 1999) and 8-20 (Jaikaran et al. 2001) in IAPP have been identified as capable o f forming amyloid fibrils. The thioflavin T binding assay that we used to examine rates of fibril formation by I A P P 1 -37, 11-37, and 12-37 is dependent on the binding of thioflavin T to fibrils and changing fluorescence emission wavelength. A weakness of this method is that components of the reaction being tested may interfere with detection of the change in fluorescence. For example, in our experiments we found that the antibiotic rifampicin completely ablated the thioflavin T signal without inhibiting fibril formation. We presume therefore that rifampicin displaces the thioflavin T dye from the fibrils, thereby giving a false negative signal. Our data on the stimulation of human IAPP 11-37 and 12-37 fibrillogenesis by heparin is called into question by Cohlberg et al. (Cohlberg et al. 2002), who suggest that the presence of 122 heparin in fibrils could increase the Thioflavin T signal either by increasing the amount o f dye bound or by a local effect on the Thioflavin T fluorescence. Confirmation of these findings w i l l require examination using E M , or sedimentation assays. L o w molecular weight polysulfated compounds have been used effectively in vivo to inhibit amyloid formation in a murine model o f cerebral amyloid deposition similar to Alzheimer 's Disease (Kisilevsky et al. 1995). It has been cautioned that the use o f such inhibitors may be restricted to certain amyloids, and may not have the same effect on others (Castillo et al. 1999). Similar candidate inhibitors may actually have the opposite effect (Castillo et al. 1999), i.e. to accelerate fibril formation. Monosaccharide G A G subunits have the ability to either stimulate A B fibril formation, or to encourage the accumulation of protofibrils (Fraser et al. 2001) further cautionary evidence that potential inhibitors of amyloid formation must be carefully screened (Lansbury 1999; Conway et al. 2000; Goldberg and Lansbury 2000). 4.3 Increased FibriUogenicity and Beta Cell Toxicity of S20G Mutant IAPP The increased severity and early onset of type 2 diabetes experienced by a small number o f Japanese patients bearing the S20G mutation in IAPP has been hypothesized to be influenced by increased fibrillogenicity and cytotoxicity of this molecule (Sakagashira et al. 2000; M a et al 2001). Previous work has demonstrated that C O S cells engineered to produce 123 S20G hIAPP are significantly less viable than those engineered to produce wild-type h IAPP (Sakagashira et al. 2000). We show that synthetic S20G hIAPP is more fibrillogenic and more toxic to beta cells than wild-type h i A P P . In these studies, exposure of INS-1 beta cells to 20 u M extracellular S20G hIAPP led to decreased INS-1 cell viability, whereas wild-type hIAPP caused no decrease in cell viability at the same concentration. Although apoptotic morphology was observed in the latter, it was less marked than that observed in the S20G-treated cells. When fibril formation was increased, either by increasing peptide concentration (40 uM) or by the addition o f heparin to the 20 u M treatment, both peptides were similarly cytotoxic. The WST-1 assay used in the current studies does not directly measure apoptosis, however concurrent work in our lab ( X u et al. 2001) has shown that using this model, IAPP fibril-induced beta cell death involves caspase activation, indicative of apoptosis. B y both thioflavin T and E M fibrillogenesis assays, the seeding event for S20G fibrillogenesis occurs significantly before that for wild-type hIAPP. Our data support the findings o f Sakagashira et al (2000) and M a et al (2001) who similarly showed increased fibrillogenicity of S20G hIAPP compared to wild-type hIAPP. However we have extended the observations to demonstrate by transmission E M a more rapid appearance o f fibrils with S20G hIAPP at a time when wild-type hIAPP was still in an amorphous aggregate. 124 In such studies with highly amyloidogenic peptides, the source, solubility, and purity o f the compounds are critical. Unlike previous studies (Ma et al. 2001), our peptides were synthesized in parallel to eliminate the possibility o f batch-to-batch variation. In addition, the use o f exogenous peptide in the toxicity assay allowed more precise control o f the amount o f peptide the cells were exposed to. The serine to glycine substitution at position 20 has been hypothesized to influence the folding o f hIAPP at a potential beta bend of the molecule (Jaikaran and Clark, 2001). Substitution of a glycine residue could affect formation o f intramolecular hydrogen bonds, creating a tighter bend and increased susceptibility to formation of an intramolecular beta sheet. The increased fibrillogenicity of S20G hIAPP is therefore l ikely to be a predisposing factor to amyloid formation. Other instances exist in which mutations in a gene encoding the major protein component o f a particular amyloid are thought to hasten amyloid deposition in vivo. For example, two mutant forms of alpha synuclein, the A53T and A30P mutants, are associated with early onset Parkinson's disease (Polymeropoulos et al. 1997; Kruger et al. 1998). These proteins display altered kinetics of fibrillogenesis in vitro (Conway et al. 1998; 2000). The former exhibits accelerated fibrillogenesis compared to wild-type (Conway et al. 1998); importantly, 125 both display an acceleration of oligomerization compared to wild-type alpha synuclein (Conway et al. 2000). A second such example is found in a variant, early age o f onset form of Alzheimer 's disease, termed 'Dutch -type hereditary cerebral hemorrhage with amyloidosis'. This disease is associated with a glutamine for glutamic acid substitution at position 22 of A B (Levy et al. 1990). Peptides homologous to A B bearing this substitution have considerably higher beta sheet content (Fabian et al. 1993) and accelerated amyloid fibril formation in vitro (Wisniewski et al. 1991). Although the S20G mutation similarly may increase the likelihood of islet amyloid formation, this alteration alone can not be responsible for the development o f islet amyloid or diabetes. Evidence that patients with other risk factors for type 2 diabetes are more l ikely to develop islet amyloid and beta cell dysfunction (Seino 2001) is in accordance with clinical data that not all persons with the S20G mutation in IAPP develop diabetes (Chuang et al. 1998; Yamadae ta l . 1998). Our use of beta cells exposed to extracellular amyloid fibrils more closely mimics the occurrence o f islet amyloid in vivo than previous toxicity studies using S20G hIAPP (Sakagashira et al. 2000). Although the concentrations used in our fibrillogenesis studies are much higher than the picomolar amounts of IAPP found in the circulation (Percy et al. 1996), they may reflect the concentration of IAPP in the interstitial space adjacent to beta cells 126 following granule exocytosis. In situations of increased secretory demand, such as type 2 diabetes, the local concentration of IAPP outside the beta cell may be extremely high. Recent work (Potter-Perigo et al. 2003) has shown that hIAPP binds to proteoglycans produced by beta cells. Heparan sulfate proteoglycans have the potential to attract I A P P molecules and thereby contribute to an increase in local concentration of IAPP (Park and Verchere 2001). Addit ion o f heparin to the containing culture medium increased the toxicity o f I A P P in our study. These data suggest that i f IAPP interacts with heparan sulfate proteoglycans in vivo, its toxicity may be enhanced. In conclusion, we have shown that the S20G mutant form of h IAPP is more cytotoxic to beta cells than the wild-type form of the peptide. The differences in toxicity between to the two forms of hIAPP are apparent only at lower concentrations and are masked when fibril formation is stimulated by the addition of heparin. We speculate that S20G hIAPP has a lower concentration threshold for fibril formation and production o f toxic molecular species. The S20G mutation in combination with other risk factors for type 2 diabetes may hasten the onset and accelerate the progression of the disease. 127 4.4 Future Studies We have identified novel domains in IAPP and its precursor molecule, proIAPP, that are important for binding to heparin, and for stimulation of fibrillogenesis by heparin. Interaction of these domains with heparan sulfate moieties in the extracellular matrix could lead to the retention of this molecule and the formation of a nidus for amyloidogenesis. The heparan sulfate binding domain that we have identified in the N-terminal cleavage site of proIAPP represents a candidate target for inhibition of islet amyloid formation. B y interfering with the interaction o f this cluster of basic charge with heparan sulfate in the extracellular matrix, one could conceivably prevent a major seeding event in islet amyloidogenesis. The monomeric N-terminal proIAPP fragment we used in our affinity column assays would therefore be an example of a candidate inhibitor. It has the potential to bind to heparan sulfate molecules in the extracellular matrix, preventing unprocessed, fibrillogenic forms (i.e. possessing the 20-29 beta sheet region) of proIAPP that have been secreted from the beta cells from doing so, thereby preventing a nidus from forming. Drugs which enhance processing of proIAPP in the beta cell might also have therapeutic potential. Such activity would facilitate the destruction of the heparin binding domain in the N-terminal region o f proIAPP before secretion, preventing an interaction of this cluster of basic residues with heparan sulfate in the extracellular matrix. 128 Perlecan has been proposed to play a role in facilitating the deposition o f amyloid in a number of disease states (Kisilevsky and Fraser 1997). The perlecan present in islet amyloid has been proposed to be produced locally and has the potential to interact with I A P P (Potter-Perigo et al. 2003). In future studies, the creation of transgenic mice expressing human IAPP , but lacking beta cell expression of perlecan would be of considerable use i n determining whether or not perlecan contributes to the initiation and formation o f islet amyloid. Using a thioflavin T binding assay, we showed that lysine 1 is important for heparin stimulation of fibrillogenesis. Lysine 1 is also likely important for heparin stimulation o f toxicity in vitro. Future studies with fragments of I A P P ranging from I A P P 2-37 to I A P P 10-37 would shed light on whether the lysine 1 is the key residue responsible for this difference in toxicity and fibrillogenesis. Electron microscopy would be useful in confirming fibrillogenesis studies using such fragments in the presence or absence o f heparin. 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