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Kumar, Arun

University of British Columbia

Proteostasis is critical for cell survival. Exposure to stressors such as DNA damage, heat, mutations, or ageing can accumulate proteins in non-native conformations. The accumulation of these misfolded proteins, through either gain- or loss-of-function, disrupts vital cellular processes. This is notably evident in neurodegenerative diseases such as Alzheimer’s, and Parkinson's which are linked to protein aggregation pathology. Therefore, cells strive to maintain a healthy proteome which is achieved through a complex network of protein quality control (PQC) circuits. Traditionally, PQC was thought to rely on two major pathways: molecular chaperones for refolding and proteolytic systems for degradation. However, this perspective has been challenged with the discovery of protein sequestration. Under severe stress conditions that overwhelm PQC systems, misfolded proteins are spatially sequestered to specialized and distinct membrane-less inclusions within the cell. This spatial compartmentalization represents a crucial aspect of PQC, facilitating the storage of disassembled or non-native proteins until they can be either refolded or degraded in a regulated manner. The model organism Saccharomyces cerevisiae or budding yeast has three well-established sequestration sites – the Insoluble PrOtein Deposit (IPOD), the JUxtaNuclear Quality control (JUNQ) site and the IntraNuclear Quality control (INQ) site. Although substantial efforts have contributed to identifying key proteins involved in the formation and dissolution of these sequestration sites, our understanding of the regulation and dynamics of INQ remains incomplete. In my thesis, I provide a comprehensive analysis of all proteins that have been found at INQ. Furthermore, I explore different pathways and stressors to expand our knowledge of the factors governing INQ formation. To do so, I first characterize Rpd3, a histone deacetylase, as a novel INQ marker and use its localization as a proxy for INQ levels. Through microscopy and genetic approaches, I demonstrate that INQ formation of Rpd3 is a general response to DNA damage. Furthermore, I explore the role of the post-translational modification (PTM) SUMOylation at INQ and document the DNA damage induced SUMOylation of chaperone Btn2 and Hsp42. Additionally, I engineer a non-SUMOylatable Btn2 and dissect its role in INQ clearance, placing it at the nexus of protein degradation and refolding at INQ.

Text

2024-01-11

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/87208

Medical Genetics

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/