Autophagic Clearance of Proteasomes and CDC48 as Models for Amyloidogenic Protein Quality Control.

点击翻译按钮获取中文摘要

PROJECT SUMMARY/ABSTRACT Background − Maintenance of proteostasis is central to cellular fitness and is achieved through sophisticated protein quality control (PQC) pathways that remove dysfunctional and unwanted proteins and protein complexes that become cytotoxic if allowed to accumulate and condense. In fact, protein aggregation encouraged by PQC defects is a hallmark of aging, cancer, and numerous human ‘aggregation- prone’ pathologies, including amyotrophic lateral sclerosis, Alzheimer’s, Parkinson’s and Huntington’s diseases, and related multisystem proteinopathies. Consequently, full understandings of PQC could offer new strategies to mitigate protein aggregation and subsequent proteotoxic stress. Previous Work − We discovered mechanistically conserved PQC routes that direct the autophagic elimination of inactive proteasomes and the CDC48 segregase (p97/VCP in humans), which offer experimentally robust models for describing defective protein clearance. Notably, turnover of both protein complexes shares features with amyloidogenic protein removal, including sequestration, ubiquitylation, and subsequent recognition by dedicated autophagic receptors, which for proteasomes also requires a trio of ubiquitin ligases that likely work in concert to assemble appropriate poly-Ub chain topologies. Project Aims − This project proposes to describe in detail the autophagic clearance of proteasomes and CDC48 in both yeast and Arabidopsis, with the goal of discovering aspects central to autophagic PQC. For proteasomes, we will: (i) examine, using genetics, fluorescence microscopy, interaction studies and ubiquitin linkage mapping, where, when, and how the ligases San1, Rsp5 and Hul5 coordinately contribute to dysfunctional proteasome ubiquitylation; (ii) identify ubiquitylation linkages needed to generate autophagy competent substrates; and (iii) deduce how Hsp42-mediated sequestration into cytoplasmic membrane-less aggresomes, versus condensation into proteasome storage granules, contributes to the process. Likewise, studies on CDC48 turnover will confirm that ubiquitylation is a key signal, followed by the identification of relevant ubiquitin ligases and understanding of how CDC48 sequestration contributes to its turnover. Moreover, we will test our hypothesis that the autophagic routes used to clear dysfunctional proteasomes and CDC48 also eliminate amyloidogenic proteins that are at the heart of numerous aggregation-prone pathologies. Finally, we will further define and expand upon a new class of autophagic receptors/adaptors that use a novel interface to dock with ATG8 (LC3 in humans) lining autophagic vesicles, thus helping to increase the known reach of selective autophagy. Outcomes − Through this cumulative research, we hope to define autophagic routes relevant to aggregation-associated PQC, which will shed light on the roles of ubiquitylation, biomolecular condensation, and autophagy in mitigating proteotoxic stress and ultimately inform upon new therapeutic strategies for various amyloidogenic pathologies.