Maintenance of proteostasis is central to cellular fitness and is achieved through sophisticated protein quality control (PQC) pathways that remove dysfunctional or unwanted proteins and protein complexes that become cytotoxic if allowed to accumulate. In fact, protein aggregation encouraged by defects in PQC is a hallmark of aging, cancer, and numerous human ?aggregation-prone? pathologies, including amyotrophic lateral sclerosis, Alzheimer?s, Parkinson?s, Huntington?s, and prion-mediated diseases. Consequently, fully understanding PQC could offer new strategies to mitigate protein aggregation and subsequent proteotoxic stress, especially as they relate to these disease states. Recently, ancient and mechanistically conserved PQC pathways were discovered that direct the autophagic elimination of unwanted or inactive proteasomes and ribosomes, termed proteaphagy and ribophagy, respectively. Notably, a selective proteaphagic route triggered by inhibition shares features with amyloidogenic protein removal, including sequestration by the Hsp42 chaperone, ubiquitylation, and subsequent recognition by the autophagic receptors Cue5 or RPN10, suggesting that inhibitor-induced proteaphagy, and likely ribophagy, offer tractable models to dissect features underpinning PQC and protein aggregate clearance. This project aims to characterize proteaphagy and ribophagy induced by inhibition in both Arabidopsis and yeast with the goal of discovering components central to autophagic PQC. Specifically, we will define how, where, and why the yeast ubiquitin (Ub) ligases Hul5, Rsp5, and San1 (and their Arabidopsis orthologs) recognize and ubiquitylate dysfunctional proteasomes, and how this modification triggers autophagy via the Ub-binding Cue5/RPN10 autophagic receptors, using facile confocal fluorescence microscopic and GFP-fusion cleavage assays together with co-localization studies. A special focus will be on the mechanism(s) involving Hsp42 that coalesce inactive proteasomes into large cytoplasmic aggregates similar to those observed for various aggregation-prone proteins. We will determine which proteasome subunits become modified, where the Ubs are attached, which types of poly-Ub chains are assembled, and identify other factors/post- translational modifications that might be important, by mass spectrometric analysis of proteasomes purified before and after inhibition. Using similar methodologies, we will examine how ribosomes are degraded by autophagy after exposure to translation inhibitors, and examine the role(s) of ubiquitylation and the corresponding ligases, Hsp42, and receptors like Cue5/RPN10 in this clearance. We will also confirm proteaphagy in human cells and the involvement of the Cue5 ortholog Tollip in this process, and determine if the pathway(s) used for proteophagy and ribophagy also help clear amyloidogenic proteins. Finally, we will study a new class of autophagic receptors related to RPN10 that might substantially expand the influence of selective autophagy in plants, yeast, and humans. Through this cumulative research, we will define the autophagic routes for proteasome and ribosome clearance that should be relevant to aggregation-associated PQC, and thus will shed light on the roles of various subcellular protein deposits and their effectors in mitigating proteotoxic stress related to numerous aggregation-prone pathologies.
Part of the NIH mission is to seek fundamental knowledge about the nature and behavior of living systems, and to apply that knowledge to enhance human health and lengthen life. This proposed research is relevant to the NIH mission because it seeks to understand novel yet conserved pathways of protein degradation in plants and yeast that can be developed as models to study ageing and provide information relevant to a wide range of human aggregation-prone pathologies, including Alzheimer?s, amyotrophic lateral sclerosis, Huntington?s, Parkinson?s, and prion-mediated diseases. As such, information obtained from our studies of both proteaphagy and ribophagy should be directly relevant for efforts to improve human health and understand the process of ageing.
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