Deciphering Novel Principles of Rapid Proteostatic Control and Innovating Spatiotemporal Lysosomal Tools

PROJECT ABSTRACT Proteome integrity is maintained by a complex network that regulates protein synthesis, folding, transport, and degradation. Lysosomes are the catabolic center of a cell and central to maintaining proteome homeostasis by preventing, detecting, and removing abnormal proteins. Major knowledge gaps remain in the regulation, structural components, and substrate specificities of lysosomal substrates. Intracellular proteolysis through the ubiquitin-proteasome system has been the most well-characterized eukaryotic proteolytic pathway as the protein targeting by ubiquitin and the amino acid sequences recognized by E3 ubiquitin ligases are well-defined. In contrast, a major obstacle in understanding lysosomal processes is the incomplete knowledge of protein modifications that enable lysosomal trafficking mechanisms. Our work identified that arginine methylation leads to protein delivery into lysosomes for degradation. We showed that rapid methyl-driven delivery was essential for removing enzymes from the cytosol to promote growth and proliferation. The proposed studies examine the central hypothesis that methyl-driven lysosomal proteolysis is a widespread process that enables natural protein turnover during homeostasis and rapid protein remodeling in response to external stimuli. We address this hypothesis in three areas of research. Area 1 defines novel protein substrates and the peptide motifs required for lysosomal delivery. Area 2 determines the functional impact of rapid methyl-driven delivery as a control mechanism for fundamental cellular metabolic pathways. Area 3 leverages naturally-occurring lysosomal protein signals to develop tools for researchers to rapidly decrease protein levels in endogenous living systems. We test the conceptually novel model that selective lysosomal proteolysis is central for regulating cytosolic, short-lived proteins that were previously thought to be degraded in proteasomes. We anticipate use of our publicly available database of novel methyl-degraded lysosomal proteins will provide an essential resource for the fields studying protein control. We develop technically innovative tools to gain new mechanistic insight into lysosomal biology for the present studies while also providing a tool for the broader research community that significantly improves current strategies for endogenous protein depletion. 11