PROJECT SUMMARY/ABSTRACT Muscle diseases caused by mutations in genes that encode for (co-)chaperones, called chaperonopathies, are characterized by muscle weakness, degeneration, and the accumulation of protein aggregates. These diseases currently lack a cure, highlighting an urgent need for a better understanding of the underlying mechanisms. Protein chaperones are needed to recognize misfolded proteins and facilitate their proper folding. The recognized misfolded proteins are termed “clients.” Classically, DNAJ proteins are thought to diversify the function of Hsp70 by determining client specificity, but little is known about how DNAJ proteins bind and select clients for Hsp70 and how dysfunction of DNJ proteins causes chaperonopathies in muscle. Recently, mutations in DNAJB4 were reported to cause a chaperonopathy with similar myopathology to the known chaperonopathy caused by DNAJB6 mutations although their phenotypes are distinctive. Our central hypothesis is that distinct client proteins of DNAJ proteins and chaperone-chaperone availability contribute to the phenotypic differences and selective muscle vulnerability in chaperonopathies. This study will experimentally evaluate client proteins of DNAJ proteins in muscle, the combinatory effect of DNAJ proteins, and skeletal muscle-specific tissue vulnerability in chaperonopathies, using recently developed innovations (e.g., proximity labeling approach, proteomics, RNAi, RNA-seq, and small animal imaging). In Aim 1, I will Aim 1 test the hypothesis that different DNAJ proteins have common and distinct client proteins in skeletal muscle. To identify client proteins, I will apply proteomics in TurboID proximity labeling method in cultured cells and model mice, DNAJ knockout myotubes, and laser microdissections of inclusions from patient muscle with DNAJB4, DNAJB5, and DNAJB6 mutations. In Aim 2, I will test the hypothesis that DNAJ-DNAJ interactions play a synergetic role in muscle maintenance. I will employ a knockdown approach to investigate how this network dysfunction impacts protein homeostasis, using myoblasts and proteostatic stress (e.g., drug, heat shock). Finally, in Aim 3, I will test the hypothesis that the skeletal muscle-specific tissue vulnerability is caused by a disruption of proteostasis, where key molecules are compromised. Using chaperonopathy mouse models, I will thoroughly analyze the myopathology and proteostatic capacity of different skeletal muscle groups, and perform single nucleus RNA sequencing on the most vulnerable muscle groups in model mice to identify define vulnerable myofiber subpopulations in chaperonopathies. The proposed study will address an unmet medical need, providing insight into disease mechanisms and thereby potentially may serve as a basis for the development of novel treatment for chaperonopathies and a broad range of diseases related to protein misfolding.