Heat shock proteins (HSPs) or protein chaperones are essential to maintain cellular protein homeostasis. Mutations of proteins within this network can lead to degenerative diseases including muscular dystrophy. We have now identified mutations in two domains of an HSP40 co-chaperone, DNAJB6, in an autosomal dominantly inherited Limb Girdle Muscular Dystrophy (LGMDD1). Our central hypothesis is that LGMDD1 mutations alter the conformation of DNAJB6 such that the interface between these two domains is disrupted, leading to myofibril disorganization and muscle dysfunction. Based on our results, we further hypothesize that all LGMDD1 mutations in DNAJB6 can be rescued by perturbation of Hsp70 interaction or activity. Thus, our approach is to employ all of the tools we have developed to test these hypotheses. In order to tackle this challenging problem, we will explore DNAJB6 chaperone function and dysfunction utilizing multiple systems and the expertise of two PIs. Aim 1 will explore whether manipulating DNAJB6:HSP70 interactions rescues LGMDD1 phenotypes in mouse models and human cells. Aim 2 will determine the effect of newly identified LGMDD1-associated DNAJB6 J- domain mutations on chaperone activity in cellular models. Aim 3 will determine the effect of LGMDD1 mutations on chaperone and co-chaperone function in vivo and in vitro. Our preliminary data suggest that we have identified additional therapeutic targets that may be better tolerated in patients than global inhibitors of Hsp70. The overarching goal of this work is to leverage our transdisciplinary success to develop therapeutic intervention for patients with LGMDD1.