Elucidating the Effect of Myopathy-Causing Mutations and Second-Site Suppressors on Client Processing by J-Domain Proteins
Date of Award
Doctor of Philosophy (PhD)
Defects in protein quality control may lead to protein misfolding and aggregation often associated with protein conformational disorders such as Alzheimerճ Disease and Limb Girdle Muscular Dystrophy, among others. Molecular chaperones protect against protein misfolding and aggregation. A chaperone of interest is the ubiquitously expressed type II Hsp40 co-chaperone DNAJB6, which assists in protein folding and disaggregation. Mutations within the DNAJB6 G/F domain have been associated with the dominantly inherited disease Limb-Girdle Muscular Dystrophy type 1D (LGMD1D), now referred to as LGMDD1. Our collaborators recently discovered novel LGMDD1-associated mutations in the J-domain of DNAJB6. In the enclosed body of work, we used yeast as a model to perform phenotypic, biochemical and functional assays to elucidate the effect of the J-domain mutations on canonical chaperone function with the goal of beginning to understand how mutations in this domain may affect LGMDD1 pathogenesis. Moreover, we have identified second-site suppressors that rescue a viability defect in yeast that is associated with a myopathy-causing mutation. With this work we have begun to assess the ways in which second-site suppressors may be therapeutic for inherited myopathies such as LGMDD1. The heat shock response is a highly conserved program from yeast to mammals, thus, we have used a yeast model system to study disease-causing mutations. The yeast type II Hsp40 co-chaperone, Sis1, is homologous to DNAJB6 and has an important role in yeast for the propagation of two yeast prions, [RNQ+] and [PSI+]. The True lab has previously published work showing that when LGMDD1-associated mutations in the G/F domain are present in Sis1, its client processing function is altered. Since novel J-domain mutations have yet to be characterized, we assessed the effect of these mutations using our yeast model. Here, we provide evidence that novel variants in the Hsp40 J-domain lead to aberrant chaperone function and altered protein homeostasis in a client and conformer specific manner. Moreover, we identified a novel client-dependent viability defect when one of the J-domain mutants is expressed. This is the first time, to our knowledge, that steady-state levels of a mutated chaperone have been shown to be dependent on stabilization by a client. Lastly, we have identified and began to characterize second-site suppressors which may lead future studies into using second-site suppressors for therapeutic purposes. This body of work enables direct comparisons between disease-associated mutants in different domains so that we may begin to not only understand how LGMDD1 mutants could impact disease severity and pathogenesis, but also whether similar therapeutic avenues could be explored to treat patients with different mutations in the future.
Chair and Committee
Heather L. True
James Skeath, Yuna Ayala, Conrad Weihl, Hani Zaher,
Pullen, Melanie Y., "Elucidating the Effect of Myopathy-Causing Mutations and Second-Site Suppressors on Client Processing by J-Domain Proteins" (2020). Arts & Sciences Electronic Theses and Dissertations. 2233.