The Role of J-Domain Protein Interactions with Molecular Chaperone Hsp70 - Abstract Cellular proteins need to fold correctly to obtain a specific three-dimensional structure and maintain it despite internal and external conformational insults. The importance of maintaining protein structure is emphasized by numerous human disorders such as neurodegenerative Alzheimer's, Parkinson's, and Huntington's diseases, or prion disease, all considered protein-misfolding diseases. In all cases, proteins associated with the disease aggregate in a highly organized manner, forming large insoluble amyloid-like fibrils. The natural defense of the cell against protein misfolding and aggregation is a network of molecular chaperones, with the Hsp70s serving as a central hub in this network. Hsp70 chaperones recognize and bind short primary hydrophobic amino acid stretches, existing in virtually all cellular proteins, which become exposed during protein synthesis, folding, trafficking, membrane translocation, as well as disaggregation and refolding of misfolded proteins, including fragmentation of amyloid fibrils. This versatile activity of Hsp70 is regulated and targeted towards specific processes by a cohort of diverse J-domain proteins (JDPs), obligatory cochaperone partners of Hsp70. The paradigm is that various JDPs can recruit the same type of Hsp70 via direct interaction of their common J- domain, and at the same time, the diversity of other domains unrelated to cooperation with Hsp70 defines the specificity of individual JDPs. This model of JDP-driven specialization of JDP/Hsp70 systems extends Hsp70 biological roles beyond protein quality control to such essential processes as transcriptional regulation, ribosome biogenesis, mRNA splicing, or mitochondrial iron-sulfur cluster biogenesis. However, how JDP/Hsp70 systems actually perform so many diverse functions is not yet understood. This proposal examines a conceptually novel hypothesis that the specificity of JDP/Hsp70 systems can be determined by how JDP, through interactions other than J-domain, modulate Hsp70 activity directly rather than by their Hsp70-independent features. More specifically, we aim to investigate molecular and mechanistic details of a potentially new interaction between the J-domain adjacent region, glycine-rich region, and Hsp70 that emerged from our recent comparative NMR studies of two JDP classes. Furthermore, we will determine the arrangement and orchestration of all known molecular interactions between Hsp70 and the native dimeric form of JDP. Our chosen JDP/Hsp70 system is essential in vivo and specialized in amyloid fibril fragmentation and prion propagation, thus of significant medical relevance. Our experimental framework uses both yeast and human chaperone systems as well as utilizing a combination of in vitro and in vivo approaches for robust and comprehensive analysis and interpretations. Revealed molecular details and mechanistic insights will expand our understanding of JDP/Hsp70 systems, supporting ongoing efforts to develop chaperone-targeted therapies against misfolded diseases.
