Functional specification of HSP70 by J-domain protein co-chaperones

• Applicant: Professor Dr. Bernd Bukau
• Subject Area: Biochemistry ; Cell Biology
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 504257241

Members of the heat shock protein 70 (HSP70) family are ATP-dependent molecular chaperones that act as central hub of cellular protein homeostasis. They ensure proteins attain and maintain their correct three-dimensional fold, become disassembled when aggregated, or cleared by cellular degradation pathways. ATP hydrolysis promotes substrate trapping by Hsp70s and therefore is the critical step that initiates the functional chaperone cycle. It is allosterically regulated both by substrate binding and the action of co-chaperones of the J-domain protein (JDP) family, also referred to as Hsp40. JDPs are an extended family of proteins (>40 members in human cells) that contain a conserved J-domain which promotes ATP hydrolysis in the HSP70 nucleotide binding domain via distal interactions with the flexible linker connecting this domain with the substrate binding domain. Class A and B JDP members share a common architecture with the N-terminal J-domain, a disordered GF rich linker, 2 C-terminal substrate binding domains (CTDs) and a dimerisation domain. The key differentiating feature is the composition of the GF linker, which in the case of class A JDPs is extended by a zinc finger domain. Despite their high structural similarities, class A and class B JDPs have been implicated in distinct cellular activities, with class A JDPS primarily interacting with monomeric misfolded proteins or small oligomeric species, while class B proteins of both type I (DNAJB1/4) and type II (DNAJB6/8) preferentially interact with oligomeric species and larger aggregates. Despite three decades of research into the HSP70 chaperone machinery, the roles and diversity within the JDP co-chaperone family remains largely unexplored on a mechanistic level. In recent years, JDPs have come to the foreground as potential therapeutic targets for the selective tuning of specific HSP70 activities without perturbing (other) essential housekeeping activities. We therefore aim to perform a comprehensive analysis of JDP-mediated recruitment of the human HSP70 chaperone to cellular substrates to answer the fundamental question: how do canonical class A and class B JDPs get HSP70 to act in so many different folding processes in the human cell? Our specific aims are: 1/ Describe biochemical properties that drive substrate specificity between JDP classes and member within these classes, 2/ Identify the trigger for activation and differences in regulatory switches within the class B JDP family, 3/ Characterise how different regulatory mechanisms determine the assembly of HSP70 complexes that perform different cellular activities and, 4/ Probe the interplay between JDP classes in tuning HSP70 function.Overall, the proposed project will generate complementary datasets that combined will build a comprehensive mechanistic understanding of JDP mediated stimulation of HSP70 chaperone activity.

• DFG Programme: Research Grants