Processing of chaperone clients in C. elegans

• Applicant: Privatdozent Dr. Klaus Richter
• Subject Area: Biochemistry
• Term: from 2007 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 48759762

In this proposal we will investigate the biochemical mechanisms of client processing by the Hsp90 and Hsc70 chaperone systems and their relationship to specific phenotypes in Caenorhabditis elegans. We will address the processing of different kinases by the nematode CDC-37/Hsp90 chaperone complex to uncover interaction principles, which are common to all client kinases. We recorded interaction with nucleotide-free sB-Raf and now aim at defining to what extent nucleotide-binding also modulates chaperone interaction with other kinases. The interaction with the DYRK-homolog nematode kinase MBK-2 and phenotypic variants thereof will be used to understand how single-point variations in clients control the interaction with CDC-37/CeHsp90 and to specify the general in vivo requirements for CDC-37. The latter will be addressed by modifying specific parts of this cofactor by CRISPR/Cas9 mediated genome editing. We further will study the functionality of the Hsp90-associated phosphatase PPH-5 and the regulation of its dephosphorylating activity towards different clients. PPH-5 shows strong dependency on Hsp90 during formation of client complexes with GR and CDC-37 and during dephosphorylation of clients. We will test, whether ternary complexes are requirements also for other clients of PPH-5/CeHsp90. To understand PPH-5 in vivo activities we will investigate variations in nematode PPH-5, which produce phenotypes during cell division and evaluate new nematode strains with disrupted PPH-5 functionalities. For the client-containing ternary complexes of CDC-37/CeHsp90 and PPH-5/CeHsp90, we will also work to resolve structural features to better understand the organization of these complexes and their handling of client proteins. As a third chaperone-cofactor system, which produces strong phenotypes in C. elegans, we study the interaction between DNJ-13/Hsp40, UNC-23 and CeHsc70. Mutations in unc-23 lead to a muscle cell attachment-defect similar to muscular dystrophy, which can be suppressed if DNJ-13 is depleted additionally. We will use four CeHsc70 single-point mutants, which confer resistance against these unc-23 phenotypes and define, using interaction studies with DNJ-13 and ATP, what makes these Hsc70 variations biochemically distinct and how these mutations affect other functions of CeHsc70 in vivo. Further we will use CRISPR/Cas9 genome editing strategies to identify regions of DNJ-13, which are required for its negative influence on muscle attachment in vivo. We further will investigate whether DNJ-13 can influence the chaperoning of Hsp90 clients by an interaction between DNJ-13 and CDC-37, which we uncovered in vitro. Thus all three proposed projects will provide insight into the functions of the molecular chaperone machines and their client processing mechanisms and help to decipher specific disease-like phenotypes caused by single-point variations in chaperones, cofactors or their clients.

• DFG Programme: Research Grants