The ALS-associated E425K mutation uncouples DNAJC7 from the Hsp70 chaperone cycle.

Area of Science:

• Molecular Biology
• Neuroscience
• Biochemistry

Background:

• DNAJC7 (J-domain protein) is crucial for protein homeostasis and Hsp70 regulation.
• Mutations in DNAJC7 are associated with amyotrophic lateral sclerosis (ALS), but the underlying mechanisms are unclear.
• DNAJC7 possesses a J-domain for Hsp70 activation and TPR domains for protein interactions.

Purpose of the Study:

• To investigate the structural and functional impact of the ALS-associated E425K mutation in DNAJC7's J-domain.
• To elucidate the role of DNAJC7's TPR domains in Hsp70 interaction and client protein regulation.
• To understand the mechanistic basis of DNAJC7 dysfunction in ALS.

Main Methods:

• Nuclear Magnetic Resonance (NMR) spectroscopy to assess protein structure and interactions.
• Biochemical assays to evaluate Hsp70 activation and client protein binding.
• Analysis of TDP-43 aggregation and refolding in the presence of wild-type and mutant DNAJC7.

Main Results:

• The E425K mutation does not alter DNAJC7 structure but disrupts its primary interaction with Hsp70 via the J-domain.
• A secondary Hsp70-binding site involving DNAJC7's TPR domains and Hsp70's EEVD motif was identified and remains intact in the mutant.
• While the mutant retains holdase activity against TDP-43 aggregation, it fails to facilitate Hsp70-mediated client transfer and refolding.

Conclusions:

• DNAJC7 function relies on the coordinated action of its J-domain and TPR domains for effective Hsp70 regulation.
• Disruption of the J-domain-mediated Hsp70 activation by the E425K mutation uncouples DNAJC7 from the Hsp70 machinery.
• This study provides a mechanistic explanation for DNAJC7 dysfunction in ALS, highlighting the importance of precise chaperone interactions.