Factors Affecting Protein Recovery During Hsp40 Affinity Profiling

Abstract

Hsp40s play a central role in cellular protein homeostasis by promiscuously surveying the proteome for misfolded proteins. These misfolded client proteins are then delivered to Hsp70. Mutation of the Hsp40 J-domain blocks Hsp70 binding, inhibiting client protein release from Hsp40. We previously integrated misfolded protein recognition by Hsp40 into a platform to identify proteins that are destabilized by cellular stress. However, the dependences of Hsp40 interactions and client recovery on J-domain activity, Hsp40 identity, and crosslinking have not been addressed. Herein, we apply quantitative proteomics to systematically characterize the interactions networks of human Hsp40s DNAJB8 and DNAJB1 with intact or inactivated J-domains. We find that DNAJB8 irreversibly binds over a thousand protein interactors even in the absence of stress. Inactivation of the J-domain decreases interaction with Hsp70 family and associated proteins, but does not generally affect client binding. By contrast, J-domain inactivation and cellular crosslinking substantially increase the relative recovery of proteins from DNAJB1 co-immunoprecipitation. This advantage is completely offset by loss of DNAJB1 recovery under these conditions, making DNAJB1 a poor bait for client protein recovery as compared to DNAJB8. The J-domain inactivated DNAJB8H31Q has increased affinity to its client proteins under heat stress, while no such change in affinity is observed for the wild-type protein, despite their similar client binding profiles under basal conditions. Hence, we find that DNAJB8H31Q is an effective recognition element for the recovery of destabilized client proteins following cellular stress.

Profiling protein targets of cellular toxicant exposure

“…170 Even brief, mild heat shock induces monotonic increased association, consistent with DNAJB8 H31Q affinity reflecting protein destabilization. 171…”

“…After surveying various J-domain proteins, we found that the human J-domain protein mutant, DNAJB8 H31Q , strongly binds destabilized proteins allowing their affinity purification. 170,171 This binding is resistant to strong detergent buffer, allowing stringent purification conditions prior to quantitative proteomics. The DNAJB8 H31Q associated proteome is destabilized compared to the bulk proteome.…”

“…170 Even brief, mild heat shock induces monotonic increased association, consistent with DNAJB8 H31Q affinity reflecting protein destabilization. 171 We have applied our assay to several major agents of exposure. 146,172 Just 15 min of cellular exposure to arsenic (sodium arsenite; 100 mM) is adequate to induce destabilization of several proteins, primarily RNA binding proteins such as TDP43 and HNRNPA0 and including the pyruvate dehydrogenase component PDHA1 146 (Fig.…”

Cellular Exposure to Chloroacetanilide Herbicides Induces Distinct Protein Destabilization Profiles

“…We previously developed an affinity purification mass spectrometry (AP-MS) approach to profile the misfolded proteome based on its affinity to the human Hsp40 DNAJB8 (Figure B) . This assay combines affinity purification of overexpressed Flag DNAJB8 H31Q with quantitative proteomics to identify hundreds of coisolating cellular proteins with high reproducibility and statistical confidence. , The H31Q mutation blocks the release of misfolded proteins from DNAJB8, making it a thermodynamic sink for its clients. This binding is highly detergent-resistant, allowing immunoprecipitates to be stringently washed.…”

Cellular Exposure to Chloroacetanilide Herbicides Induces Distinct Protein Destabilization Profiles

“…We previously developed an affinity purification mass spectrometry (AP-MS) approach to profile the misfolded proteome using human the Hsp40 DNAJB8 37 . This assay combines affinity purification of overexpressed Flag DNAJB8 H31Q with quantitative proteomics to identify hundreds of co-isolating cellular proteins with high reproducibility and statistical confidence 38,39 . The H31Q mutation blocks the release of misfolded protein clients from DNAJB8, making it a thermodynamic sink for its clients.…”