Cellular Protein Client Recovery of Human HSP40s
- Author(s): Montoya, Maureen R
- Advisor(s): Genereux, Joseph C
- et al.
The integrity of the proteome must be maintained to ensure normal protein function and prevent cellular toxicity. Proteostasis factors scan the proteome to promote protein folding, trafficking, disassembly, and degradation. In all forms of life and cellular environments, the Hsp70/Hsp40 chaperone machinery rescues misfolded proteins, with over 40 human Hsp40s responsible for identifying and recruiting misfolded protein substrates to Hsp70. This Hsp40 diversity could be responsible for matching client recognition to Hsp70 functional diversity. To evaluate the client diversity of class B Hsp40s, we applied tandem mass tag-affinity purification-mass spectrometry (TMT-AP-MS) to characterize the interactor profiles of human Hsp40s. To increase interactor recovery yield, we used mutations that inactivate handoff of clients to Hsp70s. We found > 400 high-confidence interactors of DNAJB8 in HEK293T cells by using crosslinking with high stringency washing conditions to decrease false positives, TMT labeling to allow head-to-head comparison across different samples, and identification of specific interactors based on integrated TMT intensity correlation between prey and bait levels.
With bait correlation coupled with TMT-AP-MS we could assess the substrate profiles of different Hsp40 members. DNAJB1 and DNAJB8 are two Hsp40s that differ in size, structure, and functionality, that are ideal candidates for probing differential substrate binding across human class B Hsp40s. We first evaluated the proteome-wide effect of crosslinking and J-domain activity on interactor recovery for each Hsp40. After finding crosslinking to be unnecessary and J-domain activity to be neutral for recovery of interactors, we profiled substrates of DNAJB1WT and DNAJB8WT. Only 19 interactors, largely Hsp70 chaperones, were identified for DNAJB1WT. Approximately half of the 249 interactors for DNAJB8WT were shared with DNAJB8H31Q clients. However, heat shock, which induces protein misfolding, increases global client binding to DNAJB8H31Q but not to DNAJB8WT, suggesting that the J-domain mutation is necessary to maintain increased client binding under stress conditions. We demonstrate an effective technique that could probe client interactions for Hsp40 co-chaperones, which could lead to insight into how proteostasis machineries subdivide their large substrate pool between different fates and minimize the gap in understanding of the role of Hsp40s in Hsp70 functional diversity.