Evaluating Client Protein Recovery by the Hsp40s DNAJB8 and DNAJB1 with AP-MS
Hsp40s, also termed J-domain proteins, play a central role in cellular protein homeostasis by promiscuously surveying the proteome for misfolded proteins. We have exploited this property to develop Hsp40 affinity profiling as a method for identifying proteins that misfold in response to cellular stresses. In this assay, we use the Hsp40 FlagDNAJB8H31Q as our recognition element for misfolded proteins. This protein is exogenously introduced into cells, promoting interactions without regard for native protein clients. Herein, we evaluate potential approaches to improve the performance of this assay. We find that although intracellular crosslinking increases recovery of protein interactors, this is not enough to overcome the relative drop in DNAJB8 recovery. While the J-domain promotes Hsp70 association, it does not affect the yield of protein association with DNJAB8 under basal conditions. By contrast, crosslinking and J-domain ablation both substantially increase relative protein interactor recovery with the structurally distinct Class B Hps40 DNAJB1 but are completely compensated by poorer yield of DNAJB1 itself. Cellular thermal stress promotes increased affinity between DNAJB8H31Q and interacting proteins, as expected for interactions driven by recognition of misfolded proteins. DNAJB8WT does not demonstrate such a property, suggesting that under stress misfolded proteins are handed off to Hsp70. Hence, we find that DNAJB8H31Q is still our most effective recognition element for the recovery of destabilized client proteins following cellular stress. Raw data is accessible through the PRIDE Archive at PXD030633.