Folding state as the Decision Maker in Detecting Misfolded Proteins by Hsp70

Abstract

Maintaining cellular proteostasis is challenging due to the constantly changing and crowded cellular environment. Mutations, transcriptional and translational errors, and chemical and pathological stresses in a cell lead to protein misfolding and eventual formation of insoluble toxic aggregates. These aggregates typically result in loss of native protein function and are often associated with conformational diseases such as Alzheimer's, Parkinson's, Huntington's, and various types of cancer. The cellular protein quality control systems are essential for creating a stable cellular environment by resolving the misfolded proteins to uphold a stable proteome. The triage decision for a misfolded protein is regulated by Heat shock protein 70 (Hsp70), the central hub of protein quality control machinery, along with the E3 ubiquitin ligase C‐terminus of Hsc70‐interacting protein (CHIP). Hsp70 itself helps misfolded “client” proteins to refold and directs protein clients to downstream refolding pathways, whereas Hsp70 interaction with CHIP can lead to CHIP‐mediated ubiquitination, ultimately directing the proteins to proteasomal degradation. Understanding the molecular mechanisms that enable the Hsp70/CHIP complex to triage client proteins would allow for the identification of new avenues of treatment for the above‐mentioned diseases. With an objective to illustrate how the varying degrees of folding in a model client protein dictate the interaction between Hsp70 and the client, we have designed a spectrum of client proteins we named folding sensor TPRs (FSTPRs). We initially hypothesized that the more unfolded the client higher the affinity towards Hsp70. Circular dichroism (CD) spectroscopy was used to assess the folding states of each FSTPR variant, and biolayer interferometry (BLI) was utilized to quantify the affinities of each FSTPR towards the Hsp70 substrate‐binding domain (SBD). During our BLI experiments, we observed a range of threshold unfolding percentages that correspond to the highest affinity. Above and below the threshold range, the affinity decreased. This behavior was recapitulated with a disulfide‐locked FSTPR that is folded in the oxidized state and predominantly unfolded in the reduced state. Even though support from further data is necessary, these studies provide us preliminary insight into how Hsp70 interacts with a misfolded client and the role played by folding state in dictating Hsp70/client interactions.