The Misfolded Protein Client as a Determinant of Substrate Ubiquitination by the Hsp70/CHIP Complex

Abstract

The E3 ubiquitin ligase CHIP (carboxy terminus of Hsc70 interacting protein) targets misfolded proteins chaperoned by heat shock protein 70 (Hsp70) for ubiquitination and subsequent proteasomal degradation. Hsp70 recruits and attempts to refold misfolded proteins, or “clients”, in iterative, ATP‐dependent cycles. In contrast, binding of Hsp70 to the tetratricopeptide repeat (TPR) domain of CHIP, allows for client proteins to be recruited for ubiquitination by CHIP. Ubiquitination of lysine residues on a client protein allows CHIP/Hsp70 complexes to promote the proteasomal degradation of the client. Thus, the CHIP/Hsp70 complex stands at the crossroads of degradative and refolding pathways of client proteins which is crucial for cellular protein quality control. The current knowledge in the field lacks a clear understanding of how the CHIP/Hsp70 complex executes its triage mechanism that directs misfolded proteins for either refolding or degradation. Recent models of ATP and ADP bound Hsp70 revealed the distances between the substrate binding domain (SBD) and the E2~ubiquitin conjugate active site as ~120 Å and ~60 Å respectively. This implies, with the dynamic conformational changes of Hsp70 complex, the ADP‐bound state of Hsp70 organizes the chaperoned ubiquitination complex by properly orienting the client with respect to CHIP which allows the client protein to span this 60 Å gap and accept the ubiquitin from the bound E2‐ubiquitin conjugate. Therefore, the distance between Hsp70 binding site and the active site of the E2~ubiquitin conjugate bound to CHIP plays a significant role as a determinant in the triage mechanism of CHIP/Hsp70. In this study, we focus on investigating the relationship between key distances within the CHIP/Hsp70/client complex and the ubiquitination activity of the CHIP/Hsp70 complex by utilizing in‐vitro ubiquitination assays of modified substrates.Support or Funding InformationNSF 15‐52113This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.