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Biophysical Study of the Ubiquitin Ligase CHIP and Interactions with the Molecular Chaperones Hsp70 and Hsp90

Zhang, Huaqun
http://orcid.org/0000-0001-9627-3422
http://rave.ohiolink.edu/etdc/view?acc_num=miami1511192922525393

Abstract Details

2017, Doctor of Philosophy, Miami University, Cell, Molecular and Structural Biology (CMSB).
Protein folding and protein degradation are critical for protein homeostasis within eukaryotic cells. Protein folding and protein degradation are mainly mediated by molecular chaperones and the ubiquitin proteasome system (UPS), respectively. Heat shock proteins (Hsp) 70 and 90 are two important chaperones that assist protein folding and refolding. The C-terminus of Hsc70 interacting protein (CHIP), a co-chaperone, is a ubiquitin ligase involved in regulating protein triage through interaction with Hsp70 and Hsp90. CHIP is found to preferentially target Hsp70-bound client proteins for ubiquitination. Another co-chaperone the Hsp70 and Hsp90 organizing protein (HOP) competes with CHIP for binding to both chaperones and aids client transfer from Hsp70 to Hsp90 for refolding. Therefore, CHIP and HOP mediate two opposite directions for clients, folding and degradation. Both CHIP and HOP bind to Hsp70 and Hsp90 using tetratricopeptide repeat (TPR) domains and C-terminus EEVD motif, respectively. Although the structures of CHIP-TPR in complex with Hsp70 C-terminus peptides are already available, our data suggested that there is a second binding site between CHIP and Hsp70 in addition to the EEVD mediated interaction. This dissertation characterized a bipartite interaction between CHIP-TPR and Hsc70-lid-tail using X-ray crystallography. The interaction between CHIP-TPR and Hsc70 lid segment is required for CHIP ligase activity, as demonstrated by in vitro ubiquitination assays. This finding facilitates our understanding of how CHIP binds to Hsp70 and how it target substrates for ubiquitination. CHIP-TPR is found to be highly flexible and dynamic, and its N-terminus is thought to be not fully-folded. We assigned backbone and side chain chemical shifts for free CHIP-TPR, and the data suggest that CHIP-TPR is comprised of seven a helices, consistent with its crystal structure with a bound EEVD motif peptide. The NMR data therefore indicate the TPR domain is well folded before Hsp70 or Hsp90 binding. It has been found that a C-terminal threonine residue of Hsp70, and both threonine and serine residues of Hsp90 could be phosphorylated by protein kinases. The phosphorylation leads to binding switch of the two chaperones from CHIP to HOP, thus favoring protein refolding. In the dissertation, we also determined how phosphorylation reduced Hsp70 and Hsp90 binding to CHIP through NMR and X-ray crystallography. Additionally, we found that the weak binding between CHIP and its substrates may be sufficient for CHIP's ligase activity.
Rick Page (Advisor)
126 p.
Biochemistry; Biophysics
Ubiquitin ligase, CHIP, HOP, TPR domain, Hsp70, Hsp90

Recommended Citations

Citations

  • Zhang, H. (2017). Biophysical Study of the Ubiquitin Ligase CHIP and Interactions with the Molecular Chaperones Hsp70 and Hsp90 [Doctoral dissertation, Miami University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=miami1511192922525393

    APA Style (7th edition)

  • Zhang, Huaqun. Biophysical Study of the Ubiquitin Ligase CHIP and Interactions with the Molecular Chaperones Hsp70 and Hsp90. 2017. Miami University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=miami1511192922525393.

    MLA Style (8th edition)

  • Zhang, Huaqun. "Biophysical Study of the Ubiquitin Ligase CHIP and Interactions with the Molecular Chaperones Hsp70 and Hsp90." Doctoral dissertation, Miami University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=miami1511192922525393

    Chicago Manual of Style (17th edition)

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This open access ETD is published by Miami University and OhioLINK.