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Full text release has been delayed at the author's request until May 12, 2025

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Study of Hsp70/CHIP mediated Protein Quality Control by Folding Sensors

Karunanayake, Chamithi Samadharshi
http://orcid.org/0000-0002-8502-5615
http://rave.ohiolink.edu/etdc/view?acc_num=miami1683899932999732

Abstract Details

2023, Doctor of Philosophy, Miami University, Chemistry and Biochemistry.
Proteins can misfold in a crowded cellular environment due to physiological, pathological, and environmental stress. The fate of such misfolded proteins to either refolding by chaperones or degradation by the proteasome is determined by a protein-protein complex comprising the molecular chaperone Hsp70 and the E3 ubiquitin ligase CHIP. The decision to shunt proteins to the refolding or degradative pathways is known as the triage decision, and sufficient mechanistic details are still lacking. We hypothesized that the ensemble of different folding states of a single misfolded protein could be represented by a library of model proteins, each with a varying degree of thermodynamic stability. We termed the novel library of proteins we devised as “Folding Sensors”. Structure-guided protein engineering, computational modeling, biophysical techniques, and structural biology were utilized in this process. According to thermal denaturation data, the spectrum of folding sensors showed varying degrees of quantitative thermal stability at a given temperature. The crystal structure of the most stable folding sensor closely resembled the structure of the TPR domain, which served as the initial basis for the folding sensors, while also forming a disulfide bond as expected. We further speculate that Hsp70 recognized this variant as a natively folded state protein due to its’ low affinity towards Hsp70. We further discovered that Hsp70 tightly binds to the moderately stable folding sensors. We further observed that CHIP differentially ubiquitinated substrates based on the thermal stability and the folding state. The process was chaperone dependent. The folding sensor mimicking a natively folded protein was ubiquitinated the least while the variants with more unfolding showed more ubiquitination. Altogether we developed folding sensors with varying thermodynamic stability to serve as a model to study chaperone-mediated triage by the Hsp70/CHIP. This preliminary study provided insights into the thermodynamic stability of folding status as a determining factor in the triage decision for a misfolded protein.
Richard Page (Advisor)
Carole Dabney-Smith (Committee Chair)
Timothy Wilson (Committee Member)
Dominik Konkolewicz (Committee Member)
Ann Hagerman (Committee Member)
171 p.
Biochemistry; Biology; Biophysics; Chemistry
Hsp70, CHIP, STUB1, Molecular Chaperones, Protein Quality Control, Model proteins, Misfolded Proteins, HSP70/CHIP Triage decision, Cochaperones, Ubiquitination, Protein Folding, Hsp40, Hsp70 T13G, client protein, protein engineering, neurodegenerative diseases, SCA48

Recommended Citations

Citations

  • Karunanayake, C. S. (2023). Study of Hsp70/CHIP mediated Protein Quality Control by Folding Sensors [Doctoral dissertation, Miami University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=miami1683899932999732

    APA Style (7th edition)

  • Karunanayake, Chamithi. Study of Hsp70/CHIP mediated Protein Quality Control by Folding Sensors. 2023. Miami University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=miami1683899932999732.

    MLA Style (8th edition)

  • Karunanayake, Chamithi. "Study of Hsp70/CHIP mediated Protein Quality Control by Folding Sensors." Doctoral dissertation, Miami University, 2023. http://rave.ohiolink.edu/etdc/view?acc_num=miami1683899932999732

    Chicago Manual of Style (17th edition)

miami1683899932999732
© 2023, all rights reserved.
This open access ETD is published by Miami University and OhioLINK.