Inhibiting Protein-Protein Interactions in the Hsp70 Complex as a Means to Regulate Protein Homeostasis.

Molecular Chaperone;Protein-Protein Interation;Small Molecule Inhibitor;Hsp70;Stub1;Protein Quality Control;Pathology;Health Sciences;Molecular & Cellular Pathology

Smith, Matthew ConradCierpicki, Tomasz ;

University of Michigan

关键词: Molecular Chaperone;    Protein-Protein Interation;    Small Molecule Inhibitor;    Hsp70;    Stub1;    Protein Quality Control;    Pathology;    Health Sciences;    Molecular & Cellular Pathology;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/93955/mcsmitty_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

The molecular chaperone, heat shock protein 70 (Hsp70), is an importantregulator of protein homeostasis that has roles in both primary protein folding andclearance of misfolded proteins. Several studies have suggested that it is theinteraction between Hsp70 and a network of co-chaperones that might determinethe fate Hsp70-bound substrates. For example, a complex formed betweenHsp70 and the E3 ubiquitin ligase CHIP, is thought to guide substrates to theproteasome. However the mechanism governing how co-chaperones assembleon Hsp70 remains unresolved. We wanted to explore how interactions betweenHsp70 and CHIP govern the ubiquitin-mediated degradation of chaperonesubstrates. Moreover, we hypothesize that chemical disruption of the Hsp70-CHIP contact might decrease turnover of these substrates and, thereby, alterprotein homeostasis.This thesis work involves the biophysical and structural characterization of theHsp70-CHIP complex and synthesis of chemical probes that block CHIP bindingto Hsp70. Using truncations and biochemical assays, we determined that thexvitetratricopeptide repeat (TPR) domain of CHIP, along with an importantsecondary interaction in the charged coiled-coil domain govern binding of CHIPto Hsp70. In addition, we developed a dramatically improved synthesis of thepromising natural product, spergualin, and created a collection of more potentand stable derivatives. We then showed that these compounds disrupt theHsp70-CHIP interaction in vitro and in cells. Together, these findings provide aquantitative exploration of the CHIP-Hsp70 complex and the first set of chemicaltools that can be used to regulate Hsp70 interactions with CHIP. We expect thatthese probes will be useful in further studying Hsp70-mediated protein qualitycontrol.