Characterization of Human Hsp70 Chaperone Complexes and Chemical Control Over Their Formation.

[摘要] Heat shock protein 70 (Hsp70) is a molecular chaperone that plays a central role in cellular protein homeostasis by assisting in the folding and/or degradation of protein substrates. In these tasks, Hsp70 is assisted by associated co-chaperones, each of which is thought to contribute to specialized chaperone functions. However, it isn’t yet clear why specific co-chaperones are recruited to the Hsp70 complex and how each distinct complex is able to carry out specific tasks. Further, imbalances in this system are thought to be involved in human diseases, such as cancer and neurodegenerative disorders. Thus, chemically controlling the formation/function of Hsp70-based chaperone complexes could provide new insights into the logic of protein quality control and also provide new avenues for drug discovery. The goals of this work are (a) biochemically characterize distinct Hsp70 complexes to permit insights into their specialized functions and (b) identify and characterize chemical compounds that promote the formation of specific Hsp70 complexes. Towards these goals, we devised a new approach termed ;;gray-box” screening, in which high throughput chemical screens are performed against reconstituted components of the Hsp70 chaperone family, facilitating the identification of compounds that target specific protein-protein interfaces in the Hsp70 complex. Using this approach, we found unexpected allosteric mechanisms for some of these compounds. For example, we found that 115-7c stimulates the ATPase and protein-folding activities of Hsp70 by promoting the action of the co-chaperone Hsp40. To further extend this screening method, we biochemically characterized ~12 distinct Hsp70 complexes formed from reconstitution of recombinant, human co-chaperones. These findings are expected to facilitate a large number of additional ;;gray box” screens. Finally, we extended this work to the Hsp70 complex with CHIP, a co-chaperone that is emerging as a critical regulator of the pro-degradation activities of Hsp70. Together, these studies have provided important new insights into the molecular mechanisms of Hsp70 chaperone system, including key differences between the prokaryotic and human chaperones. This work has also provided chemical probes that will be useful in understanding the roles of protein-protein interactions in the Hsp70 complexes.

[发布机构] University of Michigan

[效力级别] Gray Box"

[关键词] "Gray Box"Screening Against Hsp70 Chaperone Multi-protein Complexes;Small Molecule Modulators for Hsp70;Biochemical Characterization of Hsp70 Chaperone Complexes;Biological Chemistry;Pathology;Pharmacy and Pharmacology;Science (General);Health Sciences;Science;Molecular & Cellular Pathology