Date of Award

1-1-2015

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Biological Sciences

Content Description

1 online resource (x, 109 pages) : color illustrations

Dissertation/Thesis Chair

Hua Shi

Committee Members

Ben Szaro, Robert Osuna, Jayanti Pande

Keywords

Aptamer, Chaperone, CHIP, Hsp70, Protein triage, RNA, Protein folding, Heat shock proteins, Molecular chaperones

Subject Categories

Biochemistry | Biology

Abstract

Protein quality control involves refolding of damaged proteins and facilitating degradation of irreparable proteins. Understanding the protein quality control mechanism is critical, since defects in it has been implicated in a number of age-related diseases like neurodegenerative diseases and also in cancer. A vast network of molecular chaperones and proteolytic systems collaborate to maintain protein quality control. The 70 kilodalton Heat shock protein (Hsp70) is a highly conserved and ubiquitous chaperone, which interacts with a variety of protein substrates including newly synthesized polypeptides, unfolded, partially misfolded and native proteins to maintain protein quality control. Hsp70 chaperone function is coupled to its ATPase activity that regulates its substrate binding and release cycle. The Hsp70 chaperone system plays an important role in protein quality control by assisting in the folding and clearance of misfolded proteins, but the mechanism and regulation that enable it to choose between folding and degradation pathways are not clear. I hypothesized that chemical tools capable of modulating the ATPase activity of Hsp70, can be used in the mechanistic dissection of this system. To test this hypothesis, a conformation-specific RNA aptamer for Hsp70-ATP, which inhibited the ATPase activity of Hsp70 was developed. The aptamer was used as a chemical probe to show that Hsp70's ATPase activity enables both folding and degradation pathways to proceed in parallel. But the degradation pathway is subject to an inbuilt negative feedback, which would prevent an onrush of protein destruction. By means of an engineered bi-functional aptamer this negative feedback could be reversed for a specific Hsp70 substrate protein, the estrogen receptor α, to promote its specific diversion into the degradation pathway.

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