Citation
Blythe, Emily Elizabeth (2019) Characterization of Adaptor Binding and Substrate Processing by VCP/p97. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/9K4M-0C48. https://resolver.caltech.edu/CaltechTHESIS:05222019-125958370
Abstract
Valosin-containing protein (VCP/p97) is an essential AAA+ ATPase that is critical to numerous important cellular pathways, such as ER-associated degradation. p97 works in concert with a repertoire of adaptor proteins to extract ubiquitylated proteins from membranes or complexes and, often, target them for degradation by the protesome. The nature of the p97 system—dependent upon a complex network of accessory proteins and targeted to substrates that are unstable and heterogeneous—makes the mechanism of substrate processing challenging to study. Here, we developed in vitro biochemical assays to reconstitute two important steps in the p97 pathway for mechanistic study: adaptor binding and substrate processing. We showed that p97-adaptor complexes are highly dynamic, recapitulating observations made in cell lysate. Using a model p97 substrate, we demonstrated for the first time that p97 processes its substrates through unfolding, a fact long presumed but never explicitly proven. Finally, with these model systems in hand, we explored the effects of p97 mutations that cause the neurodegenerative disease multisystem proteinopathy (MSP) on p97-adaptor-substrate complexes. MSP mutations cause faster substrate unfolding, and we hypothesize that this increase is due to a higher affinity for the requisite adaptors Ufd1-Npl4. Our biochemical data presents evidence for a gain of function model for MSP pathology and suggests new avenues for treating MSP.
| Item Type: | Thesis (Dissertation (Ph.D.)) | |||||||||
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| Subject Keywords: | Protein quality control, p97, multisystem proteinopathy | |||||||||
| Degree Grantor: | California Institute of Technology | |||||||||
| Division: | Chemistry and Chemical Engineering | |||||||||
| Major Option: | Biochemistry and Molecular Biophysics | |||||||||
| Thesis Availability: | Public (worldwide access) | |||||||||
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| Defense Date: | 15 May 2019 | |||||||||
| Record Number: | CaltechTHESIS:05222019-125958370 | |||||||||
| Persistent URL: | https://resolver.caltech.edu/CaltechTHESIS:05222019-125958370 | |||||||||
| DOI: | 10.7907/9K4M-0C48 | |||||||||
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| Default Usage Policy: | No commercial reproduction, distribution, display or performance rights in this work are provided. | |||||||||
| ID Code: | 11529 | |||||||||
| Collection: | CaltechTHESIS | |||||||||
| Deposited By: | Emily Blythe | |||||||||
| Deposited On: | 23 May 2019 20:28 | |||||||||
| Last Modified: | 25 Nov 2019 17:08 |
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