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i
THE ROLE OF THE PROTEASOME & ITS REGULATORS IN ADAPTATION TO OXIDATIVE STRESS
by
Andrew Michael Pickering
A Dissertation Presented To The
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR BIOLOGY)
May 2012
Copyright 2012 Andrew Michael Pickering
Object Description
| Title | The role of the proteasome & its regulators in adaptation to oxidative stress |
| Author | Pickering, Andrew Michael |
| Author email | pickering.andrew@gmail.com;ampicker@usc.edu |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Molecular Biology |
| School | College of Letters, Arts And Sciences |
| Date defended/completed | 2012-03-06 |
| Date submitted | 2012-05-01 |
| Date approved | 2012-05-02 |
| Restricted until | 2012-05-02 |
| Date published | 2012-05-02 |
| Advisor (committee chair) | Davies, Kelvin J. A. |
| Advisor (committee member) |
Finch, Caleb E. Cadenas, Enrique Finkel, Steven E. |
| Abstract | Oxidized cytoplasmic and nuclear proteins are normally degraded by the proteasome, but accumulate with age and disease. I demonstrate the importance of various forms of the proteasome during transient (reversible) adaptation (hormesis), to oxidative stress in murine embryonic fibroblasts. Adaptation was achieved by 'pre-treatment' with acute oxidative stress exposure (e.g. H₂O₂, peroxynitrite, menadione, paraquat), and tested by measuring inducible resistance to a subsequent much higher 'challenge' dose of H₂O₂. ❧ Oxidative stress adaptation causes an initial direct physical activation of pre-existing proteasomes, then a subsequent de novo synthesis of 20S proteasome, immunoproteasome and Pa28αβ during over the next 24 h. Cellular capacity to degrade oxidatively damaged proteins increased with 20S proteasome, immunoproteasome and Pa28αβ synthesis, and was mostly blocked by the 20S proteasome, immunoproteasome and Pa28 siRNA knockdown treatments. Direct comparison of purified 20S proteasome and immunoproteasome demonstrated that the immunoproteasome can selectively degrade oxidized proteins. Cell proliferation and DNA replication both decreased and oxidized proteins accumulated, during high H₂O₂ challenge. However H₂O₂ adaptation was protective against such H₂O₂ challenge. Importantly, siRNA knockdown of the 20S proteasome, immunoproteasome or Pa28αβ regulator blocked 50-100% of these adaptive increases in cell division and DNA replication. Immunoproteasome knock-down also largely abolished protection against protein oxidation. ❧ I show that the adaptative increase in oxidative stress tolerance and capacity to degrade oxidized proteins is dependent induction of the Nrf2 transcription factor. Furthermore I show that adaptation causes an increase in cellular levels of Nrf2, and translocation of Nrf2 from the cytoplasm to the nucleus. It also causes increased binding of Nrf2 to antioxidant response elements (ARE) or electrophile response elements (EpRE) in the 5-untranslated region of the Proteasome β5 subunit gene [demonstrated by chromatin immunoprecipiation (or ChIP) assay]. I go on to show that this induction of Nrf2 is a necessary requirement for increased Proteasome/Pa28αβ levels, and for maximal increases in proteolytic capacity and stress resistance. The oxidative stress induced increase in immunoproteasome however did not appear to be Nrf2 dependent. ❧ I show that Pa28αβ and Pa28γ have increased expression under mild oxidant exposure. I also demonstrate that both of the proteasome regulators enhance the capacity of the proteasome to selectively degrade oxidized proteins. In conjunction with their increased expression, there is an increase in binding of the Pa28αβ regulator to 20S proteasome. I show that the Pa200 proteasome regulator is also induced by H₂O₂ exposure. The Pa200 regulator however does not enhance the capacity of proteasome to degrade oxidized proteins. However, it does appear to enhance the capacity of the proteasome to degrade histones. ❧ I also demonstrate that this adaptive response is highly conserved. Exposure to a mild dose of an oxidant can increase oxidative stress tolerance in both Drosophila and C. elegans. There is also, in both of these animals, an increase in proteolytic capacity and a corresponding increase in 20S proteasome levels. If the increase in 20S proteasome or induction by Nrf2 homologues is blocked then the adaptive response is either blunted or completely lost in both animals. |
| Keyword | oxidative stress; hormesis; adaptation; Nrf2; Skn-1; Cnc-C; Drosophila; C. elegans; mouse; free radical; proteasome; immunoproteasome; Pa200; Pa28; cell culture ❧ |
| Language | English |
| Part of collection | University of Southern California dissertations and theses |
| Publisher (of the original version) | University of Southern California |
| Place of publication (of the original version) | Los Angeles, California |
| Publisher (of the digital version) | University of Southern California. Libraries |
| Provenance | Electronically uploaded by the author |
| Type | texts |
| Legacy record ID | usctheses-m |
| Rights | Pickering, Andrew Michael |
| Access conditions | The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the author, as the original true and official version of the work, but does not grant the reader permission to use the work if the desired use is covered by copyright. It is the author, as rights holder, who must provide use permission if such use is covered by copyright. The original signature page accompanying the original submission of the work to the USC Libraries is retained by the USC Libraries and a copy of it may be obtained by authorized requesters contacting the repository e-mail address given. |
| Repository name | University of Southern California Digital Library |
| Repository address | USC Digital Library, University of Southern California, University Park Campus MC 7002, 106 University Village, Los Angeles, California 90089-7002, USA |
| Repository email | cisadmin@usc.edu |
| Archival file | uscthesesreloadpub_Volume4/etd-PickeringA-712.pdf |
Description
| Title | Page 1 |
| Full text | i THE ROLE OF THE PROTEASOME & ITS REGULATORS IN ADAPTATION TO OXIDATIVE STRESS by Andrew Michael Pickering A Dissertation Presented To The FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (MOLECULAR BIOLOGY) May 2012 Copyright 2012 Andrew Michael Pickering |
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