Ghosh, Hiyaa Singhee
(2008)
REGULATION OF TRANSLATION AND TRANSCRIPTION BY SIRT1: POTENTIAL NOVEL MECHANISMS FOR REGULATING STRESS RESPONSE AND AGING.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
SIRT1 is a NAD+ dependent deacetylase that targets many histone and non histone proteins, thereby regulating a broad range of physiological processes such as metabolism, reproduction, development, and cell survival. In this study, we have identified novel binding partners for SIRT1 and documented three different cellular processes that are affected by these novel interactions.Using a yeast two-hybrid screen, we have identified several potential binding partners for SIRT1. Transducin like Enhancer of split 1 (TLE1) and eIF2-alpha (eIF2) are two such proteins identified in the screen whose interaction with SIRT1 was further confirmed by co-immunoprecipitation. TLE1 is co-repressor for several transcriptional factors including NF-κB. We demonstrate that SIRT1 and TLE1 repress NF-κB activity and that the catalytic activity of SIRT1 may not be critical for this. Using knock-out cell lines, we further demonstrate that both SIRT1 and TLE1 are required for the down-regulation of NF-κB activity. Our results suggest that the interaction between SIRT1 and TLE1 is important for mediating repression of NF-κB activity, potentially through a deacetyalse independent mechanism. SIRT1 protects cells from genetoxic and oxidative stress, whereas phosphorylation of eIF2 is critical for translation attenuation and preferential expression of stress related genes under stress conditions. We demonstrate that SIRT1 depleted cells show higher levels of phosphorylated eIF2 and delayed expression of the stress response protein, CHOP. Furthermore, SIRT1 deficient cells show higher sensitivity to stress treatments and a delayed recovery of protein systhesis. SIRT1 associates with eIF2 regardless of stress condition, SIRT1's catalytic activity or the phosphorylation state of eIF2. These observations suggest a novel aspect of SIRT1 mediated regulation of cellular stress response.Both SIRT1 and the target of rapamycin (TOR) are involved in age related diseases and lifespan. We demonstrate for the first time that these two pathways are interconnected. We show that SIRT1 null mouse embryonic fibroblasts (MEFs) have larger cell morphology and upregulated mTOR signaling. Furthermore, SIRT1 activator reduces, whereas inhibitor activates the mTOR pathway. Rapamycin is effective in inhibiting mTOR activity in both SIRT1 positive and deficient cells. Finally, we show that SIRT1 physically associates with TSC2 in HeLa cells. These observations demonstrate that SIRT1 negatively regulates mTOR pathway upstream of mTOR complex-1 (TORC1), potentially, by regulating the TSC1/2 complex.
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Details
| Item Type: |
University of Pittsburgh ETD
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| Status: |
Unpublished |
| Creators/Authors: |
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| ETD Committee: |
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| Date: |
28 March 2008 |
| Date Type: |
Completion |
| Defense Date: |
19 December 2007 |
| Approval Date: |
28 March 2008 |
| Submission Date: |
25 March 2008 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Medicine > Biochemistry and Molecular Genetics |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
aging; eIF2-alpha; mTOR; SIRT1; TLE |
| Other ID: |
http://etd.library.pitt.edu/ETD/available/etd-03252008-114622/, etd-03252008-114622 |
| Date Deposited: |
10 Nov 2011 19:32 |
| Last Modified: |
15 Nov 2016 13:37 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/6582 |
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