Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. / Includes bibliographical references. / (cont.) phosphoproteomics technology, IMAC/LC/MS/MS, [approximately] 200 phosphosites were identified from HT-29 cells, some of which were detected only from insulin-treated cells. Our phosphoproteomics approach also enabled us to detect alteration of both known and unknown phosphorylation states of apoptosis-related proteins at two time points during early apoptosis induced by tumor necrosis factor-α / Apoptosis, a physiologically regulated cell death, plays critical roles in development and immune system by maintaining tissue homeostasis. The thesis project investigates regulations of apoptosis in a human colon adenocarcinoma cell line, HT-29, exposed to diverse cellular stimuli, focusing on a specific protein as well as global level of proteins. The first part of the thesis demonstrated S-nitrosation of procaspase-9. S-nitrosation is a novel protein modification to regulate protein-protein interaction or protein activity. This modification has been implied to inactivate caspases. We could visualize S-nitrosation of an initiator caspase, procaspase-9, by enriching low-abundant procaspase-9 with immunoprecipitation and stabilizing S-nitroso-cysteine with biotin labeling. Nitric oxide synthase inhibitors and tumor necrosis factor-α (TNF-α) reduced the S-nitrosation level of procaspase-9, suggesting that S-nitrosation may be regulated by a nitric oxide synthase and denitrosation is likely a mechanism of apoptosis. The second part of the thesis is to examine survival effects of insulin on cells undergoing TNF-α-induced apoptosis. Insulin decreased the TNF-α-induced cleavage of key apoptotic mediators, caspases, and their substrates as well as apoptosis, in part, depending on phosphatidylinositol-3 kinase (PI-3K)/Akt pathway. One of protective mechanisms by insulin is likely to decrease the TNF-α-induced dissociation of a potent inhibitor of caspases, X-chromosome linked inhibitor of apoptosis protein (XIAP), from procaspase-9 via PI-3K/Akt pathway. Lack of phosphoproteomics data in HT-29 cells led the third part of the thesis to focus on investigating global level regulation of phosphoproteins during apoptosis. With a / by Ji-Eun Kim. / Ph.D.
| Identifer | oai:union.ndltd.org:MIT/oai:dspace.mit.edu:1721.1/28865 |
| Date | January 2004 |
| Creators | Kim, Ji-Eun, 1974- |
| Contributors | Steven R. Tannenbaum., Massachusetts Institute of Technology. Biological Engineering Division., Massachusetts Institute of Technology. Biological Engineering Division. |
| Publisher | Massachusetts Institute of Technology |
| Source Sets | M.I.T. Theses and Dissertation |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
| Format | 165 p., 8638393 bytes, 8659094 bytes, application/pdf, application/pdf, application/pdf |
| Rights | MIT theses are protected by copyright. They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission., http://dspace.mit.edu/handle/1721.1/7582 |
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