ISBN:  Unavailable

Category: Unavailable

Page count: 165

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-[alpha] (TNF-[alpha]) 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-[alpha]-induced apoptosis. Insulin decreased the TNF-[alpha]-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-[alpha]-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.