All ETDs from UAB

Advisory Committee Chair

Jeffrey B Smith

Advisory Committee Members

Martin R Johnson

Xu Cao

Weei-Chin Lin

Kevin A Roth

Document Type

Dissertation

Date of Award

2010

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Apoptosis, or programmed cell death, is essential for the development and maintenance of mammalian tissues. Activation of cysteinyl aspartate specific proteases, called caspases, is crucial to the implementation apoptosis. During apoptosis, the second mito-chondrial derived activator of caspase (Smac), augments caspase activity by antagonizing the inhibitor of apoptosis proteins (IAPs) down-regulation of caspase function. Smac protein synthesis occurs in the cytosol from a nuclear gene. Mitochondrial import of Smac leads to proteolytic removal of the first 55 amino acids, exposing a novel amino-terminus composed Ala56-Val-Pro-Ile59, which is an inhibitor of apoptosis binding motif (IBM). The IBM facilitates the interactions with IAPs by docking with their baculovirus inhibitor of apoptosis repeats, which also interact with and can inhibit caspases. During apoptosis, Smac homodimer exits the mitochondria and counteracts IAP inhibition of caspases. This study utilizes a ubiquitin fusion model to express wild type and mutant forms of Smac in the cytosol of mammalian cells. Smac60, which lacks the IBM and has a re-duced ability to bind XIAP, potentiated higher levels of caspase activation and apoptosis evoked by proteasome inhibition than cytosolic wild type Smac56. Therefore, the IBM is not sufficient or required for XIAP interaction and proapoptotic function of Smac. Cytosolic Smac56 but not Smac60, lost the ability to homodimerize and interact with XIAP after persistent cytosolic expression, possibly due to a posttranslational modification(s) in the dimeric interface. Missense mutations that disrupted the dimeric inter-face of Smac56 abolished homodimerization and IAP interaction without abrogating the proapoptotic function. Furthermore, cell lines that expressed Smac56 or monomeric Smac mutants in the cytosol underwent apoptosis more readily than a mock transfected cell line supporting the idea that Smac has an IAP independent pro-apoptotic function. An additional study demonstrated a novel anti-apoptotic activity of cIAP1, which is specific to the caspase-9 apoptosome. In a reconstituted assay, cIAP1 potently prevented the caspase-9 apoptosome holoenzyme from processing and activating caspase-3. Overall, these studies improved the understanding of apoptosis regulation: by unveiling a new inhibitory function of cIAP1, showing Smac maintains pro-apoptotic function without the IBM, and uncovering a mechanism of disarming of Smac triggered by the IBM.

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