Student Theses and Dissertations

Date of Award

2009

Document Type

Thesis

RU Laboratory

Steller Laboratory

Keywords

programmed cell death, apoptosis proteins (IAP's), XIAP ubiquitin ligase activity

Abstract

Virtually all animal cells have the known ability to self-destruct by undergoing apoptosis, a morphologically distinct form of programmed cell death. The proper regulation of apoptosis is critical for both development and tissue homeostasis, and inhibition of apoptosis contributes to the development and progression of cancer. Inhibitor of Apoptosis Proteins (IAPs) can bind to and inhibit caspases, the key executioners of apoptosis. Because IAPs are frequently over-expressed in human tumors, they have become major pharmacological targets for developing new cancer therapeutics. Many IAPs contain RING domains that function as E3 ubiquitin-ligases to regulate the abundance of IAPs themselves and their binding partners by engaging the ubiquitin system. The precise physiological function of individual mammalian IAPs and their role as E3- ubiquitin ligases in situ remain largely obscure. Here, we investigated the function of XIAP ubiquitin-ligase activity by deleting the RING motif via gene targeting in the mouse. Mice expressing XIAP ΔRING were fertile, born in expected proportions, and were not obviously prone to disease in a pathogen-free environment. Removing the RING finger motif stabilized XIAP protein in apoptotic thymocytes, demonstrating that XIAP ubiquitin-ligase activity is a major determinant of XIAP protein stability. However, consistent with earlier reports on XIAP-null mice, we found no detectable abnormalities in apoptosis of mutant thymocytes. On the other hand, ΔRING embryonic stem cells and fibroblasts had elevated caspase-3 enzyme activity and impaired ubiquitination of active caspase-3 during apoptosis. Furthermore, XIAP ΔRING embryonic fibroblasts were strongly sensitized to TNF-α-induced apoptosis. Similar results were obtained with XIAP-null mice. Finally, deletion of the RING also improved the survival of mice in the Eμ-Myc lymphoma model. The improved prognosis corresponded to increased apoptosis and decreased abundance of proliferating B-cells in the bone marrow, and a curtailed incidence of leukemia. This demonstrates a physiological requirement of XIAP E3 ubiquitin-ligase activity for the inhibition of apoptosis and for tumor suppression in vivo.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Permanent URL

http://hdl.handle.net/10209/422

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