Date of Award

5-2012

Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Chemistry

First Advisor

Jeanne A. Hardy

Second Advisor

Lila M. Gierasch

Third Advisor

Peter Chien

Subject Categories

Chemistry

Abstract

Tight regulation of caspase-9, a key initiator of apoptosis, is required to uphold cellular homeostasis. Although it is controlled on a multifactorial level, misregulation of this process does occur, which is a characteristic of a variety of diseases from ischemic injury to cancer. Therefore it remains important to gain a detailed understanding of the mechanisms behind native caspase-9 regulatory pathways and harness these mechanisms for therapeutic purposes.

Based on known mechanisms, such as the unique inhibitory complex of caspase-9 and XIAP-BIR3, development of synthetic regulators can be envisioned, while other mechanisms such as zinc-mediated inhibition and CARD activation of caspse-9 remain undefined. Intrigued by the multiple ways to control caspase-9’s activity, we sought after designing synthetic caspase-9 inhibitors in addition to defining the mechanistic details metal regulation and CARD domain activation. We report the first stabilized α-helical peptides that harness the native regulatory mechanism of caspase-9 and the BIR3 domain which lead to the understanding of the importance of exosites in inhibitory complexes. Our studies also revealed that there are two distinct zinc binding sites, one at the active site and another at a novel zinc binding site of yet unknown function in caspase-9 however this site may have the potential to control caspase-6 based on its regulatory mechanism. Furthermore, an interaction was discovered between CARD and the catalytic core of caspase-9 in the presence of a properly formed substrate binding groove, a potential mechanism utilized by the apoptosome for activation of the enzyme.

All in all, the regulation of caspase-9 occurs on a variety of levels that requires almost every surface of the enzyme. Through exploring these underlying molecular details behind the various mechanisms, not only has the field of caspase-9 regulation mechanisms been extended, essential information was gained for further pursuit in an advancement towards the design of caspase-9 activators and inhibitors.

Included in

Chemistry Commons

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