Date of Award

Spring 1-1-2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Leslie Leinwand

Second Advisor

Ding Xue

Third Advisor

Tom Blumenthal

Abstract

The nematode Caenorhabditis elegans has become a successful animal model for biomedical research, particularly in studying mechanisms of cell death and human disease. The conservation of critical biological pathways between C. elegans and higher organisms, together with the simplicity and cost-effectiveness of cultivation, make for an effective in vivo animal model that is amenable to genetic dissection and pharmacological manipulation. My thesis describes how I used C. elegans as an animal model to advance our understanding of caspase regulation and HBV pathogenesis.

First I report that inactivation of the C. elegans csp-3 and csp-2 genes, which encode two proteins similar to the CED-3 caspase, causes somatic and germ cells that normally live to undergo apoptosis in a CED-3-dependent manner, respectively. Biochemical analysis reveals that both CSP-3 and CSP-2 associate with the CED-3 zymogen and inhibit zymogen auto-activation. However, neither CSP-3 nor CSP-2 blocks CED-3 activation induced by CED-4 nor inhibit the activity of the activated CED-3 protease. Therefore CSP-3 and CSP-2 employ a novel mechanism to protect somatic and germ cells from inadvertent apoptosis.

Then I used C. elegans as an animal model to study the key pathogenic protein of HBV, HBx. I show that expression of HBx in C. elegans induces both necrotic and apoptotic cell death, mimicking an early event of liver infection by HBV. Genetic and biochemical analyses indicate that HBx interacts directly with the Bcl-2 homolog CED-9, and triggers an increase in cytosolic Ca2+ and induction of both apoptosis and necrosis. Moreover, Bcl-2 binds HBx in human hepatic cells and can substitute for CED-9 in mediating HBx-induced cell killing in C. elegans, suggesting that CED-9 and Bcl-2 are conserved cellular targets of HBx. In addition, a genetic screen has been carried out to identify genes important for HBx-induced cell death. I report the identification of the vps-29 gene, which encodes a retromer component, as an important regulator of HBx induced cell death in C. elegans. Loss of vps29 as well as other retromer components strongly and specifically reduced HBx induced ectopic cell death, suggesting that the retromer plays an important regulatory role in HBV replication and pathogenesis.

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