Date

September 2012

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Cell and Developmental Biology

Institution

Oregon Health & Science University

Abstract

Following DNA damage, the sequence-specific transcription factor p53 functions as a node for orchestrating biological responses such as cell-cycle arrest, apoptosis, and senescence by promoting the transactivation of various target genes. Importantly, the ability of p53 to orchestrate these diverse processes critically depends on the regulation of p53 by cellular cofactors and modifying enzymes. Notably, p53 acetylation is essential for its transcriptional activation following DNA damage. p53 is deacetylated by Sirtuin 1 (SIRT1), anicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylase, thereby repressing p53 transcriptional activation. Therefore, understanding how the SIRT1-p53 axis is regulated during the DNA damage response is essential for gaining insight into the mechanisms controlling p53 activation. Here we demonstrate that the membrane trafficking protein PACS-2 inhibits SIRT1-mediated deacetylation of p53 to modulate the cellular response to DNA damage. We demonstrate that PACS-2 directly interacts with SIRT1 and inhibits SIRT1-mediated deacetylation of p53 in vivo. Moreover, PACS-2 deficient cells have aSIRT1-dependent reduction in p53 acetylation and p21 expression, resulting in increased apoptosis and reduced clonogenic survival in a p53- and p21-dependent manner following DNA damage. Based on these observations, I propose that PACS-2 is a critical regulator of the SIRT1-p53-p21 axis to modulate the DNA damage response. In addition, the studies in this dissertation describe how the identification of PACS-2 as essential for HIV-1 Nef-mediated immune evasion lead to the discovery of a small molecule inhibitor that targets a critical PACS-2-dependentstep in HIV-1 Nef action. To evade CD8+ T-cell destruction, HIV-1 Nef assembles a Src family kinase (SFK)-ZAP-70-PI3K complex to trigger MHC-I downregulation. These studies demonstrate that chemical inhibition of the Nef-SFK interaction disrupts formation of the multi-kinase complex and represses Nef-mediated MHC-I downregulation in primary CD4+ T-cells. Moreover, transport studies reveal Nef assembles the multi-kinase complex to trigger downregulation of cell-surface MHC-I early following infection. By three days post-infection, Nefswitches to a stoichiometric mode that prevents surface delivery of newly synthesized MHC-I. Together, these studies suggest Nef orchestrates a regulated molecular program to evade immune surveillance and provides new insights into the mechanism of Nef action. Together, these studies significantly contribute to the understanding of how a membrane trafficking protein can have diverse cellular roles linking viral pathogenesis to DNA damage and tumor suppression pathways. Future studies must systematically determine how PACS-2 coordinates these various functional roles and the cellular and biological contexts for which these regulatory networks are essential. Given the diverse nature of the cellular proteins and processes modulated by PACS-2, the implications of this data are far reaching and suggest that PACS-2 may function in various cellular processes such as gene regulation, cell growth and survival as well as biological processes such as cancer, viral immunity, aging, and metabolism.

Identifier

doi:10.6083/M4MG7MH2

School

School of Medicine

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