Publication Date

2013-11-21

Availability

Embargoed

Embargo Period

2015-11-21

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Cancer Biology (Medicine)

Date of Defense

2013-11-07

First Committee Member

Edward W. Harhaj

Second Committee Member

Xiangxi M. Xu

Third Committee Member

Mark Pegram

Fourth Committee Member

John R. Bethea

Fifth Committee Member

Wasif Khan

Abstract

Upon virus infection, the innate immune response provides the first line of protection and rapidly induces type I interferons (IFN alpha/beta), which mediate potent antiviral effects. To maintain homeostasis and prevent autoimmunity, IFN production is tightly regulated; however, the mechanisms of negative regulation are poorly understood. Herein, we demonstrate that the A20 binding inhibitor of NF-kappaB 1 (ABIN1) is a novel negative regulator of antiviral signaling. Overexpression of ABIN1 inhibited IFN-beta promoter activation in response to virus infection or poly(I:C) transfection, whereas siRNA-mediated knockdown of ABIN1 enhanced IFN-beta production upon virus infection. ABIN1 interacted with the A20 regulatory molecule TAX1BP1 and was essential for the recruitment of TAX1BP1 and A20 to the noncanonical IkappaB kinases TBK1 and IKKi in response to poly(I:C) transfection. ABIN1 and TAX1BP1 together disrupted the interactions between the E3 ubiquitin ligase TRAF3 and TBK1/IKKi to attenuate lysine 63-linked polyubiquitination of TBK1/IKKi. Finally, an intact ubiquitin binding domain of ABIN1 was essential for ABIN1 to interact with TBK1/IKKi and inhibit IFN-beta production upon poly(I:C) transfection or virus infection. Together, these results suggest that ABIN1 requires its ubiquitin binding domain and cooperates with TAX1BP1 and A20 to restrict antiviral signaling. Viruses have diverse strategies to evade the host immune response, replicate and transform host cells, some of which is under the help of the host proteins. Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 genome encodes a critical protein, Tax, that plays essential regulatory roles in HTLV-1 replication and oncogenic transformation of T lymphocytes. Despite intensive study of Tax, our knowledge of how Tax interfaces with host signaling pathways to regulate virus replication and drive T-cell proliferation and immortalization remains poorly understood. To gain new insight into the mechanisms of Tax function and regulation, we used tandem affinity purification and mass spectrometry to identify novel cellular Tax interacting proteins. This screen identified heat shock protein 90 (HSP90) as a new binding partner of Tax. The interaction between HSP90 and Tax was validated by co-immunoprecipitation assays and co-localization between the two proteins was observed by confocal microscopy. Treatment of HTLV-1 transformed cells with the HSP90 inhibitor 17-DMAG elicited proteasomal degradation of Tax in the nuclear matrix with concomitant inhibition of NF-kappaB and HTLV-1 long terminal repeat (LTR) activation. Knockdown of HSP90 by lentiviral shRNAs similarly provoked a loss of Tax protein in HTLV-1 transformed cells. Finally, treatment of HTLV-1 transformed cell lines with 17-DMAG suppressed HTLV-1 replication and promoted apoptotic cell death. Taken together, our results reveal that Tax is a novel HSP90 client protein and HSP90 inhibitors may exert therapeutic benefits for ATL and HAM/TSP patients. Nuclear factor kappaB (NF-kappaB) is a family of transcription factors that play diverse roles in innate and adaptive immunity, cell survival and development. Persistent activation of NF-kappaB by Tax leads to transformation of T lymphocytes. Under normal conditions, activation of NF-kappaB is transient due to tight regulation by inhibitors of NF-kappaB (IkappaBs), A20, TAX1BP1 and other negative regulators. Zinc finger, AN1-type domain 6 (ZFAND6) contains an A20-like zinc finger (ZF) and an AN1 ZF, domains that are both found in a group of stress associated proteins in plants. Although ZFAND6 has been shown to function as a negative regulator of NF-kappaB through overexpression strategies, its physiological role is unclear. We generated a Zfand6 knockout mouse model to study the effect of genetic deletion of ZFAND6 on the host immune system. Zfand6–/– mice were born at normal Mendelian ratios and T-cell and B-cell development appeared normal. Activation and proliferation of knockout B cells was also similar to wild-type B cells. However, there was a significant increase in IFN-gama production and T helper 1 (Th1) differentiation in vitro of Zfand6–/– CD4+ T cells compared to wild-type CD4+ T cells. Zfand6–/– mice also appeared to be more sensitive to sub-lethal doses of bacterial endotoxin. After intraperitoneal injection with LPS, Zfand6–/– mice produced more IL-1 and exhibited increased mortality compared to the wild-type mice. Furthermore, in response to LPS stimulation, reactive oxygen species (ROS) were upregulated in Zfand6–/– murine embryonic fibroblasts (MEFs) and bone marrow-derived macrophages (BMDMs) compared to wild-type counterparts. In conclusion, these experiments have revealed novel roles of ZFAND6 in regulating endotoxin signaling and Th1 differentiation.

Keywords

ABIN1; HSP90; ZFAND6

Share

COinS