Mechanisms of HTLV-I Tax-mediated NF-kB activation

描述(申请人提供)：人类T细胞白血病病毒1型(HTLV-1)与成人T细胞白血病(ATL)的发生有关，ATL是一种侵袭性的CD4+CD25+恶性肿瘤。Tax是一种编码在HTLV-1基因组Px区域的癌蛋白，它调节病毒和细胞基因的表达，并促进细胞转化，部分是通过激活NF-κB转录因子来实现的。Tax需要NF-κB使原代T细胞永生化，而转化的HTLV-1细胞依赖于NF-κB生存，从而为研究Tax激活NF-κB的机制提供了强有力的理论基础。Tax通过与IKK调节亚基NEMO相互作用持久地激活IκB激酶(IKK)和NF-κB，但其潜在机制尚不清楚。我们的集体研究强化了这样的概念，即Tax劫持宿主泛素机制以激活NF-κB。Tax是通过Lys63(K63)连接的多泛素链实现多泛素化的，需要E2泛素结合酶UBC13来实现Tax泛素化、NEMO结合和NF-κB激活。在我们的初步研究中，我们进行了泛素蛋白质组学筛选，确定了抗凋亡蛋白Mcl-1和E3连接酶TRAF6是Tax的下游靶点。税收诱导的K63连接的Mcl-1泛素化使Mcl-1具有更高的稳定性，并阻止了化疗药物依托泊苷对其的降解。通过酵母双杂交筛选，我们还鉴定了E3/E4酶UBE4B是一种新的Tax相互作用蛋白，它可能调节Tax K63连锁的多泛素化和NF-κB的激活。我们还证明了TAX已经篡夺了TAX1BP1的适配功能，TAX1BP1是NF-κB抑制剂和泛素编辑酶A20的骨架分子，促进NF-κB信号转导。在这场竞争性更新中，我们将继续建立税收劫持泛素机械组件的机制，以促进持续的NF-κB激活和细胞生存。该提案的具体目的是：(1)确定税收稳定Mcl-1的机制；(2)确定宿主泛素机制在税收诱导的NF-κB激活中的作用；(3)确定TAX1BP1在税收激活NF-κB和白血病发生中的作用。这些研究将阐明逆转录病毒癌蛋白如何劫持宿主泛素化机制，以抑制细胞死亡和促进细胞转化。

DESCRIPTION (provided by applicant): Human T-cell leukemia virus type 1 (HTLV-1) is linked to the genesis of adult T-cell leukemia (ATL), an aggressive CD4+CD25+ malignancy. Tax is an oncoprotein encoded in the pX region of the HTLV-1 genome that regulates viral and cellular gene expression and promotes cell transformation, in part, by constitutive activation of the NF-κB transcription factor. Tax requires NF-κB for the immortalization of primary T cells and HTLV-1 transformed cells are dependent on NF-κB for their survival, thereby providing a strong rationale for studying the mechanisms used by Tax to activate NF-κB. Tax activates the IκB kinase (IKK) and NF-κB persistently by interacting with the IKK regulatory subunit NEMO, however the underlying mechanisms remain poorly understood. Our collective studies reinforce the notion that Tax hijacks the host ubiquitin machinery to activate NF-κB. Tax is polyubiquitinated via Lys63 (K63)-linked polyubiquitin chains and requires the E2 ubiquitin conjugating enzyme Ubc13 for Tax ubiquitination, NEMO binding and NF-κB activation. In our preliminary studies we have conducted ubiquitin proteomics screening which has identified the anti-apoptotic protein Mcl-1 as a downstream target of Tax and the E3 ligase TRAF6. Tax-induced K63-linked polyubiquitination of Mcl-1 endowed Mcl-1 with enhanced stability and prevented its degradation by the chemotherapy drug etoposide. Using a yeast two-hybrid screen we have also identified the E3/E4 enzyme UBE4B as a novel Tax interacting protein that may regulate Tax K63-linked polyubiquitination and NF-κB activation. We have also demonstrated that Tax has usurped the adaptor function of TAX1BP1, a scaffold molecule for the NF-κB inhibitor and ubiquitin-editing enzyme A20, to promote NF-κB signaling. In this competitive renewal, we will continue to establish the mechanisms by which Tax hijacks ubiquitin machinery components to promote persistent NF-κB activation and cell survival. The specific aims of the proposal are to: (1) determine the mechanisms of Mcl-1 stabilization by Tax; (2) determine the role of the host ubiquitin machinery in Tax-induced NF-κB activation and (3) determine the role of TAX1BP1 in Tax activation of NF-κB and leukemogenesis. These studies will elucidate how a retroviral oncoprotein hijacks host ubiquitination machinery to inhibit cell death and promote cell transformation.