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

描述（由申请人提供）：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CD4+CD25+恶性肿瘤。Tax是HTLV-1基因组pX区域编码的一种癌蛋白，通过NF-κB转录因子的组成性激活，调节病毒和细胞基因表达，促进细胞转化。Tax需要NF-κB来实现原代T细胞的永生化，HTLV-1转化的细胞依赖NF-κB存活，因此为研究Tax激活NF-κB的机制提供了强有力的理论依据。Tax通过与IKK调控亚基NEMO相互作用持续激活ikb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税收劫持宿主泛素机制来激活NF-κB的概念。Tax通过Lys63 （K63）连接的多泛素链被多泛素化，并且需要E2泛素偶联酶Ubc13来实现Tax泛素化、NEMO结合和NF-κB活化。在我们的初步研究中，我们进行了泛素蛋白质组学筛选，发现抗凋亡蛋白Mcl-1是Tax和E3连接酶TRAF6的下游靶点。税收诱导的K63-linked Mcl-1多泛素化增强了Mcl-1的稳定性，并阻止了其被化疗药物依托泊苷降解。通过酵母双杂交筛选，我们还发现E3/E4酶UBE4B是一种新的税收相互作用蛋白，可能调节税收k63相关的多泛素化和NF-κB激活。我们还证明，tax1取代了TAX1BP1 （NF-κB抑制剂和泛素编辑酶A20的支架分子）的接头功能，以促进NF-κB信号传导。在这种竞争性更新中，我们将继续建立Tax劫持泛素机制组件以促进持续NF-κB激活和细胞存活的机制。该建议的具体目标是：(1)确定通过税收稳定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来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.