HTLV-1 TAX 持续性激活 IKK-NF-κB的机制探讨

Human T-cell lymphotropic virus type 1(HTLV-1) has been characterized as the etiological agent of adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 encodes a 40-KD regulatory protein, TAX, that can activate various intracellular signaling pathways including IKK/NF-kB, PI3K/AKT and SRF pathways, among which IKK/NF-kB signaling pathway is thought to be critical in onset of ATL. A lot of studies have been focused on the mechanism of TAX-mediated activation of IKK/NF-kB; however, exactly how TAX activates IKK is still a mystery and further studies are needed.. To understand exactly how IKK is activated by TAX, we used an in vitro cell-free system to reconstitute TAX-dependent IKK activation. Using this assay, our fractionation studies revealed that several factors are required for IKK activation by TAX. One factor was further fractionated, purified and identified by mass spectrometry as the ubiquitin conjugating enzyme E2 UBE2L3 (UbcH7). Screening of recombinant E2s using the cell-free system showed that two other E2s, UBE2H (UbcH2) and UBE2D3 (UbcH5c), can confer TAX-dependent IKK activation. The importance of these E2s in supporting TAX-dependent IKK activation was further verified in vivo by using dominant negative forms of these E2s, which inhibit TAX-dependent NF-kB activation when overexpressed.. We also characterized the requirement of TAK1, Ubc13 and TRAF6 for IKK activation by TAX, which have been controversial in the literature. Our both in vitro and in vivo studies revealed that neither TAK1, TRAF6 nor Ubc13 is involved in TAX-dependent IKK activation.. TAX-dependent IKK activation might provide probably the simplest system to understand biochemical mechanism of IKK activation and understanding the mechanism might also help us find therapies to ATL. Further studies are underway to elucidate the detailed biochemical mechanism of IKK activation by TAX.

人类T淋巴细胞1型病毒HTLV-1 TAX通过持续性激活IKK-NF-κB信号通路导致成人T细胞白血病ATL。TAX持续性激活IKK的生化机制函待深入研究。为避免现有细胞和动物水平上研究的局限，我们发展了基于TAX激活IKK的体外无细胞系统。该系统避免了TAX激活的多种信号通路和IKK-NF-κB正反馈通路叠加在一起而无法分割的情况。利用该系统，我们将1）使用经典液相色谱蛋白纯化系统来分离纯化TAX激活IKK所需的宿主因子，2）探讨这些因子与TAX共同激活IKK的深入机制，3）探讨TAX自身的生化功能，4）TAX抑制A20、CYLD去泛素化酶活性以及A20、CYLD的诱导表达达到持续性激活IKK的机制。对TAX激活IKK机制的研究将有助于我们一方面深入理解TAX以及HTLV-1病毒引起肿瘤的原因，另一方面深入理解慢性炎症NF-κB通路持续性激活在肿瘤生成中扮演的重要功能。

人T细胞白血病病毒Ⅰ型（HTLV-1）是第一种被发现的与人类疾病相关的逆转录病毒。它感染着全球超过1500万人，并能够引起包括成年人T细胞白血病（ATL）、HTLV-1相关性脊髓病/热带痉挛性瘫痪（HAM/TSP）以及HTLV-1葡萄膜炎（HU）在内的诸多严重疾病。HTLV-1感染后一旦发展成为ATL，由于其病程发展快且预后差，85%的患者会在发病后4年内死亡。遗憾的是，针对HTLV-1，至今仍没有疫苗或者有效的治疗方法。..在HTLV-1的基因组上，除了编码逆转录病毒所必须的结构和功能蛋白以外，还编码着一系列的调节蛋白，包括Tax、Rex、HBZ等。其中，最重要并且是研究最为广泛的，就是Tax。已有的研究表明，Tax激活IKK-NF-κB通路的能力，对于HTLV-1引起ATL等疾病是必要的。但是，其具体激活的机制并不明了。..为了探究Tax激活IKK-NF-κB的具体的生化机制，本项目组首先在体外无细胞体系中建立了一个Tax激活IKK的生化反应。利用这个反应，结合经典生物化学分离纯化的方法，他们鉴定到宿主细胞内的几种特定的E2泛素转移酶——UbcH2、UbcH5c和UbcH7——对于Tax的活力是必要的。进一步的，他们发现Tax是一个E3泛素连接酶，它能够利用上述E2合成非锚定的混合型多聚泛素链（free mixed-linkage polyubiquitin chains）。而这种泛素链在体外可以直接地激活IKK。..这项研究揭示了Tax的新型生化功能以及它在激活IKK-NF-κB通路中具体的生化机制，阐明了前人研究中的诸多矛盾和疑问，为后续相关研究以及HTLV-1感染的治疗提供了理论依据。