Effect of HIV Tat on endothelial cell function

描述(申请人提供)：感染HIV-1的患者表现出明显的内皮细胞形态和功能异常。内膜组织、增殖、细胞凋亡和分化的紊乱可见于许多艾滋病相关的病理情况，涉及异常的血管功能和血管生成行为。病毒辅助蛋白TAT被认为是一种潜在的内皮原细胞因子，因为它能够连接内皮整合素和血管内皮生长因子受体-2。虽然HIV-1TAT的促血管生成作用已经被密切关注，但涉及的信号通路却知之甚少。上一次资助期的研究表明，TAT引起内皮细胞NADPH氧化酶的快速激活，导致下游墨水MAPK的激活。在鉴定内皮细胞中该氧化酶的关键激活亚基p47Phox时，我们发现该蛋白与内皮细胞骨架有结构性的联系，这表明该氧化酶的物理靶向是氧化剂敏感的信号复合体，如那些含有酪氨酸激酶和磷酸酶的复合体。对p47结合伙伴的搜索得到了孤立的适配器TRAF4，而p47Phox与TRAF4的结合似乎是TAT激活墨水所必需的。对TRAF4相互作用蛋白的二次筛选产生了富含Pro的酪氨酸激酶-2(PYK2)支架Hic-5。第三次筛选以开放形式的p47Phox为诱饵，发现了一种额外的蛋白--内皮衍生基因-1(EG-1)，它可能与肿瘤血管生成有关。我们假设TAT通过定点产生氧化剂来启动与Hic-5和EG-1相关的信号复合体的信号传递。这项资助的广泛目标是进一步确定内皮细胞中通过Hic-5和EG-1依赖TAT的氧化剂信号的性质和意义。

DESCRIPTION (provided by applicant): Patients infected with HIV-1 display marked abnormalities in endothelial morphology and function. Derangements in intimal organization, proliferation, apoptosis, and differentiation are seen in a number of AIDS-related pathologic conditions involving aberrant vascular function and angiogenic behavior. The viral accessory protein Tat has been implicated as a potential endothelial protocytokine, given its ability to ligate endothelial integrins and Vascular Endothelial Growth Factor Receptor-2. While the pro-angiogenic effects of HIV-1 Tat have been closely scrutinized, the signal pathways involved are poorly understood. Studies in the previous funding period showed that Tat caused rapid activation of an endothelial cell NADPH oxidase, leading to downstream INK MAPK activation. During characterization of a key activating subunit of the oxidase, p47phox, in endothelial cells, we found constitutive association of this protein with the endothelial cytoskeleton, suggesting physical targeting of the oxidase to oxidant-sensitive signaling complexes such as those containing tyrosine kinases and phosphatases. A search for p47-binding partners yielded the orphan adapter TRAF4, and the association ofp47phox with TRAF4 appeared necessary for activation of INK by Tat. A secondary screen for TRAF4-interacting proteins yielded the Proline-rich Tyrosine Kinase-2 (Pyk2)scaffold Hic-5. A third screen using an open form ofp47phox as bait revealed an additional protein, Endothelial-derived Gene-1 (EG-1), which may be associated with tumor angiogenesis. We hypothesize that Tat initiates signaling through site-directed production of oxidants to Hic-5 and EG-1-related signaling complexes. The broad objectives of this grant are to further define the nature and significance of Tat dependent oxidant signaling through Hic-5 and EG-1 in endothelial cells.