Effect of HIV Tat on endothelial cell function

描述（由申请方提供）：感染HIV-1的患者显示内皮形态和功能明显异常。内膜组织、增殖、凋亡和分化的紊乱见于许多与AIDS相关的病理状态，包括异常的血管功能和血管生成行为。病毒辅助蛋白达特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-1达特的促血管生成作用已被仔细研究，但对所涉及的信号通路知之甚少。上一个资助期的研究表明，达特引起内皮细胞NADPH氧化酶的快速激活，导致下游INK MAPK激活。在表征的一个关键活化亚基的氧化酶，p47 phox，在内皮细胞中，我们发现这种蛋白质与内皮细胞骨架的组成性协会，这表明物理靶向的氧化酶氧化剂敏感的信号复合物，如那些含有酪氨酸激酶和磷酸酶。对p47结合配偶体的搜索产生了孤儿衔接子TRAF 4，并且p47 phox与TRAF 4的结合似乎是达特激活INK所必需的。TRAF 4相互作用蛋白的二次筛选产生了富含脯氨酸的酪氨酸激酶-2（Pyk 2）支架Hic-5。第三次筛选使用p47 phox的开放形式作为诱饵，发现了一种额外的蛋白质，内皮衍生基因-1（EG-1），它可能与肿瘤血管生成有关。我们假设达特通过定点产生氧化剂来启动Hic-5和EG-1相关信号复合物的信号传导。这项资助的主要目的是进一步确定内皮细胞中通过Hic-5和EG-1的达特依赖性氧化剂信号传导的性质和意义。

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.