REGULATION OF ADP-RIBOSYLATION FACTOR

ADP-核糖基化因子的调节

• 批准号: 5201369
• 负责人: P RANDAZZO
• 金额: --
• 依托单位: DIVISION OF CANCER TREATMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5201369
• 关键词: ADP ribosylation; binding proteins; cofactor; detergents; guanine nucleotide binding protein; myristates; phosphatidylinositols; protein purification; protein structure function; protein transport

The main objective of this work is to elucidate the biochemical mechanisms regulating the GTP-binding protein ARF and, in so doing, to further our understanding of the role of ARF in membrane traffic. We found that the amino terminal alpha helix of ARF and covalently bound myristate form a GTP- and phospholipid-sensitive switch and that this domain is necessary for interaction with ARF GAP and Gs. We are currently testing the hypothesis that this is an effector domain of the protein. ARF's interaction with target proteins is dependent on phospholipids. We found that ARF has both nonspecific interactions with lipids and detergents and a specific interaction with phosphatidylinositol 4,5- bisphosphate (PIP2). These phospholipid interactions could be dissociated. The amino terminus of ARF was necessary for the nonspecific association with phospholipids but was not involved in specific interactions with PIP2. Using mutational analysis we have preliminary data implicating several specific residues of ARF in PIP2 binding. We are further testing the hypothesis that ARF is a PIP2 binding protein and the related hypothesis that PIP2 acts as a cofactor for ARF interaction with target proteins. Having found that PIP2 and PA were required for ARF - ARF GAP interaction, we have been able to make some progress in the purification of the latter protein. ARF GTPase activating protein copurified with clathrin assembly protein complex 2. We are currently testing the hypothesis that AP-2 is ARF GAP. These data support the hypothesis that the binding and hydrolysis of GTP by ARF regulates the assembly of coat proteins on transport vesicles.

这项工作的主要目的是阐明生化机制 调节GTP结合蛋白ARF，并在这样做的过程中，进一步我们 了解ARF在膜运输中的作用。 我们发现，ARF的氨基末端α螺旋与 肉豆蔻酸酯形成GTP和磷脂敏感开关， 结构域是与ARF GAP和Gs相互作用所必需的。 我们目前正在 测试这是蛋白质的效应域的假设。 ARF与靶蛋白的相互作用依赖于磷脂。 我们 发现ARF与脂质有非特异性相互作用， 洗涤剂和与磷脂酰肌醇4，5- 二磷酸盐（PIP 2）。 这些磷脂相互作用可能是 分离 ARF的氨基端是非特异性的 与磷脂结合，但不涉及特定的 与PIP 2的互动 通过突变分析，我们初步 这些数据暗示了PIP 2结合中ARF的几个特定残基。 我们 进一步检验了ARF是PIP 2结合蛋白的假设， PIP 2作为ARF相互作用的辅因子与 靶蛋白。 已经发现PIP 2和PA是ARF - ARF GAP相互作用所必需的， 我们已经能够在后者的纯化方面取得一些进展 蛋白 与网格蛋白组装体共纯化的ARF GT3活化蛋白 蛋白质复合物2. 我们目前正在测试AP-2是 ARF间隙。 这些数据支持这样的假设，即结合和 ARF对GTP的水解调节了蛋白质外壳的组装， 运输囊泡