THE REGULATION OF GOLGI COAT PROTEIN BY ARF AND BREFELDIN A

ARF和Brefeldin A对高尔基体外壳蛋白的调节

• 批准号: 3838155
• 负责人: R A KAHN
• 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/3838155
• 关键词: ADP ribosylation; Golgi apparatus; X ray crystallography; antineoplastic antibiotics; clone cells; exocytosis; gene expression; human tissue; intracellular membranes; intracellular transport; macrolide antibiotics; membrane fusion; molecular cloning; protein structure function

The main objectives of this project are the elucidation of the cellular functions of the family of ADP-ribosylation factors (ARFs) and the mechanism of their regulation. As Brefeldin A (BFA) has recently been shown to affect ARF functions and cellular distribution, we have recently expanded our efforts to include a better understanding of the mechanism of BFA action. Specific inhibitors of ARF activities have been exploited to provide evidence for the role of ARF proteins in a variety of intracellular membrane events. Anti-ARF peptides were found to be potent inhibitors of ER-Golgi transport, intra-Golgi transport, and endosome-endosome fusion. In addition, sensitivity of each of these assays, as well as nuclear membrane fusion, to GTPgammaS was shown to be mediated by ARF proteins. A search for regulators of ARF activity has identified an activity on Golgi membranes which appears to activate ARF. This activity is being investigated as a potential site of action of Brefeldin A and for its potential role as regulator of the protein secretory pathway. The ability of Brefeldin A to alter the structure of the Golgi and location of specific coat proteins (ARF and beta-COP) is being investigated in concert with the development of Brefeldin A as a potential anticancer agent. We have used inducible mammalian expression vectors to control the expression of ARF genes. These cell lines will be used to analyze the effects of varied expression of wild-type and mutant ARF proteins on the secretory pathway and on the integrity of intracellular organelles, e.g. Golgi complex. The three dimensional crystal structure of ARF is also being solved to aid in the determination of critical functional domains and ultimate design of inhibitors.

该项目的主要目标是阐明细胞的 ADP-核糖基化因子（ARF）家族的功能和 其调节机制。 Brefeldin A（BFA） 显示影响ARF功能和细胞分布，我们最近 扩大了我们的努力，包括更好地了解该机制 BFA的行动。 ARF活性的特异性抑制剂已被开发用于提供 ARF蛋白在多种细胞内 膜事件 抗ARF肽被发现是ARF的有效抑制剂。 内质网-高尔基体转运、高尔基体内转运和内体-内体融合。 此外，这些检测中的每一种的灵敏度以及核 膜融合，GTP γ S显示由ARF蛋白介导。 对ARF活性调节剂的研究已经确定了一种活性， 似乎激活ARF的高尔基体膜。 这项活动正在 作为布雷菲德菌素A潜在作用位点进行研究， 作为蛋白质分泌途径的调节剂的潜在作用。 的 Brefeldin A改变高尔基体结构和位置的能力 特定外壳蛋白（ARF和β-COP）的研究正在进行中。 与布雷菲德菌素A作为潜在抗癌药物的发展相一致 剂 我们已经使用可诱导的哺乳动物表达载体来控制 ARF基因的表达。 这些细胞系将用于分析 野生型和突变型ARF蛋白的不同表达对 分泌途径和细胞内细胞器的完整性， 例如高尔基复合体。 ARF的三维晶体结构也正在解决， 有助于确定关键功能域和最终的 抑制剂的设计