THE REGULATION OF GOLGI COAT PROTEIN BY ARF AND BREFELDIN A

这个项目的主要目标是阐明细胞 腺苷二磷酸核糖化因子家族的功能 它们的调控机制。正如Brefeldin A(BFA)最近 被证明影响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 博鳌亚洲论坛的行动。 已经开发了ARF活性的特定抑制剂来提供 ARF蛋白在多种细胞内的作用的证据 膜事件。抗ARF多肽被发现是一种有效的抑制 ER-高尔基体转运、高尔基体内转运和内体-内体融合。 此外，每种化验方法的灵敏度以及核 与GTP-GammaS的膜融合是由ARF蛋白介导的。 对ARF活动监管机构的搜索发现了一项活动 高尔基体膜似乎能激活ARF。这项活动正在进行 作为Brefeldin A潜在作用部位的研究及其 蛋白质分泌途径的潜在调节作用。这个 Brefeldin A改变高尔基体结构和位置的能力 正在对特定外壳蛋白(ARF和β-COP)的 与Brefeldin A作为潜在抗癌药物的发展相一致 探员。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。这些细胞系将被用来分析 野生型和突变型ARF蛋白的不同表达对血管紧张素转换酶的影响 分泌途径和细胞内细胞器的完整性， 例如高尔基复合体。 ArF的三维晶体结构也被解算为 帮助确定关键功能域和最终 抑制剂的设计。

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.