CHARACTERIZATION OF THE ARF FAMILY OF REGULATORY GTP-BINDING PROTEINS

GTP 调节蛋白 ARF 家族的表征

• 批准号: 3774556
• 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/3774556
• 关键词: Golgi apparatus; antineoplastic antibiotics; cell growth regulation; cellular respiration; endoplasmic reticulum; genetic regulation; guanine nucleotide binding protein; human tissue; macrolide antibiotics; molecular cloning; nucleic acid sequence; protein structure function; suppressor mutations; tissue /cell culture; yeasts

The ARF proteins are a sub-family of structurally and functionally conserved 21 kDa GTP binding proteins. ARF has now been shown to have essential functions in the maintenance of the integrity and proper functioning of the endoplasmic reticulum, Golgi complex, and endosome compartments. These activities are the targets for the actions of Brefeldin A, a fungal metabolite under development in the the Developmental Therapeutics Program as a potential anti-neoplastic agent in man. Work described in this project focuses on characterizing the members of the ARF family with regard to sequence, cellular location, and functions. We have discovered that in humans this family consists not only of the five previously identified gene products, but of more than 10 structurally related genes and their products. We are engaged in cloning and sequencing this newly enlarged family to begin to document its size, functions and importance to cell physiology and pathophysiology. In addition, as sequence information becomes available we continue to produce more specific antisera for diagnostic purposes and immunolocalization. We have continued to exploit simpler eukaryotic organisms, particularly yeast, to identify new functions for ARF proteins. We will perform functional screens to identify extragenic suppressors of ARF mutations in efforts to identify other components of the ARF pathway and potential regulators of ARF activities. Expression of a number of mutants in yeast has also served as background for similar studies now underway to express wild-type and mutant ARF proteins in mammalian cells in culture. An exhaustive screen has now identified at least one temperature sensitive allele of ARF1 which will be used to assist in defining functions of ARF, consequences of an arf null cell, and functional screens for extragenic suppressors of the arf null cell. The three dimensional crystal structure of ARF is also being solved to aid in the determination of critical functional domains and ultimate design of specific inhibitors.

ARF 蛋白是结构和功能上的一个亚家族 保守的 21 kDa GTP 结合蛋白。 ARF现已被证明具有 维护完整性和正确性的基本职能 内质网、高尔基复合体和内体的功能 隔间。 这些活动是行动的目标 Brefeldin A，一种正在开发的真菌代谢物 作为潜在抗肿瘤药物的发展治疗计划 在人身上。 该项目中描述的工作重点是描述 ARF 家族成员的序列、细胞位置和 功能。 我们发现，在人类中，这个家族不仅由 之前已鉴定出 5 种基因产物，但数量超过 10 种 结构相关的基因及其产物。 我们从事克隆 对这个新扩大的家族进行测序以开始记录其规模， 功能以及对细胞生理学和病理生理学的重要性。 在 此外，随着序列信息的出现，我们将继续 生产用于诊断目的的更特异的抗血清 免疫定位。 我们继续开发更简单的真核生物，特别是 酵母，以确定 ARF 蛋白的新功能。 我们将表演 识别 ARF 突变的基因外抑制因子的功能筛选 努力确定 ARF 途径的其他组成部分和潜力 ARF 活动的监管机构。 多个突变体的表达 酵母也作为目前正在进行的类似研究的背景 在培养的哺乳动物细胞中表达野生型和突变型 ARF 蛋白。 详尽的屏幕现已识别出至少一个温度 ARF1 的敏感等位基因将用于协助定义 ARF 的功能、arf 无效细胞的后果以及功能 筛选arf无效细胞的外源抑制因子。 ARF的三维晶体结构也正在被解决 帮助确定关键功能域和最终 特异性抑制剂的设计。