HUMAN ARF1 STRUCTURE--SECRETORY GTP BINDING PROTEIN PATH

人 ARF1 结构--分泌型 GTP 结合蛋白路径

• 批准号: 2188280
• 负责人: DAGMAR RINGE
• 金额: $ 12.53万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-22 至 1998-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2188280
• 关键词: ADP ribosylation; X ray crystallography; brefeldin A; computer program /software; conformation; crystallization; divalent cations; endocytosis; exocytosis; fatty acylation; guanine nucleotide binding protein; guanosine diphosphate; guanosine triphosphate; guanosinetriphosphatases; intracellular transport; magnesium; membrane fusion; mutant; myristates; nucleotide analog; posttranslational modifications; protein isoforms; protein structure function; protein transport; site directed mutagenesis; vesicle /vacuole

GTP-binding proteins are molecular switches that regulate a host of cellular processes including cell growth and division. One class of small, monomeric GTP-binding proteins, the ARF family, plays an essential role in the organization and movement of a wide array of intracellular membranes in all eukaryotic cells. Newly synthesized proteins destined for export must pass from the endoplasmic reticulum through the various compartments of the Golgi, trans-Golgi network (TEN) and secretory vesicles; this transport is accomplished by a vectorial series of vesicle fusions. Vesicles bud off the host membrane and then fuse selectively with the appropriate target membrane. Budding requires the binding of a high molecular weight protein complex (coatomer) to the membrane. And coatomer binding requires prior binding of ARF. ARF proteins (ADP-ribosylation factor; required for cholera toxin dependent ADP-ribosylation of the regulatory component of adenylate cyclase, Gs) are ubiquitous in eukaryotes. The ARF1 gene encodes a highly conserved protein of 181 amino acids in mammals and in the yeast S. cerevisiae. Variants are found in all eukaryotes examined thus far, including plants and insects. The function of ARF has been conserved between yeast and man. Deletion of the two ARF genes in yeast is lethal. Myristoylation of the amino terminus of ARF is essential for functions in vivo. A number of in vitro assays of ARF functions have been described, most related to activities as a regulator of intracellular vesicle movement (e.g. budding and fusion). ARF has been shown to regulate multiple steps in both the exocytic and endocytic pathways. In addition to regulation of ER-Golgi, intra-Golgi transport and endosome fusion, ARF proteins have also been implicated in nuclear vesicle fusion, coatomer recruitment onto ER and Golgi membranes and as the direct regulator of the intrinsic membrane effector, phospholipase D. Further, the inhibition of activation of ARF has been shown to be a critical component in the actions of both Brefeldin A and ilimaquinone; each is currently being evaluated at NCI as a novel anti-tumor agent. The ARF cloned from Giardia lamblia is sufficiently different from mammalian ARF that the design of specific inhibitors of this essential protein can be envisaged as a potential anti- parasitic agent. A better understanding of the structure and functional domains of this essential regulatory molecule would greatly assist both basic cell biology studies and the design of selective inhibitors of critical cellular processes for potential chemotherapeutic advantage. Recombinant human ARF1 with GDP bound has been crystallized in a form diffracting to better than 2A resolution. The crystals are monoclinic, space-group C2 (a=122.8, b=45.8, c=89.3, beta=131.l ), with two ARF molecules in the assymetric unit. The aim of this proposal is to obtain the structures of ARF1 in its GDP-bound and GTP-bound states by X-ray crystallography. An interpretable electron density map of ARF1-GDP has been calculated at 2A resolution. The structures of the myristoylated enzyme in both states will also be determined.

GTP 结合蛋白是调节许多细胞的分子开关 细胞过程包括细胞生长和分裂。小一班， 单体 GTP 结合蛋白 ARF 家族在 多种细胞内膜的组织和运动 在所有真核细胞中。新合成的蛋白质将用于出口 必须从内质网穿过各个隔室 高尔基体、跨高尔基体网络 (TEN) 和分泌囊泡；这 运输是通过一系列矢量囊泡融合来完成的。 囊泡从宿主膜上出芽，然后选择性地与宿主细胞融合 合适的目标膜。萌芽需要高的结合 膜上的分子量蛋白质复合物（涂层异构体）。和涂层剂 绑定需要事先绑定 ARF。 ARF 蛋白（ADP-核糖基化因子；霍乱毒素所需 腺苷酸调节成分的依赖性 ADP-核糖基化 环化酶（Gs）在真核生物中普遍存在。 ARF1基因编码一个高度 哺乳动物和酵母 S 中含有 181 个氨基酸的保守蛋白。 酿酒酵母。迄今为止，在所有检查过的真核生物中都发现了变异， 包括植物和昆虫。 ARF的功能被保留 酵母和人之间。酵母中两个 ARF 基因的缺失是致命的。 ARF 氨基末端的肉豆蔻酰化对于以下功能至关重要 体内。已经描述了许多 ARF 功能的体外测定， 与细胞内囊泡调节剂的活性最相关 运动（例如萌芽和融合）。 ARF 已被证明可以调节 胞吐和内吞途径中的多个步骤。此外 ER-高尔基体、高尔基体内运输和内体融合、ARF 的调节 蛋白质也与核囊泡融合、衣壳异构体有关 募集到内质网和高尔基体膜上，并作为 内在膜效应物磷脂酶 D。此外，抑制 ARF 的激活已被证明是行动中的关键组成部分 Brefeldin A 和 ilimaquinone 两者；目前正在对每个项目进行评估 NCI 作为一种新型抗肿瘤药物。从贾第鞭毛虫克隆的 ARF 是 与哺乳动物 ARF 完全不同的是，特定的设计 这种必需蛋白质的抑制剂可以被设想为一种潜在的抗 寄生剂。更好地理解结构和功能 这种重要调节分子的域将极大地帮助 基础细胞生物学研究和选择性抑制剂的设计 潜在化疗优势的关键细胞过程。 与 GDP 结合的重组人 ARF1 已以一种形式结晶 衍射分辨率优于 2A。晶体为单斜晶系， 空间群 C2 (a=122.8, b=45.8, c=89.3, beta=131.l )，具有两个 ARF 不对称单元中的分子。该提案的目的是获得 X 射线显示 ARF1 在 GDP 结合和 GTP 结合状态下的结构 晶体学。 ARF1-GDP 的可解释电子密度图 以2A分辨率计算。肉豆蔻酰化物的结构 两种状态下的酶也将被测定。