HUMAN ARF1 STRUCTURE--SECRETORY GTP BINDING PROTEIN PATH

GTP结合蛋白是一种分子开关， 包括细胞生长和分裂的细胞过程。一类小的， 单体GTP结合蛋白，ARF家族，在 一系列细胞内膜的组织和运动 在所有真核细胞中。用于出口的新合成蛋白质 必须从内质网通过各个隔室 高尔基体，trans-Golgi网络（TEN）和分泌囊泡;这 通过一系列载体囊泡融合完成转运。 囊泡从宿主膜上出芽，然后选择性地与宿主细胞融合。 合适的靶膜。发芽需要一种高浓度的 分子量蛋白质复合物（涂层）的膜。和coatomer 绑定需要ARF的优先绑定。 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。ARF 1基因编码一种高度 哺乳动物和酵母中的181个氨基酸的保守蛋白。 啤酒。到目前为止，在所有的真核生物中都发现了变异体， 包括植物和昆虫。ARF的功能是保守的 在酵母中，两个ARF基因的缺失是致命的。 ARF氨基末端的豆蔻酰化对于ARF的功能是必需的， vivo.已经描述了许多ARF功能的体外测定， 与作为胞内囊泡调节剂的活性最相关 运动（例如出芽和融合）。ARF已被证明可以调节 胞吐和胞吞途径中的多个步骤。除了 内质网-高尔基体、高尔基体内转运和核内体融合的调节 蛋白质还涉及核囊泡融合、外被体融合、核融合和核融合。 募集到ER和高尔基体膜，并作为直接调节剂， 内源性膜效应物磷脂酶D。此外，抑制 ARF的激活已被证明是行动的关键组成部分， Brefeldin A和伊利马醌;目前正在对每一种进行评估， NCI是一种新型抗肿瘤药物。从蓝氏贾第鞭毛虫中克隆的ARF是 与哺乳动物ARF有足够的不同， 这种必需蛋白的抑制剂可以被设想为潜在的抗- 寄生虫更好地理解结构和功能 这种重要的调节分子的结构域将大大有助于 基础细胞生物学研究和选择性抑制剂的设计 关键的细胞过程，以获得潜在的化疗优势。 与GDP结合的重组人ARF 1已结晶为 分辨率优于2A。晶体是单斜晶， 空间群C2（a=122.8，B=45.8，c=89.3，β = 131.1），具有两个ARF 不对称单位中的分子。该提案的目的是获得 ARF 1在GDP结合态和GTP结合态的结构 结晶学一个可解释的ARF 1-GDP电子密度图 分辨率为2A。肉豆蔻酰化的结构 还将测定两种状态下的酶。

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.