HUMAN ARF1 STRUCTURE--SECRETORY GTP BINDING PROTEIN PATH

GTP结合蛋白是一种分子开关，调节一系列 包括细胞生长和分裂在内的细胞过程。一班小的， 单体GTP结合蛋白，ARF家族，在 广泛的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(10个)和分泌囊泡； 运输是通过一系列的囊泡融合来完成的。 囊泡从宿主膜上发芽，然后选择性地与 合适的靶膜。萌芽需要一个高的 分子量蛋白质复合体(辅酶)到膜上。和辅助者 绑定需要ARF的事先绑定。 ARF蛋白(ADP-核糖化因子；霍乱毒素所必需 腺苷调节成分的依赖ADP-核糖化 在真核生物中普遍存在。ARF1基因编码高度的 哺乳动物和酵母菌中181个氨基酸的保守蛋白质。 酿酒。到目前为止，在所有被检查的真核生物中都发现了变体， 包括植物和昆虫。ARF的功能得到了保留 在酵母和人类之间。酵母中这两个ARF基因的缺失是致命的。 ARF氨基末端的肉豆蔻酰化是重要的功能 活着。已经描述了许多ARF功能的体外检测， 大多数与作为细胞内小泡调节器的活动有关 运动(如萌芽和融合)。ARF已被证明可以调节 胞外和胞内途径都有多个步骤。除了……之外 内质网高尔基体、高尔基体内转运和内体融合的调控 蛋白质也与核泡融合有关 在内质网和高尔基体膜上的募集，并作为内质网和高尔基体膜的直接调节因子 内源性膜效应器，磷脂酶D，进一步，抑制 激活ARF已被证明是行动中的一个关键组成部分 Brefeldin A和ILIMAQUONE；目前正在进行评估 NCI作为一种新型抗肿瘤药物。从蓝氏贾第鞭毛虫克隆的ARF是 与哺乳动物ARF足够不同的是，特定的设计 这种基本蛋白质的抑制剂可以被认为是一种潜在的抗 寄生虫剂。对结构和功能有更好的理解 这一重要调控分子的结构域将极大地帮助 细胞生物学基础研究及其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以一种形式结晶 衍射率高于2倍分辨率。晶体是单斜晶系， C2空间群(a=122.8，b=45.8c=89.3，β=131.1)，有两个ARF 不对称单位中的分子。这项提议的目的是获得 ARF1在GDP结合态和GTP结合态的X射线结构 结晶学。一张可解释的ARF1-GDP电子密度图 在2a分辨率下进行了计算。肉豆蔻酰化的结构 还将测定这两种状态下的酶。

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.