RIN1, A NOVEL RAS-INHIBITORY PROTEIN IN YEAST

描述(申请人提供)：RAS家族的小鸟嘌呤3‘-三磷酸酶(GTP酶)包括一组分子开关，连接细胞表面的受体与调节细胞增殖和分化的信号通路。RAS蛋白在真核生物中高度保守。在30%的人类肿瘤中发现了RAS的突变形式，这些突变形式锁定在鸟氨酸三磷酸(GTP)结合的状态，从而通过与其靶蛋白相互作用来持续推动细胞增殖。本申请描述了一种在萌芽酵母中的新型蛋白质，它似乎具有两种不同的功能，尽管它的长度只有68个氨基酸。编码这种蛋白的基因命名为RIN1(RAS抑制物1)，在遗传上是RAS的抑制因子，Rin1蛋白在体内与GTP结合的RAS以一种表明它竞争与RAS靶蛋白相同的结合位点的方式结合。由于Rin1位于内质网的膜上，而不是与大多数RAS一起位于细胞表面，因此推测Rin1的一个功能是在其翻译后修饰内质网的过程中保持RAS的非活性状态。该项目的长期目标是开发一种形式的Rin1，有效地抑制细胞表面GTP结合的RAS，用于治疗RAS相关的恶性肿瘤。Rin1的第二个功能是处理或运输内质网中糖基磷脂酰肌醇(GPI)锚定的蛋白。据推测，这一功能是通过与参与蛋白质分泌的RAS家族的第二个成员相关联来实现的。本项目的具体目的是：1)了解Rin1的膜拓扑结构，并确定RAS相互作用区域。这是开发一种针对细胞表面RAS抑制进行优化的Rin1形式的第一步。2)了解Rin1在GPI蛋白锚定或分泌中的作用。这将涉及识别Rin1的第二个目标。这一目标也可能有助于ER中蛋白质分类的新领域。3)确定酵母RAS是否能从内质网发出信号，并创造一种更有效的基于Rin1的RAS抑制剂。本研究的目的是评估在内质网中表达的RAS的功能，以及在细胞表面表达的Rin1的RAS相互作用结构域的功能。这些实验将有助于测试申请人的Rin1模型，作为内质网中RAS信号的抑制因子。

DESCRIPTION (provided by applicant): The Ras family of small guanine 5'-triphosphatases (GTPases) comprise a group of molecular switches that link receptors on the cell surface to signaling pathways that regulate cell proliferation and differentiation. Ras proteins are highly conserved among eukaryotic species. Mutant forms of Ras that are locked in the guanine 5'-triphosphate (GTP)-bound state, and thus continuously drive cell proliferation by interacting with their target proteins, are found in 30 percent of human tumors. This application describes a novel protein in budding yeast that appears to serve two separate functions, despite the fact that it is only 68 amino acids in length. The gene encoding this protein, designated RIN1 (for Ras inhibitor 1) behaves genetically as an inhibitor of Ras, and the Rin1 protein associates in vivo with GTP-bound Ras in a manner that suggests it competes for the same binding site as Ras target proteins. Because Rin1 resides in the membrane of the ER, rather than on the cell surface with the majority of Ras, it is hypothesized that one function of Rin1 is to maintain Ras in an inactive state during its posttranslational modification in the ER. The long-term objective of this project is to develop a form of Rin1 that effectively inhibits GTP-bound Ras on the cell surface for the purpose of treating Ras-involved malignancies. The second function of Rin1 is in the processing or transport of glycosyl-phosphatidylinositol (GPI)-anchored proteins from the ER. It is hypothesized that this function is carried out through association with a second member of the Ras family that is involved in protein secretion. The specific aims of this project are: 1) To understand the membrane topology of Rin1, and to identify the Ras-interaction region. This is the first step toward developing a form of Rin1 that is optimized for Ras inhibition at the cell surface. 2) To understand the function of Rin1 in GPI-protein anchoring or secretion. This will involve identification of the second target of Rin1. It is also likely that this aim will contribute to the new area of protein sorting in the ER. 3) To determine if yeast Ras can signal from the ER, and to create a more effective Rin1-based inhibitor of Ras. This aim is focused on assessing the function of Ras expressed in the ER, and the function of the Ras-interacting domain of Rin1 expressed at the cell surface. These experiments will help to test the applicant's model of Rin1 function as an inhibitor of Ras signaling in the ER.