RIN1, A NOVEL RAS-INHIBITORY PROTEIN IN YEAST

RIN1，酵母中一种新型 RAS 抑制蛋白

• 批准号: 6890919
• 负责人: DAVID E. LEVIN
• 金额: $ 25.34万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-06-01 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6890919
• 关键词: binding sites; biological signal transduction; endoplasmic reticulum; fungal proteins; guanine nucleotide binding protein; membrane proteins; protein protein interaction; protein structure function; protein transport; site directed mutagenesis

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.

描述（由申请人提供）：Ras家族的小鸟嘌呤5'-三磷酸酶（GTPases）由一组分子开关组成，这些分子开关将细胞表面的受体与调节细胞增殖和分化的信号通路连接起来。Ras蛋白在真核生物中高度保守。Ras的突变形式被锁定在鸟嘌呤5'-三磷酸（GTP）结合状态，从而通过与靶蛋白相互作用不断驱动细胞增殖，在30%的人类肿瘤中被发现。本申请描述了一种在出芽酵母中的新型蛋白质，尽管它只有68个氨基酸的长度，但它似乎具有两种不同的功能。编码该蛋白的基因被命名为RIN1 (Ras inhibitor 1)，在遗传上表现为Ras的抑制剂，RIN1蛋白在体内与gtp结合的Ras结合，其方式表明它与Ras靶蛋白竞争相同的结合位点。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，因此假设Rin1的一个功能是在内质网翻译后修饰期间维持Ras处于非活性状态。该项目的长期目标是开发一种有效抑制细胞表面gtp结合的Ras的Rin1形式，以治疗Ras相关的恶性肿瘤。Rin1的第二个功能是处理或运输来自内质网的糖基磷脂酰肌醇（GPI）锚定蛋白。据推测，这种功能是通过与Ras家族中参与蛋白质分泌的第二个成员的关联来实现的。本课题的具体目的是：1)了解Rin1的膜拓扑结构，确定ras -相互作用区域。这是开发一种对细胞表面的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功能。这将涉及确定Rin1的第二个目标。这一目标也可能有助于内质网蛋白质分类的新领域。3)确定酵母Ras是否可以从内质网发出信号，并建立更有效的基于rin1的Ras抑制剂。本研究的目的是评估内质网中表达的Ras的功能，以及细胞表面表达的Rin1的Ras相互作用域的功能。这些实验将有助于测试申请人的Rin1在内质网中作为Ras信号抑制剂的功能模型。