BIOCHEMISTRY OF PROTEIN PRENYLATION

蛋白质异戊二烯化的生物化学

• 批准号: 2095049
• 负责人: Michael H Gelb
• 金额: $ 20.29万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-20 至 1999-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2095049
• 关键词: alkyltransferase; chemical reaction; endopeptidases; enzyme activity; enzyme mechanism; farnesyl compound; geranyl compound; guanine nucleotide binding protein; isoprenoid; laboratory rat; microorganism mass culture; palmitates; posttranslational modifications; protein purification; protein sequence; protein structure function; recombinant proteins; tissue /cell culture; transfection

The prenylation of proteins in mammalian cells is a recently discovered cellular event. Both 15-carbon farnesyl and 20-carbon geranylgeranyl groups are found at the C-termini of proteins such as ras proteins, heterotrimeric G proteins, GTP-binding proteins, and rhodopsin kinase. It has been shown that the prenylation of ras proteins is absolutely required for the transformation of cells expressing oncogenic forms of this protein. Thus, the study of the protein prenylation pathway may lead to better treatments for controlling human malignancies. A protein geranylgeranyltransferase that attaches geranylgeranyl groups to the cysteine residues near the C-terminal end of proteins using geranylgeranyl pyrophosphate as a substrate has been purified to homogeneity. The kinetic properties and substrate specificities of this enzyme will be further characterized. Inhibition of this enzyme as well as the inhibition of the protein farnesyltransferase by limonene and its oxidized metabolites will also be studied. Many prenylated proteins are further modified by proteolysis which removes the C-terminal three amino acids adjacent to the prenylated cysteine residue. This protease has been detected in cell membranes and continued work may lead to the purification of the relevant enzyme. Substrate specificity studies will be carried out with the protease in membranes or in purified form, if possible. Both H-ras and N-ras proteins are modified by farnesylation and palmitoylation. A protein palmitoyltransferase that acts on ras proteins has been partially purified. Efforts will be made to purify the enzyme to homogeneity so that it can be characterized. Attempts will also be made to clone the enzyme from a cDNA library and to express large amounts of this protein using the baculovirus/insect cell system. Protein prenyl groups probably serve as membrane anchors but very little is known about the function of these elements. The farnesyl group of ras is required for the activation of its downstream components in the cytosol of cells suggesting that the prenyl group may dictate protein-protein interactions. This possible role of protein prenyl groups will be explored by using ras proteins that are modified with farnesyl analogs. Such information may be the most useful for designing strategies for the treatment of ras-dependent tumors. Similar studies will be carried out with rhodopsin kinase.

哺乳动物细胞中蛋白质的蛋白质原化是最近发现的 蜂窝事件。 15-碳法尼和20-碳乙酰基凝酰基基因 在蛋白质（例如Ras蛋白）的C-末端发现组， 异三聚体G蛋白，GTP结合蛋白和视紫红质激酶。它 已经证明了RAS蛋白的原始化是绝对需要的 为了转化表达肿瘤形式的细胞 蛋白质。因此，对蛋白质原苯途径的研究可能导致 更好地控制人类恶性肿瘤的治疗方法。 一种将黄烷基凝酰基基团连接到 使用蛋白质C末端附近的半胱氨酸残基使用 黄烷基果氰基焦磷酸作为底物已被纯化为 同质性。动力学特性和底物特异性 酶将进一步表征。抑制该酶以及 limonene及其抑制蛋白质Farnesylsylansxylansferase及其抑制 还将研究氧化的代谢产物。 许多蛋白水解进一步修饰了许多蛋白质的蛋白质蛋白 邻近婚前半胱氨酸的C末端三氨基酸 残留物。该蛋白酶已在细胞膜中检测到 工作可能导致相关酶的净化。基材 特异性研究将使用膜中的蛋白酶进行或 如果可能的话，以纯化的形式。 H-RAS和N-RAS蛋白均通过Farnesylation和 棕榈酰化。作用于RAS蛋白的蛋白质棕榈转移酶 已被部分纯化。将努力净化酶 同质性，以便可以表征它。也将尝试 从cDNA库中克隆酶并表达大量 使用杆状病毒/昆虫细胞系统的蛋白质。 蛋白质蛋白质基可能用作膜锚，但很少 已经知道这些元素的功能。 RAS的Farnesyl群 激活其在细胞质中的下游成分需要 细胞表明蛋白酶可能决定蛋白质蛋白 互动。将探索蛋白质蛋白质基团的这种可能作用 通过使用用Farnesyl类似物修饰的RAS蛋白。这样的 信息可能是设计策略的最有用的 治疗依赖RAS的肿瘤。将进行类似的研究 与视紫红质激酶。