REGULATION OF CYCLIC NUCLEOTIDE METABOLISM

环核苷酸代谢的调节

• 批准号: 3843260
• 负责人: J MOSS
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3843260
• 关键词: ADP ribosylation; Escherichia coli; G protein; adenine phosphoribosyltransferase; adenylate cyclase; biological signal transduction; chemical binding; chimeric proteins; cholera toxin; cyclic nucleoside monophosphate; enterotoxins; enzyme activity; enzyme structure; human tissue; hydrolase; laboratory mouse; laboratory rat; molecular cloning; nucleotide metabolism; protein purification

Cholera toxin (CT), an etiologic agent in cholera, exerts its effect on cells through the ADP-ribosylation of guanine nucleotide-binding (G) proteins that are critical for signaling from cell surface receptors to their intracellular targets (e.g., adenylyl cyclase). The toxin consists of one A subunit, which is an ADP-ribosyltransferase and five B subunits, which bind the toxin to cell surface ganglioside GM1. The enzymatic activity of the A subunit is latent, and activity requires proteolysis and reduction of a single disulfide resulting in the formation of a catalytically active A1 protein and a much smaller A2 protein derived from the carboxy terminus of the A subunit. The activity of the A1 protein is enhanced by 20 kDa ADP-ribosylation factors or ARFs, in a GTP-dependent manner. (1) To determine if the latent form of the toxin expresses an ARF activation site, an inactive mutant form of E. coli heat-labile enterotoxin (LT), with a critical glutamate to lysine substitution at position 112 of the A subunit, was utilized as a competitor in a cholera toxin assay containing limiting ARF. The holotoxin did not significantly inhibit the ARF-stimulated ADP-ribosyltransferase activity of CT A subunit. Trypsinization and reduction of LT resulted in a potent inhibitor; reduction alone did not. Trypsinized LT did not inhibit ARF stimulation of a purified, alkylated CTA1 protein, which is not dependent on reduction for activity. These studies are consistent with the hypothesis that the ARF site on CT is not accessible in the latent toxin. Release of the CT A1 protein, which is necessary for expression of ADP-ribosyltransferase activity, is also required for appearance of the ARF binding site. (2) The cholera toxin A1 protein ADP-ribosylates arginine residues in G proteins. Mammalian cells contain enzymes, termed ADP-ribosylarginine hydrolases, that cleave the ADP-ribosyl-arginine bond, releasing free arginine and could play a role in recovery from cholera. To examine the structural features and conservation of hydrolases, they were cloned from rat, mouse and human sources. The hydrolases from these species exhibited considerable amino acid identity.

霍乱毒素（CT）是霍乱的一种病原体，对 细胞通过鸟嘌呤核苷酸结合 (G) 的 ADP 核糖基化 对细胞表面受体的信号传递至关重要的蛋白质 它们的细胞内靶标（例如腺苷酸环化酶）。该毒素由 一个 A 亚基（ADP-核糖基转移酶）和 5 个 B 亚基 亚基，将毒素与细胞表面神经节苷脂 GM1 结合。这 A亚基的酶活性是潜在的，活性需要 单个二硫键的蛋白水解和还原导致 形成具有催化活性的 A1 蛋白和更小的 A2 源自 A 亚基羧基末端的蛋白质。活动内容 A1 蛋白的 20 kDa ADP 核糖基化因子增强或 ARF，以 GTP 依赖的方式。 (1) 确定潜在形式是否 该毒素表达 ARF 激活位点，这是一种非活性突变体形式 大肠杆菌不耐热肠毒素 (LT)，具有关键的谷氨酸 A 亚基 112 位的赖氨酸取代被用作 含有限制 ARF 的霍乱毒素测定中的竞争者。这 Holotoxin 并没有显着抑制 ARF 刺激 CTA 亚基的 ADP-核糖基转移酶活性。胰蛋白酶消化和 LT 的减少产生了有效的抑制剂；仅靠减少并不能。 胰蛋白酶消化的 LT 不会抑制纯化的、烷基化的 ARF 刺激 CTA1 蛋白，其活性不依赖于降低。这些 研究与 CT 上的 ARF 部位是 无法接触到潜伏的毒素。 CT A1 蛋白的释放， 对于 ADP-核糖基转移酶活性的表达是必需的，也是 ARF 结合位点出现所必需的。 (2)霍乱毒素 A1 蛋白 ADP 核糖基化 G 蛋白中的精氨酸残基。 哺乳动物 细胞含有称为 ADP-核糖精氨酸水解酶的酶， 裂解 ADP-核糖基-精氨酸键，释放游离精氨酸，并且可以 在霍乱康复中发挥作用。 来检查结构 水解酶的特征和保守性，它们是从大鼠中克隆的， 小鼠和人类来源。 这些物种的水解酶表现出 相当大的氨基酸同一性。