ADP-ribosylation Cycles

ADP-核糖基化循环

• 批准号: 6809647
• 负责人: Joel 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/6809647
• 关键词: ADP ribosylation; NAD nucleosidase; T lymphocyte; adenine phosphoribosyltransferase; bacterial toxins; enzyme activity; glycosylphosphatidylinositols; laboratory rat; nicotinamide adenine dinucleotide; pentosyltransferase; posttranslational modifications; protein sequence; protein structure function; pyrophosphatase; tissue /cell culture

ADP-ribosylation, in which the ADP-ribose moiety of NAD is transferred to a target protein, is catalyzed by a family of bacterial toxins and mammalian enzymes. Some toxin transferases appear to be responsible for the diseases caused by the bacterium. The mammalian ADP-ribosyltransferases (ARTs) are located both within the cell and on the cell surface, sometimes, linked through a glycosylphosphatidylinositol anchor (ART1). Other mammalian ADP-ribosyltransferases (ART5) apppear to be secreted. A family of the mammalian enzymes has been cloned in the laboratory. ART2a (RT6.1) and ART2b(RT6.2) are NAD glycohydrolases (NADases) that are linked to T lymphocytes by glycosylphosphatidylinositol (GPI) anchors. Although both mature proteins possess three conserved regions (I, II, III) that form the NAD-binding site and differ by only ten amino acids, only ART2b is auto-ADP-ribosylated and only ART2a is glycosylated. To investigate the structural basis for these differences, wild-type and mutant ART2a and ART2b were expressed in rat mammary adenocarcinoma (NMU) cells and released with phosphatidylinositol-specific phospholipase C. All mutants were immunoreactive NADases. Arginine 204 (Arg204), amino-terminal to essential glutamate 209 in Region III, is found in ART2b, but not ART2a. Replacement of Arg204 in ART2b with lysine, tyrosine, or glutamate abolished auto-ADP-ribosylation. Unlike wild-type ART2a, ART2a(Y204R) was auto-ADP-ribosylated. The tryptophan mutant ART2b(R204W) was auto-ADP-ribosylated and exhibited an enhanced NADase activity compared to the wild-type ART2b. Incubation with NAD with auto-ADP-ribosylation decreased the NADase activities of wild-type ART2b and ART2b(R204W), whereas activity of ART2b(R204K), which is not auto-modified, was unchanged by NAD. Facilitation of auto-ADP-ribosylation by tryptophan 204 suggests that the hydrophobic amino acid mimics an ADP-ribosylated arginine and may be involved in the regulation of ART activity. . Thus, Arg204 in ART2b serves as a regulatory switch whose presence is required for additional auto-ADP-ribosylation and regulation of catalytic activity.

ADP-核糖基化，即 NAD 的 ADP-核糖部分转移到靶蛋白上，由细菌毒素和哺乳动物酶家族催化。一些毒素转移酶似乎是由细菌引起的疾病的原因。哺乳动物 ADP-核糖基转移酶 (ART) 位于细胞内和细胞表面，有时通过糖基磷脂酰肌醇锚 (ART1) 连接。其他哺乳动物 ADP-核糖基转移酶 (ART5) 似乎是分泌的。哺乳动物酶家族已在实验室被克隆。 ART2a (RT6.1) 和 ART2b (RT6.2) 是 NAD 糖水解酶 (NADase)，通过糖基磷脂酰肌醇 (GPI) 锚与 T 淋巴细胞连接。尽管两种成熟蛋白都具有形成 NAD 结合位点的三个保守区域（I、II、III），并且仅相差 10 个氨基酸，但只有 ART2b 是自动 ADP 核糖基化的，并且只有 ART2a 是糖基化的。为了研究这些差异的结构基础，野生型和突变型 ART2a 和 ART2b 在大鼠乳腺癌 (NMU) 细胞中表达，并与磷脂酰肌醇特异性磷脂酶 C 一起释放。所有突变体都是免疫反应性 NAD 酶。精氨酸 204 (Arg204) 位于 III 区必需谷氨酸 209 的氨基末端，存在于 ART2b 中，但在 ART2a 中未发现。用赖氨酸、酪氨酸或谷氨酸替换 ART2b 中的 Arg204 可消除自动 ADP 核糖基化。与野生型 ART2a 不同，ART2a(Y204R) 是自动 ADP 核糖基化的。色氨酸突变体 ART2b(R204W) 被自动 ADP 核糖基化，并与野生型 ART2b 相比表现出增强的 NADase 活性。与具有自动 ADP-核糖基化的 NAD 一起孵育会降低野生型 ART2b 和 ART2b(R204W) 的 NADase 活性，而未自动修饰的 ART2b(R204K) 的活性不受 NAD 的影响。色氨酸 204 促进自动 ADP 核糖基化表明疏水性氨基酸模拟 ADP 核糖基化精氨酸，并可能参与 ART 活性的调节。 。因此，ART2b 中的 Arg204 充当调节开关，其存在是额外的自动 ADP 核糖基化和催化活性调节所必需的。