ADP-RIBOSYLATION CYCLES

ADP-核糖基化循环

• 批准号: 5203499
• 负责人: A ZOLKIEWSKA
• 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/5203499
• 关键词: ADP ribosylation; SDS polyacrylamide gel electrophoresis; adenine phosphoribosyltransferase; affinity chromatography; arginine; enzyme structure; immunoprecipitation; integrins; intracellular; laboratory mouse; laminin; myoblasts; nicotinamide adenine dinucleotide; posttranslational modifications; protein purification; protein sequence; striated muscles

Integrin alpha 7 is a major substrate in skeletal muscle cells for the cell surface, glycosylphosphatidylinositol (GPI)-anchored, arginine- specific ADP-ribosyltransferase. Since ADP-ribosylarginine hydrolase, the enzyme responsible for cleavage of the ADP-ribosylarginine bond and a component with the transferase of a putative ADP-ribosylation cycle, is cytosolic, the processing of ADP-ribosylated integrin alpha 7 was investigated. Following incubation of differentiated mouse C2C12 myoblasts with [adenylate-32P]NAD and analysis by SDS-PAGE under reducing conditions, two [32P]ADP-ribosylated forms of integrin alpha 7 were resolved. By pulse-chase and purification of the radiolabeled proteins on a laminin affinity column, it was demonstrated that a 105- kDa ADP-ribosylated form originated from a mono-ADP-ribosylated 102-kDa form and represented integrin alpha 7 modified at more than one site. The additional site(s) of modification, utilized at higher NAD concentrations, were located in the 63-kDa N-terminal segment of integrin alpha 7. Both [32P]ADP-ribosylated integrins were loosely associated with the cytoskeleton, bound to laminin affinity columns, and immunoprecipitated with antibodies to integrin beta1. 32P label was rapidly removed from [32P]ADP-ribosylated integrin alpha 7 at either site of modification, a process inhibited by free ADP-ribose or p- nitrophenylthymidine-5'-monophosphate, an alternative substrate of 5'- nucleotide phosphodiesterase. The processed integrin alpha 7 was unavailable for subsequent ADP-ribosylation, although the amount of surface integrin alpha 7 remained constant. During the processing, no loss of label was observed from integrin alpha 7 radiolabeled with [14C]NAD, containing 14C in the nicotinamide proximal ribose, consistent with the degradation of ADP-ribose moiety by a cell surface 5'- nucleotide phosphodiesterase. Thus, cell surface ADP-ribosylation, in contrast to intracellular ADP-ribosylation, is not readily reversed by ADP-ribosylarginine hydrolase and seems to operate outside the postulated ADP-ribosylation cycle.

整合素α7是骨骼肌细胞的主要底物 细胞表面，糖基磷脂酰肌醇 (GPI)-锚定，精氨酸- 特异性 ADP-核糖基转移酶。由于 ADP-核糖精氨酸水解酶， 负责裂解 ADP-核糖精氨酸键的酶和 具有假定的 ADP-核糖基化循环的转移酶的组分， 是胞质的，ADP-核糖基化整合素α7的加工是 调查了。分化小鼠 C2C12 孵育后 具有 [腺苷酸-32P]NAD 的成肌细胞并通过 SDS-PAGE 进行分析 还原条件，整合素 α 的两种 [32P]ADP-核糖基化形式 7 已解决。通过脉冲追踪和放射性标记的纯化 105- 层粘连蛋白亲和柱上的蛋白质 kDa ADP-核糖基化形式源自单 ADP-核糖基化 102-kDa 形式并代表在多个位点修饰的整合素α7。 额外的修饰位点，在较高的 NAD 下使用 浓度，位于 63 kDa N 末端片段 整合素 α 7。两种 [32P]ADP-核糖基化整合素都是松散的 与细胞骨架相关，与层粘连蛋白亲和柱结合，并且 用整合素 beta1 抗体进行免疫沉淀。 32P 标签为 快速从 [32P]ADP-核糖基化整合素 α 7 中去除 修饰位点，受游离 ADP-核糖或 p- 抑制的过程 硝基苯基胸苷-5'-单磷酸，5'-的替代底物 核苷酸磷酸二酯酶。加工后的整合素α7是 无法用于后续的 ADP-核糖基化，尽管 表面整合素α7保持不变。处理过程中，不 从用放射性标记的整合素α7观察到标记丢失 [14C]NAD，烟酰胺近端核糖中含有14C，一致 随着 ADP-核糖部分被细胞表面 5'- 降解 核苷酸磷酸二酯酶。因此，细胞表面 ADP-核糖基化，在 与细胞内 ADP-核糖基化相反，不易被逆转 ADP-核糖精氨酸水解酶似乎在 假定的 ADP-核糖基化循环。