Wang Tao, Simbulan-Rosenthal Cynthia M, Smulson Mark E, Chock P Boon, Yang David C H
Department of Chemistry, Georgetown University, Washington, District of Columbia 20057, USA.
J Cell Biochem. 2008 May 1;104(1):318-28. doi: 10.1002/jcb.21624.
Poly(ADP-ribose) polymerase-1 (PARP-1) is the most abundant and the best-studied isoform of a family of enzymes that catalyze the polymerization of ADP-ribose from NAD(+) onto target proteins. PARP-1 is well known to involve in DNA repair, genomic stability maintenance, transcription regulation, apoptosis, and necrosis. Polyubiquitylation targets proteins towards degradation and regulates cell cycle progression, transcription, and apoptosis. Here we report polyubiquitylation of PARP-1 in mouse fibroblasts in the presence of proteasome inhibitor and in full-length recombinant PARP-1 in vitro under standard ubiquitylation assay conditions by immunoprecipitation and immunoblotting. Mutation of ubiquitin K48R but not ubiquitin K63R abolishes polyubiquitylation of PARP-1, indicating that K48 of ubiquitin was used in the formation of polyubiquitin chain and that ubiquitylated PARP-1 is likely destined for degradation. Full-length PARP-1 was ubiquitylated most likely at the N-terminal 24 kDa domain of PARP-1 as suggested by the inhibition of ubiquitylation by activated DNA and the absence of polyubiquitin in the C-terminal 89 kDa PARP-1 derived from caspase-catalyzed cleavage. NAD(+) inhibited ubiquitylation of PARP-1, while dipeptides ArgAla and LeuAla enhanced ubiquitylation of PARP-1. ATP inhibited the synthesis of poly(ADP-ribose) by PARP-1 and affinity purified polyubiquitylated PARP-1 was active in PAR synthesis. The results suggest polyubiquitylation of PARP-1 could regulate poly(ADP-ribosyl)ation of nuclear proteins by PARP-1 and consequently apoptosis and PARP-1 regulated cellular processes through ubiquitin-dependent degradation pathways.
聚（ADP - 核糖）聚合酶 -1（PARP -1）是一类酶中含量最丰富且研究最深入的亚型，该类酶催化从NAD⁺将ADP - 核糖聚合到靶蛋白上。众所周知，PARP -1参与DNA修复、基因组稳定性维持、转录调控、细胞凋亡和坏死。多聚泛素化将蛋白质靶向降解并调节细胞周期进程、转录和细胞凋亡。在此，我们通过免疫沉淀和免疫印迹报告了在蛋白酶体抑制剂存在的情况下小鼠成纤维细胞中PARP -1的多聚泛素化，以及在标准泛素化测定条件下体外全长重组PARP -1的多聚泛素化。泛素K48R突变而非泛素K63R突变消除了PARP -1的多聚泛素化，表明泛素的K48用于多聚泛素链的形成，并且泛素化的PARP -1可能注定要被降解。如活化DNA对泛素化的抑制以及源自半胱天冬酶催化切割的C末端89 kDa PARP -1中不存在多聚泛素所表明的，全长PARP -1最有可能在PARP -1的N末端24 kDa结构域被泛素化。NAD⁺抑制PARP -1的泛素化，而二肽ArgAla和LeuAla增强PARP -1的泛素化。ATP抑制PARP -1合成聚（ADP - 核糖），并且亲和纯化的多聚泛素化PARP -1在PAR合成中具有活性。结果表明，PARP -1的多聚泛素化可通过PARP -1调节核蛋白的聚（ADP - 核糖基）化，从而通过泛素依赖性降解途径调节细胞凋亡和PARP -1调控的细胞过程。
