ADP-ribosylation Cycles

ADP-核糖基化循环

• 批准号: 8557900
• 负责人: Joel Moss
• 金额: $ 266.41万
• 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/8557900
• 关键词: ADP Ribose Transferases; ADP ribosylation; ADP-Ribosylation Pathway; Accounting; Adenosine Diphosphate Ribose; Affect; Alternative Splicing; Amino Acids; Antithymoglobulin; Arginine; Bacterial Toxins; Catalytic Domain; Cell Differentiation process; Cell Nucleus; Cells; Cholera; Cholera Toxin; Cleaved cell; Complementary DNA; Cysteine; Cytosol; DNA Repair; Down-Regulation; Embryo; Enzymes; Event; Exhibits; Exons; Family; Frequencies; Gene Expression; Genes; Genetic; Goals; Human; Hydrolase; In Vitro; Knock-out; Knockout Mice; Lead; Length; Mammalian Cell; Masks; Metabolism; Mitochondria; Mitochondrial Matrix; Modification; Mus; N-terminal; Niacinamide; Nuclear; Open Reading Frames; Pathway interactions; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerases; Polymers; Post-Translational Protein Processing; Protein Isoforms; Proteins; RNA Splicing; Reaction; Recombinant DNA; Regulation; Reporting; Role; Signal Transduction; Site; Structure; Tissues; Transcript; Transferase; Translating; Triplet Multiple Birth; Wild Type Mouse; biological systems; carcinogenesis; clinical phenotype; enzyme activity; member; mutant; poly ADP-ribose glycohydrolase; protein function; tumor

Explanation The activity of poly-ADP-ribose polymerases (PARPs) is reversed by poly-ADP-ribose (PAR)-degrading enzymes, of which poly-ADP-ribose glycohydrolase (PARG) and ADP ribosylhydrolase 3 (ARH3) catalyze PAR-degradation in vitro and in cells. In humans and mice, four PARG isoforms result from alternative splicing of a single PARG gene. Of them, only the protein encoded by the full-length open reading frame (hPARG111 and mPARG110) localizes to the nucleus. Most PARG activity is detected in the cytosol, which is in apparent contradiction to the nuclear localization of PARP1, the most abundant and most active PARP. Knockout of the PARG gene is embryonic lethal, whereas mice that express PARG from a gene deleted in exons 2 and 3 (PARGexon2-3), which lack nuclear mPARG110 as well as the two cytosolic isoforms mPARG101 and mPARG98, are viable. This suggests a vital role of the small murine PARG isoform mPARG63. The human counterpart of mPARG63 is a 60 kDa-protein (hPARG60) that, in contrast to mice, lacks exon 5-encoded amino acids. Owing to alternative translational start sites, the transcripts encoding the small human and murine PARG isoforms were suggested to encode proteins, in which the N-terminal amino acids constitute a mitochondrial targeting sequence (MTS) that is masked in all other PARGs. We established the genetic background of a fifth human PARG isoform hPARG55, which results from a hitherto unrecognized alternative splicing event of the primary PARG transcript. In-depth localization analysis revealed hPARG55 to be the only isoform that is targeted to the mitochondrial matrix, where the presence of poly-ADP-ribose metabolism is debated. Surprisingly, hPARG55 is catalytically inactive both in vitro and in cells. The PAR-degrading activity of hPARG55 could be restored by reintroducing exon 5-encoded amino acids. These findings could be applied to hPARG60 that also lacks exon 5, but localizes to the cytosol. In wild-type mice, we identified a splicing event leading to a cytosolic 52 kDa-PARG isoform (mPARG52), which was so far only reported in the PARGexon2-3 mutant. This isoform lacks exon 4 (encoding the MTS) and a significant portion of exon 5 supporting the conclusion that the human and the murine PARG genes encode PARG isoforms with functions different from PAR-degradation. Given these findings, we investigated the role of ARH3 in PAR degradation in mitochondria. In cells from ARH3-/- mice, targeted expression of PARP1 activity in mitochondria lead to greater accumulation of PAR. Matrix-accumulated PAR in ARH3-/- cells was no longer subject to degradation. Downregulation of PARG gene expression in ARH3-/- cells did not affect the PAR-degrading activity in mitochondria. Thus, the PAR-degrading activity we previously identified within mitochondria is carried out by ARH3, suggesting a role of this enzyme in PAR metabolism.

解释 聚腺苷二磷酸核糖聚合酶(PARP)的活性被聚腺苷二磷酸核糖(PAR)降解酶逆转，其中聚ADP核糖水解酶(PARG)和ADP核糖水解酶3(ARH3)在体外和细胞内催化PAR的降解。 在人类和小鼠中，四种PARG亚型是由单个PARG基因的选择性剪接产生的。其中，只有全长开放阅读框(hPARG111和mPARG110)编码的蛋白质定位于细胞核。大多数PARG活性都在胞浆中检测到，这与PARP1的核定位明显相反，PARP是含量最丰富、活性最高的PARP。PARG基因的敲除是胚胎致死的，而从外显子2和3(PARGexon2-3)缺失的基因(PARGexon2-3)表达PARG的小鼠是可行的，该基因缺失核内mPARG110以及两种胞质异构体mPARG101和mPARG98。这表明小鼠PARG亚型mPARG63具有重要作用。人类的mPARG63是一个60 kDa的蛋白质(HPARG60)，与小鼠不同，它缺乏外显子5编码的氨基酸。由于不同的翻译起始点，编码人和小鼠PARG亚型的转录本被认为是编码蛋白质的，其中N-末端氨基酸构成一个线粒体靶向序列(MTS)，该序列在所有其他PARG中都被掩盖。 我们建立了第五个人类PARG亚型hPARG55的遗传背景，它是由初级PARG转录本的一个迄今未被识别的选择性剪接事件引起的。深入的定位分析显示，hPARG55是唯一针对线粒体基质的异构体，在线粒体基质中是否存在多聚ADP-核糖代谢是有争议的。令人惊讶的是，hPARG55在体外和细胞内都没有催化活性。通过重新引入外显子5编码的氨基酸可以恢复hPARG55的PAR降解活性。这些发现可以应用于hPARG60，它也没有外显子5，但定位于胞浆。在野生型小鼠中，我们发现了导致胞液52 kDa-PARG异构体(MPARG52)的剪接事件，到目前为止，这只在PARGexon2-3突变体中被报道。该异构体缺少外显子4(编码MTS)和外显子5的很大一部分，支持人和小鼠PARG基因编码具有不同于PAR降解功能的PARG异构体的结论。 鉴于这些发现，我们研究了ARH3在线粒体PAR降解中的作用。在ARH3-/-小鼠的细胞中，靶向表达PARP1活性的线粒体导致PAR的更大积累。ARH3-/-细胞中积累的基质PAR不再被降解。下调ARH3-/-细胞中PARG基因的表达并不影响线粒体中PAR的降解活性。因此，我们以前在线粒体中发现的PAR降解活性是由ARH3进行的，这表明该酶在PAR代谢中发挥了作用。