权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

ADP-ribosylation Cycles

ADP-核糖基化循环

基本信息

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
项目类别：
财政年份：
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

项目摘要

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ADP-核糖聚合酶（PARP）的活性被聚ADP-核糖（PAR）降解酶逆转，其中聚ADP-核糖糖水解酶（PARG）和ADP核糖基水解酶3（ARH 3）在体外和细胞中催化PAR降解。在人类和小鼠中，四种PARG同种型由单个PARG基因的选择性剪接产生。其中，只有全长开放阅读框编码的蛋白质（hPARG 111和mPARG 110）定位于细胞核。大多数PARG活性在胞质溶胶中检测到，这与PARP 1的核定位明显矛盾，PARP 1是最丰富和最活跃的PARP。PARG基因的敲除是胚胎致死性的，而从外显子2和3中缺失的基因（PARG外显子2 -3）表达PARG的小鼠是存活的，其缺乏核mPARG 110以及两种胞质同种型mPARG 101和mPARG 98。这表明小鼠PARG同种型mPARG 63的重要作用。mPARG 63的人类对应物是一种60 kDa的蛋白质（hPARG 60），与小鼠相反，它缺乏外显子5编码的氨基酸。由于替代的翻译起始位点，编码小的人类和小鼠PARG同种型的转录物被认为编码蛋白质，其中N-末端氨基酸构成在所有其他PARG中被掩蔽的线粒体靶向序列（MTS）。我们建立了第五种人类PARG亚型hPARG 55的遗传背景，该亚型由初级PARG转录物的迄今未被识别的选择性剪接事件引起。深入的定位分析显示，hPARG 55是唯一的亚型，是针对线粒体基质，其中存在的聚ADP-核糖代谢的争论。令人惊讶的是，hPARG 55在体外和细胞中均无催化活性。hPARG 55的PAR降解活性可以通过重新引入外显子5编码的氨基酸来恢复。这些发现可以应用于也缺乏外显子5但定位于胞质溶胶的hPARG 60。在野生型小鼠中，我们鉴定了导致胞质52 kDa PARG同种型（mPARG 52）的剪接事件，迄今为止仅在PARGexon 2 -3突变体中报道。该亚型缺乏外显子4（编码MTS）和外显子5的显著部分，支持人和鼠PARG基因编码的PARG亚型具有不同于PAR降解的功能的结论。鉴于这些发现，我们研究了ARH 3在线粒体PAR降解中的作用。在来自ARH 3-/-小鼠的细胞中，PARP 1活性在线粒体中的靶向表达导致PAR的更大积累。ARH 3-/-细胞中基质积累的PAR不再降解。ARH 3-/-细胞中PARG基因表达下调不影响线粒体中PAR降解活性。因此，我们先前在线粒体内鉴定的PAR降解活性是由ARH 3进行的，表明这种酶在PAR代谢中的作用。