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.

解释多聚ADP-核糖（PAR） - 降解酶逆转多聚ADP-核糖聚合酶（PARPS）的活性，其中多聚ADP-核糖糖糖酶（PARG）和ADP核糖基水解酶3（ARH3）催化了催化酶。体外和细胞中。在人类和小鼠中，单个PARG基因的替代剪接引起了四个PARG同工型。其中，只有由全长开放阅读框（HPARG111和MPARG110）编码的蛋白质定位于细胞核。大多数PARG活性在细胞质中检测到，这显然与PARP1的核定位相矛盾，PARP1是最丰富，最活跃的PARP。 PARG基因的敲除胚胎致死，而从外显子2和3中删除的基因表达PARG的小鼠（缺乏核MPARG110以及两个胞质同工型MPARG101和MPARGG98）是可行的。这表明小鼠Parg同工型MPARG63的重要作用。 MPARG63的人类对应物是60 kDa蛋白（HPARG60），与小鼠相比，它缺乏外显子5编码的氨基酸。由于替代转化起始位点，建议编码小型人和鼠PARG同工型的转录本编码蛋白质，其中N末端氨基酸构成线粒体靶向序列（MTS），在所有其他PARG中都掩盖了线粒体靶向序列（MTS）。我们建立了第五人PARG同工型HPARG55的遗传背景，这是由迄今未识别的主要PARG转录物的替代剪接事件引起的。深入的定位分析表明，HPARG55是唯一针对线粒体基质的同工型，在该线粒体基质中存在多聚ADP-核糖代谢的存在。出乎意料的是，HPARG55在体外和细胞中均催化活性。可以通过重新引入外显子5编码的氨基酸来恢复HPARG55的分类活性。这些发现可以应用于也缺乏外显子5的HPARG60，但本地化为细胞质。在野生型小鼠中，我们确定了一个剪接事件，导致52 kDa-parg同工型（MPARG52），迄今仅在Pargexon2-3突变体中报道了这一事件。这种同工型缺乏外显子4（编码MTS），外显子5的很大一部分支持人类和鼠Parg基因编码具有不同功能与降解的功能的结论。鉴于这些发现，我们研究了ARH3在线粒体中PAR降解中的作用。在来自ARH3 - / - 小鼠的细胞中，线粒体中PARP1活性的靶向表达导致PAR的积累更大。在ARH3 - / - 细胞中矩阵蓄积的PAR不再受到降解。 ARH3 - / - 细胞中PARG基因表达的下调不会影响线粒体中的分裂活性。因此，我们先前在线粒体中确定的分析活性是通过ARH3进行的，这表明该酶在PAR代谢中的作用。