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.

Project Start
Project End
Budget Start
Budget End
Support Year
20
Fiscal Year
2012
Total Cost
$2,664,079
Indirect Cost
Name
National Heart, Lung, and Blood Institute
Department
Type
DUNS #
City
State
Country
Zip Code
Ida, Chieri; Yamashita, Sachiko; Tsukada, Masaki et al. (2016) An enzyme-linked immunosorbent assay-based system for determining the physiological level of poly(ADP-ribose) in cultured cells. Anal Biochem 494:76-81
Tsutsuki, Hiroyasu; Yahiro, Kinnosuke; Ogura, Kohei et al. (2016) Subtilase cytotoxin produced by locus of enterocyte effacement-negative Shiga-toxigenic Escherichia coli induces stress granule formation. Cell Microbiol 18:1024-40
Yamashita, Sachiko; Tanaka, Masakazu; Sato, Teruaki et al. (2016) Effect of mild temperature shift on poly(ADP-ribose) and γH2AX levels in cultured cells. Biochem Biophys Res Commun 476:594-9
Mashimo, Masato; Moss, Joel (2016) Functional Role of ADP-Ribosyl-Acceptor Hydrolase 3 in poly(ADPRibose) Polymerase-1 Response to Oxidative Stress. Curr Protein Pept Sci :
Yahiro, Kinnosuke; Hirayama, Toshiya; Moss, Joel et al. (2016) New Insights into VacA Intoxication Mediated through Its Cell Surface Receptors. Toxins (Basel) 8:
Kato, J; Vekhter, D; Heath, J et al. (2015) Mutations of the functional ARH1 allele in tumors from ARH1 heterozygous mice and cells affect ARH1 catalytic activity, cell proliferation and tumorigenesis. Oncogenesis 4:e151
Nakashima, Shota; Kakugawa, Tomoyuki; Yura, Hirokazu et al. (2015) Identification of Helicobacter pylori VacA in human lung and its effects on lung cells. Biochem Biophys Res Commun 460:721-6
Nakano, Masayuki; Yamasaki, Eiki; Moss, Joel et al. (2015) Study of the stn protein in salmonella; a regulator of membrane composition and integrity. Methods Mol Biol 1225:127-38
Akazawa, Yuko; Matsuda, Katsuya; Isomoto, Hajime et al. (2015) BH3-only protein Bim is associated with the degree of Helicobacter pylori-induced gastritis and is localized to the mitochondria of inflammatory cells in the gastric mucosa. Int J Med Microbiol 305:553-62
Stevens, Linda A; Barbieri, Joseph T; Piszczek, Grzegorz et al. (2014) Nonenzymatic conversion of ADP-ribosylated arginines to ornithine alters the biological activities of human neutrophil peptide-1. J Immunol 193:6144-51

Showing the most recent 10 out of 25 publications