Isolation and Cloning of Rat Poly(ADP-Ribose) Glycohydrolase: Presence of a Potential Nuclear Export Signal Conserved in Mammalian Orthologs

Abstract

Poly(ADP-ribose) glycohydrolase (Parg) is the main enzyme of poly(ADP-ribose) degradation. To understand its structure-and-function relationship, we purified Parg from rat testis 9,740-fold using an improved affinity column; the purified product was a 60 kDa protein. Based on the determined sequences of three peptide fragments, degenerated primers were synthesized and a Parg cDNA comprising 3,974 nucleotides, encoding a 109 kDa protein, was isolated. The 60 kDa Parg purified from rat testes corresponded to the C-terminal half of the 109 kDa deduced peptide. When recombinant rat Parg was expressed as a glutathione S-transferase fusion protein in Escherichia coli, Parg activity was observed for the full-length and C-terminal half proteins but not in for the N-terminal half protein. Taken together, these data indicate that the catalytic domain of Parg is located in the C-terminal half. Further, we newly identified the presence of a potential nuclear export signal in the N-terminal half in addition to the previously reported nuclear localization signals in rat and other mammalian Pargs. Northern blot analysis showed the ubiquitous expression of a single 4.0 kb Parg mRNA in various rat tissues. The findings suggest that the 60 kDa Parg is produced by post-transcriptional processing.

Depletion of the 110-Kilodalton Isoform of Poly(ADP-Ribose) Glycohydrolase Increases Sensitivity to Genotoxic and Endotoxic Stress in Mice

“…The observation that PARG ⌬2-3/⌬2-3 cells have approximately 28% of normal PARG activity in the nucleus suggests the possibility of a weak nuclear localization signal in the C-terminal portion of PARG, which has been proposed previously (43). An intriguing finding is that PARG activity was elevated more than threefold in the mitochondria of PARG ⌬2-3/⌬2-3 cells, suggesting that the loss of PARG 110 may target or upregulate the PARG 60 isoform in the mitochondria in order to support cell viability.…”

Two small enzyme isoforms mediate mammalian mitochondrial poly(ADP-ribose) glycohydrolase (PARG) activity

“…We hypothesize that hPARG60 may be capable of shuttling between cellular compartments, e. g. in response to DNA damage-induced, PARP-1 and PARP-2 dependent formation of PAR. The fact that PARG may have a second, weak NLS near the C-terminus [43,48] which would facilitate this type of nucleo-mitochondrial shuttling supports this hypothesis. However, results obtained from overexpression of PARG proteins in the present study may require further confirmatory experiments involving the endogenously expressed proteins to verify the discussed hypotheses of small PARG isoform subcellular localization.…”

“…(iii) Both, hPARG60 and hPARG55 are associated with the mitochondria, making them potentially important factors in the nuclear-mitochondrial crosstalk involving PAR metabolism. While PARG111 is a nuclear enzyme carrying a nuclear localization signal (NLS) in exon I near the N-terminus [8], PARG 102 and PARG 99 are mainly extranuclear proteins [7,47] carrying putative nuclear export signals (NES), [43,48] in the N-terminal A-domain. The presence of the bulk of PARG activity in the cytoplasm remains puzzling as the vast majority of PAR synthesis following genotoxic stress is catalyzed by the predominantly nuclear enzymes PARP-1 and PARP-2 and PAR is therefore detectable mainly in the nucleus after DNA damage.…”

Global Analysis of Transcriptional Regulation by Poly(ADP-ribose) Polymerase-1 and Poly(ADP-ribose) Glycohydrolase in MCF-7 Human Breast Cancer Cells

“…The catalytically inactive human PARP-1 contained a change at Glu-988 to Lys (E988K) (38,39), whereas the catalytically inactive rat PARG contained changes at Tyr-788 and -791 to Phe and Ala, respectively (Y788F/Y791A) (40). Cytomegalovirus-based mammalian expression constructs for full-length wild-type or catalytically inactive human PARP-1 (with a carboxyl-terminal His 6 /FLAG tag) or rat PARG (with a carboxylterminal FLAG tag) were generated by PCR-based cloning of the tagged cDNAs into pCMV5.…”