Research ArticleTwo small enzyme isoforms mediate mammalian mitochondrial poly(ADP-ribose) glycohydrolase (PARG) activity☆
Introduction
Poly(ADP-ribosyl)ation is a transient, dynamic and reversible posttranslational modification of proteins involved in a host of biological functions [1], [2]. Poly(ADP-ribose) (PAR) is formed by all PARP enzymes by cleavage of NAD+ into nicotinamide and ADP-ribose which becomes polymerized and transferred to specific target proteins. Genome-wide sequence analyses in human suggest the existence of up to 17 different genes encoding enzymes capable of synthesizing poly(ADP-ribose) (PAR) [3], [4], [5], and, so far, ten of them have been shown to be catalytically active. In contrast, only three human poly(ADP-ribose) glycohydrolase (PARG) proteins, PARG111, PARG102 and PARG99, have been described, which are all expressed from one single PARG gene by alternative splicing [6], [7], [8]. PARG proteins have been the only enzymes known to efficiently catalyze hydrolysis of poly(ADP-ribose) (PAR) produced by enzymes of the poly(ADP-ribose) polymerase (PARP) family [9] until recently a novel gene encoding a 39 kDa enzyme with low poly(ADP-ribose) glycohydrolase activity has been discovered [10], [11], [12]. However, the extent to which this enzyme, (ADP-ribose) hydrolase (ARH3), that is structurally unrelated to PARG, participates in the rapid, DNA damage dependent PAR turnover within the cell has not been elucidated yet.
PARG rapidly cleaves PAR to form monomeric ADP-ribose by both exoglycosidic and endoglycosidic activity. Following DNA damage, PAR metabolism is mediated by PARP-1 and PARP-2 in concert with PARG, and PAR turnover can be extremely rapid with a half-life of the biopolymer of only ∼ 1 min [13]. Cellular accumulation of PAR beyond a short period of time has been shown to be deleterious [14], [15] and is involved in caspase-independent cell death regulation ([16] reviewed in [17]). Alterations of PAR metabolism are causally involved in the pathogenesis of inflammatory [19] and autoimmune disease [20], ischemia–reperfusion injury in brain [21], heart and intestine [22], [23], [24], neuronal degeneration and neurotoxicity [25], genetic and genomic instability (reviewed in [26]) and cancer (reviewed in [27], [28]). Targeting of the poly(ADP-ribose) metabolic pathway has therefore become an important focus in research on cancer therapy (recent reviews in [29], [30], [31]), cardiovascular disease intervention [32], [33], [34], [35] and a number of other pathophysiological conditions [36], [37]. Because of the potential of PAR metabolism as a drug target, identification of the key players appears to be crucial. While several PARP enzymes have been studied extensively, comparatively little is known about regulation of the catabolic arm of the pathway. However, the function of PARG in the PAR pathway is essential as deletion of the PARG gene results in a chronic, lethally toxic accumulation of cellular PAR [14], [38].
In contrast, a hypomorphic PARGΔ2–3/Δ2–3 mutant mouse that is devoid of exons II and III [18] was shown to be viable and expressing a residual PARG of 63 kDa by alternative splicing of the disrupted gene. This protein, designated mPARG63, is catalytically active and has unique properties [18]. In the present study we tested the hypothesis that the residual PARG proteins in the hypomorphic mouse may in fact be proteins that are naturally also expressed in wildtype mice as well as human. We show that mPARG63 is a naturally occurring protein that is expressed not only in the hypomorphic mutant but also in wildtype mouse tissues. More significantly, we demonstrate the existence of a human homolog, hPARG60, that is expressed in all tissues tested (skin, liver, testis, HeLa). At mRNA level, human PARG60 (hPARG60) is characterized as missing exon V, making it slightly smaller in size than the mouse homolog mPARG63. The data presented support the hypothesis that alternative translation initiation allows for expression of a protein with a consensus mitochondrial targeting sequence of the presequence type with strictly mitochondrial localization, termed hPARG55 (mPARG58 in mouse). The identification of hPARG60 (mPARG63) and hPARG55 (mPARG58) as novel PARG isoforms that associate with the mitochondria is of particular interest with regard to the emerging view that ADP-ribose metabolism may be involved in mitochondria-mediated, caspase-independent cell death pathways of biomedical interest [15], [39].
Section snippets
Cloning of human PARG cDNA by 5′RACE and construction of expression vectors
Human total RNA preparations from skin, liver and testis were purchased from Stratagene (La Jolla, CA). Mouse total RNA from liver and testis was also purchased or isolated from PARGΔ2–3/Δ2–3 mouse embryonic fibroblast culture, reversely transcribed and used in subsequent polymerase chain reaction (PCR) amplification reactions as described earlier [6], [7]. A mouse or human PARG gene specific primer which binds in the distal portion of the PARG 5′-UTR (human: hex0for:
Isolation of a cDNA encoding a novel hPARG60 isoform
PCR amplification of hPARG cDNA (Fig. 1A) from reversely transcribed total RNA yielded numerous bands rather than a single product. In order to amplify specifically the 5′ ends of cDNA derived from PARG transcripts, a forward primer in the 5′ untranslated region (hex0for) in combination with a reverse primer in exon VI (hex6rev) was used (5′UTR, Fig. 1A, left hand side of the gel). These primers produced three bands corresponding to the cDNAs of hPARG111 (band 1, 1880 bp), hPARG102 (band 2,
Discussion
The results of the study presented here suggest the presence of an additional level of complexity of cellular PAR metabolism due to the catalytic activity of novel small PARG isoforms associated with the mitochondria. This view is supported by several conclusions that can be drawn from this study: (i) two novel protein isoforms, hPARG60 and hPARG55, were discovered which are capable of PAR hydrolysis. They are conserved from mouse to human and are ubiquitously expressed by alternative splicing
Acknowledgments
We thank Donna Coyle for preparing 32P-labeled PAR and Zhao-Qi Wang, Universitaet Jena, for providing PARGΔ2–3/Δ2–3 3T3 cells. This work was supported by NIH grants CA-43894 (MKJ) and HD048837 (RGM).
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This work was supported by NIH grants CA-43894 (MKJ) and HD048837 (RGM).