Identification of a Regulatory Segment of Poly(ADP-ribose) Glycohydrolase

Abstract

Coordinate regulation of PARPs-1/2 and PARG is required for cellular responses to genotoxic stress. While PARPs-1/2 are regulated by DNA breaks and covalent modifications, mechanisms of PARG regulation are poorly understood. We report here discovery of a PARG regulatory segment far removed linearly from residues involved in catalysis. Expression and analysis of human PARG segments identified a minimal catalytically active C-terminal PARG (hPARG59) containing a 16-residue N-terminal mitochondrial targeting sequence (MTS). Deletion analysis and site-directed mutagenesis revealed that the MTS, specifically hydrophobic residues L473 and L474 were required for PARG activity. This region of PARG was termed "Regulatory Segment/MTS" (REG/ MTS). The overall α-helical composition of hPARG59, determined by circular dichroism (CD), was unaffected by mutation of the REG/MTS leucine residues, suggesting that activity loss was not due to incorrect protein folding. REG/MTS was predicted to be in a loop conformation since the CD spectra of mutant Δ1-16 lacking the REG/MTS showed a higher α-helical content compared to hPARG59, indicating a secondary structure other than α-helix for this segment. Deletion of the REG/MTS from full-length hPARG111 also resulted in complete loss of activity, indicating that all PARG isoforms are subject to regulation at this site. Presence of the REG/MTS raises the possibility that PARG activity is regulated by interactions of PARPs-1/2 and other proteins at this site, raises interesting questions concerning mitochondrial PARG since MTS residues are often removed after transport, and offers a potentially novel site for drug targeting of PARG.The synthesis of polymers of ADP-ribose (PAR)1 is an immediate cellular response to DNA strand breaks caused by oxidative stress, ionizing radiation or alkylating agents (1-3). A primary enzyme involved in this post-translational modification is poly (ADP-ribose) polymerase-1 (PARP-1), which belongs to a growing family of PARPs that includes a second member (PARP-2) that also responds to DNA strand breaks (4,5). Upon binding to DNA strand breaks, PARPs-1/2 use nicotinamide adenine dinucleotide (NAD + ) as a substrate to synthesize PAR targeted to the PARPs themselves and other nuclear acceptor proteins, such as histones, p53 and enzymes involved in DNA repair (6). PARP-1 is inactivated and released from the DNA strand break by its automodification. Poly(ADP- † Supported by research grant CA 43894 from the National Institutes of Health to M.K.J.To whom correspondence should be addressed: Arizona Cancer Center Room 3985, University of Arizona, 1515 North Campbell Avenue, Tucson, AZ 85724, USA. Tel: (520) 626-5957. Fax: (520) 626-8657. mjacobson@pharmacy.arizona.edu. 1 Abbreviations: ADP, adenosine-5′-diphosphate; PAR, poly(ADP-ribose); PARG, poly(ADP-ribose) glycohydrolase; hPARG, human PARG; PARP-1/2, poly(ADP-ribose) polymerase 1 and 2; ADPR, ADP-ribose; ADPRT, ADP-ribosyltransferase; NAD + , nicotinamide adenine dinucleotide; MTS, mitochondrial targeting...

ADP-ribosylhydrolase 3 (ARH3), Not Poly(ADP-ribose) Glycohydrolase (PARG) Isoforms, Is Responsible for Degradation of Mitochondrial Matrix-associated Poly(ADP-ribose)

“…This isoform completely lacks exon 4 (and thus, the MTS) as well as a substantial amount of exon 5-encoded amino acids, whose importance for efficient PAR-degradation was demonstrated in this study. Indeed, lack of PARdegrading activity was shown for an N-terminally truncated human PARG isoform that would reflect mPARG52 with regard to translational initiation and exon composition (43). Taken together, these findings suggest that the mammalian PARG gene encodes cytosolic and mitochondrial isoforms that do not display PAR-degrading activity.…”

“…These findings extend the critical role of amino acids within the MTS for catalysis (43) by the adjacent amino acids encoded by exon 5. Amino acid exchanges within the MTS affected the catalytic activity of PARG55(ex5) as well as of N-terminally extended PARG variants (43). However, the MTS is part of the primary structures of both PARG55 and PARG60, and deletion of the MTS caused PARG55(ex5) to become inactive.…”

Structure and mechanism of a canonical poly(ADP-ribose) glycohydrolase

“…In contrast, the N-terminal accessory domain of the TTPARG accounts for approximately half of the enzyme structure and is predominantly α-helical in nature, with the exception of a few β-strands that form an extension to Supplementary Fig. S4), demonstrating that the RS/MTS motif 14 and a large part of the N-terminal domain are not absolutely essential for the enzymatic activity of canonical PARGs.…”

“…Despite this progress, structural information on the canonical PARGs is still lacking. Recent studies identified a minimal region in human PARG required for catalytic activity in vitro, which extends beyond the macrodomain alone 14 . This amino-terminal extension contains a short motif called the Regulatory Segment/MTS (RS/MTS) that was suggested to be essential for PARG activity 14 .…”

“…Recent studies identified a minimal region in human PARG required for catalytic activity in vitro, which extends beyond the macrodomain alone 14 . This amino-terminal extension contains a short motif called the Regulatory Segment/MTS (RS/MTS) that was suggested to be essential for PARG activity 14 . Additionally, a conserved tyrosine only present in canonical-type PARGs (Y795 in human PARG) was suggested to be involved in PAR binding 15 .…”