Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate.
ABSTRACT: The post-translational modification (PTM) and signaling molecule poly(ADP-ribose) (PAR) has an impact on diverse biological processes. This PTM is regulated by a series of ADP-ribosyl glycohydrolases (PARG enzymes) that cleave polymers and/or liberate monomers from their protein targets. Existing methods for monitoring these hydrolases rely on detection of the natural substrate, PAR, commonly achieved via radioisotopic labeling. Here we disclose a general substrate for monitoring PARG activity, TFMU-ADPr, which directly reports on total PAR hydrolase activity via release of a fluorophore; this substrate has excellent reactivity, generality (processed by the major PARG enzymes), stability, and usability. A second substrate, TFMU-IDPr, selectively reports on PARG activity only from the enzyme ARH3. Use of these probes in whole-cell lysate experiments has revealed a mechanism by which ARH3 is inhibited by cholera toxin. TFMU-ADPr and TFMU-IDPr are versatile tools for assessing small-molecule inhibitors in vitro and probing the regulation of ADP-ribosyl catabolic enzymes.
Project description:Poly (ADP ribose) (PAR) formation catalyzed by PAR polymerase 1 in response to genotoxic stress mediates cell death due to necrosis and apoptosis. PAR glycohydrolase (PARG) has been thought to be the only enzyme responsible for hydrolysis of PAR in vivo. However, we show an alternative PAR-degradation pathway, resulting from action of ADP ribosyl-acceptor hydrolase (ARH) 3. PARG and ARH3, acting in tandem, regulate nuclear and cytoplasmic PAR degradation following hydrogen peroxide (H2O2) exposure. PAR is responsible for induction of parthanatos, a mechanism for caspase-independent cell death, triggered by apoptosis-inducing factor (AIF) release from mitochondria and its translocation to the nucleus, where it initiates DNA cleavage. PARG, by generating protein-free PAR from poly-ADP ribosylated protein, makes PAR translocation possible. A protective effect of ARH3 results from its lowering of PAR levels in the nucleus and the cytoplasm, thereby preventing release of AIF from mitochondria and its accumulation in the nucleus. Thus, PARG release of PAR attached to nuclear proteins, followed by ARH3 cleavage of PAR, is essential in regulating PAR-dependent AIF release from mitochondria and parthanatos.
Project description:Important cellular processes are regulated by poly(ADP-ribosyl)ation. This protein modification is catalyzed mainly by nuclear poly(ADP-ribose) polymerase (PARP) 1 in response to DNA damage. Cytosolic PARP isoforms have been described, whereas the presence of poly(ADP-ribose) (PAR) metabolism in mitochondria is controversial. PAR is degraded by poly(ADP-ribose) glycohydrolase (PARG). Recently, ADP-ribosylhydrolase 3 (ARH3) was also shown to catalyze PAR-degradation in vitro. PARG is encoded by a single, essential gene. One nuclear and three cytosolic isoforms result from alternative splicing. The presence and origin of a mitochondrial PARG is still unresolved. We establish here the genetic background of a human mitochondrial PARG isoform and investigate the molecular basis for mitochondrial poly(ADP-ribose) degradation. In common with a cytosolic 60-kDa human PARG isoform, the mitochondrial protein did not catalyze PAR degradation because of the absence of exon 5-encoded residues. In mice, we identified a transcript encoding an inactive cytosolic 52-kDa PARG lacking the mitochondrial targeting sequence and a substantial portion of exon 5. Thus, mammalian PARG genes encode isoforms that do not catalyze PAR degradation. On the other hand, embryonic fibroblasts from ARH3(-/-) mice lack most of the mitochondrial PAR degrading activity detected in wild-type cells, demonstrating a potential involvement of ARH3 in PAR metabolism.
Project description:ADP-ribosylation of proteins plays key roles in multiple biological processes, including DNA damage repair. Recent evidence suggests that serine is an important acceptor for ADP-ribosylation, and that serine ADP-ribosylation is hydrolyzed by ADP-ribosylhydrolase 3 (ARH3 or ADPRHL2). However, the structural details in ARH3-mediated hydrolysis remain elusive. Here, we determined the structure of ARH3 in a complex with ADP-ribose (ADPR). Our analyses revealed a group of acidic residues in ARH3 that keep two Mg2+ ions at the catalytic center for hydrolysis of Ser-linked ADP-ribosyl group. In particular, dynamic conformational changes involving Glu41 were observed in the catalytic center. Our observations suggest that Mg2+ ions together with Glu41 and water351 are likely to mediate the cleavage of the glycosidic bond in the serine-ADPR substrate. Moreover, we found that ADPR is buried in a groove and forms multiple hydrogen bonds with the main chain and side chains of ARH3 residues. On the basis of these structural findings, we used site-directed mutagenesis to examine the functional roles of key residues in the catalytic pocket of ARH3 in mediating the hydrolysis of ADP-ribosyl from serine and DNA damage repair. Moreover, we noted that ADPR recognition is essential for the recruitment of ARH3 to DNA lesions. Taken together, our study provides structural and functional insights into the molecular mechanism by which ARH3 hydrolyzes the ADP-ribosyl group from serine and contributes to DNA damage repair.
Project description:Poly(ADP-ribosyl)ation (PAR) is a versatile and complex posttranslational modification composed of repeating units of ADP-ribose arranged into linear or branched polymers. This scaffold is linked to the regulation of many of cellular processes including the DNA damage response, alteration of chromatin structure and Wnt signalling. Despite decades of research, the principles and mechanisms underlying all steps of PAR removal remain actively studied. In this work, we synthesise well-defined PAR branch point molecules and demonstrate that PARG, but not ARH3, can resolve this distinct PAR architecture. Structural analysis of ARH3 in complex with dimeric ADP-ribose as well as an ADP-ribosylated peptide reveal the molecular basis for the hydrolysis of linear and terminal ADP-ribose linkages. We find that ARH3-dependent hydrolysis requires both rearrangement of a catalytic glutamate and induction of an unusual, square-pyramidal magnesium coordination geometry.
Project description:Poly(ADP-ribosyl)ation refers to the covalent attachment of ADP-ribose to protein, generating branched, long chains of ADP-ribose moieties, known as poly(ADP-ribose) (PAR). Poly(ADP-ribose) polymerase 1 (PARP1) is the main polymerase and acceptor of PAR in response to DNA damage. Excessive intracellular PAR accumulation due to PARP1 activation leads cell death in a pathway known as parthanatos. PAR degradation is mainly controlled by poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribose-acceptor hydrolase 3 (ARH3). Our previous results demonstrated that ARH3 confers protection against hydrogen peroxide (H2O2) exposure, by lowering cytosolic and nuclear PAR levels and preventing apoptosis-inducing factor (AIF) nuclear translocation. We identified a family with an ARH3 gene mutation that resulted in a truncated, inactive protein. The 8-year-old proband exhibited a progressive neurodegeneration phenotype. In addition, parthanatos was observed in neurons of the patient's deceased sibling, and an older sibling exhibited a mild behavioral phenotype. Consistent with the previous findings, the patient's fibroblasts and ARH3-deficient mice were more sensitive, respectively, to H2O2 stress and cerebral ischemia/reperfusion-induced PAR accumulation and cell death. Further, PARP1 inhibition alleviated cell death and injury resulting from oxidative stress and ischemia/reperfusion. PARP1 inhibitors may attenuate the progression of neurodegeneration in affected patients with ARH3 deficiency.
Project description:Protein poly(ADP-ribosyl)ation (PARylation) regulates a number of important cellular processes. Poly(ADP-ribose) glycohydrolase (PARG) is the primary enzyme responsible for hydrolyzing the poly(ADP-ribose) (PAR) polymer in vivo. Here we report crystal structures of the mouse PARG (mPARG) catalytic domain, its complexes with ADP-ribose (ADPr) and a PARG inhibitor ADP-HPD, as well as four PARG catalytic residues mutants. With these structures and biochemical analysis of 20 mPARG mutants, we provide a structural basis for understanding how the PAR polymer is recognized and hydrolyzed by mPARG. The structures and activity complementation experiment also suggest how the N-terminal flexible peptide preceding the PARG catalytic domain may regulate the enzymatic activity of PARG. This study contributes to our understanding of PARG catalytic and regulatory mechanisms as well as the rational design of PARG inhibitors.
Project description:ADP-ribosylation is a reversible and site-specific post-translational modification that regulates a wide array of cellular signaling pathways. Regulation of ADP-ribosylation is vital for maintaining genomic integrity, and uncontrolled accumulation of poly(ADP-ribosyl)ation triggers a poly(ADP-ribose) (PAR)-dependent release of apoptosis-inducing factor from mitochondria, leading to cell death. ADP-ribosyl-acceptor hydrolase 3 (ARH3) cleaves PAR and mono(ADP-ribosyl)ation at serine following DNA damage. ARH3 is also a metalloenzyme with strong metal selectivity. While coordination of two magnesium ions (Mg<sup>A</sup> and Mg<sup>B</sup>) significantly enhances its catalytic efficiency, calcium binding suppresses its function. However, how the coordination of different metal ions affects its catalysis has not been defined. Here, we report a new crystal structure of ARH3 complexed with its product ADP-ribose and calcium. This structure shows that calcium coordination significantly distorts the binuclear metal center of ARH3, which results in decreased binding affinity to ADP-ribose, and suboptimal substrate alignment, leading to impaired hydrolysis of PAR and mono(ADP-ribosyl)ated serines. Furthermore, combined structural and mutational analysis of the metal-coordinating acidic residues revealed that Mg<sup>A</sup> is crucial for optimal substrate positioning for catalysis, whereas Mg<sup>B</sup> plays a key role in substrate binding. Our collective data provide novel insights into the different roles of these metal ions and the basis of metal selectivity of ARH3 and contribute to understanding the dynamic regulation of cellular ADP-ribosylations during the DNA damage response.
Project description:ARH3/ADPRHL2 and PARG are the primary enzymes reversing ADP-ribosylation in vertebrates, yet their functions in vivo remain unclear. ARH3 is the only hydrolase able to remove serine-linked mono(ADP-ribose) (MAR) but is much less efficient than PARG against poly(ADP-ribose) (PAR) chains in vitro. Here, by using ARH3-deficient cells, we demonstrate that endogenous MARylation persists on chromatin throughout the cell cycle, including mitosis, and is surprisingly well tolerated. Conversely, persistent PARylation is highly toxic and has distinct physiological effects, in particular on active transcription histone marks such as H3K9ac and H3K27ac. Furthermore, we reveal a synthetic lethal interaction between ARH3 and PARG and identify loss of ARH3 as a mechanism of PARP inhibitor resistance, both of which can be exploited in cancer therapy. Finally, we extend our findings to neurodegeneration, suggesting that patients with inherited ARH3 deficiency suffer from stress-induced pathogenic increase in PARylation that can be mitigated by PARP inhibition.
Project description:Poly(ADP-ribosyl)ation is a post-translational modification of proteins involved in regulation of many cellular pathways. Poly(ADP-ribose) (PAR) consists of chains of repeating ADP-ribose nucleotide units and is synthesized by the family of enzymes called poly(ADP-ribose) polymerases (PARPs). This modification can be removed by the hydrolytic action of poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylhydrolase 3 (ARH3). Hydrolytic activity of macrodomain proteins (MacroD1, MacroD2 and TARG1) is responsible for the removal of terminal ADP-ribose unit and for complete reversion of protein ADP-ribosylation. Poly(ADP-ribosyl)ation is widely utilized in eukaryotes and PARPs are present in representatives from all six major eukaryotic supergroups, with only a small number of eukaryotic species that do not possess PARP genes. The last common ancestor of all eukaryotes possessed at least five types of PARP proteins that include both mono and poly(ADP-ribosyl) transferases. Distribution of PARGs strictly follows the distribution of PARP proteins in eukaryotic species. At least one of the macrodomain proteins that hydrolyse terminal ADP-ribose is also always present. Therefore, we can presume that the last common ancestor of all eukaryotes possessed a fully functional and reversible PAR metabolism and that PAR signalling provided the conditions essential for survival of the ancestral eukaryote in its ancient environment. PARP proteins are far less prevalent in bacteria and were probably gained through horizontal gene transfer. Only eleven bacterial species possess all proteins essential for a functional PAR metabolism, although it is not known whether PAR metabolism is truly functional in bacteria. Several dsDNA viruses also possess PARP homologues, while no PARP proteins have been identified in any archaeal genome. Our analysis of the distribution of enzymes involved in PAR metabolism provides insight into the evolution of these important signalling systems, as well as providing the basis for selection of the appropriate genetic model organisms to study the physiology of the specific human PARP proteins.
Project description:ADP-ribosylation (ADPr) is a posttranslational modification (PTM) of proteins that controls many cellular processes, including DNA repair, transcription, chromatin regulation and mitosis. A number of proteins catalyse the transfer and hydrolysis of ADPr, and also specify how and when the modification is conjugated to the targets. We recently discovered a new form of ADPr that is attached to serine residues in target proteins (Ser-ADPr) and showed that this PTM is specifically made by PARP1/HPF1 and PARP2/HPF1 complexes. In this work, we found by quantitative proteomics that histone Ser-ADPr is reversible in cells during response to DNA damage. By screening for the hydrolase that is responsible for the reversal of Ser-ADPr, we identified ARH3/ADPRHL2 as capable of efficiently and specifically removing Ser-ADPr of histones and other proteins. We further showed that Ser-ADPr is a major PTM in cells after DNA damage and that this signalling is dependent on ARH3.