PARG has a robust endo-glycohydrolase activity that releases protein-free poly(ADP-ribose) chains
Introduction
The transient, posttranslational modification of proteins through the addition of poly(ADP-ribose) (PAR) by PAR polymerase 1 (PARP1) is required for the maintenance of genomic integrity [1,2]. However, the uncontrolled accumulation of PAR polymers is cytotoxic [[3], [4], [5], [6]]. Upon excessive DNA damage, PARP1 becomes hyper-PARylated and a nuclear-to-cytoplasmic translocation of protein-free PAR chains activates a PAR-dependent cell death pathway (called parthanatos) by triggering a PAR-mediated release of apoptosis-inducing factor (AIF) from mitochondria [5,6]. However, it remains unclear how the protein-free PAR is generated.
The cellular PAR levels are dynamically regulated by PAR glycohydrolase (PARG), a predominant PAR turnover enzyme in mammals [7,8]. A knock-out of PARG in mice results in early embryonic lethality due to the concomitant PAR accumulation [4]. While excessive PAR synthesis by PARP1 can deplete cellular ATP stores, PAR turnover by PARG enables the recycling of nucleotides to fuel DNA repair enzymes [9,10] and restores the activities of PARylated proteins [11].
Historically, it has been suggested that PARG has both exo- and endo-glycohydrolase activities (Fig. S1) [9,12,13]. Although this is still debated [14], the endo-glycohydrolase activity enables PARG to cleave internal sites of PAR, generating protein-free PAR chains that signal DNA damage and activate parthanatos [1,5,15]. Consistent with the endo-glycosidic activity, PARG-deficient cells showed decreased parthanatos when exposed to oxidative stresses [5,16].
The structure and mechanism of PARG have been studied extensively [[17], [18], [19], [20], [21], [22]]. PARG specifically hydrolyzes α(1′′-2′) O-glycosidic linkages in PAR chains (Fig. S1) [23]. However, PARG is unable to trim the last ADP-ribose unit linked to target proteins, leaving mono(ADP-ribosyl)ated proteins as a substrate for mono-ADP-ribosyl-acceptor hydrolases, such as ARH3, MacroD1, MacroD2, and TARG1 [[24], [25], [26], [27]]. Structures of mammalian PARGs revealed a substantially expanded macrodomain fold, which is elaborated with the unique Tyr-clasp that is structurally buttressed by the mitochondrial targeting sequence [[18], [19], [20],28]. Importantly, the open substrate-binding pocket near the chain-elongation point (2′-OH group of the ribose’; Fig. S1) in PAR polymers suggests that this enzyme would likely engage internal sites of PAR chains for endo-glycosidic cleavage [19].
In line with the endo-glycohydrolase activity, PARG exhibits a biphasic mode of action depending on the length of PAR chains [9,12,13]. Whereas long PAR polymers are efficiently hydrolyzed (Km = ∼1 μM), presumably by a combination of endo- and exo-glycosidic activity [13], smaller PAR chains are poor substrates for PARG (Km > 10 μM). Therefore, this failure to completely degrade PAR polymers into ADP-ribose may help to preserve PAR chains as ligands for the pro-apoptotic protein AIF [15,29]. However, it has been debated whether or not PARG possesses a robust endo-glycohydrolase.
Here, we quantitatively analyzed PARG metabolites and revealed that human PARG releases a significant amount of protein-free PAR chains longer than three ADP-ribose units from PARylated PARP1, which strongly supports its endo-glycohyrolase activity. The released PAR chains are transient and degraded into monomeric ADP-ribose by PARG. Bacterial PARG similarly produces protein-free PAR chains with a remarkably higher ratio of PAR over ADP-ribose, suggesting that endo-glycohydrolase activity is evolutionary conserved and is likely predominant in the early stage of PARG reactions. Our collective results demonstrate the endo-glycohydrolase activity of PARG and establish bacterial PARG as a useful tool for enriching short PAR chains that emerge as an important tool for biomedical research.
Section snippets
Plasmids and protein purification
A gene encoding human PARG389 (residues 389–976) was cloned into a modified pET21b vector with an N-terminal 6X histidine tag and a subsequent preScission protease cleavage site (pET21b-his6-pps). hPARG389 was overexpressed in E. coli Rosetta 2 (DE3) cells and purified by a Ni-NTA (GE healthcare) chromatography. After elution with imidazole (250 mM), the protein was loaded onto a heparin column (GE healthcare) and eluted with a NaCl gradient (0.1–1 M). Fractions with hPARG389 were pooled, and
Human PARG has a robust endo-glycohydrolase activity
Historically, direct measurement of the endo-glycohydrolase activity of PARG has been challenging, and conflicting results have been published [9,[12], [13], [14]]. Recently, it was proposed that PARG acts predominantly as an exo-glycohydrolase that primarily releases monomeric ADPR as a reaction product [14]. However, the open substrate-binding platform found in mammalian PARGs readily exposes the 2′-OH group of the adenosine ribose’ of n ADPR, a chain-elongation point of PAR (Fig. S1) [19].
Discussion
Although it has been shown that protein-free PAR chains signal DNA damage and activate parthanatos through direct interactions with AIF [5,6], the primary source for the protein-free PAR during DNA damage responses remains unclear. Here, we demonstrated that PARGs from mammals to bacteria have an evolutionarily conserved endo-glycohydrolase activity that releases protein-free PAR chains (Fig. 1, Fig. 2). This endo-glycohydrolase activity appears to function in the early reaction stage,
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was supported by V foundation V scholar Grant (V2018-25 to I.K.K), American Cancer Society RSG Grant (133405-RSG-19-200-01-DMC to I.K.K.), and Marlene Harris Ride Cincinnati Breast Cancer Pilot Grant program (to I.K.K).
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2023, Journal of Biological ChemistryThe expanding universe of PARP1-mediated molecular and therapeutic mechanisms
2022, Molecular CellCitation Excerpt :ADPRylation is a dynamic and reversible PTM, which is maintained and regulated by a network of “writers” (PARPs), “readers” (ADPR binding domains), and “erasers” (ADP-ribosyl hydrolases) (Gupte et al., 2017; Figure 2B). The relevance of the readers and erasers to ADPRylation and PARP function is discussed in detail for the examples presented below as well as elsewhere (Kliza et al., 2021; Palazzo et al., 2017; Pourfarjam et al., 2020; Rack et al., 2020). ADPRylation has been linked to a wide variety of molecular and cellular processes through its ability to alter the properties and functions of substrate proteins as well as their network of interacting proteins (Gibson and Kraus, 2012).
Poly(ADP-ribose) drives condensation of FUS via a transient interaction
2022, Molecular CellCitation Excerpt :In vitro data indicate that PAR can be released by PARG through its endo-glycosidic activity or by the ADP-ribosylhydrolase TARG1, which removes entire PAR chains from PARylated proteins (Sharifi et al., 2013). However, definitive evidence of long-lived soluble PAR in cells is currently lacking (Mashimo et al., 2013; Pourfarjam et al., 2020). Second, PARP5a may PARylate FUS, which then becomes proficient at condensation.
Targeting SARS-CoV-2 Nsp3 macrodomain structure with insights from human poly(ADP-ribose) glycohydrolase (PARG) structures with inhibitors
2021, Progress in Biophysics and Molecular BiologyCitation Excerpt :PARG has been the focus of concentrated inhibitor development for cancer, both as a complement to and substitute for clinical PARP inhibitors (PARPi) (Chen and Yu, 2019; Houl et al., 2019; Slade, 2020), which act in part by trapping PARP1 on damaged DNA (Zandarashvili et al., 2020) and by acting synergistically to kill cancer cells with defective homology-directed repair (Syed and Tainer, 2018) or alternative end-joining (Eckelmann et al., 2020). Following DNA damage, PARG reverses the signaling response initiated by PARP1 at ssDNA breaks by hydrolyzing the ‘cloud’ of poly(ADP-ribose) (PAR) into mono-nucleotide ADP-ribose (ADPr) (Pourfarjam et al., 2020; Slade et al., 2011). The dispersion of the PAR cloud enables subsequent progression of DNA repair at the damage site.
DNA replication stress and emerging prospects for PARG inhibitors in ovarian cancer therapy
2021, Progress in Biophysics and Molecular BiologyCitation Excerpt :A single PARG gene has been identified which encodes five different transcripts generated by alternative splicing (Meyer-Ficca et al., 2004) Three of these isoforms are catalytically active, cleaving the O-Glycosidic bond between ADP-ribose subunits (Fig. 1) (Haince et al., 2006; Hatakeyama et al., 1986). PARG exoglycosidase activity at the termini of PAR chains releases ADP-ribose, whereas PARG endoglycosidase activity releases intact PAR chains (Barkauskaite et al., 2013a; Pourfarjam et al., 2020). However, PARG is unable to remove the final ADP-ribose subunit attached to proteins because of steric hindrance with the acceptor amino acid side chains, and hence other ADP-hydrolases perform this function (Barkauskaite et al., 2013a).
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These authors equally contributed to this work.