PARG has a robust endo-glycohydrolase activity that releases protein-free poly(ADP-ribose) chains

https://doi.org/10.1016/j.bbrc.2020.04.120Get rights and content

Highlights

  • Human PARG has a robust endo-glycohydrolase activity.

  • The endo-glycohydrolase activity is conserved across organisms.

  • Released protein-free PAR chains are highly transient.

  • Bacterial PARG is a useful tool to efficiently enrich short PAR chains.

Abstract

Poly(ADP-ribosyl)ation (PARylation) regulates DNA damage response, chromatin structure, and cell-fate. Dynamic regulation of cellular PAR levels is crucial for the maintenance of genomic integrity and excessive cellular PAR activates a PAR-dependent cell death pathway. Thus, PAR serves as a cell-death signal; however, it has been debated how the protein-free PAR is generated. Here, we demonstrate that PAR glycohydrolases (PARGs) from mammals to bacteria have a robust endo-glycohydrolase activity, releasing protein-free PAR chains longer than three ADP-ribose units as early reaction products. Released PAR chains are transient and rapidly degraded to monomeric ADP-ribose, which is consistent with a short half-life of PAR during DNA damage responses. Computational simulations using a tri-ADP-ribose further support that PARG can efficiently bind to internal sites of PAR for the endo-glycosidic cleavage. Our collective results suggest PARG as a key player in producing protein-free PAR during DNA damage signaling and establish bacterial PARG as a useful tool to enrich short PAR chains that emerge as important reagents for biomedical research.

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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