ENPP1 processes protein ADP‐ribosylation in vitro

Abstract

ADP‐ribosylation is a conserved post‐translational protein modification that plays a role in all major cellular processes, particularly DNA repair, transcription, translation, stress response and cell death. Hence, dysregulation of ADP‐ribosylation is linked to the physiopathology of several human diseases including cancers, diabetes and neurodegenerative disorders. Protein ADP‐ribosylation can be reversed by the macrodomain‐containing proteins PARG, TARG1, MacroD1 and MacroD2, which hydrolyse the ester bond known to link proteins to ADP‐ribose as well as consecutive ADP‐ribose subunits; targeting this bond can thus result in the complete removal of the protein modification or the conversion of poly(ADP‐ribose) to mono(ADP‐ribose). Recently, proteins containing the NUDIX domain – namely human NUDT16 and bacterial RppH – have been shown to process in vitro protein ADP‐ribosylation through an alternative mechanism, converting it into protein‐conjugated ribose‐5′‐phosphate (R5P, also known as pR). Though this protein modification was recently identified in mammalian tissues, its physiological relevance and the mechanism of generating protein phosphoribosylation are currently unknown. Here, we identified ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) as the first known mammalian enzyme lacking a NUDIX domain to generate pR from ADP‐ribose on modified proteins in vitro. Thus, our data show that at least two enzyme families – Nudix and ENPP/NPP – are able to metabolize protein‐conjugated ADP‐ribose to pR in vitro, suggesting that pR exists and may be conserved from bacteria to mammals. We also demonstrate the utility of ENPP1 for converting protein‐conjugated mono(ADP‐ribose) and poly(ADP‐ribose) into mass spectrometry‐friendly pR tags, thus facilitating the identification of ADP‐ribosylation sites.

Disruption of Macrodomain Protein SCO6735 Increases Antibiotic Production in Streptomyces coelicolor

“…Recombinant PARG was purified as described previously (54). pGEX-4T1 GST-PARP10cd (amino acids 818–1025) was purified as previously described (55) with slight modifications. Briefly, after binding of GST-tagged PARP10 on glutathione-Sepharose beads (GE Healthcare), the protein was extensively washed in lysis buffer and equilibrated in PARP10 reaction buffer (50 m m Tris-HCl, pH 7.5, 75 m m KCl, 4 m m MgCl 2 , 0.25 m m DTT).…”

“…After 30 min, the reactions were split in two, and SCO6735 was added to one aliquot at 5 μ m concentration and incubated for additional 30 min. Auto-ADP-ribosylation of PARP1 E988Q mono-mutant was performed as previously described (55, 74), using 0.5 μ m of recombinant PARP1 E988Q. After 30 min, reaction were split and treated or not with 1 μ m of SCO6735 or SCO6735 G128E mutant.…”

“…The beads were pelleted again, and 1 μl of the liquid was phase-loaded on the thin layer chromatography plate (TLC; Macherey-Nagel, Polygram CEL 300 PEI/UV254). As [ 32 P]ADP-ribose marker, PARP1 (Trevigen) was automodified, passed three times through G25 columns (GE Healthcare), and treated with recombinant PARG as previously described (55, 74). The plates were developed in 0.15 m LiCl and 0.15 m formic acid.…”

Insights into the biogenesis, function, and regulation of ADP-ribosylation

“…By contrast, enzymes that specifically cleave the lysine–ADPr or the cysteine–ADPr bond have not yet been identified, although the Nudix hydrolase NUDT16 (refs. 64,65) or the ectonucleotide pyrophosphatase (ENPP1) 66 , which can cleave the phosphodiester bond in protein-bound ADPr, might be responsible for their removal in cells. It is not well-studied whether specific aa-ADPr linkages impact recognition by specific ADPr hydrolases or alter the ability of specific ADPr hydrolases to remove the modification.…”

ADP-Ribosylated Peptide Enrichment and Site Identification: The Phosphodiesterase-Based Method

“…In the case of MAR and PAR degradation, the field has traditionally used snake venom phosphodiesterase (SVP) [25], an enzyme which is commercially available but must be purified from snake venom and therefore is subject to high lot-to-lot variability. As it is the simplest option, we have outlined its preparation and use in this example, but it is worth knowing that three alternatives have recently been proposed: the bacterial protein RppH [26] and the human protein NudT16 [27] from the Nudix family of enzymes, and the murine protein ENPP1 from the NPP enzyme family [28]. These proteins can be recombinantly expressed and purified from E. coli , making them an attractive replacement for SVP.…”