Nudix hydrolases degrade protein-conjugated ADP-ribose

Abstract

ADP-ribosylation refers to the transfer of the ADP-ribose group from NAD+ to target proteins post-translationally, either attached singly as mono(ADP-ribose) (MAR) or in polymeric chains as poly(ADP-ribose) (PAR). Though ADP-ribosylation is therapeutically important, investigation of this protein modification has been limited by a lack of proteomic tools for site identification. Recent work has demonstrated the potential of a tag-based pipeline in which MAR/PAR is hydrolyzed down to phosphoribose, leaving a 212 Dalton tag at the modification site. While the pipeline has been proven effective by multiple groups, a barrier to application has become evident: the enzyme used to transform MAR/PAR into phosphoribose must be purified from the rattlesnake Crotalus adamanteus venom, which is contaminated with proteases detrimental for proteomic applications. Here, we outline the steps necessary to purify snake venom phosphodiesterase I (SVP) and describe two alternatives to SVP—the bacterial Nudix hydrolase EcRppH and human HsNudT16. Importantly, expression and purification schemes for these Nudix enzymes have already been proven, with high-quality yields easily attainable. We demonstrate their utility in identifying ADP-ribosylation sites on Poly(ADP-ribose) Polymerase 1 (PARP1) with mass spectrometry and discuss a structure-based rationale for this Nudix subclass in degrading protein-conjugated ADP-ribose, including both MAR and PAR.

PARPs and ADP-ribosylation: recent advances linking molecular functions to biological outcomes

“…The investigators showed that different NUDIX enzymes, including the RNA 5 ′ pyrophosphohydrolase (RppH) from Escherichia coli (EcRppH) and the human NUDIX enzyme HsNudT16, can be used for digestion of PAR chains for MS (Table 2). However, in a direct comparison with SVP, only two of the 14 phosphoribosylated peptides detected from automodified PARP-1 by Daniels et al (2015b) were found in all three enzyme-treated samples (SVP vs. EcRppH and HsNudT16). Palazzo et al (2015) detected only two distinct sites of ADP-ribosylation on PARP-1, but these sites overlapped those identified by Daniels et al (2015b).…”

“…a See Kalisch et al (2012), Feijs et al (2013), Krietsch et al (2013), Hottiger (2015), Teloni and Altmeyer (2016), and Rack et al (2016) as well as references therein for a more comprehensive listing of ARBDs. 2006), PAR glycohydrolase (PARG) (Slade et al 2011), TARG/C6orf130 (Sharifi et al 2013), MacroD1 and MacroD2 Rosenthal et al 2013), and the NUDIX family of hydrolases (Daniels et al 2015b). Many of these enzymes contain a macrodomain fold, which allows them to interact with ADP-ribosylated substrates.…”

“…In contrast, TARG, MacroD1, and MacroD2 can hydrolyze the ester bond between the ribose and acceptor amino acids (aspartates or glutamates), thus facilitating the complete removal of the ADP-ribose moiety Rosenthal et al 2013;Sharifi et al 2013). The NUDIX family of hydrolases can hydrolyze PAR chains by targeting the phosphodiester bond in the protein-proximal ADP-ribose unit, which results in the formation of a phosphoribose moiety attached to the acceptor amino acid (Daniels et al 2015b). Not surprisingly, these "erasers" exhibit different specificities and different modes of binding with different targets (Kistemaker et al 2016).…”

“…Enzymatic cleavage Enzymatic cleavage of PAR and ADP-ribose to reduce complexity using enzymes, such as snake venom phosphodiesterase (SVP), ARH3, PARG, or NUDIX hydrolases, is becoming a popular approach as a prelude to MS (Messner et al 2010;Chapman et al 2013;Sharifi et al 2013;Daniels et al 2014Daniels et al , 2015bMartello et al 2016). A number of groups have used SVP, a pyrophosphatase that cleaves ADP-ribose subunits to phosphoribose (also known as ribose-5 ′ -phosphate), which remains covalently attached to the amino acid in the ADP-ribosylated protein, and 5 ′ -AMP, which is released (Chapman et al 2013;Daniels et al 2014).…”

“…To address this issue, Palazzo et al (2015) and Daniels et al (2015b) explored the use of NUDIX hydrolase enzymes instead of SVP. Enzymes in the NUDIX superfamily of hydrolases catalyze the hydrolysis of nucleoside diphosphates linked to other moieties, including ADP-ribose (Fig.…”