Nudix hydrolases degrade protein-conjugated ADP-ribose.

Casey M Daniels,Anthony K L Leung,Sandra B Gabelli,Shao-En Ong,Puchong Thirawatananond

doi:10.1038/srep18271

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.

Highlights

Nudix hydrolase activity, free ADPr would amass during the breakdown of PAR23,24, as a side product of tRNA synthesis[25], following NAD+ glycohydrolysis[26], following deacetylation of O-acetyl-ADPr27, or through the breakdown of the signaling molecule cyclic ADPr28
ADPr degrading Nudix enzymes are broadly conserved, with humans possessing at least six distinct ADPr pyrophosphatases (ADPrases) responsible for hydrolyzing ADPr to AMP and phosphoribose[18,29]. In light of this known ADPrase activity, we reasoned that Nudix hydrolases could potentially replace snake venom phosphodiesterase I (SVP) for generating phosphoribose tags at ADP-ribosylation sites in the proteomics pipeline described above, and screened bacterial Nudix hydrolases for comparable hydrolysis activity of protein-conjugated ADPr
We provide a structure-based rationale for the inability of Nudix ADPrases to degrade protein-conjugated ADPr, in contrast to Nudix RNA decapping enzymes, and demonstrate the use of both EcRppH and HsNudT16 in the identification of protein ADP-ribosylation sites by mass spectrometry

Summary

Introduction

Free ADPr would amass during the breakdown of PAR23,24, as a side product of tRNA synthesis[25], following NAD+ glycohydrolysis[26], following deacetylation of O-acetyl-ADPr27, or through the breakdown of the signaling molecule cyclic ADPr28. ADPr degrading Nudix enzymes are broadly conserved, with humans possessing at least six distinct ADPr pyrophosphatases (ADPrases) responsible for hydrolyzing ADPr to AMP and phosphoribose[18,29] In light of this known ADPrase activity, we reasoned that Nudix hydrolases could potentially replace SVP for generating phosphoribose tags at ADP-ribosylation sites in the proteomics pipeline described above, and screened bacterial Nudix hydrolases for comparable hydrolysis activity of protein-conjugated ADPr. In light of this known ADPrase activity, we reasoned that Nudix hydrolases could potentially replace SVP for generating phosphoribose tags at ADP-ribosylation sites in the proteomics pipeline described above, and screened bacterial Nudix hydrolases for comparable hydrolysis activity of protein-conjugated ADPr These efforts lead to the identification of the RNA 5′ pyrophosphohydrolase (RppH) from Escherichia coli (EcRppH) as capable of degrading protein-conjugated ADPr, including both MAR and PAR. We provide a structure-based rationale for the inability of Nudix ADPrases to degrade protein-conjugated ADPr, in contrast to Nudix RNA decapping enzymes, and demonstrate the use of both EcRppH and HsNudT16 in the identification of protein ADP-ribosylation sites by mass spectrometry

Methods

Results

Conclusion