Expression, purification, crystallization and preliminary X-ray analysis of rat ecto-ADP-ribosyltransferase 2 (ART2.2)

Mueller-Dieckmann, Christoph; Scheuermann, Till; Wursthorn, Karsten; Schröder, Jörg; Haag, Friedrich; Schulz, Georg E.; Koch-Nolte, Friedrich

doi:10.1107/s090744490200700x

Cited by 10 publications

(6 citation statements)

References 19 publications

(17 reference statements)

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“…In the case of ALC1 and histone MACROH2A proteins only the macrodomains were purified rather than full length proteins. Mouse ARTC2.2 protein was purified as previously described ( Mueller-Dieckmann et al, 2002 ). PARP1 wild type and the E988Q mutant were expressed and purified as previously reported ( Langelier et al, 2011 ).…”

Section: Methodsmentioning

confidence: 99%

Serine ADP-ribosylation reversal by the hydrolase ARH3

Fontana

Bonfiglio

Palazzo

et al. 2017

eLife

199

265

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ADP-ribosylation (ADPr) is a posttranslational modification (PTM) of proteins that controls many cellular processes, including DNA repair, transcription, chromatin regulation and mitosis. A number of proteins catalyse the transfer and hydrolysis of ADPr, and also specify how and when the modification is conjugated to the targets. We recently discovered a new form of ADPr that is attached to serine residues in target proteins (Ser-ADPr) and showed that this PTM is specifically made by PARP1/HPF1 and PARP2/HPF1 complexes. In this work, we found by quantitative proteomics that histone Ser-ADPr is reversible in cells during response to DNA damage. By screening for the hydrolase that is responsible for the reversal of Ser-ADPr, we identified ARH3/ADPRHL2 as capable of efficiently and specifically removing Ser-ADPr of histones and other proteins. We further showed that Ser-ADPr is a major PTM in cells after DNA damage and that this signalling is dependent on ARH3.DOI: http://dx.doi.org/10.7554/eLife.28533.001

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Section: Methodsmentioning

confidence: 99%

Serine ADP-ribosylation reversal by the hydrolase ARH3

Fontana

Bonfiglio

Palazzo

et al. 2017

eLife

199

265

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“…Sequences of Pma ARH3 (GenBank: XP_015504659; aa residues 19-370), Xtr ARH3 (GenBank: CAJ81573.1; full length) and Lch ARH3 (GenBank: XP_005988572; aa residues 10-362) were codon optimized for expression in E. coli , gene synthesized (GeneArt™; Thermo Fisher Scientific) and cloned into pET-9H 3 ( Rack et al., 2015 ) using the vector’s NcoI/BamHI sites. Expression vectors for h ARH3, HPF1, PARP1, PARG and m ARTC2.2 were described earlier ( Fontana et al., 2017 , Gibbs-Seymour et al., 2016 , Lambrecht et al., 2015 , Langelier et al., 2011 , Mueller-Dieckmann et al., 2002 , Tucker et al., 2012 ). All indicated mutations were introduced via PCR based site-directed mutagenesis.…”

Section: Methodsmentioning

confidence: 99%

(ADP-ribosyl)hydrolases: Structural Basis for Differential Substrate Recognition and Inhibition

Rack

Ariza

Drown

et al. 2018

Cell Chemical Biology

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SummaryProtein ADP-ribosylation is a highly dynamic post-translational modification. The rapid turnover is achieved, among others, by ADP-(ribosyl)hydrolases (ARHs), an ancient family of enzymes that reverses this modification. Recently ARHs came into focus due to their role as regulators of cellular stresses and tumor suppressors. Here we present a comprehensive structural analysis of the enzymatically active family members ARH1 and ARH3. These two enzymes have very distinct substrate requirements. Our data show that binding of the adenosine ribose moiety is highly diverged between the two enzymes, whereas the active sites harboring the distal ribose closely resemble each other. Despite this apparent similarity, we elucidate the structural basis for the selective inhibition of ARH3 by the ADP-ribose analogues ADP-HPD and arginine-ADP-ribose. Together, our biochemical and structural work provides important insights into the mode of enzyme-ligand interaction, helps to understand differences in their catalytic behavior, and provides useful tools for targeted drug design.

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“…The fractions containing the ENPP1 protein were concentrated to 2.1 mg·mL −1 , based on an extinction coefficient of 1Au = 1 mg·mL −1 (0.63 mg final yield) before use. pASK60‐OmpA‐mARTC2.2 6xHis‐Flag tag was a gift from Friedrich Koch‐Nolte (Universitätsklinikum Hamburg‐Eppendorf) and purified as previously described .…”

Section: Methodsmentioning

confidence: 99%

ENPP1 processes protein ADP‐ribosylation in vitro

et al. 2016

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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.

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Expression, purification, crystallization and preliminary X-ray analysis of rat ecto-ADP-ribosyltransferase 2 (ART2.2)

Cited by 10 publications

References 19 publications

Serine ADP-ribosylation reversal by the hydrolase ARH3

Serine ADP-ribosylation reversal by the hydrolase ARH3

(ADP-ribosyl)hydrolases: Structural Basis for Differential Substrate Recognition and Inhibition

ENPP1 processes protein ADP‐ribosylation in vitro

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