Engineering the substrate specificity of ADP-ribosyltransferases for identifying direct protein targets

Ian Carter-O'Connell, Haihong Jin, Rory K. Morgan, Larry David, Michael Cohen

Research output: Contribution to journalArticle

52 Citations (Scopus)

Abstract

Adenosine diphosphate ribosyltransferases (ARTDs; ARTD1-17 in humans) are emerging as critical regulators of cell function in both normal physiology and disease. These enzymes transfer the ADP-ribose moiety from its substrate, nicotinamide adenine dinucleotide (NAD+), to amino acids of target proteins. The functional redundancy and overlapping target specificities among the 17 ARTDs in humans make the identification of direct targets of individual ARTD family members in a cellular context a formidable challenge. Here we describe the rational design of orthogonal NAD+ analogue-engineered ARTD pairs for the identification of direct protein targets of individual ARTDs. Guided by initial inhibitor studies with nicotinamide analogues containing substituents at the C-5 position, we synthesized an orthogonal NAD+ variant and found that it is used as a substrate for several engineered ARTDs (ARTD1, -2, and -6) but not their wild-type counterparts. Comparing the target profiles of ARTD1 (PARP1) and ARTD2 (PARP2) in nuclear extracts highlighted the semi-complementary, yet distinct, protein targeting. Using affinity purification followed by tandem mass spectrometry, we identified 42 direct ARTD1 targets and 301 direct ARTD2 targets. This represents a powerful new technique for identifying direct protein targets of individual ARTD family members, which will facilitate studies delineating the pathway from ARTD activation to a given cellular response.

Original languageEnglish (US)
Pages (from-to)5201-5204
Number of pages4
JournalJournal of the American Chemical Society
Volume136
Issue number14
DOIs
StatePublished - Apr 9 2014

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ADP Ribose Transferases
Administrative data processing
Substrate Specificity
NAD
Proteins
Substrates
Adenosine Diphosphate Ribose
Forensic Anthropology
Niacinamide
Physiology
Protein Transport
Tandem Mass Spectrometry
Adenosine Diphosphate
Purification
Mass spectrometry
Redundancy
Amino acids
Enzymes
Chemical activation
Amino Acids

ASJC Scopus subject areas

  • Chemistry(all)
  • Catalysis
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

Engineering the substrate specificity of ADP-ribosyltransferases for identifying direct protein targets. / Carter-O'Connell, Ian; Jin, Haihong; Morgan, Rory K.; David, Larry; Cohen, Michael.

In: Journal of the American Chemical Society, Vol. 136, No. 14, 09.04.2014, p. 5201-5204.

Research output: Contribution to journalArticle

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