Combining affinity purification by ADP-ribose-binding macro domains with mass spectrometry to define the mammalian ADP-ribosyl proteome.
Mono-ADP-ribosylation is a reversible posttranslational modification that modulates the function of target proteins. The enzymes that catalyze this reaction in mammalian cells are either bacterial pathogenic toxins or endogenous cellular ADP-ribosyltransferases. For the latter, both the enzymes and their targets have largely remained elusive, mainly due to the lack of specific techniques to study this reaction. The recent discovery of the macro domain, a protein module that interacts selectively with ADP-ribose, prompted us to investigate whether this interaction can be extended to the identification of ADP-ribosylated proteins. Here, we report that macro domains can indeed be used as selective baits for high-affinity purification of mono-ADP-ribosylated proteins, which can then be identified by mass spectrometry. Using this approach, we have identified a series of cellular targets of ADP-ribosylation reactions catalyzed by cellular ADP-ribosyltransferases and toxins. These proteins include most of the known targets of ADP-ribosylation, indicating the validity of this method, and a large number of other proteins, which now need to be individually validated. This represents an important step toward the discovery of new ADP-ribosyltransferase targets and an understanding of the physiological role and the pharmacological potential of this protein modification.
Dani
Nadia
N
Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Via Nazionale, 66030 Santa Maria Imbaro (Chieti), Italy.
Stilla
Annalisa
A
Marchegiani
Adriano
A
Tamburro
Antonio
A
Till
Susanne
S
Ladurner
Andreas G
AG
Corda
Daniela
D
Di Girolamo
Maria
M
D036002:ADP Ribose Transferases / Q000378:metabolism; D000246:Adenosine Diphosphate Ribose / Q000737:chemistry*; D000818:Animals; D019611:Archaeoglobus fulgidus; D001427:Bacterial Toxins / Q000378:metabolism; D001665:Binding Sites; D002846:Chromatography, Affinity; D006801:Humans; D013058:Mass Spectrometry; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*
D001427:Bacterial Toxins; D011506:Proteins; D000246:Adenosine Diphosphate Ribose; D036002:ADP Ribose Transferases