Combining affinity purification by ADP-ribose-binding macro domains with mass spectrometry to define the mammalian ADP-ribosyl proteome

Abstract

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.

Fig. 1.

Macro module pull-down assay of mono-ADP-ribosylated G protein β subunit from CHO cells. (A–C) Plasma membrane proteins (10 μg) were ADP-ribosylated with [³²P]-NAD⁺ (1 h at 37°C) in the absence (A–C) or presence (C) of the purified βγ dimer (250 ng) (see Materials and Methods), and solubilized and pulled down with 200 pmol of the indicated macro modules. The pulled-down samples (A, C) and the unbound fractions (B; one-eighth of the total volume) were analyzed by autoradiography (AR) and Western blot analysis (WB) with an anti-β subunit antibody. The Ponceau red staining (A, lower) of the pulled-down samples shows that equivalent amounts of the different macro modules were used. (D) Plasma membrane proteins (10 μg) were incubated with unlabeled β-NAD⁺ or α-NAD⁺ (12 h, 30°C). His₆-mAf1521-pulled-down proteins and the corresponding input material were analyzed by Western blot with an antibody to the β subunit. When β-NAD⁺ was replaced with α-NAD⁺, the residual labeling of substrates can be ascribed to the contaminating β-NAD⁺ present in the commercial α-NAD⁺ preparations. The data shown are representative of two to six experiments.

Fig. 2.

Efficiency and reversibility of binding to His₆-mAf1521 of the ADP-ribosylated βγ dimer. CHO plasma membrane proteins (10 μg) and 250 ng of purified βγ dimer were ADP-ribosylated in the presence of [³²P]-NAD⁺ for 12 h at 30°C. After solubilization, they were pulled down with His₆-mAf1521. (A) Increasing concentrations of His₆-mAf1521 in the [³²P]-ADP-ribosylated β subunit pull-down assay, shown as percentage of the input of [³²P]-ADP-ribosylated β subunit (≈4 pmol). Data are means ± SD of five independent experiments. (B) As for (A), showing pulled-down samples and unbound fractions (one-eighth of the total volume) from a representative experiment, as analyzed by autoradiography (AR) and by Western blotting (WB) with an antibody against the β subunit that recognizes both the unmodified (36 kDa) and the ADP-ribosylated (36.5 kDa) β subunit. The disappearance of the [³²P]-ADP-ribosylated β subunit from the unbound fraction was also followed in parallel (B, lower). (C) Pull-down of the [³²P]-ADP-ribosylated β subunit after its incubation with 30 pmol of His₆-mAf1521 in the presence of increasing concentrations of ADP-ribose (1 nM to 1 mM) (“competition” conditions). Data are expressed as percentages of [³²P]-ADP-ribosylated β subunit pulled down in the absence of ADP-ribose and are means ± SD from three independent experiments. Inset: Autoradiography of the pulled-down samples from a representative experiment. (D) As for (C), but with the addition of ADP-ribose after the pull-down assay of the [³²P]-ADP-ribosylated β subunit by 30 pmol of His₆-mAf1521 (“displacement” conditions). At 100 μM ADP-ribose (≈400 pmol under these incubation conditions), all of the His₆-mAf1521-bound [³²P]-ADP-ribosylated β subunit (≈4 pmol) was displaced. A ≈100-fold excess of ADP-ribose can displace the immobilized ADP-ribosylated β subunit from the His₆-mAf1521-resin complex. Data are means ± SD from three independent experiments. Curve fitting analyses in (C) and (D) were performed with GraphPad PRISM, giving ADP-ribose EC₅₀ values of 5 and 10 μM, respectively.

Fig. 3.

(A, B) GST-mAf1521 pull-down assay of proteins from CHO cell membranes that are either ADP-ribosylated by PT or endogenously ADP-ribosylated. (A) Cells were either untreated (control) or intoxicated with 5 nM PT for 15 h (PT), and then their solubilized membrane proteins (25 μg) were ADP-ribosylated with [³²P]-NAD⁺ and in the absence (−) or presence of PT (+) (see Materials and Methods). The samples were analyzed by SDS/PAGE and visualized by autoradiography (AR). (B) Cells were untreated (−) or intoxicated with PT (+), and their solubilized membrane proteins (2 mg) were pulled down with 2 nmol of GST-mAf1521. The samples were analyzed by SDS/PAGE and visualized by silver staining. The indicated band was identified by MALDI-TOF-MS (see Materials and Methods and Table 1). (C, D) His₆-mAf1521 pull-down assay of endogenous ADP-ribosyltransferase substrates. (C) Solubilized HL60 cell membrane proteins (2 mg) were pulled down with 2 nmol of His₆-mAf1521. The samples were analyzed by SDS/PAGE followed by Western blot analysis (WB) with an anti-β subunit antibody. The β subunit relative content in the input was revealed by Western blot analysis (C, lower, WB). (D) Solubilized CHO postnuclear membrane proteins (5 mg) were pulled down with 2 nmol of His₆-mAf1521, resolved by SDS/PAGE, and visualized by silver staining. The indicated bands were identified by MALDI-TOF-MS (see Materials and Methods and Table 1). The data shown are representative of two to six experiments.

Fig. 4.

GST-mAf1521 pull-down assay and protein identification from HL60 cells. Solubilized HL60 cell proteins (20 mg) were pulled down with resin-bound GST-tagged mAf1521/G42E or mAf1521, as indicated, eluted with 20 mM ADP-ribose, then resolved by SDS/PAGE, and visualized by Coomassie blue staining. The indicated bands (in the sample and in the control lanes) were identified by LC-MS/MS analyses (see Materials and Methods and Table 1). The data shown are representative of two experiments.