PMC:6172693 / 9739-15169 JSONTXT

{"project":"2_test","denotations":[{"id":"30257210-27723750-20058700","span":{"begin":686,"end":690},"obj":"27723750"},{"id":"30257210-27723750-20058702","span":{"begin":1087,"end":1091},"obj":"27723750"},{"id":"30257210-27067600-20058703","span":{"begin":1850,"end":1854},"obj":"27067600"},{"id":"30257210-27067600-20058704","span":{"begin":4164,"end":4168},"obj":"27067600"},{"id":"30257210-22847107-20058705","span":{"begin":4825,"end":4829},"obj":"22847107"},{"id":"30257210-28035797-20058706","span":{"begin":4869,"end":4873},"obj":"28035797"},{"id":"30257210-27686526-20058707","span":{"begin":4948,"end":4952},"obj":"27686526"},{"id":"30257210-27067600-20058784","span":{"begin":4164,"end":4168},"obj":"27067600"}],"text":"Discovery of Tyrosine as a Target Residue for ADPr\nADPr of Ser led us to question whether a hydroxyl group is sufficient and necessary to target an amino acid for ADPr when adjacent to Lys. We therefore decided to substitute H3S10 with threonine (Thr) and tyrosine (Tyr), the two other residues that contain hydroxyl groups, and additionally Glu and Asp as further controls. Not only were we unable to detect ADPr on Glu and Asp but also on Thr residues (Figure 2A). This suggests that although chemically similar to Ser, the additional methyl group on Thr interferes with the ADPr reaction mediated by PARP1/HPF1. In fact, in none of our previous proteomic analyses (Leidecker et al., 2016, Bonfiglio et al., 2017b) were we able to detect Thr-ADPr. Conversely, we identified a reproducible modification of Tyr when we introduced this amino acid instead of Ser10 (Figure 2A). Because Tyr has not previously been described as a substrate for ADPr, we sought mass spectrometric evidence for Tyr-ADPr. Although we could not detect Tyr-ADPr in our histone proteomics data (Leidecker et al., 2016), we confidently identified Tyr-ADPr of HPF1 in an in vitro reaction containing PARP1 (Figures 2B and S2B). We could also identify Ser97 in HPF1 as another site modified in this reaction (Figure 2C). These data suggested that PARP1 was the enzyme responsible for HPF1 Tyr-ADPr modification. To follow up on this point, we modified recombinant HPF1 using a panel of different PARPs and radioactively labeled NAD. We could observe a low but reproducible modification by PARP1 and possibly by PARP2 (Figures 2D, S2A, and S2E). This modification is at least partly dependent on the assembly of the PARP1/HPF1 complex, because the modification of the HPF1 R239A mutant protein (previously shown to be deficient in interacting with PARP; see Gibbs-Seymour et al., 2016) was significantly reduced (Figure 2E).\nFigure 2 Discovery of Tyrosine as a Target Residue for ADPr\n(A) Autoradiogram showing ADPr of H3 peptide (1–20aa) with Ser10 substituted by Ala, Thr, Tyr, Glu, and Asp, alongside Lys9 substituted by Arg and Ala. Coomassie staining of the SDS-PAGE is included.\n(B) High-resolution ETD fragmentation spectrum of an HPF1 peptide modified by ADP-ribose on tyrosine 238. The chemical structure of ADP-ribose is depicted (see also Figure S2B). ∗1, peaks corresponding to co-isolated species in their original charge state. Multiple species in charge states 2–5 passed through the quadrupole and could not be completely deconvoluted.\n(C) High-resolution ETD fragmentation spectrum of an HPF1 peptide modified by ADP-ribose on serine 97. The chemical structure of ADP-ribose is depicted.\n(D) Autoradiogram showing a panel of PARPs incubated with HPF1 protein. Reaction with mono(ADP-ribosyl)ating PARP1 E988Q (EQ) mutant enhances detection of the HPF1 ADPr. Coomassie staining of the SDS-PAGE is included.\n(E) Autoradiogram showing PARP1 E988Q-mediated ADPr of HPF1 WT, HPF1 R239A, and GST-HPF1 proteins. Coomassie staining of the SDS-PAGE is included.\n(F) 293T cells were transfected with the same amount of EV or plasmid expressing WT, S97A, or Y238A FLAG-tagged HPF1 protein and left untreated or treated for 10 or 120 min with H2O2. Inputs and FLAG-IPs were analyzed by western blotting. CMV, cytomegalovirus.\n(G) 293T parental or PARP1 KO cells were transfected with the same amount of EV or plasmid expressing WT FLAG-tagged HPF1 protein and left untreated or treated for 10 or 120 min with H2O2. Inputs and FLAG-IPs were analyzed by western blotting.\nTo confirm the ADPr of HPF1 in vivo, we overexpressed and immunoprecipitated FLAG-tagged HPF1 WT, S97A, and Y238A mutant proteins from 293T cells, as was described above for histone H3. We observed that HPF1 is significantly modified in cells even in undamaged conditions (Figure 2F). We did not detect a major effect of the S97A mutation on the modification of HPF1. However, mutation of the Tyr238 site to Ala had a profound effect on the HPF1 ADPr signal (Figure 2F). This defect may be at least partly due to a reduced ability of the Y238A mutant to interact with PARP1 and to stimulate ADPr (Gibbs-Seymour et al., 2016). To further prove that HPF1 ADPr is dependent on PARP1, we performed FLAG-IP in PARP1 knockout (KO) 293T cells. As can be seen in Figure 2G, HPF1 ADPr is largely missing in PARP1 KO cells. It is likely that the remaining HPF1 modification is due to PARP2 activity. ADPribosylation of Tyr238 is not essential for the global HPF1-dependent ADPr of histones because the non-modifiable Y238F HPF1 mutant supports this activity both in cells and in vitro (Figures S3A and S3B).\nWhile it appears that there may be multiple ADPr sites on HPF1, we were able to confirm the ADPr of Y238 on HPF1 in cell extracts by ADPr mapping through reprocessing (Matic et al., 2012) of a published dataset (Bilan et al., 2017) (Figure S2D). Reanalysis of a large-scale ADPr dataset (Martello et al., 2016) revealed four additional high-certainty Tyr-ADPr target proteins (Figures S2C and S2E–S2G). Although the type of mass spectrometric analysis used to generate the latter dataset is suboptimal (for additional information about the inadequacies of the higher-energy collisional dissociation [HCD] technology for ADPr site mapping, please refer to Bonfiglio et al., 2017a), our discovery of a Tyr-ADPr diagnostic peak (Figure S2C) enhances the confidence of Tyr-ADPr site mapping."}