PMC:6172693 / 5426-9737
Annnotations
{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/6172693","sourcedb":"PMC","sourceid":"6172693","source_url":"https://www.ncbi.nlm.nih.gov/pmc/6172693","text":"Factors Influencing Ser-ADPr of Histone Peptides\nTo analyze the substrate properties that influence Ser-ADPr, we investigated sequence features that may affect the efficiency of in vitro histone peptide ADPr reactions. Our previous proteomics data provided a short consensus motif for in vivo Ser-ADPr with either Lys or Arg N-terminal to the target Ser (Leidecker et al., 2016, Bonfiglio et al., 2017b). Based on these observations, we incubated PARP1 and HPF1 with a variety of histone peptides, each containing an Lys-Ser (KS) motif known to be the modification site in vivo (Leidecker et al., 2016). Similar to what we reported before (Bonfiglio et al., 2017b), we observed that two different histone H3 peptides as well as H2A and H4 peptides were modified by the HPF1/PARP1 complex in vitro (Figure 1A). The Ser-ADPr glycosylhydrolase ARH3 (Fontana et al., 2017) was able to efficiently remove the ADP-ribose on all of the analyzed peptides (Figure 1A). We also compared the efficiency of H3 peptide 1–20 modification to that of the H3/H4 tetramer and the whole nucleosome. As shown in Figure S1A, peptide modification is not dramatically lower, especially considering the additional ADPr sites on the histone proteins and that this H3 peptide is mostly mono-ADPr in vitro (Bonfiglio et al., 2017b). These experiments establish that KS motifs in a variety of histone peptides can be modified efficiently and reversibly, demonstrating the utility of the histone peptide as a tractable in vitro assay for histone Ser-ADPr.\nFigure 1 Modifiers of Serine-ADP-Ribosylation of Histone Peptides\n(A) Autoradiogram showing ADPr, and subsequent ARH3-mediated glycohydrolysis of H3 1–20aa, H3 27–45aa, H2A 1–17aa, and H4 1–23aa peptides. Coomassie staining of the SDS-PAGE is included and represents the loading control.\n(B) Autoradiogram showing PARP1/2 + HPF1-mediated ADPr of H3 peptide with Lys9 substituted by Ala and Arg, and Ser10 substituted by Ala. Coomassie staining of the SDS-PAGE is included.\n(C) 293T cells were transfected with the same amount of empty vector (EV) or plasmid expressing WT, K9A, K9R, or S10A FLAG-tagged histone H3 protein and treated for 10 min with H2O2. Inputs (A) and FLAG-IPs (B) were analyzed by western blotting.\nNext, we opted to focus on H3 Ser10 (H3S10) ADPr, because this site was previously shown to be the primary ADPr site on H3 in vivo (Palazzo et al., 2018). We investigated how alterations of the key KS residues affect the modification profile of the H3 histone peptide in vitro. Based on our previous finding that both PARP1 and PARP2 can modify this H3 peptide in the presence of HPF1 (Bonfiglio et al., 2017b), we examined both PARPs with variations on the KS motif. Substitution of Ser10 with alanine (Ala) led to a complete loss of the modification (Figure 1B), as we have previously shown (Bonfiglio et al., 2017b). Changing the neighboring Lys residue into Arg or Ala had varying effects on histone Ser-ADPr. The H3 peptide containing the K9R mutation was still modified, albeit to a lesser extent than wild-type (WT) peptide. In contrast, the H3K9A mutation strongly (but not completely) inhibited histone H3 Ser-ADPr (Figure 1B), highlighting the importance of a basic residue preceding the Ser. Both PARP1 and PARP2 modified the peptide panel with similar profiles, although PARP1 catalyzed the reactions more efficiently under the conditions used.\nWe further confirmed the importance of the consensus KS motif for Ser-ADPr in vivo. We transfected 293T cells with FLAG-tagged histone H3 WT, K9A, K9R, K9Q, or S10A mutant H3 and assessed ADPr efficiency, as described previously (Palazzo et al., 2018). DNA damage was induced by the treatment with 2 mM hydrogen peroxide (H2O2), followed by FLAG-immunoprecipitation (FLAG-IP). Western blotting was performed using a pan-ADPr reagent that recognizes all forms of cellular ADPr (Figures 1C and S1B). The ADPr patterns obtained were similar to those observed in our in vitro reactions. To note, by using a specific anti-H3K9ac antibody, we show that the KS motif is also important for K9 acetylation in vivo (Figure 1C, FLAG-IP).\nThese data extend our previous findings that the KS and RS motifs are preferred targets for Ser-ADPr and exclude the possibility that Lys rather than Ser is the modification target.","divisions":[{"label":"Title","span":{"begin":0,"end":48}},{"label":"Figure caption","span":{"begin":1527,"end":2245}}],"tracks":[{"project":"2_test","denotations":[{"id":"30257210-27723750-20058690","span":{"begin":373,"end":377},"obj":"27723750"},{"id":"30257210-27723750-20058692","span":{"begin":597,"end":601},"obj":"27723750"},{"id":"30257210-28650317-20058694","span":{"begin":863,"end":867},"obj":"28650317"},{"id":"30257210-29480802-20058696","span":{"begin":2394,"end":2398},"obj":"29480802"},{"id":"30257210-29480802-20058699","span":{"begin":3649,"end":3653},"obj":"29480802"}],"attributes":[{"subj":"30257210-27723750-20058690","pred":"source","obj":"2_test"},{"subj":"30257210-27723750-20058692","pred":"source","obj":"2_test"},{"subj":"30257210-28650317-20058694","pred":"source","obj":"2_test"},{"subj":"30257210-29480802-20058696","pred":"source","obj":"2_test"},{"subj":"30257210-29480802-20058699","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#ec9b93","default":true}]}]}}