Family-wide analysis of poly(ADP-ribose) polymerase activity

Abstract

The poly(adenosine diphosphate (ADP)-ribose) polymerase (PARP) protein family generates ADP-ribose (ADPr) modifications onto target proteins using NAD(+) as substrate. Based on the composition of three NAD(+) coordinating amino acids, the H-Y-E motif, each PARP is predicted to generate either poly(ADPr) (PAR) or mono(ADPr) (MAR). However, the reaction product of each PARP has not been clearly defined, and is an important priority since PAR and MAR function via distinct mechanisms. Here we show that the majority of PARPs generate MAR, not PAR, and demonstrate that the H-Y-E motif is not the sole indicator of PARP activity. We identify automodification sites on seven PARPs, and demonstrate that MAR and PAR generating PARPs modify similar amino acids, suggesting that the sequence and structural constraints limiting PARPs to MAR synthesis do not limit their ability to modify canonical amino-acid targets. In addition, we identify cysteine as a novel amino-acid target for ADP-ribosylation on PARPs.

Figure 1. H-Y-E motif is not the sole indicator for PAR synthesis activity

GFP-PARPs were immunoprecipitated from 293F cells and subjected to NAD⁺ incorporation reactions in vitro with 5 or 10μM cold NAD⁺ supplemented with a constant ratio of ³²P-NAD⁺. Automodifed PARPs were resolved on SDS-PAGE gels and subjected to autoradiography. Representative coomassie stained gels for each PARP purification are shown to the left of the autoradiogram and the expected molecular weight of the PARP indicated by an asterisk. Assays were repeated at least twice. Of the H-Y-E motif containing PARPs, PARP1, 2, 5a and 5b generated polymer, as evidenced by the smear of signal starting from the molecular weight of the PARP. The remaining PARPs resolve as a discreet band, indicating that they do not generate poly(ADP-ribose). See also Supplementary Fig. 1-3.

Figure 2. Enzymatic and chemical release of H-Y-E PARPs indicates that additional features of PARP catalytic domain impact to enzymatic activity

Automodified PARPs were treated with CHES or the indicated wild type and catalytically inactive ADPr hydrolytic enzyme. Signal remaining attached to protein was analyzed by SDS-PAGE (top row), coomassies of either IgG (PARPs 1, 2, 5a and 5b) or PARP (PARP3 and 4) are shown below. Released product was analyzed by TLC (middle row) and sequencing gel (bottom row). Assays were repeated at least twice. PARPs 1, 2, 5a and 5b release PAR ladders upon CHES treatment and are sensitive to T.c. PARG hydrolysis. In contrast, PARP3 and 4 release ADPr upon CHES, MacroD1 and T.c. PARG treatment, indicative of MAR synthesis activity. See also Supplementary Fig. 4-7.

Figure 3. Enzymatic and chemical release shows that MAR synthesis is the primary activity of non-H-Y-E PARPs

Automodified PARPs were treated with CHES or the indicated wild type and catalytically inactive ADPr hydrolytic enzyme. Signal remaining attached to protein was analyzed by SDS-PAGE (top row), coomassies for each PARP are shown below. Released product was analyzed by TLC (middle row) and sequencing gel (bottom row). Assays were repeated at least twice. CHES, MacroD1 and T.c. PARG treatment of non-H-Y-E PARPS results in ADPr release, indicative of MAR synthesis activity. See also Supplementary Fig. 4, 5 and 8.

Figure 4. Comparison of chemical treatments to release ADPr linkages

Automodified PARP1, 7, 10 and 11 were treated with 100mM CHES, pH9, 2mM EDTA or 0.4M Hydroxylamine, pH 7.5, 2mM EDTA or 100mM Tris, pH12, 1mM EDTA. Released products were analyzed both on 20% TBE-polyacrylamide sequencing gels (left) and TLC (right). Hydroxylamine treatment does not release PAR ladders from PARP1 and high pH treatment results in the degradation of ADPr to AMP.

Figure 5. DNA dependence of PARP enzymatic activity

GFP-PARPs 1, 2, 3 and 10 were immunoprecipitated from 293F cells and subjected to NAD⁺ incorporation reactions in vitro with 10μM cold NAD⁺ supplemented with a constant ratio of 32P-NAD⁺, with or without the addition of activated DNA. Automodified PARPs were resolved on SDS-PAGE gels and subjected to coomassie staining and autoradiography. Addition of DNA to PARPs 1, 2, and 3 results in upregulation of enzymatic activity whereas PARP10 activity is slightly decreased.

Figure 6. Bovine PARG treatment of poly and mono(ADP-ribosyl)ating PARPs

Automodified GFP-PARPs 1, 3, 4 and 10 were treated with a titration of bovine PARG ranging from 1-10ng/mL. GFP-PARP1, which produces PAR, is sensitive to bovine PARG treatment whereas GFP-PARPs 3, 4 and 10, which produce MAR, do not show any decrease in signal upon bovine PARG treatment. Assay was repeated twice.

Figure 7. Impact of donor and acceptor loops on enzymatic activity

a) Wild type and chimeric GFP-PARP1 and 16 constructs were expressed in 293F cells, immunoprecipitated and subject to in vitro NAD⁺ incorporation assays. Assays were repeated twice. Replacement of PARP1 donor loop with PARP16’s (PARP1^{P16 D-loop}) inhibited PAR synthesis activity instead resulting in MAR synthesis. In contrast replacement with the acceptor loop of PARP16 (PARP1^{P16 A-loop}) retained PAR synthesis, but decreased total PAR activity. PARP16 activity was unchanged after replacement with either the PARP1 donor or acceptor loop. In contrast, PARP16^Y254E inhibits the endogenous MAR synthesis activity that is restored by replacement with either the donor or acceptor loops of PARP1. b, c) Wild type, PARP1^{D-loop Pro} and chimeric GFP-PARP1 and 3 constructs were expressed in 293F cells, immunoprecipitated and subject to in vitro NAD⁺ incorporation assays either with (b) or without (c) DNA. Assays were repeated twice. Mutation of the 3 proline residues in the PARP1 D-loop results in an enrichment for PAR that resolves higher on the SDS-PAGE gel whereas replacement with the PARP-3 donor loop results in MAR synthesis activity in the absence and presence of DNA. The GFP-PARP3^{P1 D-loop} construct is not active. d) Wild type GFP-PARP1 and GFP-PARP1^{D-loop Pro} were automodified and attached PAR chains were released with either CHES or NaOH and analyzed by sequencing gel. Assay was repeated twice. GFP-PARP1^{D-loop Pro} is enriched for longer polymer. e) NAD⁺ incorporation kinetics of wild type GFP-PARP1 and GFP-PARP1^{D-loop Pro} indicate that, although both proteins have similar initial reaction rates, the PARP1^{D-loop Pro} incorporation reaction does not plateau with similar kinetics, n≥3, error bars represent standard deviation, data fit to logarithmic regression. Representative coomassie stain of input shown to right. See also Supplementary Figures 6 and 7.