Proteome-wide microarray-based screening of PAR-binding proteins

Abstract

Poly (ADP-ribose) (PAR) plays a crucial role in intracellular signaling and scaffolding through covalent modification or non-covalent binding to target proteins. The non-covalent PAR binding proteome (PARylome) has not been extensively characterized. Here we performed a PAR-binding screen using a human protein microarray that covers most of the human proteome to characterize the non-covalent binding PARylome. A total of 356 PAR-binding proteins were identified. The PAR-binding PARylome suggests that PAR binding regulates a variety of biological processes beyond DNA damage signaling and DNA repair. Proteins that may be reprogrammed by PAR binding include signaling molecules, transcription factors, nucleic acid binding proteins, calcium binding proteins, ligases, oxidoreductases, enzymes, transferases, hydrolases, and receptors. The global database of PAR-binding proteins that we established will be a valuable tool for further in-depth analysis of the role of PARylation in a wide range of biological contexts.

Graphical Abstract

Figure 1.

Identification and characterization of 356 unique PAR-binding proteins using 17k human protein microarray. (A) Workflow illustration of PAR-binding detection via protein microarray using biotin-PAR. (B) Representative scanned images of biotin-Poly A tails as (a) non-specific negative control background, (b) PAR-binding detection using anti-streptavidin antibodies, and (c) anti-PAR based detection for direct PAR-binding confirmation. (C) The 356 PAR-binding proteins were statistically filtered by signal intensity. The 15 representative proteins showing the highest affinity out of the 356 PAR-binding proteins are listed at the top-right corner. (D) Subcellular localization of the 356 PAR-binding proteins. (E) Functional classification of the 356 PAR-binding proteins. (F) Hub proteins that connects to a high fraction of PAR-binding proteins based on the BioGRID analysis (Biological General Repository for Interaction Datasets). (G) Classification of the most frequently occurring sequence features found in PAR-binding proteins according to UniProt annotations..

Figure 2.

Identification of new PBMs and computationally refined KR-based motifs. (A) Dot plot depicting the distribution of domains and motifs identified from the 17K human protein array and four major screening methods: Photoaffinity-based proteomics, GST-fusion macrodomain screen, boronate-affinity chromatography screen, and PAR antibody-based proteomics. (B) A bar graph showing 12 most common Pfam domain families based on the number of occurrences within the 356 PAR-binding proteins. (C) Two computationally refined versions of previously proposed motifs. Modifications were made to further specify certain regions that were previously unknown, especially the KR-based sequence motifs. (D) Amino Acid sequence representations for the two most significant motifs, CPxC and CNxC, which are novel PAR-binding domains, using MEME de novo amino acid alignment protocol. Associated E-values were 2.4e-003 and 3.6e-007 for each CPxC and CNxC motif. Amino Acid sequence representation for the most significant motif observed across PAR-binding proteins using MEGA7 protocol. Associated E-value was 8.2e-696. (E) A venn diagram comparing human proteins containing new CPxC/CNxC motifs and two refined KR motifs, showing that 34 proteins contain all three motifs. (F) A native electromobility gel shift assay for the validation of PAR-binding of newly predicted proteins based on the new CPxC/CNxC motifs, and (G) a graph of corresponding intensities of the gel shift assay.

Figure 3.

Identification of novel PAR-binding kinase and acetyltransferase, and functional alteration. (A) A Venn diagram comparing human kinases with new motifs and two refined motifs, showing that 1 kinase contain all three motifs. (B) A native electromobility gel shift assay for the validation of PAR-binding of newly predicted kinases based on the new motifs, and (C) a graph of corresponding intensities of the gel shift assay. (D) Each of GFP-fused WT- or PBMs for VRK3 (TOP) and GSG2 (Bottom) was expressed in HeLa cells and microirradiated. Cumulative GFP signals at laser strips for VRK3 and GSG2 were measured and plotted, ****P< 0.0001, by one-way ANOVA when compared between indicated groups by Bonferroni's post-test, Data represent mean ± S.E.M. from six independent cells (n = 6). (E) In vitro acetylation assay of p300 on Histone H3 was performed in the presence or absence of pADPr. Total levels of acetylated lysine (left) or site-specific acetylated lysine on Lysine at 27 (middle) or at 36 (right) were probed and normalized with the levels of total Histone. Intensity of the acetylated lysine was plotted from three independent experiments (n = 3). ***P< 0.001, ****P< 0.0001, by two-tailed unpaired t-test with Welch's correction.

Figure 4.

Characterization of the novel PBMs, CPxC and CNxC. (A) A Venn diagram comparing human proteins containing the two new PBMs, CPxC and CNxC, showing a 127-protein overlap across the two. (B) A bar chart showing the three most significant biological processes, molecular functions, and KEGG pathways for both CPxC and CNxC motifs. (C) A distance-based graph of motifs CPxC and CNxC with respect to two protein domains, Zinc finger (RING-HC) and E3 Ligase (RING-H2). (D) A Venn diagram of 614 human protein E3 Ligases containing one or more of PBMs: Refined KR motif 1, Refined KR motif 2, and CPxC/CNxC Motif. 4 E3 Ligases contained all three motifs. (E) A native electromobility gel shift assay for the validation of PAR-binding of E3 ligases, and (C) a graph of corresponding intensities of the gel shift assay. Intensity of each free [³²P]-PAR was quantified and plotted. * P< 0.05 by two tailed student t-test when compared GST binding to each of E3 ligases. n.s. not significant.

Figure 5.

Identification of the PBMs in PAR-binding ubiquitin E3 Ligases. (A) Schematic illustrations of PBMs in DTX1, DTX2, and RNF4. Each amino acid indicated in red bar were replaced with Alanine by site-directed mutagenesis. (B) Each of recombinant WT, PBM, and CC mutant was incubated with [³²P]-PAR and separated in 20% TBE gel. Both [³²P]-PAR bound signal and unbound free [³²P]-PAR signal were visualized by autoradiography. (C) Intensity of each [³²P]-PAR bound protein was quantified and plotted. Gel shift assays were performed in triplicate. *P< 0.05, **P< 0.01, ****P< 0.001. ****P< 0.0001, by one-way ANOVA when compared between indicated groups by Bonferroni's post-test and two-tailed unpaired t-test when compared between WT and each mutant, n.s. not significant. (D) Schematic illustrations of PBMs in RNF8, RNF138, and RNF168. Each amino acid indicated under each illustration was replaced with Alanine by site-directed mutagenesis. (E) Both [³²P]-PAR bound signal and unbound free [³²P]-PAR signal were visualized by autoradiography. (F) Intensity of each [³²P]-PAR bound protein was quantified and plotted. Each signal was normalized by GST binding signal. Gel shift assays were performed in triplicate. *P< 0.05, **P< 0.01, ****P< 0.001. ****P< 0.0001, by one-way ANOVA when compared between indicated groups by Bonferroni's post-test and two-tailed unpaired t-test when compared between WT and each mutant, n.s. not significant. (G-L) GFP-fused WT and PBM E3s were expressed in either WT or PARP1 KO HeLa cells and microirradiated. Cumulative GFP signals at laser strips for (G) DTX1, (H) DTX2, (I) RNF4, (J) RNF8, (K) RNG138, and (L) RNF168 were measured and plotted., ****P< 0.0001, by one-way ANOVA when compared between indicated groups by Bonferroni's post-test, Data represent mean ± S.E.M. from six independent cells (n = 6).

Figure 6.

PAR-binding to PAR-binding ubiquitin E3 Ligases is required for the translocation into the damaged region of DNA. (A) Identification of co-localization between mCherry-FOKI nuclease and each of GFP-fused WT- or PBM of (a) DTX1 (b) DTX2, and (c) RNF4 at DSB site. (B) The kinetics of GFP-fused (a) DTX1, (b) DTX2 and (c) RNF4 to DNA lesions in a PARP1 dependent manner. Each of WT form of GFP-fused E3s were tested in the presence or absence of PARP inhibitor, PJ34 or ATM inhibitor, KU55933. For PBM mutants for DTX1 and DTX2, gRNA-DTX1 and gRNA-DTX2 was applied before the assay, respectively. Each of PBM form of GFP-fused E3s were tested in the absence of inhibitor. Fluorescence intensity at the laser strips were quantified and plotted as mean ± S.E.M of six independent cells for DTX1 and DTX2 (N = 6) or five independent cells for RNF4 (N = 5). ****P< 0.0001 by one-way ANOVA when compared between indicated groups by Bonferroni's posttest. Scale bars, 10 μm.

Figure 7.

PAR-binding E3 Ligases are essential for cell survival and involved in both NHEJ and HR pathway. (A) Clonogenic analysis. (Aa) HeLa cells were transfected with DOX-inducible gRNA-resistant constructs (gRNAResDTX1 WT or gRNAResDTX1 Dmut) and induced in the presence or absence of DOX and then, cells were infected with Lentiviral gRNA for DTX1. Followed by ionizing irradiation with different dosages, the colony formation assay was performed as indicated. (Ab) HeLa cells were transfected with DOX-inducible gRNA resistant constructs (gRNAResDTX2 WT or gRNAResDTX2 Dmut) and induced in the presence or absence of DOX and then, cells were infected with Lentiviral gRNA for DTX2. Followed by ionizing irradiation with different dosages, the colony formation assay was performed as indicated. (Ac) HeLa cells were transfected with siRNA resistant RNF4 constructs (siResRNF4 WT or siResRNF4 Qmut) and then transfected with siRNA. Followed by ionizing irradiation with different dosages, the colony formation assay was performed as indicated. *P< 0.05, **P< 0.01, ****P< 0.0001, by one-way ANOVA when compared between indicated groups by Bonferroni's post-test, n.s. not significant. (B) Representative images or frequency of foci formation of 53BP1 (Ba, Bc, Be) and BRCA1 (Bb, Bd, Bf) at DNA break sites after irradiation. Depletion of endogenous E3s and ectopic expression gRNA- or siRNA-resistant WT or PBM form of E3s were prepared as indicated. Followed by immunocytochemistry, numbers of cells with at least 10 foci were counted and plotted. ***P< 0.001, ****P< 0.0001, by one-way ANOVA when compared between indicated groups by Bonferroni's posttest, n.s. not significant. Scale bars, 10 μm.