Identifying Poly(ADP-ribose)-Binding Proteins with Photoaffinity-Based Proteomics

Article abstract of DOI:10.1021/jacs.0c12246

Post-translational modification of proteins with poly(ADP-ribose) (PAR) is an important component of the DNA damage response. Four PAR synthesis inhibitors have recently been approved for the treatment of breast, ovarian, and prostate cancers. Despite the clinical significance of PAR, a molecular understanding of its function, including its binding partners, remains incomplete. In this work, we synthesized a PAR photoaffinity probe that captures and isolates endogenous PAR binders. Our method identified dozens of known PAR-binding proteins and hundreds of novel candidates involved in DNA repair, RNA processing, and metabolism. PAR binding by eight candidates was confirmed using pull-down and/or electrophoretic mobility shift assays. Using PAR probes of defined lengths, we detected proteins that preferentially bind to 40-mer versus 8-mer PAR, indicating that polymer length may regulate the outcome and timing of PAR signaling pathways. This investigation produces the first census of PAR-binding proteins, provides a proteomics analysis of length-selective PAR binding, and associates PAR binding with RNA metabolism and the formation of biomolecular condensates.

Full text of DOI:10.1021/jacs.0c12246

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Identifying Poly(ADP-ribose)-Binding Proteins with Photoaffinity-
Based Proteomics
Morgan Dasovich, Morgan Q. Beckett, Scott Bailey, Shao-En Ong, Marc M. Greenberg,*
and Anthony K. L. Leung*
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ABSTRACT: Post-translational modification of proteins with poly(ADP-ribose) (PAR) is an important component of the DNA
damage response. Four PAR synthesis inhibitors have recently been approved for the treatment of breast, ovarian, and prostate
cancers. Despite the clinical significance of PAR, a molecular understanding of its function, including its binding partners, remains
incomplete. In this work, we synthesized a PAR photoaffinity probe that captures and isolates endogenous PAR binders. Our method
identified dozens of known PAR-binding proteins and hundreds of novel candidates involved in DNA repair, RNA processing, and
metabolism. PAR binding by eight candidates was confirmed using pull-down and/or electrophoretic mobility shift assays. Using
PAR probes of defined lengths, we detected proteins that preferentially bind to 40-mer versus 8-mer PAR, indicating that polymer
length may regulate the outcome and timing of PAR signaling pathways. This investigation produces the first census of PAR-binding
proteins, provides a proteomics analysis of length-selective PAR binding, and associates PAR binding with RNA metabolism and the
formation of biomolecular condensates.
oly(ADP-ribose) (PAR) is an NAD+-dependent post-
translational modification (PARylation) synthesized by
advantageous since it covalently traps binders, allowing for
stringent washes that remove indirect interactions. Since PAR
and RNA are structurally similar, we envisioned employing a
cross-linking strategy to identify PAR-binding proteins. We
synthesized a photoaffinity probe (PARprolink, Figure 1a)
consisting of: PAR of defined length, a biotin handle for
enrichment, and a single, randomly incorporated photo-
inducible cross-linker to stabilize PAR−protein interactions.
PARs of defined length were purified from an in vitro
enzymatic reaction using anion exchange chromatography.25
The biotin handle was incorporated at the 2′-OH-terminus of
PAR using the ELTA bioconjugation technique.26 We took
advantage of the selective modification of RNA hydroxyl
groups by activated carboxylic acids to randomly incorporate
the benzophenone tethered photo-inducible cross-linker on
PAR via a nicotinic acid imidazolide.27−29 Polymers containing
a single benzophenone modification were purified from a PAR
mixture composed mostly of 0, 1, and 2 conjugated nicotinic
acid analogues by C18 reverse-phase HPLC (Figures 1b and
S1).
P
PAR polymerases (PARPs).1−6 PARP inhibitors, which have
garnered four FDA approvals in the past 6 years, are used to
treat cancers.7 Preclinical data also support repurposing these
anticancer drugs as therapeutics for neurodegeneration, cardiac
failure, and inflammation.8 A major function of PARylation is
the recruitment of proteins through non-covalent interactions.
For instance, PARP1 synthesizes PAR within seconds
following DNA strand scission.9 These PAR chains in turn
recruit DNA repair proteins to the damaged sites, locally
concentrating repair processes precisely where and when they
are needed on the chromatin.10 Understanding of PAR-
dependent processes has been bolstered by proteomics
methods that have identified thousands of ADP-ribosylated
proteins during genotoxic stress and other contexts.10−16
However, complementary proteomics methods that identify
non-covalent PAR interactions are currently lacking. Here, we
describe a photo-cross-linking-based approach that identifies
PAR-binding proteins.
Antibody-based approaches have been used to characterize
the PAR interactome, which includes PAR binders, PARylated
proteins, and indirect interactors, making it difficult to identify
direct PAR−protein interactions.17,18 To date, 92 proteins
have been shown to bind PAR directly (Table S1).19,20 This
number is relatively small compared to RNA- and DNA-
binding proteins (1541 and 2765, respectively).21,22 Therefore,
we reasoned that many PAR-binding proteins remain
undiscovered. A census of the PAR-binding proteome would
provide greater insight into PAR-dependent pathways such as
DNA repair and may reveal novel biology.
The specificity of PARprolink for PAR-binding proteins was
examined by incubating the probe with an increasing amount
of either the PAR-binding WWE domain from human RNF146
or bovine serum albumin (BSA). Mixtures were then irradiated
(350 nm, 10 min), separated by SDS-PAGE, and transferred to
Received: November 23, 2020
Published: February 17, 2021
Photo-cross-linking strategies have been used widely to
identify proteins that bind to RNA.23,24 Cross-linking is
https://dx.doi.org/10.1021/jacs.0c12246
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Given that most characterized PAR-dependent processes
occur in the nucleus, we tested whether our probe can identify
endogenous PAR binders in nuclear extracts. We irradiated
HeLa nuclear extract incubated with either an 8-mer
PARprolink or biotinylated 8-mer PAR lacking benzophenone
(negative control) in the presence of an inhibitor against PAR
glycohydrolase. PAR−protein cross-links were isolated with
streptavidin, then subjected to on-bead trypsin digestion
(Figures 2a and S3a and Table S1). LC-MS/MS analysis of
the pull-downs identified 798 proteins with two unique
peptides in two replicates, and their abundance was quantified
using the label-free quantification technology MaxLFQ (Figure
S3b).31 The majority (743, 93%) were at least 2-fold more
abundant in the pull-down with PARprolink than in the no-
cross-linker control (Figure 2b), demonstrating that the
stringent wash removed most non-covalent interactions.
There was no correlation between LFQ intensity and protein
copy number, further indicating a specific enrichment by
PARprolink (Figure S3c). The identified proteins overlap
significantly with those identified by two antibody-based PAR
Figure 1. PARprolink selectively captures PAR-binding proteins in
vitro. (a) PARprolink structure, where each PAR molecule contains
one randomly incorporated benzophenone cross-linker. (b) PAGE
analyses of PARprolink at each synthetic step. (c) PARprolink (100
nM) was mixed with the indicated concentrations of protein and
irradiated at 350 nm, and PAR−protein cross-link formation was
detected with streptavidin. (d) Quantification of streptavidin signal
from (c); values represent mean s.d. (n = 3).
interactome studies (P = 6.05 × 10−111 and 1.10 × 10−103
;
Figure S3d and Table S1).17,18 Although PARprolink identified
a similar number of overall proteins as either antibody-based
approach, our cross-linking approach captured a greater
percentage of known PAR binders (39 out of 92 (42%);
Figure S3e and Table S1).19,20
To further validate that PARprolink identifies direct PAR−
protein interactions, we expressed and purified eight
candidates (AK2, CAPRIN1, DDX6, G3BP2, UHRF1,
G3BP1, GAPDH, and PARP1) and subjected them to two
PAR-binding assays. Initially, the candidate proteins were
incubated with a mixture of biotinylated PAR of different
lengths, followed by streptavidin pull-down. The specificity of
the assay was validated with the WWE domain and BSA as a
negative control.
We confirmed a direct interaction between PAR and seven
out of eight candidates (Figures 2c and S4). The affinities of
these candidates for 16-mer PAR were determined by
electromobility shift assays (EMSAs) (Table 1 and Figure
S5). Consistent with our qualitative pull-down assay, the same
seven candidates had affinities for PAR within the range
nitrocellulose, which selectively retains protein but not PAR.30
Irradiation of PARprolink incubated with WWE, but not BSA,
formed a protein cross-link that could be detected with
streptavidin in a dose-dependent manner (Figures 1c,d and
S2a). This signal was dependent on UV irradiation and the
presence of PARprolink, consistent with covalent conjugation
of the PAR-binding domain to the biotinylated probe.
PARprolink specificity was further demonstrated in a complex
background by cross-linking WWE domain dosed in cell
extracts (Figure S2b). Addition of unlabeled PAR reduced the
streptavidin signal on WWE (Figure S2c), suggesting that
cross-link formation depends on the PAR−WWE interaction.
Figure 2. Photoaffinity-based isolation of the endogenous PAR-binding proteome. (a) Proteomics workflow schematic. (b) Volcano plot of protein
Log2 enrichment ratios and −Log10(P-values) from proteomics experiments (n = 2). (c) Non-covalent biotin-PAR pull-downs with recombinantly
expressed PAR-binding candidates.
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Table 1. Summary of PAR−Protein Affinities Measured with
EMSAs
protein
AK2
CAPRIN1
G3BP2
DDX6
UHRF1
G3BP1
GAPDH
PARP1
KD (nM)
95% CI (nM)
R2
∼46 000
∼3 400
∼313
∼199
∼162
∼93
33 000−65 000
1 500−7 700
197−497
134−291
115−230
73−124
0.88
0.82
0.91
0.94
0.92
0.95
0.94
0.93
∼54
∼27
37−79
20−38
reported for other PAR-binding proteins (KD = 1 nM−10
μM).20,32 The dissociation constant for AK2, which was not
detected in the pull-down experiment, was estimated to be 33−
65 μM, suggesting the AK2−PAR interaction may not be
physiologically relevant. Taken together, these results indicate
that PARprolink captured a substantial fraction of the known
PAR-binding proteome and enabled the discovery of novel
PAR binders.
Having validated that PARprolink identifies PAR-binding
proteins, we systematically investigated how endogenous
proteins bind to different lengths of PAR. Emergent data
suggest that signaling pathways are only activated when PAR
length exceeds a certain threshold. Parthanatos, a PAR-
dependent cell death pathway, is induced more strongly by
long PAR (∼60-mer) than short PAR (∼15-mer).33 In
addition, three DNA repair-related proteins (XPA, DEK,
p53) preferentially bind long PAR, and the Chk1 kinase is only
activated by long PAR.34−36 HeLa nuclear extract was cross-
linked to either 8-mer or ∼40-mer PAR photoaffinity probes,
with a bead-only pull-down as a negative control. Comparing
the intensities between these pull-downs uncovered 156
proteins that prefer ∼40-mer PAR (Log2 fold change >2;
Figures 3a and S6a and Table S1). Importantly, we observed
the long PAR-binding preference of DEK,34 validating that our
approach identifies length-selective PAR binders.
Intriguingly, our analyses revealed that the central DNA
repair protein PARP1 preferred binding to long PAR (∼40-
mer/8-mer = 13, Figure 3a). To verify this finding, EMSAs
were performed with recombinant PARP1 and PAR of varied
lengths (Figures 3b and S6b,c). We observed a 16-fold increase
in PARP1−PAR affinity as PAR length increased from 4- to
16-mer. Importantly, the affinity of the PARP1−PAR
interactions for the 16- and 32-mers (KD = 11−110 nM) is
in the same range as the reported affinities between PARP1
and nucleosomes (KD = 2−100 nM).37 These PARP1−PAR
interactions can be disrupted by PARP1 automodification
(Figure S6d). Given that PAR length is controlled temporally
during DNA damage, where long polymers (>22-mer) are
rapidly synthesized by PARP1 and then slowly degraded to
shorter lengths,27,38 our data suggest that PAR length may
control the dissociation of PARP1 from the chromatin during
DNA repair.
Our investigation represents the first census of PAR binders.
We took this opportunity to analyze properties of the identified
PAR-binding proteome. Gene ontology (GO) analysis on all
743 PAR-binding candidates (Figure 2b) revealed the expected
enrichment of several DNA repair pathways (Figure S7).17 Yet,
the enrichments of physiological processes such as RNA
splicing, RNA transport, and DNA replication were even more
significant. Notably, long PAR binders are enriched with
Figure 3. Defined-length probes reveal length-specific PAR−protein
interactions. (a) Volcano plot of protein Log2 enrichment ratios and
− Log10 P-values from proteomics experiments using either 8- or ∼40-
mer probes (n = 2). (b) The effect of PAR length on the affinity
toward PARP1 measured with EMSA (KD
95% CI; n = 3). (c)
Gene ontology analysis of proteins that were more abundant in the
∼40-mer pull-down (enrichment ratio >4).
proteins involved in nucleic acid metabolism, such as DNA
repair and RNA splicing (Figure 3c). STRING protein
association network analyses mapped two central coresone
involved in DNA repair and chromatin remodeling, and the
other in RNA splicing and translationalong with distal
clusters involved in metabolism and tRNA synthesis (Figures
4a and S8). Consistent with recent proteomics studies
identifying ADP-ribosylated substrates,10−16 our analysis of
PAR-binding candidates strengthens the view that PAR has
roles beyond DNA repair in metabolism and RNA
regulation.39,40
We next assessed the enrichment of protein domains from
the Pfam database in our dataset (Figures 4b and S9a,b and
Table S1).41−43 We observed a significant enrichment of
multiple helicase-associated domains among PAR-binding
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Figure 4. Analyses of the PAR-binding proteome. (a) STRING network analysis of high-confidence PAR-binding candidates (enrichment ratio >8,
P < 0.05, 416 genes) displaying at least one connection to another candidate. (b) Enrichment of known PAR-binding and other domains. (c) pI
distribution among PAR-, RNA-, and DNA-binding proteins.
candidates. Consistently, helicase activity is the most enriched
molecular function based on GO analyses (Figure S7). Several
known PAR-binding domains, such as WWE domain and
macrodomain, were also enriched. Among them, the most
significant was the Tri-RGG motif, with 12 out of 16 Tri-RGG-
containing proteins in the human proteome identified. In
addition, we observed the enrichment of DNA/RNA-binding
domains known to bind PAR, e.g., the RNA recognition motif
and OB-fold.44 Therefore, it is not surprising that a significant
amount of PAR-binding candidates are also known DNA- or
trigger a wave of PAR binding-dependent PARylation in their
vicinity, building extensive PAR−protein interaction networks
to form biomolecular condensates in cells.6,54
This work describes the first proteomics method developed
to identify direct PAR binders. Our census sheds lights into
PAR−protein interactions involved in DNA repair, RNA
regulation, and biomolecular condensate formation, thereby
serving as a rich resource to explore these frontiers in PAR
biology.
RNA-binding proteins(P = 3.57 × 10−6 and 2.00 × 10−136
;
ASSOCIATED CONTENT
■
Table S1).21,22 RNA- and DNA-binding proteins tend to have
higher isoelectric points (median pI = 7.93 and 7.38).
Unexpectedly, the median isoelectric point of PAR-binding
candidates was lower than the proteome (pI = 6.81 vs 7.15,
Figures 4c and S9c−f). Together, these data suggest a specific
interaction between PAR and particular protein domains,
rather than a non-specific enrichment of positively charged
proteins.
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.0c12246.
Experimental methods, Figures S1−S9, and NMR
spectra of new compounds (PDF)
Table S1, proteomics data and analysis (XLSX)
In addition to defined motifs or domains, PAR-binding
candidates were statistically enriched with proteins containing
low-complexity sequence (P ≤ 5.5 × 10−18),45,46 which is
critical for the formation of biomolecular condensates.47
Indeed, PARprolink identified proteins enriched with
components of biomolecular condensates such as DNA repair
foci, nucleoli, and stress granules (P = 8.36 × 10−21, 2.34 ×
10−57, and 5.97 × 10−94; Table S1).48−50 Notably, PARylation
of the DNA repair factor p53 and the nucleolar helicase
DDX21 is dependent on their ability to bind PAR.51,52
Consistent with these studies, comparison with proteomics
analyses of ADP-ribosylated substrates revealed that most
PAR-binding candidates are also ADP-ribosylated (647 out of
743 (87%), P = 1.43 × 10−246; Table S1).53 Taken together,
our data suggest that one or more PARylation events may
AUTHOR INFORMATION
■
Corresponding Authors
Marc M. Greenberg − Department of Chemistry, Krieger
School of Arts and Sciences, Johns Hopkins University,
Baltimore, Maryland 21218, United States; orcid.org/
0000-0002-5786-6118; Email: mgreenberg@jhu.edu
Anthony K. L. Leung − Department of Biochemistry and
Molecular Biology, Bloomberg School of Public Health,
Department of Molecular Biology and Genetics, School of
Medicine, and Department of Oncology, School of Medicine,
Johns Hopkins University, Baltimore, Maryland 21205,
United States; orcid.org/0000-0001-5569-4036;
Email: anthony.leung@jhu.edu
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Strosznajder, R. P.; Sumegi, B.; Swanson, R. A.; Szabó, C.
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Detection and Signaling of DNA Single-Strand Breaks by Human
PARP-1. Mol. Cell 2015, 60 (5), 742−754.
Authors
Morgan Dasovich − Department of Chemistry, Krieger School
of Arts and Sciences and Department of Biochemistry and
Molecular Biology, Bloomberg School of Public Health, Johns
Hopkins University, Baltimore, Maryland 21218, United
States
Morgan Q. Beckett − Department of Biophysics and
Biophysical Chemistry, School of Medicine, Johns Hopkins
University, Baltimore, Maryland 21205, United States
Scott Bailey − Department of Biophysics and Biophysical
Chemistry, School of Medicine and Department of
Biochemistry and Molecular Biology, Bloomberg School of
Public Health, Johns Hopkins University, Baltimore,
Maryland 21205, United States
(10) Zhen, Y.; Yu, Y. Proteomic Analysis of the Downstream
Signaling Network of PARP1. Biochemistry 2018, 57 (4), 429−440.
(11) Daniels, C. M.; Ong, S.-E.; Leung, A. K. L. The Promise of
Proteomics for the Study of ADP-Ribosylation. Mol. Cell 2015, 58
(6), 911−924.
(12) Buch-Larsen, S. C.; Hendriks, I. A.; Lodge, J. M.; Rykær, M.;
Shao-En Ong − Department of Pharmacology, University of
Washington, Seattle, Washington 98195, United States;
orcid.org/0000-0003-3314-5903
̈
Furtwangler, B.; Shishkova, E.; Westphall, M. S.; Coon, J. J.; Nielsen,
M. L. Mapping Physiological ADP-Ribosylation Using Activated Ion
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Complete contact information is available at:
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(13) Nowak, K.; Rosenthal, F.; Karlberg, T.; Bu
A.-G.; Dreier, B.; Grossmann, J.; Sobek, J.; Imhof, R.; Lu
Schuler, H.; Pluckthun, A.; Leslie Pedrioli, D. M.; Hottiger, M. O.
̈
tepage, M.; Thorsell,
̈
scher, B.;
̈
̈
Notes
Engineering Af1521 Improves ADP-Ribose Binding and Identification
of ADP-Ribosylated Proteins. Nat. Commun. 2020, 11 (1), 5199.
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Science 2016, 353 (6294), 45−50.
(17) Gagné, J.-P.; Pic, E.; Isabelle, M.; Krietsch, J.; Ethier, C.;
Paquet, E.; Kelly, I.; Boutin, M.; Moon, K.-M.; Foster, L. J.; Poirier, G.
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Srinivasan Yegnasubramanian for the UHRF1
plasmid; Dr. J. Paul Taylor for G3BP1 protein and plasmids for
G3BP2, DDX6, and CAPRIN1; Dr. John Pascal for the PARP1
plasmid; and Tim Mitchison for in vitro synthesized PAR. We
would like to thank Robert O’Meally, Tatiana Boronina (Johns
Hopkins), and Mark Hail (Novatia) for assistance with the
mass analysis. We thank Biorender for providing some artwork
used in the TOC graphic. We thank the Johns Hopkins
Discovery Award as well as NIH grants T32GM080189,
T32GM008403, R01GM104135 (AKLL), R35GM131736
(MMG), and RF1AG071326 (AKLL) for funding.
ABBREVIATIONS
■
RNF146, ring finger protein 146; BSA, bovine serum albumin;
AK2, adenylate kinase 2; OB, oligonucleotide/oligosaccharide-
binding
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A.; Bonet, J.; Nacht, A. S.; Samino, S.; Font-Mateu, J.; Vicent, G. P.;
Wierer, M.; Trabado, M. A.; Schelhorn, C.; Carolis, C.; Macias, M. J.;
Yanes, O.; Oliva, B.; Beato, M. ADP-Ribose-Derived Nuclear ATP
Synthesis by NUDIX5 Is Required for Chromatin Remodeling. Science
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