Enabling drug discovery for the PARP protein family through the detection of mono-ADP-ribosylation.

Published on May 7, 2019in Biochemical Pharmacology4.96
· DOI :10.1016/J.BCP.2019.05.007
Alvin Lu15
Estimated H-index: 15
Ryan Abo17
Estimated H-index: 17
+ 6 AuthorsMario Niepel28
Estimated H-index: 28
Abstract Poly-ADP-ribose polymerases (PARPs) are a family of enzymes responsible for transferring individual or chains of ADP-ribose subunits to substrate targets as a type of post-translational modification. PARPs regulate a wide variety of important cellular processes, ranging from DNA damage repair to antiviral response. However, most research to date has focused primarily on the polyPARPs, which catalyze the formation of ADP-ribose polymer chains, while the monoPARPs, which transfer individual ADP-ribose monomers, have not been studied as thoroughly. This is partially due to the lack of robust assays to measure mono-ADP-ribosylation in the cell. In this study, the recently developed MAR/PAR antibody has been shown to detect mono-ADP-ribosylation in cells, enabling the field to investigate the function and therapeutic potential of monoPARPs. In this study, the antibody was used in conjunction with engineered cell lines that overexpress various PARPs to establish a panel of assays to evaluate the potencies of literature-reported PARP inhibitors. These assays should be generally applicable to other PARP family members for future compound screening efforts. A convenient and generalizable workflow to identify and validate PARP substrates has been established. As an initial demonstration, aryl hydrocarbon receptor was verified as a direct PARP7 substrate and other novel substrates for this enzyme were also identified and validated. This workflow takes advantage of commercially available detection reagents and conventional mass spectrometry instrumentation and methods. Ultimately, these assays and methods will help drive research in the PARP field and benefit future therapeutics development.
📖 Papers frequently viewed together
1 Citations
18 Citations
54 Citations
#1Ilsa T. Kirby (OHSU: Oregon Health & Science University)H-Index: 8
#2Ana Kojic (Heidelberg University)H-Index: 3
Last. Michael S. Cohen (OHSU: Oregon Health & Science University)H-Index: 30
view all 10 authors...
Summary Poly-ADP-ribose polymerases (PARPs1-16) play pivotal roles in diverse cellular processes. PARPs that catalyze poly-ADP-ribosylation (PARylation) are the best characterized PARP family members because of the availability of potent and selective inhibitors for these PARPs. There has been comparatively little success in developing selective small-molecule inhibitors of PARPs that catalyze mono-ADP-ribosylation (MARylation), limiting our understanding of the cellular role of MARylation. Here...
27 CitationsSource
#1Alvin Gomez (U of T: University of Toronto)H-Index: 18
#2Christian Bindesbøll (University of Oslo)H-Index: 12
Last. Jason Matthews (University of Oslo)H-Index: 32
view all 14 authors...
Here we report the biochemical characterization of the mono-ADP-ribosyltransferase 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) poly-ADP-ribose-polymerase (TIPARP/ARTD14/PARP7), which is known to repress aryl hydrocarbon receptor (AHR)-dependent transcription. We found that the nuclear localization of TIPARP was dependent on a short N-terminal sequence and its zinc finger domain. Deletion and in vitro ADP-ribosylation studies identified amino acids 400-657 as the minimum catalytically active regio...
23 CitationsSource
#1Zebin Wang (AstraZeneca)H-Index: 18
#2Shaun E. Grosskurth (AstraZeneca)H-Index: 11
Last. Huawei Chen (AstraZeneca)H-Index: 28
view all 25 authors...
PARP proteins represent a class of post-translational modification enzymes with diverse cellular functions. Targeting PARPs has proven to be efficacious clinically, but exploration of the therapeutic potential of PARP inhibition has been limited to targeting poly(ADP-ribose) generating PARP, including PARP1/2/3 and tankyrases. The cancer-related functions of mono(ADP-ribose) generating PARP, including PARP6, remain largely uncharacterized. Here, we report a novel therapeutic strategy targeting P...
14 CitationsSource
#1Kabirul Islam (University of Pittsburgh)H-Index: 16
Successful mapping of the human genome has sparked a widespread interest in deciphering functional information encoded in gene sequences. However, because of the high degree of conservation in sequences along with topological and biochemical similarities among members of a protein superfamily, uncovering physiological role of a particular protein has been a challenging task. Chemical genetic approaches have made significant contributions toward understanding protein function. One such effort, du...
34 CitationsSource
#1Emily M. Schleicher (PSU: Pennsylvania State University)H-Index: 4
#2Adri M. Galvan (PSU: Pennsylvania State University)H-Index: 2
Last. Claudia M. Nicolae (PSU: Pennsylvania State University)H-Index: 17
view all 5 authors...
During carcinogenesis, cells are exposed to increased replication stress due to replication fork arrest at sites of DNA lesions and difficult to replicate genomic regions. Efficient fork restart and DNA repair are important for cancer cell proliferation. We previously showed that the ADP-ribosyltransferase PARP10 interacts with the replication protein proliferating cell nuclear antigen and promotes lesion bypass by recruiting specialized, non-replicative DNA polymerases. Here, we show that PARP1...
24 CitationsSource
#1Edward Bartlett (University of Oxford)H-Index: 5
#2Juan José Bonfiglio (MPG: Max Planck Society)H-Index: 13
Last. Ivan Matic (MPG: Max Planck Society)H-Index: 28
view all 7 authors...
Summary Serine ADP-ribosylation (Ser-ADPr) is a recently discovered protein modification that is catalyzed by PARP1 and PARP2 when in complex with the eponymous histone PARylation factor 1 (HPF1). In addition to numerous other targets, core histone tails are primary acceptors of Ser-ADPr in the DNA damage response. Here, we show that specific canonical histone marks interfere with Ser-ADPr of neighboring residues and vice versa. Most notably, acetylation, but not methylation of H3K9, is mutually...
44 CitationsSource
#1Ian Carter-O’Connell (OHSU: Oregon Health & Science University)H-Index: 7
#2Anke Vermehren-Schmaedick (OHSU: Oregon Health & Science University)H-Index: 11
Last. Michael S. Cohen (OHSU: Oregon Health & Science University)H-Index: 30
view all 6 authors...
Poly(ADP-ribose) polymerase 14 (PARP14) is a member of the PARP family of enzymes that transfer ADP-ribose from NAD+ to nucleophilic amino acids on target proteins, a process known as mono-ADP-ribosylation (MARylation). PARP14 is involved in normal immune function through the IL-4 signaling pathway and is a prosurvival factor in multiple myeloma and hepatocellular carcinoma. A mechanistic understanding of the physiological and pathophysiological roles of PARP14 has been limited by the dearth of ...
22 CitationsSource
#1Bahram Sharif-Askari (JGH: Jewish General Hospital)H-Index: 2
#2Lilian Amrein (McGill University)H-Index: 13
Last. Lawrence Panasci (McGill University)H-Index: 48
view all 4 authors...
Purpose PARP-3 is member of the PARP family of poly (ADP-ribose) polymerases involved in ADPribosylation. PARPs are involved in the basic mechanisms of DNA repair. PARP3, a critical player for efficient mitotic progression, is required for the stabilization of the mitotic spindle by regulation of the mitotic components, NuMA and Tankyrase 1.
14 CitationsSource
#1Deena M. Leslie Pedrioli (UZH: University of Zurich)H-Index: 4
#2Mario Leutert (UZH: University of Zurich)H-Index: 9
Last. Michael O. Hottiger (UZH: University of Zurich)H-Index: 74
view all 9 authors...
Abstract Despite recent mass spectrometry (MS)‐based breakthroughs, comprehensive ADP‐ribose (ADPr)‐acceptor amino acid identification and ADPr‐site localization remain challenging. Here, we report the establishment of an unbiased, multistep ADP‐ribosylome data analysis workflow that led to the identification of tyrosine as a novel ARTD1/PARP1‐dependent in vivo ADPr‐acceptor amino acid. MS analyses of in vitro ADP‐ribosylated proteins confirmed tyrosine as an ADPr‐acceptor amino acid in RPS3A (Y...
43 CitationsSource
#1Jim Voorneveld (LEI: Leiden University)H-Index: 2
#2Johannes Gregor Matthias Rack (University of Oxford)H-Index: 11
Last. Dmitri V. Filippov (LEI: Leiden University)H-Index: 32
view all 6 authors...
A solid-phase methodology to synthesize oligopeptides, specifically incorporating serine residues linked to ADP-ribose (ADPr), is presented. Through the synthesis of both α- and β-anomers of the phosphoribosylated Fmoc-Ser building block and their usage in our modified solid-phase peptide synthesis protocol, both α- and β-ADPr peptides from a naturally Ser-ADPr containing H2B sequence were obtained. With these, and by digestion studies using the human glycohydrolase, ARH3 (hARH3), compelling evi...
22 CitationsSource
Cited By16
#1Joseph M. GozgitH-Index: 5
#2Melissa VasbinderH-Index: 8
Last. Swinger Kerren KalaiH-Index: 7
view all 28 authors...
PARP7 is a monoPARP that catalyzes the transfer of single units of ADP-ribose onto substrates to change their function. Here, we identify PARP7 as a negative regulator of nucleic acid sensing in tumor cells. Inhibition of PARP7 restores type I interferon (IFN) signaling responses to nucleic acids in tumor models. Restored signaling can directly inhibit cell proliferation and activate the immune system, both of which contribute to tumor regression. Oral dosing of the PARP7 small-molecule inhibito...
1 CitationsSource
#1Antonio Ginés García-Saura (KI: Karolinska Institutet)
#2Laura K. Herzog (Umeå University)H-Index: 3
Last. Herwig Schüler (KI: Karolinska Institutet)H-Index: 34
view all 4 authors...
Enzymes in the PARP family partake in the regulation of vital cellular signaling pathways by ADP-ribosylating their targets. The roles of these signaling pathways in disease development and the de-regulation of several PARP enzymes in cancer cells have motivated the pursuit of PARP inhibitors for therapeutic applications. In this rapidly expanding research area, availability of simple research tools will help assess the functions of ADP-ribosylation in a wider range of contexts. Here, we generat...
#1Lisa Weixler (RWTH Aachen University)H-Index: 1
#2Katja Schäringer (RWTH Aachen University)H-Index: 1
Last. R. Žaja (RWTH Aachen University)H-Index: 2
view all 6 authors...
The functionality of DNA, RNA and proteins is altered dynamically in response to physiological and pathological cues, partly achieved by their modification. While the modification of proteins with ADP-ribose has been well studied, nucleic acids were only recently identified as substrates for ADP-ribosylation by mammalian enzymes. RNA and DNA can be ADP-ribosylated by specific ADP-ribosyltransferases such as PARP1-3, PARP10 and tRNA 2'-phosphotransferase (TRPT1). Evidence suggests that these enzy...
6 CitationsSource
Adenosine diphosphate (ADP)-ribosylation is a nicotinamide adenine dinucleotide (NAD+)-dependent post-translational modification that is found on proteins as well as on nucleic acids. While ARTD1/PARP1-mediated poly-ADP-ribosylation has extensively been studied in the past 60 years, comparably little is known about the physiological function of mono-ADP-ribosylation and the enzymes involved in its turnover. Promising technological advances have enabled the development of innovative tools to dete...
2 CitationsSource
Summary PARP14 has been implicated by genetic knockout studies to promote protumor macrophage polarization and suppress the antitumor inflammatory response due to its role in modulating interleukin-4 (IL-4) and interferon-γ signaling pathways. Here, we describe structure-based design efforts leading to the discovery of a potent and highly selective PARP14 chemical probe. RBN012759 inhibits PARP14 with a biochemical half-maximal inhibitory concentration of 0.003 μM, exhibits >300-fold selectivity...
4 CitationsSource
#1Sridevi Challa (UTSW: University of Texas Southwestern Medical Center)H-Index: 7
#2Mi Kayla S. Stokes (UTSW: University of Texas Southwestern Medical Center)H-Index: 1
Last. W. Lee Kraus (UTSW: University of Texas Southwestern Medical Center)H-Index: 64
view all 3 authors...
Mono(ADP-ribosyl)ation (MARylation) is a regulatory post-translational modification of proteins that controls their functions through a variety of mechanisms. MARylation is catalyzed by mono(ADP-ribosyl) transferase (MART) enzymes, a subclass of the poly(ADP-ribosyl) polymerase (PARP) family of enzymes. Although the role of PARPs and poly(ADP-ribosyl)ation (PARylation) in cellular pathways, such as DNA repair and transcription, is well studied, the role of MARylation and MARTs (i.e., the PARP 'm...
7 CitationsSource
#1Kelsie M Rodriguez (OHSU: Oregon Health & Science University)H-Index: 2
#2Sara C. Buch-Larsen (UCPH: University of Copenhagen)H-Index: 3
Last. Michael S. Cohen (OHSU: Oregon Health & Science University)H-Index: 30
view all 11 authors...
Poly(ADP-ribose) polymerase 7 (PARP-7) has emerged as a critically important member of a large enzyme family that catalyzes ADP-ribosylation in mammalian cells. PARP-7 is a critical regulator of the innate immune response. What remains unclear is the mechanism by which PARP-7 regulates this process, namely because the protein targets of PARP-7 mono-ADP-ribosylation (MARylation) are largely unknown. Here, we combine chemical genetics, proximity labeling, and proteome-wide amino acid ADP-ribosylat...
8 CitationsSource
#1Palmiro Poltronieri (National Research Council)H-Index: 16
#2Angela Celetti (National Research Council)H-Index: 20
Last. Luca Palazzo (National Research Council)H-Index: 17
view all 3 authors...
Mono(ADP-ribose) transferases and mono(ADP-ribosyl)ating sirtuins use NAD+ to perform the mono(ADP-ribosyl)ation, a simple form of post-translational modification of proteins and, in some cases, of nucleic acids. The availability of NAD+ is a limiting step and an essential requisite for NAD+ consuming enzymes. The synthesis and degradation of NAD+, as well as the transport of its key intermediates among cell compartments, play a vital role in the maintenance of optimal NAD+ levels, which are ess...
3 CitationsSource
#1Nicola J. Curtin (University of Newcastle)H-Index: 70
#2Csaba Szabó (University of Fribourg)H-Index: 143
The process of poly(ADP-ribosyl)ation and the major enzyme that catalyses this reaction, poly(ADP-ribose) polymerase 1 (PARP1), were discovered more than 50 years ago. Since then, advances in our understanding of the roles of PARP1 in cellular processes such as DNA repair, gene transcription and cell death have allowed the investigation of therapeutic PARP inhibition for a variety of diseases — particularly cancers in which defects in DNA repair pathways make tumour cells highly sensitive to the...
39 CitationsSource
#1Tim J. WigleH-Index: 25
Last. Kevin Wayne KuntzH-Index: 19
view all 15 authors...
Summary Poly(ADP-ribose) polymerase (PARP) enzymes use nicotinamide adenine dinucleotide (NAD+) to modify up to seven different amino acids with a single mono(ADP-ribose) unit (MARylation deposited by PARP monoenzymes) or branched poly(ADP-ribose) polymers (PARylation deposited by PARP polyenzymes). To enable the development of tool compounds for PARP monoenzymes and polyenzymes, we have developed active site probes for use in in vitro and cellular biophysical assays to characterize active site-...
8 CitationsSource