The transfer of an adenosine diphosphate (ADP) ribose moiety to a nucleophilic side chain by consumption of nicotinamide adenine dinucleotide is referred to as ADP-ribosylation, which allows for... Show moreThe transfer of an adenosine diphosphate (ADP) ribose moiety to a nucleophilic side chain by consumption of nicotinamide adenine dinucleotide is referred to as ADP-ribosylation, which allows for the spatiotemporal regulation of vital processes such as apoptosis and DNA repair. Recent mass-spectrometry based analyses of the "ADP-ribosylome" have identified histidine as ADP-ribose acceptor site. In order to study this modification, a fully synthetic strategy towards α-configured N(τ)- and N(π)-ADP-ribosylated histidine-containing peptides has been developed. Ribofuranosylated histidine building blocks were obtained via Mukaiyama-type glycosylation and the building blocks were integrated into an ADP-ribosylome derived peptide sequence using fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase peptide synthesis. On-resin installation of the ADP moiety was achieved using phosphoramidite chemistry, and global deprotection provided the desired ADP-ribosylated oligopeptides. The stability under various chemical conditions and resistance against (ADP-ribosyl) hydrolase-mediated degradation has been investigated to reveal that the constructs are stable under various chemical conditions and non-degradable by any of the known ADP-ribosylhydrolases. Show less
Đukić, N.; Strømland, Ø.; Elsborg, J.D.; Munnur, D.; Zhu, K.; Schuller, M.; ... ; Ahel, I. 2023
PARP14 is a mono-ADP-ribosyl transferase involved in the control of immunity, transcription, and DNA replication stress management. However, little is known about the ADP-ribosylation activity of... Show morePARP14 is a mono-ADP-ribosyl transferase involved in the control of immunity, transcription, and DNA replication stress management. However, little is known about the ADP-ribosylation activity of PARP14, including its substrate specificity or how PARP14-dependent ADP-ribosylation is reversed. We show that PARP14 is a dual-function enzyme with both ADP-ribosyl transferase and hydrolase activity acting on both protein and nucleic acid substrates. In particular, we show that the PARP14 macrodomain 1 is an active ADP-ribosyl hydrolase. We also demonstrate hydrolytic activity for the first macrodomain of PARP9. We reveal that expression of a PARP14 mutant with the inactivated macrodomain 1 results in a marked increase in mono(ADP-ribosyl)ation of proteins in human cells, including PARP14 itself and antiviral PARP13, and displays specific cellular phenotypes. Moreover, we demonstrate that the closely related hydrolytically active macrodomain of SARS2 Nsp3, Mac1, efficiently reverses PARP14 ADP-ribosylation in vitro and in cells, supporting the evolution of viral macrodomains to counteract PARP14-mediated antiviral response. Show less
Minnee, H.; Chung, H.W.; Rack, J.G.M.; Marel, G.A. van der; Overkleeft, H.S.; Codee, J.D.C.; ... ; Filippov, D.V. 2023
Adenosine diphosphateribosylation (ADP-ribosylation) is a crucialpost-translational modification involved in important regulatory mechanismsof numerous cellular pathways including histone... Show moreAdenosine diphosphateribosylation (ADP-ribosylation) is a crucialpost-translational modification involved in important regulatory mechanismsof numerous cellular pathways including histone maintenance and DNAdamage repair. To study this modification, well-defined ADP-ribosylatedpeptides, proteins, and close analogues thereof have been invaluabletools. Recently, proteomics studies have revealed histidine residuesto be ADP-ribosylated. We describe here the synthesis of a completeset of triazole-isosteres of ADP-ribosylated histidine to serve asprobes for ADP-ribosylating biomachinery. By exploiting Cu(I)- andRu(II)-catalyzed click chemistry between a propargylglycine buildingblock and an & alpha;- or & beta;-configured azidoribose, we have successfullyassembled the & alpha;- and & beta;-configured 1,4- and 1,5-triazoles,mimicking N(& tau;)- and N(& pi;)-ADP-ribosylated histidine, respectively.The ribosylated building blocks could be incorporated into a peptidesequence using standard solid-phase peptide synthesis and transformedon resin into the ADP-ribosylated fragments to provide a total offour ADP-ribosyl triazole conjugates, which were evaluated for theirchemical and enzymatic stability. The 1,5-triazole analogues mimickingthe N(& pi;)-substituted histidines proved susceptible to base-inducedepimerization and the ADP-ribosyl & alpha;-1,5-triazole linkage couldbe cleaved by the (ADP-ribosyl)hydrolase ARH3. Show less
Madern, J.M.; Voorneveld, J.; Rack, J.G.M.; Kistemaker, H.A.V.; Ahel, I.; Marel, G.A. van der; ... ; Filippov, D.V. 2023
Adenosine diphosphate (ADP) ribosylation is an importantpost-translationalmodification (PTM) that plays a role in a wide variety of cellularprocesses. To study the enzymes responsible for the... Show moreAdenosine diphosphate (ADP) ribosylation is an importantpost-translationalmodification (PTM) that plays a role in a wide variety of cellularprocesses. To study the enzymes responsible for the establishment,recognition, and removal of this PTM, stable analogues are invaluabletools. We describe the design and synthesis of a 4-thioribosyl APRrpeptide that has been assembled by solid phase synthesis. The key4-thioribosyl serine building block was obtained in a stereoselectiveglycosylation reaction using an alkynylbenzoate 4-thioribosyl donor. Show less
We report here chemoenzymatic and fully synthetic methodologiesto modify aspartate and glutamate side chains with ADP-ribose at specificsites on peptides. Structural analysis of aspartate and... Show moreWe report here chemoenzymatic and fully synthetic methodologiesto modify aspartate and glutamate side chains with ADP-ribose at specificsites on peptides. Structural analysis of aspartate and glutamateADP-ribosylated peptides reveals near-quantitative migration of theside chain linkage from the anomeric carbon to the 2 ''- or 3 ''-ADP-ribosehydroxyl moieties. We find that this linkage migration pattern isunique to aspartate and glutamate ADP-ribosylation and propose thatthe observed isomer distribution profile is present in biochemicaland cellular environments. After defining distinct stability propertiesof aspartate and glutamate ADP-ribosylation, we devise methods toinstall homogenous ADP-ribose chains at specific glutamate sites andassemble glutamate-modified peptides into full-length proteins. Byimplementing these technologies, we show that histone H2B E2 tri-ADP-ribosylationis able to stimulate the chromatin remodeler ALC1 with similar efficiencyto histone serine ADP-ribosylation. Our work reveals fundamental principlesof aspartate and glutamate ADP-ribosylation and enables new strategiesto interrogate the biochemical consequences of this widespread proteinmodification. Show less
Smith, R.; Zentout, S.; Rother, M.; Bigot, N.; Chapuis, C.; Mihut, A.; ... ; Huet, S. 2023
Poly(ADP-ribose) polymerase 1 (PARP1) activity is regulated by its co-factor histone poly(ADP-ribosylation) factor 1 (HPF1). The complex formed by HPF1 and PARP1 catalyzes ADP-ribosylation of... Show morePoly(ADP-ribose) polymerase 1 (PARP1) activity is regulated by its co-factor histone poly(ADP-ribosylation) factor 1 (HPF1). The complex formed by HPF1 and PARP1 catalyzes ADP-ribosylation of serine residues of proteins near DNA breaks, mainly PARP1 and histones. However, the effect of HPF1 on DNA repair regulated by PARP1 remains unclear. Here, we show that HPF1 controls prolonged histone ADP-ribosylation in the vicinity of the DNA breaks by regulating both the number and length of ADP-ribose chains. Furthermore, we demonstrate that HPF1-dependent histone ADP-ribosylation triggers the rapid unfolding of chromatin, facilitating access to DNA at sites of damage. This process promotes the assembly of both the homologous recombination and non-homologous end joining repair machineries. Altogether, our data highlight the key roles played by the PARP1/HPF1 complex in regulating ADP-ribosylation signaling as well as the conformation of damaged chromatin at early stages of the DNA damage response.Smith, Zentout et al. investigate the role of HPF1 in DNA repair using live-cell imaging methods and find that HPF1-dependent histone ADP-ribosylation drives early process in DNA repair, including chromatin relaxation and repair factor recruitment. Show less
Minnee, H.; Rack, J.G.M.; Marel, G.A. van der; Overkleeft, H.S.; Codee, J.D.C.; Ahel, I.; Filippov, D.V. 2022
A convergent synthesis provided nearly perfect tau-ADP-ribosylated histidine isosteres (His*-tau-ADPr) via a copper(I)-catalyzed cycloaddition between an azido-ADP-ribosyl analogue and an... Show moreA convergent synthesis provided nearly perfect tau-ADP-ribosylated histidine isosteres (His*-tau-ADPr) via a copper(I)-catalyzed cycloaddition between an azido-ADP-ribosyl analogue and an oligopeptide carrying a propargyl glycine. Both alpha- and beta-configured azido-ADP-ribosyl analogues have been synthesized. The former required participation of the C-2 ester functionality during glycosylation, while the latter was obtained in high stereoselectivity from an imidate donor with a nonparticipating para-methoxy benzyl ether. Four His*-tau-ADPr peptides were screened against a library of human ADP-ribosyl hydrolases. Show less
Luscher, B.; Ahel, I.; Altmeyer, M.; Ashworth, A.; Bai, P.T.; Chang, P.; ... ; Ziegler, M. 2021
ADP-ribosylation, a modification of proteins, nucleic acids, and metabolites, confers broad functions, including roles in stress responses elicited, for example, by DNA damage and viral infection... Show moreADP-ribosylation, a modification of proteins, nucleic acids, and metabolites, confers broad functions, including roles in stress responses elicited, for example, by DNA damage and viral infection and is involved in intra- and extracellular signaling, chromatin and transcriptional regulation, protein biosynthesis, and cell death. ADP-ribosylation is catalyzed by ADP-ribosyltransferases (ARTs), which transfer ADP-ribose from NAD(+) onto substrates. The modification, which occurs as mono- or poly-ADP-ribosylation, is reversible due to the action of different ADP-ribosylhydrolases. Importantly, inhibitors of ARTs are approved or are being developed for clinical use. Moreover, ADP-ribosylhydrolases are being assessed as therapeutic targets, foremost as antiviral drugs and for oncological indications. Due to the development of novel reagents and major technological advances that allow the study of ADP-ribosylation in unprecedented detail, an increasing number of cellular processes and pathways are being identified that are regulated by ADP-ribosylation. In addition, characterization of biochemical and structural aspects of the ARTs and their catalytic activities have expanded our understanding of this protein family. This increased knowledge requires that a common nomenclature be used to describe the relevant enzymes. Therefore, in this viewpoint, we propose an updated and broadly supported nomenclature for mammalian ARTs that will facilitate future discussions when addressing the biochemistry and biology of ADP-ribosylation. This is combined with a brief description of the main functions of mammalian ARTs to illustrate the increasing diversity of mono- and poly-ADP-ribose mediated cellular processes. Show less
Poly(ADP-ribosyl)ation (PAR) is a versatile and complex posttranslational modification composed of repeating units of ADP-ribose arranged into linear or branched polymers. This scaffold is linked... Show morePoly(ADP-ribosyl)ation (PAR) is a versatile and complex posttranslational modification composed of repeating units of ADP-ribose arranged into linear or branched polymers. This scaffold is linked to the regulation of many of cellular processes including the DNA damage response, alteration of chromatin structure and Wnt signalling. Despite decades of research, the principles and mechanisms underlying all steps of PAR removal remain actively studied. In this work, we synthesise well-defined PAR branch point molecules and demonstrate that PARG, but not ARH3, can resolve this distinct PAR architecture. Structural analysis of ARH3 in complex with dimeric ADP-ribose as well as an ADP-ribosylated peptide reveal the molecular basis for the hydrolysis of linear and terminal ADP-ribose linkages. We find that ARH3-dependent hydrolysis requires both rearrangement of a catalytic glutamate and induction of an unusual, square-pyramidal magnesium coordination geometry. Show less
ADP-ribosylation (ADPr), as a post-translational modification, plays a crucial role in DNA-repair, immunity and many other cellular and physiological processes. Serine is the main acceptor for ADPr... Show moreADP-ribosylation (ADPr), as a post-translational modification, plays a crucial role in DNA-repair, immunity and many other cellular and physiological processes. Serine is the main acceptor for ADPr in DNA damage response, whereas the physiological impact of less common ADPr-modifications of cysteine and threonine side chains is less clear. Generally, gaining molecular insights into ADPr recognition and turn-over is hampered by the availability of homogeneous, ADP-ribosylated material, such as mono-ADP-ribosylated (MARylated) peptides. Here, a new and efficient solid-phase strategy for the synthesis of Ser-, Thr- and Cys-MARylated peptides is described. ADP-ribosylated cysteine, apart from being a native post-translational modification in its own right, proved to be suitable as a stabile bioisostere for ADP-ribosylated serine making it a useful tool to further biochemical research on serine ADP-ribosylation. In addition, it was discovered that the Streptococcus pyogenes encoded protein, SpyMacroD, acts as a Cys-(ADP-ribosyl) hydrolase. Show less
ADP-ribosylation (ADPr), as a post-translational modification, plays a crucial role in DNA-repair, immunity and many other cellular and physiological processes. Serine is the main acceptor for ADPr... Show moreADP-ribosylation (ADPr), as a post-translational modification, plays a crucial role in DNA-repair, immunity and many other cellular and physiological processes. Serine is the main acceptor for ADPr in DNA damage response, whereas the physiological impact of less common ADPr-modifications of cysteine and threonine side chains is less clear. Generally, gaining molecular insights into ADPr recognition and turn-over is hampered by the availability of homogeneous, ADP-ribosylated material, such as mono-ADP-ribosylated (MARylated) peptides. Here, a new and efficient solid-phase strategy for the synthesis of Ser-, Thr- and Cys-MARylated peptides is described. ADP-ribosylated cysteine, apart from being a native post-translational modification in its own right, proved to be suitable as a stabile bioisostere for ADP-ribosylated serine making it a useful tool to further biochemical research on serine ADP-ribosylation. In addition, it was discovered that the Streptococcus pyogenes encoded protein, SpyMacroD, acts as a Cys-(ADP-ribosyl) hydrolase. Show less
Voorneveld, J.; Rack, J.G.M.; Ahel, I.; Overkleeft, H.S.; Marel, G.A. van der; Filippov, D.V. 2018
