Mimetics of ADP-ribosylated histidine through copper(I)-catalyzed click chemistry

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 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.Bio-organic Synthesi

Solid-phase synthesis and biological evaluation of peptides ADP-Ribosylated at histidine

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 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.Bio-organic Synthesi

4-thioribose analogues of adenosine diphosphate ribose (ADPr) peptides

This is the final version. Available from the American Chemical Society via the DOI in this record. Data Availability Statement: The data underlying this study are available in the published article and its Supporting Information.Adenosine diphosphate (ADP) ribosylation is an important post-translational modification (PTM) that plays a role in a wide variety of cellular processes. To study the enzymes responsible for the establishment, recognition, and removal of this PTM, stable analogues are invaluable tools. We describe the design and synthesis of a 4-thioribosyl APRr peptide that has been assembled by solid phase synthesis. The key 4-thioribosyl serine building block was obtained in a stereoselective glycosylation reaction using an alkynylbenzoate 4-thioribosyl donor.Biotechnology and Biological Sciences Research CouncilWellcome TrustWellcome TrustOvarian Cancer Research AllianceNetherlands Organization for Scientific Research (NWO

Molecular basis for the reversible ADP-ribosylation of guanosine bases

Modification of nucleic acids by ADP-ribosylation is catalyzed by various ADP-ribosyltransferases, including the DarT enzyme. The latter is part of the bacterial toxin-antitoxin (TA) system DarTG, which was shown to provide control of DNA replication and bacterial growth as well as protection against bacteriophages. Two subfamilies have been identified, DarTG1 and DarTG2, which are distinguished by their associated antitoxins. While DarTG2 catalyzes reversible ADP-ribosylation of thymidine bases employing a macrodomain as antitoxin, the DNA ADP-ribosylation activity of DarTG1 and the biochemical function of its antitoxin, a NADAR domain, are as yet unknown. Using structural and biochemical approaches, we show that DarT1-NADAR is a TA system for reversible ADP-ribosylation of guanosine bases. DarT1 evolved the ability to link ADP-ribose to the guanine amino group, which is specifically hydrolyzed by NADAR. We show that guanine de-ADP-ribosylation is also conserved among eukaryotic and non-DarT-associated NADAR members, indicating a wide distribution of reversible guanine modifications beyond DarTG systems

Serine ADP-ribosylation in Drosophila provides insights into the evolution of reversible ADP-ribosylation signalling

In the mammalian DNA damage response, ADP-ribosylation signalling is of crucial importance to mark sites of DNA damage as well as recruit and regulate repairs factors. Specifically, the PARP1:HPF1 complex recognises damaged DNA and catalyses the formation of serine-linked ADP-ribosylation marks (mono-Ser-ADPr), which are extended into ADP-ribose polymers (poly-Ser-ADPr) by PARP1 alone. Poly-Ser-ADPr is reversed by PARG, while the terminal mono-Ser-ADPr is removed by ARH3. Despite its significance and apparent evolutionary conservation, little is known about ADP-ribosylation signalling in non-mammalian Animalia. The presence of HPF1, but absence of ARH3, in some insect genomes, including Drosophila species, raises questions regarding the existence and reversal of serine-ADP-ribosylation in these species. Here we show by quantitative proteomics that Ser-ADPr is the major form of ADP-ribosylation in the DNA damage response of Drosophila melanogaster and is dependent on the dParp1:dHpf1 complex. Moreover, our structural and biochemical investigations uncover the mechanism of mono-Ser-ADPr removal by Drosophila Parg. Collectively, our data reveal PARP:HPF1-mediated Ser-ADPr as a defining feature of the DDR in Animalia. The striking conservation within this kingdom suggests that organisms that carry only a core set of ADP-ribosyl metabolising enzymes, such as Drosophila, are valuable model organisms to study the physiological role of Ser-ADPr signalling

Mechanistic insights into the three steps of poly(ADP-ribosylation) reversal

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 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. Bio-organic Synthesi

Synthetic alpha- and beta-Ser-ADP-ribosylated Peptides Reveal alpha-Ser-ADPr as the Native Epimer

Bio-organic Synthesi

Chemoenzymatic and synthetic approaches to investigate aspartate- and glutamate-ADP-ribosylation

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 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.Bio-organic Synthesi

Molecular Tools for the Study of ADP-Ribosylation: A Unified and Versatile Method to Synthesise Native Mono-ADP-Ribosylated Peptides

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 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.Bio-organic Synthesi