Arginine ADP-Ribosylation: Chemical Synthesis of Post-Translationally Modified Ubiquitin Proteins

Abstract

We describe the development and optimization of a methodology to prepare peptides and proteins modified on the arginine residue with an adenosine-di-phosphate-ribosyl (ADPr) group. Our method comprises reacting an ornithine containing polypeptide on-resin with an α-linked anomeric isothiourea N-riboside, ensuing installment of a phosphomonoester at the 5′-hydroxyl of the ribosyl moiety followed by the conversion into the adenosine diphosphate. We use this method to obtain four regioisomers of ADP-ribosylated ubiquitin (UbADPr), each modified with an ADP-ribosyl residue on a different arginine position within the ubiquitin (Ub) protein (Arg42, Arg54, Arg72, and Arg74) as the first reported examples of fully synthetic arginine-linked ADPr-modified proteins. We show the chemically prepared Arg-linked UbADPr to be accepted and processed by Legionella enzymes and compare the entire suite of four Arg-linked UbADPr regioisomers in a variety of biochemical experiments, allowing us to profile the activity and selectivity of Legionella pneumophila ligase and hydrolase enzymes.

Chemoenzymatic and Synthetic Approaches To Investigate Aspartate- and Glutamate-ADP-Ribosylation

“…Many of the proteins involved in ADPr regulation and function have been identified as therapeutic targets for various human diseases, , with poly­(ADP-ribose) polymerase 1/2 (PARP1/2) inhibitors being successfully employed to treat homology repair-deficient cancers . The translational potential of PARP inhibition has inspired extensive efforts to develop PARP isoform-specific small molecule inhibitors and decipher mechanisms of ADP-ribosyltransferase activity and substrate selectivity. , More recently, innovative synthetic and chemoenzymatic technologies have been developed to install ADP-ribose at specific sites on synthetic peptides and full-length proteins. ,− These approaches have been largely focused on serine-ADPr (Ser-ADPr), a DNA damage-induced modification that is catalyzed by the PARP1:HPF1 complex. , Semisynthetic ADP-ribosylated proteins, including core histones and other chromatin architecture proteins, have been employed to determine important mechanistic principles by which specific Ser-ADPr sites and corresponding polymer chain lengths impact chromatin structure at DNA damage sites. ,, Serine mono-ADP-ribosylated peptides have also enabled the development of the first site-specific ADP-ribose antibodies . Building upon these successes, strategies to prepare synthetic peptides bearing ADP-ribose at additional side chain functionalities, such as the guanidinium group of arginine, have been recently developed …”

“…12 Building upon these successes, strategies to prepare synthetic peptides bearing ADP-ribose at additional side chain functionalities, such as the guanidinium group of arginine, have been recently developed. 16 Aspartate and glutamate (Asp/Glu) ADP-ribose linkages, which are commonly observed in biological systems, present unique chemical complexities that have prevented the preparation of homogenously modified molecules. Here, we have developed efficient and scalable chemoenzymatic and fully synthetic strategies to insert ADP-ribose at specific Asp/ Glu residues on synthetic peptides.…”

Four of a Kind: A Complete Collection of ADP-Ribosylated Histidine Isosteres Using Cu(I)- and Ru(II)-Catalyzed Click Chemistry

“…The most common amino acid residue to be ADP-ribosylated is serine, , but glutamate, aspartate, ,− arginine, cysteine, , lysine, and more recently tyrosine , and histidine , have been found to be ADP-ribosylated as well. Synthetic, well-defined MARylated peptides and ADPr-oligomers have been shown to be valuable molecular tools to investigate ADP-ribosylation, informing on the exact structure of ADPr polymers and modified peptides, the chemical and enzymatic stability of the PTMs, and the binding with interaction partners. − Various isosteres of ADP-ribosylated amino acids have been introduced as ADPr chemical biology tools with special attention being paid to stabilizing the glycosidic linkage that connects the ADPr moiety to a protein and to expedite synthetic accessibility. Examples of the isosteric replacements for native ADPr-peptides include ADP-ribosylated glutamine and asparagine and N -methyl aminooxy functionalized peptides serving as base-stable substitutes for their glutamate and aspartate counterparts.…”

PARP14 is a PARP with both ADP-ribosyl transferase and hydrolase activities

“…The ADP-ribosylated peptides were chemically synthesized (table S1). Serine-ADPr and arginine-ADPr peptides were synthesized as previously described ( 71 , 72 ). Chemical synthesis of the glutamate-ADPr peptide was previously described ( 73 ).…”