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

Voorneveld, J.; Kloet, M.S.; Wijngaarden, S.; Kim, R.Q.; Moutsiopoulou, A.; Verdegaal, M.; Misra, M.; Dikic, I.; Marel, G.A. van der; Overkleeft, H.S.; Filippov, D.V.; Noort, G.J.V. van

doi:doi:10.1021/jacs.2c06249

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 alpha-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...Show moreWe 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 alpha-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.Show less

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