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

作者：Jim Voorneveld, Max S. Kloet, Sven Wijngaarden, Robbert Q. Kim, Angeliki Moutsiopoulou, Marnix Verdegaal, Mohit Misra, Ivan Đikić, Gijsbert A. van der Marel, Herman S. Overkleeft, Dmitri V. Filippov, Gerbrand J. van der Heden van Noort · 发表于：Journal of the American Chemical Society · 年份：2022 · DOI：10.1021/jacs.2c06249 · 被引用次数：28 · 研究领域：Calcium signaling and nucleotide metabolism、Biochemical and Molecular Research、Autophagy in Disease and Therapy

High Resolution Image Download MS PowerPoint Slide 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 (Ub ADPr ), 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 Ub ADPr to be accepted and processed by Legionella enzymes and compare the entire suite of four Arg-linked Ub ADPr regioisomers in a variety of biochemical experiments, allowing us to profile the activity and selectivity of Legionella pneumophila ligase and hydrolase enzymes.