Androgen signaling in prostate cancer cells involves multisite cysteine ADP-ribosylation of the androgen receptor (AR) by PARP7. The AR modification is read by ADP-ribosyl binding macrodomains in... Show moreAndrogen signaling in prostate cancer cells involves multisite cysteine ADP-ribosylation of the androgen receptor (AR) by PARP7. The AR modification is read by ADP-ribosyl binding macrodomains in PARP9, but the reason that multiple cysteines are modified is unknown. Here, we use synthetic peptides to show that dual ADP-ribosylation of closely spaced cysteines mediates recognition by the DTX3L/PARP9 complex. Mono and dual ADP-ribosylated cysteine peptides were prepared using a novel solid-phase synthetic strategy utilizing a key, Boc-protected, ribofuranosylcysteine building block. This synthetic strategy allowed us to synthesize fluorescently labeled peptides containing a dual ADP-ribosylation motif. It was found that the DTX3L/PARP9 complex recognizes the dual ADP-ribosylated AR peptide (Kd = 80.5 nM) with significantly higher affinity than peptides with a single ADP-ribose. Moreover, oligomerization of the DTX3L/PARP9 complex proved crucial for ADP-ribosyl-peptide interaction since a deletion mutant of the complex that prevents its oligomer formation dramatically reduced peptide binding. Our data show that features of the substrate modification and the reader contribute to the efficiency of the interaction and imply that multivalent interactions are important for AR-DTX3L/PARP9 assembly. 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
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
Voorneveld, J.; Kloet, M.S.; Wijngaarden, S.; Kim, R.Q.; Moutsiopoulou, A.; Verdegaal, M.; ... ; Noort, G.J.V. van 2022
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... 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
Voorneveld, J.; Kloet, M.S.; Wijngaarden, S.; Kim, R.Q.; Moutsiopoulou, A.; Verdegaal, M.; ... ; Noort, G.J.V. van 2022
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... 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
