Nicotinamide adenine dinucleotide (NAD+) is the substrate used for the introduction of the ubiquitous and highly dynamic PTM in which either one or multiple adenosine diphosphate ribose (ADPr)... Show moreNicotinamide adenine dinucleotide (NAD+) is the substrate used for the introduction of the ubiquitous and highly dynamic PTM in which either one or multiple adenosine diphosphate ribose (ADPr) moieties are covalently attached to a nucleophilic side chain of an specific amino acid in the target protein to regulate cellular pathways including adipogenesis, DNA damage repair and gene expression. A significant fraction of the nucleophilic amino acid functionalities, most recently histidine and tyrosine, have been identified as ADPr-acceptor sites. In this thesis, new methodologies have been developed to synthesize peptide fragments carrying an ADPr modification to investigate ADP-ribosylation on histidine. Show less
This thesis presents the first synthetic peptides ADP-ribosylated on serine, threonine, tyrosine, arginine and cysteine. Besides synthetic peptides, this thesis discusses the first synthetic route... Show moreThis thesis presents the first synthetic peptides ADP-ribosylated on serine, threonine, tyrosine, arginine and cysteine. Besides synthetic peptides, this thesis discusses the first synthetic route towards ADP-ribosylated nucleic acids. Furthermore, two photoaffinity probes for PARP1 have been developed and assessed in living cells and two activity based probes have been synthesized, designed for CD38. Show less
Madern, J.M.; Kim, R.Q.; Misra, M.; Dikic, I.; Zhang, Y.; Ovaa, H.; ... ; Noort, G.J.V. van 2020
Stable NAD(+)analogues carrying single atom substitutions in either the furanose ring or the nicotinamide part have proven their value as inhibitors for NAD(+)-consuming enzymes. To investigate the... Show moreStable NAD(+)analogues carrying single atom substitutions in either the furanose ring or the nicotinamide part have proven their value as inhibitors for NAD(+)-consuming enzymes. To investigate the potential of such compounds to inhibit the adenosine diphosphate ribosyl (ADPr) transferase activity of the Legionella SdeC enzyme, we prepared three NAD(+)analogues, namely carbanicotinamide adenosine dinucleotide (c-NAD(+)), thionicotinamide adenosine dinucleotide (S-NAD(+)) and benzamide adenosine dinucleotide (BAD). We optimized the chemical synthesis of thionicotinamide riboside and for the first time used an enzymatic approach to convert all three ribosides into the corresponding NAD(+)mimics. We thus expanded the known scope of substrates for the NRK1/NMNAT1 enzyme combination by turning all three modified ribosides into NAD(+)analogues in a scalable manner. We then compared the three NAD(+)mimics side-by-side in a single assay for enzyme inhibition on Legionella effector enzyme SdeC. The class of SidE enzymes to which SdeC belongs was recently identified to be important in bacterial virulence, and we found SdeC to be inhibited by S-NAD(+)and BAD with IC(50)values of 28 and 39 mu M, respectively. Show less
Phosphorylation affects all four major biomolecules – proteins, lipids, carbohydrates and nucleic acids – and plays a pivotal role in the most fundamental cellular functions. A prime example of a... Show morePhosphorylation affects all four major biomolecules – proteins, lipids, carbohydrates and nucleic acids – and plays a pivotal role in the most fundamental cellular functions. A prime example of a PTM that caught a lot of scientific interest is adenosine diphosphate ribosylation (ADP-ribosylation), carried out by the PARP family of enzymes. ADP-ribosylation is an indirect form of (pyro)phosphorylation, where a monomer or polymer of ADPr is attached to an acceptor protein. The process of ADP-ribosylation has been linked to DNA damage repair, telomere maintenance and regulation of apoptosis, highlighting it of biomedical importance, most notably in relation to cancer therapy and inflammatory disorders. Well-defined phosphorylated molecular tools have already proven to be effective at elucidating the molecular mechanisms behind both normal and pathological cell function. However, the natural pyrophosphate group is inherently susceptibility to hydrolysis, trans-esterification and enzymatic cleavage. This property limits their use through possible premature degradation or restricted synthetic accessibility. The research described in this thesis focuses on the development of new molecular tools, primarily intended to facilitate the study of adenosine diphosphate ribosylation. In this context new reagents and methodologies have been developed for the synthesis of stabilized pyrophosphorylated bioisosteres. Show less
Liu, Q.; Kistemaker, H.A.V.; Bhogaraju, S.; Dikic, I.; Overkleeft, H.S.; Marel, G.A. van der; ... ; Filippov, D.V. 2018
