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Legionnaires’ disease is caused by infection with the intracellularly replicating Gram‐negative bacterium Legionella pneumophila. This pathogen uses an unconventional way of ubiquitinating host proteins by generating a phosphoribosyl linkage between substrate proteins and ubiquitin by making use of an ADPribosylated ubiquitin (UbADPr) intermediate. The family of SidE effector enzymes that catalyze this reaction is counteracted by Legionella hydrolases, which are called Dups. This unusual ubiquitination process is important for Legionella proliferation and understanding these processes on a molecular level might prove invaluable in finding new treatments. Herein, a modular approach is used for the synthesis of triazole‐linked UbADPr, and analogues thereof, and their affinity towards the hydrolase DupA is determined and hydrolysis rates are compared to natively linked UbADPr. The inhibitory effects of modified Ub on the canonical eukaryotic E1‐enzyme Uba1 are investigated and rationalized in the context of a high‐resolution crystal structure reported herein. Finally, it is shown that synthetic UbADPr analogues can be used to effectively pull‐down overexpressed DupA from cell lysate., Modular modifications: Analogues of ADPribosylated ubiquitin, including a nonhydrolyzable methylenebisphosphonate version, are constructed and subsequently applied to investigate (de‐)ubiquitinating enzymes involved in Legionella infection. These probes are used in the study of the bacterial effector DupA and the human ubiquitin activating E1 enzyme.

Protein ADP-ribosylation is an important posttranslational modification that plays versatile roles in multiple biological processes. ADP-ribosylation is catalyzed by a group of enzymes known as ADP-ribosyltransferases (ARTs). Using nicotinamide adenine dinucleotide (NAD(+)) as the donor, ARTs covalently link single or multiple ADP-ribose moieties from NAD(+) to the substrates, forming mono ADP-ribosylation or poly ADP-ribosylation (PARylation). Novel functions of ARTs and ADPribosylation have been revealed over the past few years. Here we summarize the current knowledge on ARTs and PARylation.

The norepinephrine analogue meta-iodo-benzylguanidine (MIBG), a substrate for mono(ADP-ribosylation) and inhibitor of eukaryotic ADP-ribosyltransferases, inhibits the prooxidant-induced and spontaneous calcium release from intact rat liver mitochondria without affecting pyridine nucleotide oxidation and hydrolysis. This finding strongly suggests regulation of calcium release by ADP-ribosylation in mitochondria, and may be relevant for the cellular and pharmacological effects of MIBG.

The post-translational modification of proteins by ADPribose is widely distributed among living organisms (1). In prokaryotes, a single ADPR unit is transferred to acceptor proteins by mono-ADPR transferases (1). Many bacterial toxins are mono-ADPR transferases that modify host-cell proteins (1). Only three bacterial ADPribosylating systems, in Rodospirillum rubrum, Escherichia coli and Pseudomonas maltophilia, have been described, characterized by endogenous enzyme and substrate proteins (1, 2).

Despite the increasing interest on both the physiological and pathological role of ADP-ribosylation reactions, little appears to be known about the mechanism by which the intracellular level of NAD+ is regulated with respect to its participation in the redox metabolism on the one hand and to its utilization in poly(ADP-ribosy)lation on the other. Conditions enhancing poly(ADPribosyl)ation can significantly lower NAD+ concentration thereby influencing NAD+ dependent metabolic processes in the whole cell, while the cellular redox status can be signaled to chromatin thereby influencing the level of ADPribosylation of nuclear proteins (1).