ADP-ribosylation of arginine

Arginine adenosine-5′-diphosphoribosylation (ADP-ribosylation) is an enzyme-catalyzed, potentially reversible posttranslational modification, in which the ADP-ribose moiety is transferred from NAD+ to the guanidino moiety of arginine. At 540 Da, ADP-ribose has the size of approximately five amino acid residues. In contrast to arginine, which, at neutral pH, is positively charged, ADP-ribose carries two negatively charged phosphate moieties. Arginine ADP-ribosylation, thus, causes a notable change in size and chemical property at the ADP-ribosylation site of the target protein. Often, this causes steric interference of the interaction of the target protein with binding partners, e.g. toxin-catalyzed ADP-ribosylation of actin at R177 sterically blocks actin polymerization. In case of the nucleotide-gated P2X7 ion channel, ADP-ribosylation at R125 in the vicinity of the ligand-binding site causes channel gating. Arginine-specific ADP-ribosyltransferases (ARTs) carry a characteristic R-S-EXE motif that distinguishes these enzymes from structurally related enzymes which catalyze ADP-ribosylation of other amino acid side chains, DNA, or small molecules. Arginine-specific ADP-ribosylation can be inhibited by small molecule arginine analogues such as agmatine or meta-iodobenzylguanidine (MIBG), which themselves can serve as targets for arginine-specific ARTs. ADP-ribosylarginine specific hydrolases (ARHs) can restore target protein function by hydrolytic removal of the entire ADP-ribose moiety. In some cases, ADP-ribosylarginine is processed into secondary posttranslational modifications, e.g. phosphoribosylarginine or ornithine. This review summarizes current knowledge on arginine-specific ADP-ribosylation, focussing on the methods available for its detection, its biological consequences, and the enzymes responsible for this modification and its reversal, and discusses future perspectives for research in this field

The Salmonella enterica virulence : its role in bacterial adaption to mammalian and protozoan cells [Elektronisk resurs]

Salmonellae are Gram-negative enteric bacteria and facultative intracellular pathogens responsible for a diversity of illnesses in a wide range of hosts, including man. Many serovars of Salmonella enterica harbor a plasmid that enhances bacterial virulence in infection models, and that seems to promote extraintestinal infection in man. Consequently, the plasmid has been referred to as the"virulence plasmid". The virulence plasmid varies in its constitution among different serovars, but all these plasmids carry in common the highly conserved spv (Salmonella plasmid virulence) gene cluster. The spv gene cluster consists of five genes, spvRABCD with main promoters in front of spvR and spvA. The predicted amino acid sequence of SpvR positions it to the LysR/MetR family of prokaryotic transcriptional regulators, and functional analyses have confirmed SpvR to act as a transcriptional activator of spv expression. While the spv genes represent a major virulence trait of the plasmid, the function of the SpvABCD proteins and the mechanism by which they interact with the host are currently not understood. The work described in this thesis aims at understanding the role of Spv proteins in infection of mammalian and protozoan cells. The results presented in this thesis demonstrate that one of the Spv proteins, SpvB, functions as a mono (ADP-ribosyl) transferase. The SpvB protein uses actin as a substrate for ribosylation and thereby hinders actin polymerization in vitro. Microscopic examination of MDCK cells infected with S. enterica revealed morphological changes and disappearance of F- actin. When the ability of Salmonellae to infect and replicate in axenic Acanthamoeba was studied, electron microscopy of Salmonella-infected A. rhysodes showed the replicating bacteria to be localized within membrane-bound vacuoles. Prolonged incubation of the bacteria-amoebae cultures resulted in a gradual change in amoebae morphology, partially dependent on SpvB, and in subsequent detachment and disintegration of the host cells. In parallel, we demonstrate that in vitro SpvB-mediates label transfer from [ 32p]-NAD to a 43kDa protein that co-migrate with bovine thymus beta/gamma-actin whereas A. rhysodes cell lysate prevent SpvB-mediated [32p] -NAD-dependent labeling of thymus beta/gamma-actin Although we could not demonstrate any in situ SpvB-mediated modification of A. rhysodes proteins during infection, we did observe Spv-independent label transfer from [32p] -NAD to an 80-kDa protein. This labeling was dependent on infection, required permeabilization of the amoebae, and did not occur when infected with a laboratory strain of E. coli or when the mono (ADPribosyl) transferase inhibitor novobiocin was applied. Our findings show that a facultative intracellular parasite can apply a classical bacterial toxin activity, ADP-ribosylation, to modify mammalian actin during intracellular replication, and that the spv locus is active during bacterial intracellular infection of a protozoan host

DeActs : genetically encoded tools for perturbing the actin cytoskeleton in single cells

The actin cytoskeleton is essential for many fundamental biological processes, but tools for directly manipulating actin dynamics are limited to cell-permeable drugs that preclude single-cell perturbations. Here we describe DeActs, genetically encoded actin-modifying polypeptides, which effectively induce actin disassembly in eukaryotic cells. We demonstrate that DeActs are universal tools for studying the actin cytoskeleton in single cells in culture, tissues, and multicellular organisms including various neurodevelopmental model systems

Salmonella transcriptional signature in Tetrahymena phagosomes and role of acid tolerance in passage through the protist

Salmonella enterica Typhimurium remains undigested in the food vacuoles of the common protist, Tetrahymena. Contrary to its interaction with Acanthamoeba spp., S. Typhimurium is not cytotoxic to Tetrahymena and is egested as viable cells in its fecal pellets. Through microarray gene expression profiling we investigated the factors in S. Typhimurium that are involved in its resistance to digestion by Tetrahymena. The transcriptome of S. Typhimurium in Tetrahymena phagosomes showed that 989 and 1282 genes were altered in expression compared with that in water and in LB culture medium, respectively. A great proportion of the upregulated genes have a role in anaerobic metabolism and the use of alternate electron acceptors. Many genes required for survival and replication within macrophages and human epithelial cells also had increased expression in Tetrahymena, including mgtC, one of the most highly induced genes in all three cells types. A ΔmgtC mutant of S. Typhimurium did not show decreased viability in Tetrahymena, but paradoxically, was egested at a higher cell density than the wild type. The expression of adiA and adiY, which are involved in arginine-dependent acid resistance, also was increased in the protozoan phagosome. A ΔadiAY mutant had lower viability after passage through Tetrahymena, and a higher proportion of S. Typhimurium wild-type cells within pellets remained viable after exposure to pH 3.4 as compared with uningested cells. Our results provide evidence that acid resistance has a role in the resistance of Salmonella to digestion by Tetrahymena and that passage through the protist confers physiological advantages relevant to its contamination cycle