In Vitro ADP-Ribosylation Utilizing 2′Deoxy-NAD+ as a Substrate

1989 ◽  
pp. 53-56
Author(s):  
Rafael Alvarez-Gonzalez ◽  
Joel Moss ◽  
Claude Niedergang ◽  
Felix R. Althaus
Keyword(s):  
Adp Ribosylation

scholarly journals ADP-ribosylation of integrin α7 modulates the binding of integrin α7β1 to laminin

2004 ◽  
Vol 385 (1) ◽  
pp. 309-317 ◽  
Author(s):  
Zhefeng ZHAO ◽  
Joanna GRUSZCZYNSKA-BIEGALA ◽  
Anna ZOLKIEWSKA
Keyword(s):  
C2c12 Myotubes ◽  
Post Translational Modification ◽  
Integrin Β1 ◽  
Adp Ribosylation ◽  
In Vitro Binding ◽  
Vitro Binding ◽  
C2c12 Skeletal Muscle Cells ◽  
Adp Ribosyltransferase

The extracellular domain of integrin α7 is ADP-ribosylated by an arginine-specific ecto-ADP-ribosyltransferase after adding exogenous NAD+ to intact C2C12 skeletal muscle cells. The effect of ADP-ribosylation on the structure or function of integrin α7β1 has not been explored. In the present study, we show that ADP-ribosylation of integrin α7 takes place exclusively in differentiated myotubes and that this post-translational modification modulates the affinity of α7β1 dimer for its ligand, laminin. ADP-ribosylation in the 37-kDa ‘stalk’ region of α7 that takes place at micromolar NAD+ concentrations increases the binding of the α7β1 dimer to laminin. Increased in vitro binding of integrin α7β1 to laminin after ADP-ribosylation of the 37-kDa fragment of α7 requires the presence of Mn2+ and it is not observed in the presence of Mg2+. In contrast, ADP-ribosylation of the 63-kDa N-terminal region comprising the ligand-binding site of α7 that occurs at approx. 100 μM NAD+ inhibits the binding of integrin α7β1 to laminin. Furthermore, incubation of C2C12 myotubes with NAD+ increases the expression of an epitope on integrin β1 subunit recognized by monoclonal antibody 9EG7. We discuss our results based on the current models of integrin activation. We also hypothesize that ADP-ribosylation may represent a mechanism of regulation of integrin α7β1 function in myofibres in vivo when the continuity of the membrane is compromised and NAD+ is available as a substrate for ecto-ADP-ribosylation.

scholarly journals Recombinant protein of Haemonchus contortus small GTPase ADP-ribosylation factor 1 (HcARF1) modulate the cell mediated immune response in vitro

Oncotarget ◽  
2017 ◽  
Vol 8 (68) ◽  
pp. 112211-112221 ◽  
Author(s):  
Javaid Ali Gadahi ◽  
Muhammad Ehsan ◽  
Shuai Wang ◽  
Zhenchao Zhang ◽  
Ruofeng Yan ◽  
...  
Keyword(s):  
Immune Response ◽  
Recombinant Protein ◽  
Haemonchus Contortus ◽  
Small Gtpase ◽  
Adp Ribosylation ◽  
Cell Mediated Immune Response ◽  
Adp Ribosylation Factor ◽  
Immune Response In Vitro

ADP ribosylation factor is an essential protein in Saccharomyces cerevisiae and is encoded by two genes

1990 ◽  
Vol 10 (12) ◽  
pp. 6690-6699
Author(s):  
T Stearns ◽  
R A Kahn ◽  
D Botstein ◽  
M A Hoyt
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cold Sensitivity ◽  
Animal Cells ◽  
Gene Encoding ◽  
Phenotypic Differences ◽  
Adp Ribosylation ◽  
Protein Factor ◽  
Adp Ribosylation Factor ◽  
Sublethal Concentrations

ADP ribosylation factor (ARF) is a ubiquitous 21-kDa GTP-binding protein in eucaryotes. ARF was first identified in animal cells as the protein factor required for the efficient ADP-ribosylation of the mammalian G protein Gs by cholera toxin in vitro. A gene (ARF1) encoding a protein homologous to mammalian ARF was recently cloned from Saccharomyces cerevisiae (Sewell and Kahn, Proc. Natl. Acad. Sci. USA, 85:4620-4624, 1988). We have found a second gene encoding ARF in S. cerevisiae, ARF2. The two ARF genes are within 28 centimorgans of each other on chromosome IV, and the proteins encoded by them are 96% identical. Disruption of ARF1 causes slow growth, cold sensitivity, and sensitivity to normally sublethal concentrations of fluoride ion in the medium. Disruption of ARF2 causes no detectable phenotype. Disruption of both genes is lethal; thus, ARF is essential for mitotic growth. The ARF1 and ARF2 proteins are functionally homologous, and the phenotypic differences between mutations in the two genes can be accounted for by the level of expression; ARF1 produces approximately 90% of total ARF. Among revertants of the fluoride sensitivity of an arf1 null mutation were ARF1-ARF2 fusion genes created by a gene conversion event in which the deleted ARF1 sequences were repaired by recombination with ARF2.

scholarly journals The cross-regulation of Gi-protein by cholera toxin involves a phosphorylation by protein kinase A

1995 ◽  
Vol 306 (3) ◽  
pp. 765-769 ◽  
Author(s):  
R Levistre ◽  
M Berguerand ◽  
G Bereziat ◽  
J Masliah
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Cholera Toxin ◽  
Inhibitory Effect ◽  
Negative Control ◽  
Adp Ribosylation ◽  
Gi Proteins ◽  
Kinase A

Pretreatment of alveolar macrophages with cholera toxin inhibits the release of arachidonic acid induced by the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine. The results presented here show that cholera toxin might exert its inhibitory effect through the phosphorylation of Gi alpha by protein kinase A (PKA). (1) Gi-proteins from cells pretreated with cholera toxin showed parallel increases in their sensitivity to ADP-ribosylation by toxins in vitro and in Gi alpha phosphorylation. By contrast, the Gi alpha concentration was unchanged. (2) Cholera toxin pretreatment also decreased the functional activity of Gi, as assessed by the inhibition (80%) of agonist-induced binding of guanosine-5′-[gamma-thio]triphosphate (GTP[gamma S]). (3) These effects of cholera toxin were blocked by a specific PKA inhibitor, N-(2-[methyl-amino]ethyl)-3-isoquinolinesulphonamide dihydrochloride (H8) and mimicked by a cyclic AMP (cAMP) analogue and a phosphatase inhibitor. (4) Gi alpha was also phosphorylated in vitro by the catalytic subunit of PKA. In contrast with other cell systems, the stimulation of protein kinase C seems to have no effect on the sensitivity of Gi to ADP-ribosylation or on its phosphorylation. Therefore, the phosphorylation of Gi-proteins by PKA seems to be the actual target of the negative control of arachidonic acid release via the cAMP-mediated pathway.

scholarly journals Adp Ribosylation Factor-like Protein 2 (Arl2) Regulates the Interaction of Tubulin-Folding Cofactor D with Native Tubulin

2000 ◽  
Vol 149 (5) ◽  
pp. 1087-1096 ◽  
Author(s):  
Arunashree Bhamidipati ◽  
Sally A. Lewis ◽  
Nicholas J. Cowan
Keyword(s):  
Cultured Cells ◽  
Gtpase Activating Protein ◽  
Adp Ribosylation ◽  
Chaperone Protein ◽  
Ras Family ◽  
Mutant Forms ◽  
Adp Ribosylation Factor ◽  
Β Tubulin

The ADP ribosylation factor-like proteins (Arls) are a family of small monomeric G proteins of unknown function. Here, we show that Arl2 interacts with the tubulin-specific chaperone protein known as cofactor D. Cofactors C, D, and E assemble the α/β- tubulin heterodimer and also interact with native tubulin, stimulating it to hydrolyze GTP and thus acting together as a β-tubulin GTPase activating protein (GAP). We find that Arl2 downregulates the tubulin GAP activity of C, D, and E, and inhibits the binding of D to native tubulin in vitro. We also find that overexpression of cofactors D or E in cultured cells results in the destruction of the tubulin heterodimer and of microtubules. Arl2 specifically prevents destruction of tubulin and microtubules by cofactor D, but not by cofactor E. We generated mutant forms of Arl2 based on the known properties of classical Ras-family mutations. Experiments using these altered forms of Arl2 in vitro and in vivo demonstrate that it is GDP-bound Arl2 that interacts with cofactor D, thereby averting tubulin and microtubule destruction. These data establish a role for Arl2 in modulating the interaction of tubulin-folding cofactors with native tubulin in vivo.

scholarly journals ADP Ribosylation Factor 1 Is Required for Synaptic Vesicle Budding in PC12 Cells

1997 ◽  
Vol 138 (3) ◽  
pp. 505-515 ◽  
Author(s):  
Victor Faúndez ◽  
Jim-Tong Horng ◽  
Regis B. Kelly
Keyword(s):  
Pc12 Cell ◽  
Cell Membranes ◽  
Brefeldin A ◽  
T Antigen ◽  
Gtpase Activity ◽  
Vesicle Budding ◽  
Adp Ribosylation ◽  
Adp Ribosylation Factor

Carrier vesicle generation from donor membranes typically progresses through a GTP-dependent recruitment of coats to membranes. Here we explore the role of ADP ribosylation factor (ARF) 1, one of the GTP-binding proteins that recruit coats, in the production of neuroendocrine synaptic vesicles (SVs) from PC12 cell membranes. Brefeldin A (BFA) strongly and reversibly inhibited SV formation in vivo in three different PC12 cell lines expressing vesicle-associated membrane protein–T Antigen derivatives. Other membrane traffic events remained unaffected by the drug, and the BFA effects were not mimicked by drugs known to interfere with formation of other classes of vesicles. The involvement of ARF proteins in the budding of SVs was addressed in a cell-free reconstitution system (Desnos, C., L. Clift-O'Grady, and R.B. Kelly. 1995. J. Cell Biol. 130:1041–1049). A peptide spanning the effector domain of human ARF1 (2–17) and recombinant ARF1 mutated in its GTPase activity, both inhibited the formation of SVs of the correct size. During in vitro incubation in the presence of the mutant ARFs, the labeled precursor membranes acquired different densities, suggesting that the two ARF mutations block at different biosynthetic steps. Cell-free SV formation in the presence of a high molecular weight, ARF-depleted fraction from brain cytosol was significantly enhanced by the addition of recombinant myristoylated native ARF1. Thus, the generation of SVs from PC12 cell membranes requires ARF and uses its GTPase activity, probably to regulate coating phenomena.

scholarly journals Inactivation of the Elongation Factor Tu by Mosquitocidal Toxin-Catalyzed Mono-ADP-Ribosylation

2002 ◽  
Vol 68 (10) ◽  
pp. 4894-4899 ◽  
Author(s):  
Jörg Schirmer ◽  
Hans-Joachim Wieden ◽  
Marina V. Rodnina ◽  
Klaus Aktories
Keyword(s):  
Peptide Mapping ◽  
Elongation Factor ◽  
Bacillus Sphaericus ◽  
Elongation Factor Tu ◽  
Bacterial Cells ◽  
Single Chain ◽  
E Coli ◽  
Adp Ribosylation ◽  
Flight Mass Spectrometry

ABSTRACT The mosquitocidal toxin (MTX) produced by Bacillus sphaericus strain SSII-1 is an ∼97-kDa single-chain toxin which contains a 27-kDa enzyme domain harboring ADP-ribosyltransferase activity and a 70-kDa putative binding domain. Due to cytotoxicity toward bacterial cells, the 27-kDa enzyme fragment cannot be produced in Escherichia coli expression systems. However, a nontoxic 32-kDa N-terminal truncation of MTX can be expressed in E. coli and subsequently cleaved to an active 27-kDa enzyme fragment. In vitro the 27-kDa enzyme fragment of MTX ADP-ribosylated numerous proteins in E. coli lysates, with dominant labeling of an ∼45-kDa protein. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry combined with peptide mapping identified this protein as the E. coli elongation factor Tu (EF-Tu). ADP ribosylation of purified EF-Tu prevented the formation of the stable ternary EF-Tuaminoacyl-tRNAGTP complex, whereas the binding of GTP to EF-Tu was not altered. The inactivation of EF-Tu by MTX-mediated ADP-ribosylation and the resulting inhibition of bacterial protein synthesis are likely to play important roles in the cytotoxicity of the 27-kDa enzyme fragment of MTX toward E. coli.

scholarly journals Arginine-Specific Mono ADP-Ribosylation In Vitro of Antimicrobial Peptides by ADP-Ribosylating Toxins

PLoS ONE ◽  
2012 ◽  
Vol 7 (8) ◽  
pp. e41417 ◽  
Author(s):  
Marta Castagnini ◽  
Monica Picchianti ◽  
Eleonora Talluri ◽  
Massimiliano Biagini ◽  
Mariangela Del Vecchio ◽  
...  
Keyword(s):  
Antimicrobial Peptides ◽  
Adp Ribosylation

Distribution of elongation factor 2 between particulate and soluble fractions of the brine shrimp Artemia during early development

1983 ◽  
Vol 61 (8) ◽  
pp. 833-839 ◽  
Author(s):  
Z. Yablonka-Reuveni ◽  
J. J. Fontaine ◽  
A. H. Warner
Keyword(s):  
Early Development ◽  
Subcellular Distribution ◽  
Brine Shrimp ◽  
Elongation Factor ◽  
Stages Of Development ◽  
Adp Ribosylation ◽  
Elongation Factor 2 ◽  
Soluble Fractions

The ADP-ribosylation of elongation factor 2 (EF-2) in vitro was used to quantitate EF-2 and to determine its subcellular distribution in extracts of Artemia embryos at different stages of development. In extracts from dormant cysts of Artemia 40–45% of EF-2 is complexed to macromolecules smaller than ribosomes, whereas the remainder is soluble or free in the cytosol. During early development the amount of "complexed" EF-2 decreases markedly concomitant with an increase in the pool of soluble EF-2. Complexed EF-2 was found to be associated with macromolecules which sediment at 16S–20S and 40S–50S and not with monoribosomes or polyribosomes as reported for mammalian systems. The data show that the decrease in complexed EF-2 is associated with the resumption of development in Artemia.

Microtubule protein ADP-ribosylation in vitro leads to assembly inhibition and rapid depolymerization

Biochemistry ◽  
1992 ◽  
Vol 31 (1) ◽  
pp. 310-316 ◽  
Author(s):  
Robin M. Scaife ◽  
Leslie Wilson ◽  
Daniel L. Purich
Keyword(s):  
Adp Ribosylation ◽  
Microtubule Protein
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