Preferential ADP-ribosylation of arginine-3 in synthetic heptapeptide Leu-Arg-Arg-Ala-Ser-Leu-Gly.
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ABSTRACT:
SUBMITTER: Matsuura R
PROVIDER: S-EPMC1149392 | biostudies-other | 1988 Aug
REPOSITORIES: biostudies-other
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Project description: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.
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Project description:Sesquiterpene lactones (SL) have a wide range of applications in anti-tumor and anti-inflammatory therapy. However, the pharmacological mechanism of such substances is not clear. In this study, parthenolide (PTL) was used as an example to explore the anti-tumor effect of natural molecules and their common mechanism. We showed that PTL inhibited the proliferation and migration by reverse EMT via the ERK2/NF-κB/Snail pathway in vivo and in vitro. Interestingly, Multiple potential targets of PTL contain a Gly-Leu-Ser/Lys-"co-adaptation pocket". This inspiring us analogies of PTL may also bind to these target proteins and play a similar function. Significantly, the Concept of co-adaptation pocket may help to increase the selectivity of drug research and development.
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Project description:The Rho ADP-ribosylating C3 exoenzyme (C3bot) is a bacterial protein toxin devoid of a cell-binding or -translocation domain. Nevertheless, C3 can efficiently enter intact cells, including neurons, but the mechanism of C3 binding and uptake is not yet understood. Previously, we identified the intermediate filament vimentin as an extracellular membranous interaction partner of C3. However, uptake of C3 into cells still occurs (although reduced) in the absence of vimentin, indicating involvement of an additional host cell receptor. C3 harbors an Arg-Gly-Asp (RGD) motif, which is the major integrin-binding site, present in a variety of integrin ligands. To check whether the RGD motif of C3 is involved in binding to cells, we performed a competition assay with C3 and RGD peptide or with a monoclonal antibody binding to ?1-integrin subunit and binding assays in different cell lines, primary neurons, and synaptosomes with C3-RGD mutants. Here, we report that preincubation of cells with the GRGDNP peptide strongly reduced C3 binding to cells. Moreover, mutation of the RGD motif reduced C3 binding to intact cells and also to recombinant vimentin. Anti-integrin antibodies also lowered the C3 binding to cells. Our results indicate that the RGD motif of C3 is at least one essential C3 motif for binding to host cells and that integrin is an additional receptor for C3 besides vimentin.
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Project description:The vibrational spectrum of the Ala-Leu-Ala-Leu peptide in solution, computed from first-principles simulations, shows a prominent band in the amide I region that is assigned to stretching of carbonyl groups. Close inspection reveals combined but slightly different contributions by the three carbonyl groups of the peptide. The shift in their exact vibrational signature is in agreement with the different probabilities of these groups to form hydrogen bonds with the solvent. The central carbonyl group has a hydrogen bond probability intermediate to the other two groups due to interchanges between different hydrogen-bonded states. Analysis of the interaction energies of individual water molecules with that group shows that shifts in its frequency are directly related to the interactions with the water molecules in the first hydration shell. The interaction strength is well correlated with the hydrogen bond distance and hydrogen bond angle, though there is no perfect match, allowing geometrical criteria for hydrogen bonds to be used as long as the sampling is sufficient to consider averages. The hydrogen bond state of a carbonyl group can therefore serve as an indicator of the solvent's effect on the vibrational frequency.
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Project description:The hepatitis C virus (HCV) glycoproteins E1 and E2 form a heterodimer that mediates CD81 receptor binding and viral entry. In this study, we used site-directed mutagenesis to examine the functional role of a conserved G436WLAGLFY motif of E2. The mutants could be placed into two groups based on the ability of mature virion-incorporated E1E2 to bind the large extracellular loop (LEL) of CD81 versus the ability to mediate cellular entry of pseudotyped retroviral particles. Group 1 comprised E2 mutants where LEL binding ability largely correlated with viral entry ability, with conservative and nonconservative substitutions (W437 L/A, L438A, L441V/F, and F442A) inhibiting both functions. These data suggest that Trp-437, Leu-438, Leu-441, and Phe-442 directly interact with the LEL. Group 2 comprised E2 glycoproteins with more conservative substitutions that lacked LEL binding but retained between 20% and 60% of wild-type viral entry competence. The viral entry competence displayed by group 2 mutants was explained by residual binding by the E2 receptor binding domain to cellular full-length CD81. A subset of mutants maintained LEL binding ability in the context of intracellular E1E2 forms, but this function was largely lost in virion-incorporated glycoproteins. These data suggest that the CD81 binding site undergoes a conformational transition during glycoprotein maturation through the secretory pathway. The G436P mutant was an outlier, retaining near-wild-type levels of CD81 binding but lacking significant viral entry ability. These findings indicate that the G436WLAGLFY motif of E2 functions in CD81 binding and in pre- or post-CD81-dependent stages of viral entry.
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Project description:A solid-phase methodology to synthesize oligopeptides, specifically incorporating serine residues linked to ADP-ribose (ADPr), is presented. Through the synthesis of both α- and β-anomers of the phosphoribosylated Fmoc-Ser building block and their usage in our modified solid-phase peptide synthesis protocol, both α- and β-ADPr peptides from a naturally Ser-ADPr containing H2B sequence were obtained. With these, and by digestion studies using the human glycohydrolase, ARH3 (hARH3), compelling evidence is obtained that the α-Ser-ADPr linkage comprises the naturally occurring configuration.
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Project description:Clostridium perfringens iota-toxin (Ia) mono-ADP ribosylates Arg177 of actin, leading to cytoskeletal disorganization and cell death. To fully understand the reaction mechanism of arginine-specific mono-ADP ribosyl transferase, the structure of the toxin-substrate protein complex must be characterized. Recently, we solved the crystal structure of Ia in complex with actin and the nonhydrolyzable NAD(+) analog ?TAD (thiazole-4-carboxamide adenine dinucleotide); however, the structures of the NAD(+)-bound form (NAD(+)-Ia-actin) and the ADP ribosylated form [Ia-ADP ribosylated (ADPR)-actin] remain unclear. Accidentally, we found that ethylene glycol as cryo-protectant inhibits ADP ribosylation and crystallized the NAD(+)-Ia-actin complex. Here we report high-resolution structures of NAD(+)-Ia-actin and Ia-ADPR-actin obtained by soaking apo-Ia-actin crystal with NAD(+) under different conditions. The structures of NAD(+)-Ia-actin and Ia-ADPR-actin represent the pre- and postreaction states, respectively. By assigning the ?TAD-Ia-actin structure to the transition state, the strain-alleviation model of ADP ribosylation, which we proposed previously, is experimentally confirmed and improved. Moreover, this reaction mechanism appears to be applicable not only to Ia but also to other ADP ribosyltransferases.
| S-EPMC3600452 | biostudies-literature
Project description:The ADP-ribosylating toxins (ADPRTs) produced by pathogenic bacteria modify intracellular protein and affect eukaryotic cell function. Actin-specific ADPRTs (including Clostridium perfringens iota-toxin and Clostridium botulinum C2 toxin) ADP-ribosylate G-actin at Arg-177, leading to disorganization of the cytoskeleton and cell death. Although the structures of many actin-specific ADPRTs are available, the mechanisms underlying actin recognition and selective ADP-ribosylation of Arg-177 remain unknown. Here we report the crystal structure of actin-Ia in complex with the nonhydrolyzable NAD analog betaTAD at 2.8 A resolution. The structure indicates that Ia recognizes actin via five loops around NAD: loop I (Tyr-60-Tyr-62 in the N domain), loop II (active-site loop), loop III, loop IV (PN loop), and loop V (ADP-ribosylating turn-turn loop). We used site-directed mutagenesis to confirm that loop I on the N domain and loop II are essential for the ADP-ribosyltransferase activity. Furthermore, we revealed that Glu-378 on the EXE loop is in close proximity to Arg-177 in actin, and we proposed that the ADP-ribosylation of Arg-177 proceeds by an SN1 reaction via first an oxocarbenium ion intermediate and second a cationic intermediate by alleviating the strained conformation of the first oxocarbenium ion. Our results suggest a common reaction mechanism for ADPRTs. Moreover, the structure might be of use in rational drug design to block toxin-substrate recognition.
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Project description:Tyr-D-Ala-Phe-Leu-Arg psi (CH(2)NH) Arg-NH(2) (SK-9709) is a dynorphin derivative in which the peptide bond was replaced with a psi (CH(2)NH) bond. In the present study, the antinociceptive effects of SK-9709 were determined in an acetic acid-induced writhing test and a hot-plate test. In the acetic acid-induced writhing test, significant antinociceptive effects were observed after subcutaneous (s.c.), intracerebroventricular (i.c.v.) and intrathecal (i.t.) injection of SK-9709, with maximal effects at 120, 30 and 15 min, respectively. The antinociceptive effects were dose-dependent and ED(50) values (range of 95% confidence limits) after s.c., i.c.v. and i.t. injection were 1.36 (0.61 - 3.02) micromol kg(-1), 2.11 (1.18 - 3.79) and 0.79 (0.61 - 1.03) nmol per mouse, respectively. The effects of SK-9709 (s.c., i.c.v. and i.t.) were reversed by the opioid receptor antagonist naloxone (1.36 micromol kg(-1), s.c.). The effects of SK-9709 (s.c.) were also reversed by the selective mu-opioid receptor antagonist beta-funaltrexamine (4.7 nmol per mouse, i.c.v.), and kappa-opioid receptor antagonist nor-binaltorphimine (4.9 nmol per mouse, i.t.). In the hot-plate test, the antinociceptive effect of SK-9709 (s.c., i.c.v. and i.t.) was also dose-dependent with the maximal peak effect at 120, 15 and 15 min similarly to the acetic acid-induced writhing test. The antinociceptive effects were dose-dependent and ED(50) values (range of 95% confidence limits) after s.c., i.c.v. and i.t. injection were 39.1 (5.4 - 283.0) micromol kg(-1), 6.5 (4.0 - 10.7) and 7.4 (5.0 - 11.0) nmol per mouse, respectively. These findings indicated that systemically administered SK-9709 produced long-lasting antinociceptive effects and these effects were mediated by both supra-spinal mu- and spinal kappa-opioid receptors.
| S-EPMC1572722 | biostudies-other
Project description:The anti-hypertensive effect of processed rice bran (PRB) was recently reported, for which the novel peptide Leu-Arg-Ala (LRA) was identified as the functional substance. The purpose of this study was to assess the anti-hypertensive effects of a rice bran supplement containing PRB in individuals with high-normal blood pressure (systolic blood pressure (SBP): 130?139 mmHg and/or diastolic blood pressure (DBP): 85?89 mmHg) or grade 1 hypertension (SBP: 140?159 mmHg and/or DBP: 90?99 mmHg). One hundred individuals with high-normal blood pressure or grade 1 hypertension were recruited to participate in this double-blind, randomized, placebo-controlled study. Participants were randomly allocated to the placebo group (n = 50) or the test group (n = 50). Each group took four test tablets (43 ?g LRA/day) or four placebo tablets daily. The decrease in blood pressure in the test group compared with the placebo group was the primary outcome. Adverse events were recorded and hematological/urinary parameters measured to determine the safety of the supplement, which was the secondary outcome. In total, 87 participants completed the study. The SBP of the test group at 12 weeks was significantly lower than that of the placebo group (p = 0.0497). No serious adverse events were observed. Daily consumption of a rice bran supplement containing PRB can safely improve mildly elevated blood pressure.
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