Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0043167 (pertussis)
 19,595 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The interaction of the immunodominant pertussis toxin peptide containing residues 30-42 (p30-42) with soluble DR1 molecules and the T-cell receptor (TCR) of 12 DR1-restricted human T-cell clones has been analyzed. Peptide analogues of p30-42 containing single alanine substitutions were used in DR1-binding and T-cell proliferation assays to identify the major histocompatibility complex and TCR contact residues. Each T-cell clone was found to recognize p30-42 with a different fine specificity. However, a common core comprising amino acids 33-39 was found to be important for stimulation of all T-cell clones. Within this core two residues, Leu33 and Leu36, interact with the DR1 molecule, whereas Asp34, His35, Thr37, and Arg39 are important for TCR recognition in most of the clones. Computer modeling of the structure of p30-42 showed that an alpha-helical conformation is compatible with the experimental data. The analysis of TCR rearrangement revealed that the peptide was recognized by T-cell clones expressing different variable region alpha (V alpha) and variable region beta (V beta) chains, although a preferential use of V alpha 8-V beta 13 and V alpha 11-V beta 18 combinations was found in clones from the same donor. Understanding the details of the interaction of antigenic peptides with the major histocompatibility complex and TCR molecules should provide the theoretical basis to design T-cell epitopes and obtain more immunogenic vaccines.
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PMID:Interaction of the pertussis toxin peptide containing residues 30-42 with DR1 and the T-cell receptors of 12 human T-cell clones. 131 75

ADP-ribosylation is a posttranslational modification of proteins by amino acid-specific ADP-ribosyltransferases. Both pertussis toxin and eukaryotic enzymes ADP-ribosylate cysteine residues in proteins and also, it has been suggested, free cysteine. Analysis of the reaction mechanisms of cysteine-specific ADP-ribosyltransferases revealed that free ADP-ribose combined nonenzymatically with cysteine. L- and D-cysteine, L-cysteine methyl ester, and cysteamine reacted with ADP-ribose, but alanine, serine, lysine, arginine, N-acetyl-L-cysteine, 2-mercaptoethanol, dithiothreitol, and glutathione did not. The 1H NMR spectrum of the product, along with the requirement for both free sulfhydryl and amino groups of cysteine, suggested that the reaction produced a thiazolidine linkage. ADP-ribosylthiazolidine was labile to hydroxylamine and mercuric ion, unlike the ADP-ribosylcysteine formed by pertussis toxin and NAD in guanine nucleotide-binding (G-) proteins, which is labile to mercuric ion but stable in hydroxylamine. In the absence of G-proteins but in the presence of NAD and cysteine, pertussis toxin generated a hydroxylamine-sensitive product, suggesting that a free ADP-ribose intermediate, expected to be formed by the NADase activity of the toxin, reacted with cysteine. Chemical analysis, or the use of alternative thiol acceptors lacking a free amine, is necessary to distinguish the enzymatic formation of ADP-ribosylcysteine from nonenzymatic formation of ADP-ribosylthiazolidine, thereby differentiating putative NAD:cysteine ADP-ribosyltransferases from NAD glycohydrolases.
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PMID:Amino acid-specific ADP-ribosylation: structural characterization and chemical differentiation of ADP-ribose-cysteine adducts formed nonenzymatically and in a pertussis toxin-catalyzed reaction. 144 18

To determine the effect of protein isoprenylation with farnesyl vs geranylgeranyl groups on membrane association in vivo, COS cells were transfected with cDNAs encoding the wild-type G-protein alpha i1 (WT) subunit, the soluble nonmyristoylated G-protein alpha i1 glycine to alanine mutant (GA), a double mutant in which the carboxy-terminal residues CGLF of GA were mutated to CVLS (GA-CVLS), and a double mutant in which the carboxy terminus of GA was mutated to CALL (GA-CALL). As opposed to the WT and GA proteins, the GA-CVLS and GA-CALL proteins were not pertussis toxin substrates nor were they recognized by antibodies that recognize the nonmutated alpha i1 carboxy terminus. Only the GA-CVLS and GA-CALL proteins incorporated [3H]mevalonate in the form of a farnesyl and a geranylgeranyl moiety, respectively. Subcellular localization, as assessed by immunoblotting and immunoprecipitation, revealed that the WT protein localizes almost exclusively to the membrane fraction, whereas the GA, GA-CVLS, and GA-CALL proteins localize predominantly to the soluble fraction. The soluble GA-CVLS and GA-CALL proteins were not carboxyl methylated, but the small amount localized to the membrane was partially carboxyl methylated. These results indicate that neither farnesylation nor geranylgeranylation is sufficient alone to lead to membrane association.
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PMID:Differential isoprenylation of carboxy-terminal mutants of an inhibitory G-protein alpha-subunit: neither farnesylation nor geranylgeranylation is sufficient for membrane attachment. 151 Sep 88

The signal transduction mechanisms involved in the regulation of phagocytosis are largely unknown. We have recently shown that in neutrophils, when IgG-mediated phagocytosis is stimulated by formyl-methionyl-leucyl-phenyl-alanine (fMLP), the enhanced ingestion is dependent on the increase in [Ca2+]i which results from ligation of Fc receptors by the IgG-coated target (Rosales, C., and Brown, E. (1991) J. Immunol. 146, 3937-3944). Now, we have studied the mechanism by which this rise in [Ca2+]i occurs. Aggregated IgG, the monoclonal antibody 3G8 (which recognizes Fc receptor type III), and insoluble immune complexes caused an increase in [Ca2+]i. The rise in [Ca2+]i induced by Fc receptor ligation was resistant to pertussis toxin. In contrast, fMLP induced a rise in [Ca2+]i which was inhibited by pertussis toxin. fMLP-induced [Ca2+]i was accompanied by an accumulation of inositol 1,4,5-trisphosphate (IP3) which peaked by 15 s, and which was also abolished by pertussis toxin. IP3 accumulation after aggregated IgG, 3G8, or insoluble immune complexes was much less than after fMLP. Unlike [Ca2+]i rise induced by Fc receptor ligation, this small increase in IP3 was inhibited by pertussis toxin. These data demonstrated that the [Ca2+]i increase induced by Fc receptor ligation is not mediated by IP3. Immediate pretreatment of human polymorphonuclear neutrophils with optimal doses of fMLP also reduced subsequent increase in [Ca2+]i rise from thapsigargin, a sesquiterpene lactone tumor promoter that releases intracellular Ca2+ from IP3-sensitive stores without IP3 turnover. Similarly, to its effects on thapsigargin, fMLP inhibited the [Ca2+]i rise upon subsequent immune complex binding. Pretreatment of cells with immune complexes also prevented subsequent [Ca2+]i rise from thapsigargin and fMLP. These data demonstrate that IgG Fc receptor ligation and fMLP activation of human polymorphonuclear neutrophils use distinct signal transduction mechanisms to release Ca2+ from the same thapsigargin-sensitive intracellular pool. In contrast to fMLP, signal transduction for increased [Ca2+]i after Fc receptor stimulation does not involve a pertussis toxin-sensitive G protein, and is independent of IP3.
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PMID:Signal transduction by neutrophil immunoglobulin G Fc receptors. Dissociation of intracytoplasmic calcium concentration rise from inositol 1,4,5-trisphosphate. 153 82

Previous studies have demonstrated that mutations of highly conserved residues in the alpha subunit of Gs (alpha s) can inhibit either the intrinsic GTPase activity (glutamine-227 to leucine, Q227L) or the ability of the protein to be activated by GTP (glycine-226 to alanine, G226A). We stably transfected NIH 3T3 cells with cDNAs encoding Gi2 alpha subunit (alpha i2) containing either wild-type sequence or the homologous mutations Q205L and G204A. High expression of wild-type alpha i2, Q205L alpha i2, and G204A alpha i2 was confirmed in transfected cells by immunoblot analysis. The overexpression of all three alpha i2 proteins was accompanied by an increase in beta-subunit expression. Q205L alpha i2 was a poor substrate for ADP-ribosylation by pertussis toxin as compared with wild-type alpha i2. Expression of Q205L alpha i2 markedly decreased forskolin- or cholera toxin-stimulated intracellular cAMP levels in intact cells, confirming the constitutively activated state of the protein. In contrast, G204A alpha i2 increased intracellular cAMP and was resistant to guanosine 5'-[gamma-thio]triphosphate-induced inhibition of ADP-ribosylation by pertussis toxin, as expected for an inactive alpha i2. Transfection of wild-type, Q205L, or G204A alpha i2 cDNA did not induce focus formation of NIH 3T3 cells. However, overexpression of Q205L alpha i2 induced a decreased serum requirement, a reduced doubling time, and an 8- to 10-fold increase in [3H]thymidine incorporation. Q205L alpha i2 cells formed small colonies in soft agar, demonstrating some degree of anchorage-independent proliferation. Expression of G204A alpha i2 slowed the growth of NIH 3T3 cells. We conclude that alpha i2 plays an important role in regulation of fibroblast growth.
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PMID:Activating and inactivating mutations of the alpha subunit of Gi2 protein have opposite effects on proliferation of NIH 3T3 cells. 166 Jan 38

L-Histidine and imidazole (the histidine side chain) significantly increase cAMP accumulation in intact LLC-PK1 cells. This effect is completely inhibited by isobutylmethylxanthine (IBMX). Histidine and imidazole stimulate cAMP phosphodiesterase activity in soluble and membrane fractions of LLC-PK1 cells suggesting that the IBMX-sensitive effect of these agents to stimulate cAMP formation is not due to inhibition of cAMP phosphodiesterase. Histidine and imidazole but not alanine (the histidine core structure) increase basal, GTP-, forskolin-, and AVP-stimulated adenylate cyclase activity in LLC-PK1 membranes. Two other amino acids with charged side chains (aspartic and glutamic acids) increase AVP-stimulated but neither basal- nor forskolin-stimulated adenylate cyclase activity. This suggests that multiple amino acids with charged side chains can regulate selected aspects of adenylate cyclase activity. To better define the mechanism of histidine regulation of adenylate cyclase, membranes were detergent-solubilized which prevents histidine and imidazole potentiation of forskolin-stimulated adenylate cyclase activity and suggests that an intact plasma membrane environment is required for potentiation. Neither pertussis toxin nor indomethacin pretreatment alter imidazole potentiation of adenylate cyclase. IBMX pretreatment of LLC-PK1 membranes also prevents imidazole to potentiate adenylate cyclase activity. Since IBMX inhibits adenylate cyclase coupled adenosine receptors, LLC-PK1 cells were incubated in vitro with 5'-N-ethylcarboxyamideadenosine (NECA) which produced a homologous pattern of desensitization of NECA to stimulate adenylate cyclase activity. Despite homologous desensitization, histidine and imidazole potentiation of adenylate cyclase was unaltered. These data suggest that histidine, acting via an imidazole ring, potentiates adenylate cyclase activity and thereby increases cAMP formation in cultured LLC-PK1 epithelial cells. This potentiation requires an intact plasma membrane environment, occurs independent of a pertussis toxin-sensitive substrate and of products of cyclooxygenase, and is inhibited by IBMX. This IBMX-sensitive pathway does not involve either inhibition of cAMP phosphodiesterase activity or a stimulatory adenosine receptor coupled to adenylate cyclase.
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PMID:Histidine regulation of cyclic AMP metabolism in cultured renal epithelial LLC-PK1 cells. 168 53

Epitopes defined by monoclonal antibodies (mAb) specific for the Bordetella pertussis outer membrane protein P.69 (pertactin) were mapped using a series of amino- and carboxy-terminal deletion mutants expressed in Escherichia coli. mAb were found to bind predominantly to a region of pertactin spanning a (Pro-Gln-Pro)5 repeat motif and one mAb was found to bind to another region spanning a (Gly-Gly-Xaa-Xaa-Pro)5 repeat motif. To localize further the mAb-binding sites, a panel of synthetic peptides, a series of 94 overlapping hexameric peptides, and a P.69 30-amino acid fusion to a hepatitis B core protein (HBcAg-69), were synthesized. This combined approach has identified the binding site for the mAb BBO5: Pro-Gly-Pro-Gln-Pro-Pro; mAb BBO7, E4A8 and E4D7: Ala-Pro-Gln-Pro-Pro-Ala-Gly-Arg; and mAb BPE3: Thr-Leu-Trp-Tyr-Ala-Glu-Ser-Asn-Ala-Leu-Ser-Lys-Arg. We have used a non-lethal murine respiratory model of B. pertussis infection to investigate the ability of a peptide containing the epitope of the mAb BBO5 to elicit protective immunity. Immunization of mice with the HBcAg-69 protein prevented growth of B. pertussis in the lungs compared to mice receiving HBcAg alone, and protection correlated with high titers of anti-P.69 antibodies.
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PMID:Identification and characterization of a protective immunodominant B cell epitope of pertactin (P.69) from Bordetella pertussis. 170 65

We transfected COS cells with cDNAs for the alpha subunits of stimulatory and inhibitory GTP-binding proteins, alpha s and alpha i1, respectively, and immunoprecipitated the metabolically labeled products with specific peptide antibodies. Cells were separated into particulate and soluble fractions before immunoprecipitation; [35S]methionine-labeled alpha s and alpha i were both found primarily in the particulate fraction. [3H]Myristate was incorporated into endogenous and transfected alpha i but could not be detected in alpha s even when it was overexpressed. We converted the second residue, glycine, of alpha i1 into alanine by site-directed mutagenesis. Upon transfection of the mutant alpha i1 into COS cells, the [35S]methionine-labeled product was localized primarily to the soluble fraction, and, also unlike normal alpha i1, the mutant failed to incorporate [3H]myristate. The unmyristoylated mutant alpha i1 could still interact with the beta-gamma complex, since purified beta gamma subunits promoted pertussis toxin-catalyzed ADP-ribosylation of both the normal and mutant alpha i1 subunits. These results indicate that myristoylation is critical for membrane attachment of alpha i but not alpha s subunits.
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PMID:Myristoylation of an inhibitory GTP-binding protein alpha subunit is essential for its membrane attachment. 210 88

We transfected COS cells with expression vectors for the wild-type G protein alpha i1 subunit (pWT) and for mutated alpha i1 subunits, including the nonmyristylated glycine 2 to alanine mutant (pGA) and mutants in which the carboxyl termini of pWT and pGA were changed from CGLF to CVLS (pCVLS and pGA-CVLS, respectively). Immunoblot analysis of transfected COS cells with an antibody to residues 159-168 of the alpha i1 protein indicated that all four proteins were expressed. Unlike the WT and GA proteins, both CVLS mutant proteins failed to react with an antibody specific for the carboxyl terminus and failed to undergo pertussis toxin-catalyzed ADP-ribosylation. Analysis of COS cell lysates after [3H]mevalonic acid labeling indicated that specific incorporation of radioactivity occurred only in the alpha i1 subunits with the CVLS mutation. Immuno-precipitation of COS cell fractions after labeling with [35S]methionine indicated that both WT and CVLS mutant proteins were localized predominantly in the particulate fraction, whereas GA and GA-CVLS mutant proteins were found primarily in the soluble fraction. These results directly demonstrate that the carboxyl-terminal sequence, CGLF, is incapable of leading to isoprenylation but that alteration of two residues (glycine to valine, phenylalanine to serine) is sufficient to promote isoprenylation.
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PMID:Isoprenylation of an inhibitory G protein alpha subunit occurs only upon mutagenesis of the carboxyl terminus. 212 86

Heterotrimeric guanine nucleotide-binding proteins (G proteins) are integral to the signal transduction pathways that mediate the cell's response to many hormones, neuromodulators, and a variety of other ligands. While many signaling processes are guanine nucleotide dependent, the precise coupling between a variety of receptors, G proteins, and effectors remains obscure. We found that the family of genes that encode the alpha subunits of heterotrimeric G proteins is much larger than had previously been supposed. These novel alpha subunits could account for some of the diverse activities attributed to G proteins. We have now obtained cDNA clones encoding two murine alpha subunits, G alpha q and G alpha 11, that are 88% identical. They lack the site that is ordinarily modified by pertussis toxin and their sequences vary from the canonical Gly-Ala-Gly-Glu-Ser (GAGES) amino acid sequence found in most other G protein alpha subunits. Multiple mRNAs as large as 7.5 kilobases hybridize to G alpha q specific probes and are expressed at various levels in many different tissues. G alpha 11 is encoded by a single 4.0-kilobase message which is expressed ubiquitously. Amino acid sequence comparisons suggest that G alpha q and G alpha 11 represent a third class of alpha subunits. A member of this class was found in Drosophila melanogaster. This alpha subunit, DG alpha q, is 76% identical to G alpha q. The presence of the Gq class in both vertebrates and invertebrates points to a role that is central to signal transduction in multicellular organisms. We suggest that these alpha subunits may be involved in pertussis toxin-insensitive pathways coupled to phospholipase C.
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PMID:G protein diversity: a distinct class of alpha subunits is present in vertebrates and invertebrates. 212 49


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