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

As an alternative to parenteral administration, mucosal administration offers several advantages including the ease of administration, safety and the ability to induce mucosal immunity. As a first step towards nasal administration of important childhood vaccines, we have previously developed attenuated Bordetella pertussis strains able to protect mice against pertussis upon nasal vaccination. Since pertussis vaccines are generally combined with tetanus and diphtheria vaccines, we constructed recombinant B. pertussis strains producing the non-toxic protective tetanus toxin fragment C (TTFC). TTFC was genetically fused to the N-terminal domain of the B. pertussis filamentous haemagglutinin. The hybrid gene was introduced into B. pertussis both on a multi-copy replicative plasmid and as a single copy inserted into the chromosome of a pertussis toxin-producing strain and a toxin-deficient attenuated strain. The hybrid protein was secreted by the recombinant strains. However, the recombinant multi-copy plasmid was unstable in vivo, and immunisation could only be carried out with the strains containing the single-copy chromosomal integration. Both the toxin-producing and the toxin-deficient recombinant B. pertussis strains were able to prime mice for the production of anti-TTFC serum antibodies upon intranasal administration, suggesting the feasibility of using recombinant attenuated B. pertussis for the development of combined childhood vaccines.
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PMID:Tetanus toxin fragment C-specific priming by intranasal infection with recombinant Bordetella pertussis. 1173 59

Desensitization of G protein-coupled receptors (GPCRs) involves receptor phosphorylation and reduction in the number of receptors at the cell surface. The neuropeptide Y (NPY) Y(1) receptor undergoes fast desensitization. We examined agonist-induced signaling and internalization using NPY Y(1) receptors fused to green fluorescent protein (EGFP). When expressed in HEK293 cells, EGFP-hNPY Y(1) receptors were localized at the plasma membrane, desensitized rapidly as assessed using calcium responses, and had similar properties compared to hNPY Y(1) receptors. Upon agonist challenge, the EGFP signal decreased rapidly (t(1/2) = 107 +/- 3 s) followed by a slow recovery. This decrease was blocked by BIBP3226, a Y(1) receptor antagonist, or by pertussis toxin, in agreement with Y(1) receptor activation. Internalization of EGFP-hNPY Y(1) receptors to acidic endosomal compartments likely accounts for the decrease in the EGFP signal, being absent after pretreatment with monensin. Concanavalin A and hypertonic sucrose, which inhibit clathrin-mediated endocytosis, blocked the decrease in fluorescence. After agonist, intracellular EGFP signals were punctate and co-localized with transferrin-Texas Red, a marker of clathrin-associated internalization and recycling, but not with LysoTracker Red, a lysosomal pathway marker, supporting receptor trafficking to recycling endosomes rather than the late endosomal/lysosomal pathway. Pulse-chase experiments revealed no receptor degradation after internalization. The slow recovery of fluorescence was unaffected by cycloheximide or actinomycin D, indicating that de novo synthesis of receptors was not limiting. Use of a multicompartment model to fit our fluorescence data allows simultaneous determination of internalization and recycling rate constants. We propose that rapid internalization of receptors via the clathrin-coated pits recycling pathway may largely account for the rapid desensitization of NPY Y(1) receptors.
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PMID:Rapid internalization and recycling of the human neuropeptide Y Y(1) receptor. 1174 3

1. cDNA constructs coding for a yellow-emitting green fluorescent protein (GFP) mutant fused to the N-terminus of the G-protein subunit beta 1 (YFP-beta 1) and a cyan-emitting GFP mutant fused to the N-terminus of the G-protein subunit gamma 2 (CFP-gamma 2) were heterologously expressed in rat superior cervical ganglion (SCG) neurons following intranuclear injection of the tagged subunits. The ability of the tagged subunits to modulate effectors, form a heterotrimer and couple to receptors was characterized using the whole-cell patch-clamp technique. Fluorescent resonance energy transfer (FRET) was also measured to determine the protein-protein interaction between the two fusion proteins. 2. Similar to co-expression of untagged beta 1/gamma 2, co-expression of YFP-beta 1/gamma 2, beta 1/CFP-gamma 2, or YFP-beta 1/CFP-gamma 2 resulted in a significant increase in basal N-type Ca(2+) channel facilitation when compared to uninjected neurons. Furthermore, the noradrenaline (NA)-mediated inhibition of Ca(2+) channels was significantly attenuated. 3. Co-expression of YFP-beta 1/CFP-gamma 2 with G-protein-gated inwardly rectifying K(+) channels (GIRK1 and GIRK4) resulted in tonic GIRK currents that were blocked by Ba(2+). 4. The ability of the tagged subunits to form heterotrimers was tested by co-injecting either tagged or untagged G beta 1 and G gamma 2 with excess G alpha(oA) cDNA. Under these conditions, the NA-mediated Ca(2+) current inhibition was significantly decreased when compared to uninjected neurons. 5. Coupling to the alpha 2-adrenergic receptor was reconstituted in neurons expressing pertussis toxin (PTX)-insensitive G alpha(oA) and either tagged or untagged G beta 1 gamma 2 subunits. Application of NA to PTX-treated cells resulted in a voltage-dependent inhibition of N-type Ca(2+) currents. 6. FRET measurements in the SCG revealed an in vivo interaction between YFP-beta 1 and CFP-gamma 2. Co-expression of untagged beta 1 significantly decreased the interaction between the two fusion proteins. 7. In summary, the attachment of GFP mutants to the N-terminus of G beta 1 or G gamma 2 does not qualitatively impair their ability to form a heterotrimer, modulate effectors (N-type Ca(2+) and GIRK channels), or couple to receptors.
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PMID:Functional expression and FRET analysis of green fluorescent proteins fused to G-protein subunits in rat sympathetic neurons. 1174 47

Signaling studies in living cells would be greatly facilitated by the development of functional fluorescently tagged G-protein alpha subunits. We have designed G(i/o)alpha subunits fused to the cyan fluorescent protein and assayed their function by studying the following two signal transduction pathways: the regulation of G-protein-gated inwardly rectifying K(+) channels (Kir3.0 family) and adenylate cyclase. Palmitoylation and myristoylation consensus sites were removed from G(i/o) alpha subunits (G(i1)alpha, G(i2)alpha, G(i3)alpha, and G(oA)alpha) and a mutation introduced at Cys(-4) rendering the subunit resistant to pertussis toxin. This construct was fused in-frame with cyan fluorescent protein containing a short peptide motif from GAP43 that directs palmitoylation and thus membrane targeting. Western blotting confirmed G(i/o)alpha protein expression. Confocal microscopy and biochemical fractionation studies revealed membrane localization. Each mutant G(i/o) alpha subunit significantly reduced basal current density when transiently expressed in a stable cell line expressing Kir3.1 and Kir3.2A, consistent with the sequestration of the Gbetagamma dimer by the mutant Galpha subunit. Moreover, each subunit was able to support A1-mediated and D2S-mediated channel activation when transiently expressed in pertussis toxin-treated cells. Overexpression of tagged G(i3)alpha and G(oA)alpha alpha subunits reduced receptor-mediated and forskolin-induced cAMP mobilization.
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PMID:A novel strategy to engineer functional fluorescent inhibitory G-protein alpha subunits. 1204 13

As preferential coupling of opioid receptor to various inhibitory Galpha subunits is still under debate, we have investigated the selectivity of the human mu opioid receptor fused to a pertussis toxin insensitive C351I Gi1 alpha or C352I Gi2 alpha in stably transfected HEK 293 cells. Overall agonist binding affinities were increased for both fusion constructs when compared to the wild type receptor. [35 S]GTPgammaS binding was performed on pertussis toxin treated cells to monitor coupling efficiency of the fusion constructs. Upon agonist addition hMOR-C351I Gi1 a exhibited an activation profile similar to the non-fused receptor while hMOR-C352I Gi2 alpha was poorly activated. Interestingly no correlation could be drawn between agonist binding affinity and efficacy. Upon agonist addition, forskolin-stimulated cAMP production, as measured using a reporter gene assay, was inhibited by signals transduced via the fused Gi1 alpha and Gi2 alpha mainly. In contrast both fusion constructs were able to initiate ERK-MAPK phosphorylation via coupling to endogenous G proteins only. In conclusion our data indicate that hMOR couples more efficiently to Gi1 alpha than Gi2 alpha and that the coupling efficacy is clearly agonist-dependent.
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PMID:Agonists activate Gi1 alpha or Gi2 alpha fused to the human mu opioid receptor differently. 1206 84

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) functions by activating two receptor tyrosine kinases, Flt-1 (VEGFR-1) and KDR (VEGFR-2), both of which are selectively expressed on the primary vascular endothelium. KDR is responsible for VPF/VEGF-stimulated endothelial cell (EC) proliferation and migration, whereas Flt-1 down-modulates KDR-mediated EC proliferation. Flt-1 mediates down-regulation of EC proliferation through pertussis toxin-sensitive G proteins, betagamma subunits, small GTPase CDC42, and partly by Rac-1. However, the molecular mechanism by which KDR mediates EC migration is not clear yet. Here we show for the first time that activation of RhoA and Rac1 is fully and partially required for KDR-mediated human umbilical vein endothelial cell (HUVEC) migration, respectively, and that CDC42, however, is not involved. Furthermore, overexpression of the RhoA dominant negative mutant RhoA-19N does not affect VPF/VEGF-stimulated KDR phosphorylation, intracellular Ca(2+) mobilization, and mitogen-activated protein kinase phosphorylation. Utilizing the receptor chimeras (EGDR and EGLT) in which the extracellular domain of the epidermal growth factor receptor (EGFR) was fused to the transmembrane domain and the intracellular domains of KDR and Flt-1, respectively, we demonstrate that RhoA activation is mediated by EGDR, not by EGLT, and that EGDR mediates activation of Rac1, not CDC42. Furthermore, the EGDR-mediated RhoA and Rac1 activation is regulated by G proteins Gq/11, Gbetagamma, and phospholipase C independent of phosphatidylinositol 3-kinase and intracellular Ca(2+) mobilization. Interestingly, the RhoA activation can be partially inhibited by overexpression of Rac1-17N, but overexpression of RhoA-19N has no effect on Rac1 activation. Finally, Gq/11 and Gbetagamma subunits are also required for VPF/VEGF-stimulated HUVEC migration. Taken together, our results indicate that KDR stimulates endothelial cell migration through a heterotrimeric G protein Gq/11 and Gbetagamma-mediated RhoA pathway.
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PMID:KDR stimulates endothelial cell migration through heterotrimeric G protein Gq/11-mediated activation of a small GTPase RhoA. 1224 99

Autotransporter secretion represents a unique mechanism that Gram-negative bacteria employ to deliver proteins to their cell surface. BrkA is a Bordetella pertussis autotransporter protein that mediates serum resistance and contributes to adherence of the bacterium to host cells. BrkA is a 103 kDa protein that is cleaved to form a 73 kDa alpha-domain and a 30 kDa beta domain. The alpha domain, also referred to as the passenger domain, is responsible for the effector functions of the protein, whereas the beta domain serves as a transporter. In an effort to characterize BrkA secretion, we have shown that BrkA has a 42 amino acid signal peptide for transit across the cytoplasmic membrane, and a translocation unit made up of a short linker region fused to the beta-domain to ferry the passenger domain to the bacterial surface through a channel formed by the beta-domain. In this report, we provide genetic, biochemical and structural evidence demonstrating that a region within the BrkA passenger (Glu601-Ala692) is necessary for folding the passenger. This region is not required for surface display in the outer membrane protease OmpT-deficient Escherichia coli strain UT5600. However, a BrkA mutant protein bearing a deletion in this region is susceptible to digestion when expressed in E. coli strains expressing OmpT suggesting that the region is required to maintain a stable structure. The instability of the deletion mutant can be rescued by surface expressing Glu601-Ala692in trans suggesting that this region is acting as an intramolecular chaperone to effect folding of the passenger concurrent with or following translocation across the outer membrane.
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PMID:A conserved region within the Bordetella pertussis autotransporter BrkA is necessary for folding of its passenger domain. 1260 41

Direct contacts between dendritic cells (DCs) and T cells or natural killer T (NKT) cells play important roles in primary and secondary immune responses. SR-PSOX/CXC chemokine ligand 16 (CXCL16), which is selectively expressed on DCs and macrophages, is a scavenger receptor for oxidized low-density lipoprotein and also the chemokine ligand for a G protein-coupled receptor CXC chemokine receptor 6 (CXCR6), expressed on activated T cells and NKT cells. SR-PSOX/CXCL16 is the second transmembrane-type chemokine with a chemokine domain fused to a mucin-like stalk, a structure very similar to that of fractalkine (FNK). Here, we demonstrate that SR-PSOX/CXCL16 functions as a cell adhesion molecule for cells expressing CXCR6 in the same manner that FNK functions as a cell adhesion molecule for cells expressing CX(3)C chemokine receptor 1 (CX(3)CR1) without requiring CX(3)CR1-mediated signal transduction or integrin activation. The chemokine domain of SR-PSOX/CXCL16 mediated the adhesion of CXCR6-expressing cells, which was not impaired by treatment with pertussis toxin, a Galphai protein blocker, which inhibited chemotaxis of CXCR6-expressing cells induced by SR-PSOX/CXCL16. Furthermore, the adhesion activity was up-regulated by treatment of SR-PSOX/CXCL16-expressing cells with a metalloprotease inhibitor, which increased surface expression levels of SR-PSOX/CXCL16. Thus, SR-PSOX/CXCL16 is a unique molecule that not only attracts T cells and NKT cells toward DCs but also supports their firm adhesion to DCs.
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PMID:Cell surface-anchored SR-PSOX/CXC chemokine ligand 16 mediates firm adhesion of CXC chemokine receptor 6-expressing cells. 1463 54

In this work, two different types of supported biomimetic membranes were designed to study the membrane binding properties of two different proteins that both interact with cellular membranes in a calcium-dependent manner. The first one, neurocalcin, is a member of a subfamilly of EF-hand calcium-binding proteins that exhibit a calcium-myristoyl switch. The second protein is a bacterial toxin, the adenylate cyclase produced by Bordetella pertussis, the causative agent of whooping cough. The biomimetic membranes constructed in this study were either hybrid bilayer membranes or polymer-tethered membranes. Hemimembrane formation was obtained in two steps: a monolayer of 1-octadecanethiol or octadecyltrichlorosilane was self-assembled on top of the gold or glass surface, respectively, and then the egg-phosphatidyl choline (PC) vesicle fused on the hydrophobic alkyl layer. Polymer-tethered membranes on solid support were obtained using N-hydroxysuccinimide (NHS)-terminated-poly(ethyleneglycol) (PEG)-phospholipids as anchoring molecules. Egg-PC/1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-poly(ethyleneglycol)-N-hydroxy-succinimide (DSPE-PEG-NHS) mixture liposomes were injected on the top of an amine grafted surface (cysteamine-coated gold or silanized glass); vesicles were linked to the surface and disrupted, leading to the formation of a bilayer. The biomimetic membrane constructions were followed by surface plasmon spectroscopy, while membrane fluidity and continuity were observed by fluorescence microscopy. Protein/membrane binding properties were determined by resonance surface plasmon measurements. The tethered bilayer, designed here, is very versatile as it can be adapted easily to different types of support. The results demonstrate the potentialities of such polymer-tethered artificial membranes for the study of proteins that insert into biological membranes such as toxins and/or integral membrane proteins.
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PMID:Differential mechanisms for calcium-dependent protein/membrane association as evidenced from SPR-binding studies on supported biomimetic membranes. 1469 Apr 37

Formyl peptide receptor-like 1 (FPRL1) is a G protein-coupled receptor that binds natural and synthetic peptides as well as lipoxin A(4) and mediates important biological functions. To facilitate its pharmacological characterization, we screened a compound library and identified a substituted quinazolinone (Quin-C1, 4-butoxy-N-[2-(4-methoxy-phenyl)-4-oxo-1,4-dihydro-2H-quinazolin-3-yl]-benzamide) as a ligand for FPRL1. Quin-C1 induces chemotaxis and secretion of beta-glucuronidase in peripheral blood neutrophils with a potency of approximately 1/1000 of that of the peptide agonist WKYMVm. In studies using transfected rat basophilic leukemia (RBL) cell lines expressing either formyl peptide receptor or FPRL1, Quin-C1 induced enzyme release from RBL-FPRL1 but not RBL-FPR cells. Likewise, Quin-C1 selectively stimulates calcium mobilization in RBL-FPRL1 cells, a response that was markedly inhibited by pertussis toxin. Quin-C1 also stimulates phosphorylation of extracellular signal-regulated protein kinases 1 and 2 and induces internalization of an FPRL1 fused to green fluorescent protein. In degranulation assays, both the FPRL1-selective peptide agonist MMK1 and Quin-C1 exhibited lower efficacy and potency than WKYMVm, with EC(50) values of 7.17 x 10(-8) M and 1.88 x 10(-6) M, respectively, compared with the EC(50) value for WKYMVm (2.29 x 10(-8) M). However, Quin-C1 did not induce neutrophil superoxide generation at up to 100 microM. Based on these results, we conclude that Quin-C1 is a novel nonpeptide ligand that binds to FPRL1 and selectively stimulates FPRL1-mediated functions. Quin-C1 is a prototype of substituted quinazolinones based on which further structural modifications may be made to improve its efficacy and potency for FPRL1.
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PMID:A novel nonpeptide ligand for formyl peptide receptor-like 1. 1530 62


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