Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

We have studied the expression of G protein subtypes and the role of G protein-dependent signaling in two subclones of RED-1 cells, an erythropoetin(Epo)-sensitive, murine erythroleukemia cell line. Clone 6C8 showed terminal erythroid differentiation in response to a combined treatment with Epo and dimethylsulfoxide. Clone G3 was resistant to these inducers, but responded to Epo with enhanced proliferation. We measured G protein alpha subunit levels by toxin-catalyzed adenosine diphosphate (ADP)-ribosylation with [32P]-nicotinamide adenine dinucleotide (NAD) and by semiquantitative immunoblotting with specific antisera. Native RED-1 cells expressed G alpha i2, alpha i3, alpha s, and alpha q/11, but not alpha i1 and alpha o. Terminal differentiation was associated with a selective loss (approximately 80%) of G alpha i3 and an increase in a truncated cytosolic form of G alpha i2, while the membrane levels of alpha i2, alpha q/11, and alpha s did not change significantly. Treatment of G3 cells with the inducers was without effect on G protein abundance. However, except for alpha s, G3 cells contained significantly higher levels of the different G protein alpha subunits tested. Stimulation of G protein-coupled receptors by thrombin and ADP caused a pertussis toxin (PTX)-inhibitable transient increase in intracellular Ca2+ that was markedly reduced in differentiated cells. In G3 cells, but not in 6C8 cells, thrombin also caused a PTX-sensitive inhibition of isoprenaline-stimulated cyclic 3',5'-adenosine monophosphate (cAMP) formation. Our results show that specific alterations in G protein expression and function are associated with erythroid differentiation of erythroleukemia cells but do not prove a causal relationship. The loss of G alpha i3 may affect cellular responses that are mediated via P2T purine or thrombin receptors.
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PMID:Changes in G protein pattern and in G protein-dependent signaling during erythropoietin- and dimethylsulfoxide-induced differentiation of murine erythroleukemia cells. 799 27

Immunochemical detection of pertussis toxin-sensitive guanine-nucleotide binding proteins has been suggested to represent the most direct approach to quantitate the protein than pertussis toxin-catalysed [32P]ADP-ribosylation. The latter technique is potentially hampered by pre-existing covalent modification of the C-terminus. However, limited data exist as to whether and in what way modifications of the C-terminus affect immunoreactivity of Gi alpha (alpha-subunit of the inhibitory G-protein of adenylyl cyclase). Membranes from human myocardium, thrombocytes, adipose tissue and lung were treated with pertussis toxin or N-ethylmaleimide. Both, conditions prevented high affinity agonist binding to m-cholinoceptors and inhibited [32P]ADP-ribosylation by pertussis toxin consistent with the notion that the modifications took place at the C-terminus. Pertussis toxin treatment increased immunoreactivity to different antisera raised against the C-terminal decapeptide of transducin alpha (KENLKDCGLF, DS 1-4, AS). N-Ethylmaleimide reduced immunoreactivity towards all antisera studied. Pertussis toxin reduced the mobility of Gi alpha on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) depending on the presence of the toxin and sensitivity to inhibition of ADP-ribosylation by nicotinamide. In native membranes from none of the tissues studied, immunoreactive material comigrating with pertussis toxin-modified form of Gi alpha was detected. It is concluded that modification of the C-terminus by pertussis toxin or N-ethylmaleimide resulting in the same functional consequence, i.e. prevention of high affinity agonist receptor binding, is capable of producing opposite changes of immunoreactivity. Pertussis toxin treatment reduces the electrophoretic mobility on SDS-PAGE. Separation of the native and pertussis toxin-modified form of Gi alpha on SDS-PAGE demonstrates that endogenously ADP-ribosylated Gi alpha is lacking in membranes from human myocardium, thrombocytes, lung and adipose tissue.
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PMID:C-terminal modifications of pertussis toxin-sensitive G-protein alpha-subunits differentially affect immunoreactivity. Evidence against endogenous ADP-ribosylation in human heart, lung, thrombocytes and adipose tissue. 827 48

Pertussis toxin is a complex protein composed of five different subunits, named S1 through S5 and arranged in an A-B structure. The B oligomer, composed of S2 through S5, is the receptor-binding moiety, and the A promoter, composed of S1, is the enzymatically active moiety. S1 catalyzes the ADP-ribosylation of a cysteine in the alpha subunit of heterotrimeric G proteins. In the absence of G proteins it also catalyzes the cleavage of NAD+ into ADP-ribose and nicotinamide. Molecular dissection has indicated that the C-terminal domain of S1 is involved in G-protein binding, while the N-terminal domain, homologous to other ADP-ribosylating toxins, contains the NAD(+)-binding site and the residues involved in catalysis. By site-directed mutagenesis and kinetic analyses Glu-129 and His-35 were identified as the catalytic residues. Glutamates analogous to Glu-129 are found in all studied ADP-ribosylating toxins, while His-35 is less well conserved. This suggests that Glu-129 acts on the common substrate NAD+, whereas His-35 plays its role on the acceptor substrates. We propose a mechanism in which Glu-129 exerts its action on the 2'-OH group of the NAD+ ribose, thereby facilitating the formation of an oxocarbonium-like intermediate and the weakening of the N-glycosidic bond. His-35 could increase the nucleophilicity of the cysteine in the G protein or the water molecule to attack the weakened N-glycosidic bond of NAD+ and yield the products of the reaction.
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PMID:A proposed mechanism of ADP-ribosylation catalyzed by the pertussis toxin S1 subunit. 852 86

The catalytic, or third domain of Pseudomonas exotoxin A (PEIII) catalyzes the transfer of ADP ribose from nicotinamide adenine dinucleotide (NAD) to elongation factor-2 in eukaryotic cells, inhibiting protein synthesis. We have determined the structure of PEIII crystallized in the presence of NAD to define the site of binding and mechanism of activation. However, NAD undergoes a slow hydrolysis and the crystal structure revealed only the hydrolysis products, AMP and nicotinamide, bound to the enzyme. To better define the site of NAD binding, we have now crystallized PEIII in the presence of a less hydrolyzable NAD analog, beta-methylene-thiazole-4-carboxamide adenine dinucleotide (beta-TAD), and refined the complex structure at 2.3 angstroms resolution. There are two independent molecules of PEIII in the crystal, and the conformations of beta-TAD show some differences in the two binding sites. The beta-TAD attached to molecule 2 appears to have been hydrolyzed between the pyrophosphate and the nicotinamide ribose. However molecule 1 binds to an intact beta-TAD and has no crystal packing contacts in the vicinity of the binding site, so that the observed conformation and interaction with the PEIII most likely resembles that of NAD bound to PEIII in solution. We have compared this complex with the catalytic domains of diphtheria toxin, heat labile enterotoxin, and pertussis toxin, all three of which it closely resembles.
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PMID:Crystal structure of the catalytic domain of Pseudomonas exotoxin A complexed with a nicotinamide adenine dinucleotide analog: implications for the activation process and for ADP ribosylation. 869 16

The signaling pathway by which GnRH acts in peripheral tumors is distinct from that in the anterior pituitary. We attempted to identify the guanosine triphosphate (GTP)-binding protein (G protein) subtypes linked to GnRH receptor in the genital tract tumor membranes. Surgically removed ovarian carcinomas and uterine leiomyosarcomas were screened for GnRH receptor expression before plasma membrane isolation. The G alpha i was detected by immunoblotting of membrane extracts with specific antibody and pertussis toxincatalyzed ADP-ribosylation from nicotinamide adenine dinucleotide. Membrane phosphotyrosine phosphatase activity was determined as a GnRH-sensitive membrane event using synthetic substrate p-nitrophenyl in a spectrophotometric assay. Pertussis toxin, but not cholera toxin, brought about ADP-ribosylation of an immunodetected G alpha i of 41 kDa in the GnRH receptor-positive tumor membrane. Incubation with a GnRH analog and GTP decreased the ADP-ribosylation activity in a dose-dependent manner; a half-maximal effect occurred with 30 nmol/L buserelin (P < 0.01). The apparent inhibition by GnRH of the ADP-ribosylation demonstrated that GnRH resolved the alpha-subunit of the Gi to GTP-bound form in the membranes. The action of GnRH was neutralized by a competitive antagonist, antide. Pretreatment of the membrane with the pertussis toxin completely inhibited GnRH-sensitive phosphotyrosine phosphatase activity (P < 0.01). These data demonstrate the coupling of GnRH receptor to Gi protein subfamily. The Gi which couples GnRH receptor to the effector may define the difference of responses by peripheral tumor and the anterior pituitary.
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PMID:Coupling of gonadotropin-releasing hormone receptor to Gi protein in human reproductive tract tumors. 878 77

Pertussis toxin from Bordatella pertussis catalyzes the ADP ribosylation of several G-proteins, using NAD+ as a substrate. In the absence of an acceptor protein, the toxin acts as a NAD+ glycohydrolase. Pertussis toxin is one of the virulent factors for whooping cough and therefore a target for site-specific inhibitors based on the transition state structure. A family of kinetic isotope effects was determined for the hydrolysis reaction, using NAD+ labeled with 3H, 14C, and 15N as substrates. Primary isotope effects were 1.021 +/- 0.001 for [1'N-14C]NAD+ and 1.021 +/- 0.004 for [1N-15N]NAD+, and the double-primary effect of [1'N-14C,1N-15N]NAD+ was 1.049 +/- 0.004. Secondary kinetic isotope effects were 1.207 +/- 0.010 for the [1'N-3H]-, 1.144 +/- 0.005 for the [2'N-3H]-, 0.989 +/- 0.001 for the [4'N-3H]-, and 1.019 +/- 0.004 for the [5'N-3H]NAD+, respectively. Commitment to catalysis was excluded by isotope trapping experiments, and the experimental kinetic isotope effects were independent of pH. The measured isotope effects are therefore intrinsic. The isotope effects are remarkable because they indicate an oxocarbenium-like ribose ring at the transition state but a stiffer than expected vibrational environment for C1' at the reaction center. On the basis of these isotope effects, a bond order vibrational analysis was performed to locate a transition state structure consistent with the isotope effects. The kinetic isotope effects predict a residual bond order to the nicotinamide leaving group of 0.11, corresponding to a distance of 2.14 A. Participation of the water nucleophile is weak, consistent either with an S(N)1-like transition state with no water interaction or with the water oxygen no closer than 3.5 A from the reaction center. The positive charge of the ribose oxocarbenium is stabilized by delocalization between the C1'-O4' and C1'-C2' bonds. The enzyme contacts restrict the vibrational environment of the reaction coordinate requiring increased bonding force constants for the enzyme-stabilized transition state. NAD+ analogues with the nicotinamide ribose replaced by an iminoribitol ring, mimicking the flattened ribose ring of the transition state, are expected to be transition state inhibitors.
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PMID:Kinetic isotope effect characterization of the transition state for oxidized nicotinamide adenine dinucleotide hydrolysis by pertussis toxin. 910 61

The low resolution structure of the Pseudomonas aeroginosa exotoxin A (ETA) presented in 1986 provided the first tantalizing three-dimensional view of an ADP-ribosyl-transferase (ADPRT) catalytic domain. The major features of this protein fold have recurred in the more recently solved crystal structures of the cholera toxin-related heat-labile enterotoxin (LT), diphtheria toxin (DT) and pertussis toxin (PT). A core set of alpha + beta elements define a minimal, conserved scaffold with remarkably plastic sequence requirements-only a single glutamic acid residue critical to catalytic activity is invariant. Other interchangeable residues in locations important for catalysis and binding are suggested by the cocrystal structures of DT with the inhibitor ApUp, ETA with bound AMP and nicotinamide, and DT with substrate NAD-in close accord with labeling and mutagenic data. Faint sequence resemblances that were earlier noticed among prokaryotic ADPRTs have now been securely extended by the structural concordance between toxin folds; more recently, eukaryotic ADPRTs have surfaced and their sequences can be reliably threaded into the conserved core fold. We will briefly summarize efforts in Palo Alto and Hamburg to explore these latter relationships, and to mount a rigorous search for new ADPRT families in the growing sequence databases.
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PMID:Sequence and structural links between distant ADP-ribosyltransferase families. 919 42

Pertussis toxin from Bordetella pertussis is one of the ADP-ribosylating toxins which are the cytotoxic agents of several infectious diseases. Transition state analogues of these enzymes are expected to be potent inhibitors and may be useful in therapy. Pertussis toxin catalyzes the ADP-ribosylation of a cysteine in the synthetic peptide alphai3C20, corresponding to the C-terminal 20 amino acids of the alpha-subunits of the G-protein Gi3. A family of kinetic isotope effects was determined for the ADP-ribosylation reaction, using 3H-, 14C- and 15N-labeled NAD+ as substrates. Primary kinetic isotope effects were 1.050 +/- 0.006 for [1'N-14C] and 1.021 +/- 0.002 for [1N-15N], the double primary effect of [1'N-14C,1N-15N] was 1.064 +/- 0.002. Secondary kinetic isotope effects were 1.208 +/- 0.014 for [1'N-3H], 1.104 +/- 0.010 for [2'N-3H], 0.989 +/- 0.001 for [4'N-3H], and 1.014 +/- 0.002 for [5'N-3H]. Isotope trapping experiments yielded a commitment factor of 0.01, demonstrating that the observed isotope effects are near intrinsic. Solvent D2O kinetic isotope effects are inverse, consistent with deprotonation of the attacking Cys prior to transition state formation. The transition state structure was determined by a normal mode bond vibrational analysis. The transition state is characterized by a nicotinamide leaving group bond order of 0.14, corresponding to a bond length of 2.06 A. The incoming thiolate nucleophile has a bond order of 0.11, corresponding to 2.47 A. The ribose ring has strong oxocarbenium ion character. Pertussis toxin also catalyzes the slow hydrolysis of NAD+ in the absence of peptides. Comparison of the transition states for NAD+ hydrolysis and for ADP-ribosylation of peptide alphai3C20 indicates that the sulfur nucleophile from the peptide Cys participates more actively as a nucleophile in the reaction than does water in the hydrolytic reaction. Participation of the thiolate anion at the transition state provides partial neutralization of the cationic charge which normally develops at the transition states of N-ribohydrolases and transferases. Thus, the presence of the peptide provides increased SN2 character in a loose transition state which retains oxocarbenium character in the ribose.
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PMID:Pertussis toxin: transition state analysis for ADP-ribosylation of G-protein peptide alphai3C20. 920 66

Pertussis toxin ADP-ribosylates a specific Cys side chain in the alpha-subunit of several G-proteins. Recombinant Gialpha1-subunits were rapidly ADP-ribosylated in the absence of betagamma-subunits, with a Km of 800 microM and a kcat of 40 min-1. Addition of betagamma-subunits decreases Km to 0.3 microM with little change of kcat. Kinetic isotope effects established the transition-state structure for ADP-ribosylation of Gialpha1 subunits. The transition state is dissociative, with a 2.1 A bond to the nicotinamide leaving group and a bond of 2.5 A to the sulfur nucleophile. The nucleophilic participation of Gialpha1 at the transition state is greater than that for water in the hydrolysis of NAD+by pertussis toxin. Crystal structures for Gialpha1 show the Cys nucleophile in a disordered segment or inaccessible for attack on NAD+. Therefore, transition-state formation requires an altered Gialpha1 conformation to expose and ionize Cys. The transition state has been docked into the crystal structure of pertussis toxin in a geometry required for transition state formation.
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PMID:Transition-state structure for the ADP-ribosylation of recombinant Gialpha1 subunits by pertussis toxin. 948 25

We describe a novel application of a fragment-based ligand docking technique; similar methods are commonly applied to the de novo design of ligands for target protein binding sites. We have used several new flexible docking and superposition tools, as well as a more conventional rigid-body (fragment) docking method, to examine NAD binding to the catalytic subunits of diphtheria (DT) and pertussis (PT) toxins, and to propose a model of the NAD-PT complex. Docking simulations with the rigid NAD fragments adenine and nicotinamide revealed that the low-energy dockings clustered in three distinct sites on the two proteins. Two of the sites were common to both fragments and were related to the structure of NAD bound to DT in an obvious way; however, the adenine subsite of PT was shifted relative to that of DT. We chose adenine/nicotinamide pairs of PT dockings from these clusters and flexibly superimposed NAD onto these pairs. A Monte Carlo-based flexible docking procedure and energy minimization were used to refine the modeled NAD-PT complexes. The modeled complex accounts for the sequence and structural similarities between PT and DT and is consistent with many results that suggest the catalytic importance of certain residues. A possible functional role for the structural difference between the two complexes is discussed.
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PMID:Fragment-based modeling of NAD binding to the catalytic subunits of diphtheria and pertussis toxins. 959 99


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