Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Escherichia coli Rsd protein was previously identified on the basis of its binding to the RNA polymerase sigma(70) subunit. The Rsd-sigma(70) complex has been studied using different methods. Our data show that Rsd associates with sigma(70) to form a complex with a stoichiometry of 1:1. Alanine scanning and deletion mutagenesis were used to locate regions of sigma(70) that are required for the formation of the Rsd-sigma(70) complex.
J Mol Biol 2004 Jan 16
PMID:Studies of the Escherichia coli Rsd-sigma70 complex. 1468 66

Homologous desensitization of the micro opioid receptor (muOR) can be resolved into distinct processes that include the uncoupling of the muOR from its G-protein effectors and internalization of cell surface receptors. Using electrophysiological recordings of muOR activation of G-protein-coupled K+ channels (Kir3) in Xenopus laevis oocytes and AtT20 cells, confocal microscopy of receptor localization, and radioligand binding of cell surface receptors, we resolved these desensitization mechanisms to determine the domain of muOR important for receptor uncoupling. Activation of muOR by saturating concentrations of [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO), methadone, or fentanyl, but not morphine, produced robust internalization of a green fluorescent protein-tagged muOR. A subsaturating concentration of DAMGO (100 nM) did not cause receptor internalization but markedly reduced the subsequent responsiveness of Kir3 by uncoupling muOR. muOR desensitization in AtT20 cells was confirmed to be homologous, because desensitization by 100 nM DAMGO was blocked by dominant-negative forms of either G protein-coupled receptor kinase (GRK) or arrestin, and pretreatment with DAMGO did not affect the Kir3 response to somatostatin receptor activation. Alanine substitution of a single threonine in the second cytoplasmic loop of the muOR (Threonine 180) blocked agonist-dependent receptor uncoupling without affecting receptor internalization. These results suggest that GRK-dependent phosphorylation of muOR required threonine 180 for uncoupling but that a different GRK and arrestin-dependent mechanism controlled muOR internalization in AtT20 cells.
Mol Pharmacol 2004 Mar
PMID:Distinct domains of the mu-opioid receptor control uncoupling and internalization. 1497 26

Neuronal nicotinic acetylcholine receptors (nAChRs) both mediate direct cholinergic synaptic transmission and modulate synaptic transmission by other neurotransmitters. Novel ligands are needed as probes to discriminate among structurally related nAChR subtypes. Alpha-conotoxin MII, a selective ligand that discriminates among a variety of nAChR subtypes, fails to discriminate well between some subtypes containing the closely related alpha3 and alpha6 subunits. Structure-function analysis of alpha-conotoxin MII was performed in an attempt to generate analogs with preference for alpha6-containing [alpha6(*) (asterisks indicate the possible presence of additional subunits)] nAChRs. Alanine substitution resulted in several analogs with decreased activity at alpha3(*) versus alpha6(*) nAChRs heterologously expressed in Xenopus laevis oocytes. From the initial analogs, a series of mutations with two alanine substitutions was synthesized. Substitution at His9 and Leu15 (MII[H9A;L15A]) resulted in a 29-fold lower IC(50) at alpha6beta4 versus alpha3beta4 nAChRs. The peptide had a 590-fold lower IC(50) for alpha6/alpha3beta2 versus alpha3beta2 and a 2020-fold lower IC(50) for alpha6/alpha3beta2beta3 versus alpha3beta2 nAChRs. MII[H9A;L15A] had little or no activity at alpha2beta2, alpha2beta4, alpha3beta4, alpha4beta2, alpha4beta4, and alpha7 nAChRs. Functional block by MII[H9A;L15A] of rat alpha6/alpha3beta2beta3 nAChRs (IC(50) = 2.4 nM) correlated well with the inhibition constant of MII[H9A;L15A] for [(125)I]alpha-conotoxin MII binding to putative alpha6beta2(*) nAChRs in mouse brain homogenates (K(i) = 3.3 nM). Thus, structure-function analysis of alpha-conotoxin MII enabled the creation of novel selective antagonists for discriminating among nAChRs containing alpha3 and alpha6 subunits.
Mol Pharmacol 2004 Apr
PMID:Analogs of alpha-conotoxin MII are selective for alpha6-containing nicotinic acetylcholine receptors. 1504 24

Block of human ether-a-go-go related gene (HERG) K(+) channels by a variety of medications has been linked to acquired long QT syndrome, a disorder of cardiac repolarization that predisposes to lethal arrhythmias. The drug-binding site is composed of residues that face into the central cavity of the channel. Two aromatic residues located on the S6 domain (Tyr652 and Phe656) are particularly important structural determinants of drug block. The role of pore helix residues (Thr623, Ser624, Val625) is less clear. In this study, we compared the pharmacological properties of two structurally related compounds, ibutilide and clofilium. Both compounds are charged amines with a single phenyl ring. Clofilium, a chlorobenzene derivative, is a potent blocker of HERG channels, but has a remarkably slower time course for recovery from block than ibutilide, a methanesulfonanilide. The difference in the rate of recovery from block can be explained simply by variation in drug trapping. There is little recovery from clofilium block with D540K HERG channels that permit untrapping at hyperpolarized potentials. Alanine-scanning mutagenesis of the S6 domain and a portion of the pore helix revealed that the binding site residues were the same for both compounds. However, S624A, located at the base of the pore helix, was the only HERG mutation that enabled rapid recovery from clofilium block. In summary, the pore helix residues are important components of the HERG drug binding site, and may be particularly important for drugs with polar substituents, such as a halogen (e.g., clofilium) or a methanesulfonamide (e.g., ibutilide).
Mol Pharmacol 2004 Aug
PMID:Structural determinants of HERG channel block by clofilium and ibutilide. 1526 14

The minimal structural motif, BBXXB (where B represents a basic amino acid residue and X a non-basic residue), located in particular regions of the intracellular domains of cell surface membrane receptors is involved in the G protein-activating activity of a number of G protein-coupled receptors. The human FSH receptor (hFSHR) exhibits a reversed BBXXB motif (BXXBB) in the juxtamembrane region of the third intracellular loop (IL3) and the carboxyl terminus (Ctail) of the receptor; however the importance of this sequence on receptor function remains unclear. In the present study, we analyzed the effects of mutations in this structural motif on hFSHR expression, receptor-mediated effector activation and agonist-provoked receptor internalization. Human embryonic kidney 293 cells were transiently transfected with plasmids containing the cDNA of the wild-type (Wt) hFSHR or several hFSHR mutants in which basic amino acids of the minimal structural motif at the IL3 and Ctail were replaced with alanine (i.e. AXXAA, AXXBB, BXXAB and BXXBA mutants). Alanine substitution of the three basic residues present in the IL3-BXXBB (IL3-AXXAA mutant) yielded a < or =60 kDa possibly under-glycosylated form of the FSHR, whereas the same substitutions in the Ctail resulted in the immature >62 kDa form of the receptor; both AXXAA hFSHR mutants completely failed to bind agonist and activate effector. Individual substitutions resulted in different cAMP responses to agonist stimulation: the IL3-AXXBB and IL3-BXXBA mutant hFSHRs failed to evoke Gs protein activation, whereas agonist-stimulated cAMP production was completely normal when the IL3-BXXAB mutant was expressed. All three IL3 mutants bound [125I]-labelled FSH in a similar fashion to the Wt hFSHR. Ligand-binding, cell surface membrane receptor expression and agonist-provoked effector activation were significantly affected by the individual substitutions at the Ctail-BXXBB motif: the Ctail-AXXBB variant exhibited reduced (approximately 50%) maximal cAMP response and ability to bind ligand, whereas both ligand binding and effector activation was severely reduced or abolished by expression of the Ctail-BXXBA and -BXXAB hFSHR mutants; the expression levels of the 80 kDa form of the receptor correlated with the magnitude of ligand-provoked cAMP production and binding capability of the mutant receptors. Upon stimulation by agonist, all mutants with detectable ligand-binding activity internalized following the pattern exhibited by the Wt hFSHR species. These results indicate that the BXXBB motif at the IL3 of the hFSHR is essential for coupling the activated receptor to the Gs protein, whereas the same motif in the Ctail is apparently more important for membrane expression.
Mol Cell Endocrinol 2004 Aug 31
PMID:Functional significance of the BBXXB motif reversed present in the cytoplasmic domains of the human follicle-stimulating hormone receptor. 1527 7

Sinorhizobium meliloti DctD is an activator of sigma(54)-RNA polymerase holoenzyme and member of the AAA+ superfamily of ATPases. DctD uses energy released from ATP hydrolysis to stimulate the isomerization of a closed promoter complex to an open complex. DctD binds to upstream activation sequences (UAS) and contacts the closed complex through DNA looping to activate transcription, but the UAS is not essential for activation if DctD is expressed at higher than normal levels. Introduction of specific substitutions within or near the conserved ESELFG motif in the C3 region of a truncated, constitutively active form of DctD produced several mutant forms of the protein that had increased dependence on the UAS for activation. Removing the DNA-binding domain from one UAS-dependent mutant and from one activation-deficient mutant significantly increased transcriptional activation, indicating that the DNA-binding domain interfered with the activities of these mutant proteins. A UAS-dependent mutant with a P315L substitution in the C6 region was identified from a genetic screen. Alanine scanning mutagenesis of conserved amino acid residues around Pro-315 produced two additional UAS-dependent mutants as well as several mutants that failed to activate transcription but retained ATPase activity. In contrast to the two mutant proteins with substitutions in the C3 region, removal of the DNA-binding domain from the mutant proteins with substitutions in the C6 region did not stimulate their activity. The residues in the C6 region that were altered are in a probable hinge region between the alpha/beta and alpha-helical subdomains of the AAA+ domain. The alpha-helical subdomain contains the sensor II helix that has been implicated in other AAA+ proteins as sensing changes in the nucleotide during the hydrolysis cycle. Substitutions in the hinge region may have abolished nucleotide sensing by interfering with subdomain interactions, altering the relative orientation of the sensor II helix or interfering with oligomerization of the protein.
Mol Microbiol 2004 Oct
PMID:Novel substitutions in the sigma54-dependent activator DctD that increase dependence on upstream activation sequences or uncouple ATP hydrolysis from transcriptional activation. 1545 3

The Hms(+) phenotype of Yersinia pestis promotes the binding of haemin or Congo red (CR) to the cell surface at temperatures below 34 degrees C. We previously demonstrated that temperature regulation of the Hms(+) phenotype is not controlled at the level of transcription. Instead, HmsH, HmsR and HmsT are degraded upon a temperature shift from 26 degrees C to 37 degrees C. We used random transposon mutagenesis to identify new genes involved in the temperature-regulated expression of the Hms phenotype. One of these genes, which we designated hmsP, encodes a putative phosphodiesterase with a conserved EAL motif. Mutations in hmsP caused formation of red colonies on CR plates at 26 degrees C and 37 degrees C. Strains complemented with hmsP(+) on a plasmid form white colonies at both temperatures. We used a crystal violet assay and confocal laser scanning microscopy to demonstrate Hms-dependent biofilm formation by Y. pestis cells. Y. pestis Hms(+) strains grown at 26 degrees C but not at 37 degrees C form a biofilm on borosilicate glass surfaces. Strains that either overexpress HmsT (a GGDEF domain protein) or have a mutation in hmsP produced an extremely thick biofilm. Alanine substitutions for each of the GGEE residues (amino acids 296-299) of HmsT as well as the E506 and L508 residues of HmsP caused a loss of function. We propose that HmsT and HmsP together control the amount of biofilm produced in Y. pestis. Degradation of HmsT at 37 degrees C may be a critical factor in controlling the temperature-dependent expression of the Hms biofilm.
Mol Microbiol 2004 Oct
PMID:HmsP, a putative phosphodiesterase, and HmsT, a putative diguanylate cyclase, control Hms-dependent biofilm formation in Yersinia pestis. 1545 6

The current model for the mechanism of action of the Bacillus thuringiensis Cry delta-endotoxins involves the penetration of the alpha4-alpha5 hairpin into the target midgut epithelial cell membranes, followed by pore formation. In this study, PCR-based mutagenesis was employed to identify a critical residue within the alpha4-alpha5 loop of the 130kDa Cry4A mosquito-larvicidal protein. Alanine-substitutions of two charged (Asp-198 and Asp-200) and four polar (Asn-190, Asn-195, Tyr-201 and Tyr-202) residues in the alpha4-alpha5 loop were performed. Like the wild-type, all of the mutant toxins were over-expressed as inclusion bodies in Escherichia coli. When E. coli cells expressing each mutant toxin were bioassayed against Aedes aegypti larvae, larvicidal activity was completely abolished for the substitution of only Tyr-202, while replacements at the other positions still retained a high level of toxicity. Further replacement of Tyr-202 with an aromatic side chain, phenylalanine, did not affect the toxicity. These results revealed a crucial role in toxin activity for the conserved aromatic residue at the 202 position within the alpha4-alpha5 loop of the Cry4A toxin.
J Biochem Mol Biol 2004 May 31
PMID:Aromaticity of Tyr-202 in the alpha4-alpha5 loop is essential for toxicity of the Bacillus thuringiensis Cry4A toxin. 1546 9

The non-catalytic region of Escherichia coli RNase E contains a protein scaffold that binds to the other components of the RNA degradosome. Alanine scanning yielded a mutation, R730A, that disrupts the interaction between RNase E and the DEAD-box RNA helicase, RhlB. We show that three other DEAD-box helicases, SrmB, RhlE and CsdA also bind to RNase E in vitro. Their binding differs from that of RhlB because it is not affected by the R730A mutation. Furthermore, the deletion of residues 791-843, which does not affect RhlB binding, disrupts the binding of SrmB, RhlE and CsdA. Therefore, RNase E has at least two RNA helicase binding sites. Reconstitution of a complex containing the protein scaffold of RNase E, PNPase and RhlE shows that RhlE can furnish an ATP-dependent activity that facilitates the degradation of structured RNA by PNPase. Thus, RhlE can replace the function of RhlB in vitro. The results in the accompanying article show that CsdA can also replace RhlB in vitro. Thus, RhlB, RhlE and CsdA are interchangeable in in vitro RNA degradation assays.
Mol Microbiol 2004 Dec
PMID:The RNase E of Escherichia coli has at least two binding sites for DEAD-box RNA helicases: functional replacement of RhlB by RhlE. 1555 79

Alanine is the most effective precursor for gluconeogenesis among amino acids and the initial reaction is catalyzed by alanine aminotransferases (AlaATs). It is a less extensively studied enzyme under starvation and known to that the enzyme activity increases in liver under starvation. The present study describes the purification and characterization of two isoforms of alanine aminotransferases from starved male rat liver under starvation. The molecular mass of isoforms was found to be 17.7 and 112.2 kDa with isoelectric points of 4.2 and 5.3 respectively for AlaAT I and AlaAT II. Both the enzymes showed narrow substrate specificity for L-alanine with different Km for alanine and 2-oxoglutarate. Both the enzymes were glycoprotein in nature. Inhibition, modification and spectroscopic studies showed that both PLP and free-SH groups are directly involved in the enzymatic catalysis. PLP activated both the enzymes with a Km 0.057 mM and 0.2 mM for AlaAT I and II respectively.
Mol Cell Biochem 2004 Dec
PMID:Isolation and characterization of cytosolic alanine aminotransferase isoforms from starved rat liver. 1566 81


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