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: EC:2.7.10.2 (focal adhesion kinase)
44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The major transcription factors controlling arginine metabolism in Escherichia coli and Bacillus subtilis, ArgR and AhrC, respectively, are homologous multimeric proteins that form l -arginine-dependent DNA-binding complexes capable of repressing transcription of the biosynthetic genes (both), activating transcription of catabolic genes (AhrC only) or facilitating plasmid dimer resolution (both). Multimerisation and l -arginine binding are associated with the C-terminal 70-80 residues; the N-terminal regions contain a winged helix-turn-helix DNA-binding domain. We have constructed chimeric genes in which the sequences for the N and C-terminal domains have been swapped. The resultant chimeric proteins and their corresponding native proteins have been analysed for their ability to multimerise and bind DNA operator sites in an L-arginine-dependent fashion. Gel filtration and equilibrium sedimentation analysis are consistent with the formation of hexamers by all four proteins in the presence of L-arginine and at high protein concentrations (>100 nM monomer). The hexamer sedimentation coefficients suggest that there is a reduction in molecular volume upon binding L-arginine, consistent with a conformational change accompanying an allosteric activation of DNA-binding. In the absence of L-arginine or at lower protein concentrations, the hexamers are clearly in rapid equilibrium with smaller subunits, whose dominant species appear to be based on trimers, as expected from the crystal structure of the ArgR C-terminal fragment, with the exception of the ArgR-C chimera, which apparently dissociates into dimers, suggesting that in the intact protein the DNA-binding domains may have a significant dimeric interaction. The hexamer-trimer Kdis in the micromolar range, suggesting that trimers are the principal species at in vivo concentrations.DNA binding by all four proteins has been probed by gel retardation and DNase I footprinting analysis using all three types of naturally occurring operators: biosynthetic sites encompassing two 18 bp ARG boxes separated by 2 bp; biosynthetic sites containing two such boxes and a third 18 bp ARG box at a distance of 100 bp downstream, i.e. within the structural gene; and finally a catabolic operator which contains a single ARG box site. The data show that all four proteins bind to the operators at the expected regions in an L-arginine-dependent fashion. From the apparent affinities of the chimeras for each target site, there is no obvious sequence-specificity associated with the N-terminal domains; rather the data can be interpreted in terms of differential allosteric activation, including DNA binding in the absence of L-arginine.Remarkably, the proteins show apparent "anti-competition" in the presence of excess, specific DNA fragments in gel retardation. This appears to be due to assembly of an activated form of the protein, probably hexamers, on the operator DNA. The data are discussed in terms of the current models for the mode of action of both native proteins.
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PMID:Probing activation of the prokaryotic arginine transcriptional regulator using chimeric proteins. 1036 57

Chronic myelogenous leukemia (CML) is associated with the presence of the Philadelphia chromosome, which is generated by the reciprocal translocation of chromosomes 9 and 22. In the case of L6 (b2a2) mRNA, it is difficult to cleave the abnormal mRNA specifically because the mRNA includes no sequences that can be cleaved efficiently by conventional hammerhead ribozymes near the BCR-ABL junction. We recently succeeded in designing a novel maxizyme, which specifically cleaves BCR-ABL fusion mRNA, as a result of the formation of a dimeric structure. As an extension of our molecular engineering of maxizymes, as well as to improve their potential utility, we examined whether an analogous conformational change could be induced within a single molecule when two maxizymes were connected via a linker sequence. An active conformation was achieved by binding of the construct to the BCR-ABL junction in trans, with part of the linker sequence then acting as an antisense modulator in cis (within the complex) to adjust the overall structure. Results of studies in vitro in the presence of cetyltrimethylammonium bromide (CTAB) (but not in its absence) suggested that a certain kind of connected maxizyme (cMzB) might be able to undergo a desired conformational change and, indeed, studies in vivo confirmed this prediction. Therefore, we successfully created a fully functional, connected maxizyme and, moreover, we found that the activity and specificity of catalytic RNAs in vivo might be better estimated if their reactions are monitored in vitro in the presence of CTAB.
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PMID:Specificity of novel allosterically trans- and cis-activated connected maxizymes that are designed to suppress BCR-ABL expression. 1056

Sphingomonas paucimobilis SYK-6 can grow on several dimeric model compounds of lignin as a carbon and energy source. It has O demethylation systems on three kinds of substrates: 5, 5'-dehydrodivanillic acid (DDVA), syringate, and vanillate. We previously reported the cloning of a gene involved in the tetrahydrofolate-dependent O demethylation of syringate and vanillate. In the study reported here, we cloned the gene responsible for DDVA O demethylation. Using nitrosoguanidine mutagenesis, a mutant strain, NT-1, which could not degrade DDVA but could degrade syringate and vanillate, was isolated and was used to clone the gene responsible for the O demethylation of DDVA by complementation. Sequencing analysis showed an open reading frame (designated ligX) of 1,266 bp in this fragment. The deduced amino acid sequence of LigX had similarity to class I type oxygenases. LigX was involved in O demethylation activity on DDVA but not on vanillate and syringate. DDVA O demethylation activity in S. paucimobilis SYK-6 cell extracts was inhibited by addition of the LigX polyclonal antiserum. Thus, LigX is an essential enzyme for DDVA O demethylation in SYK-6. S. paucimobilis SYK-6 has two O demethylation systems: one is an oxygenative demethylase system, and the other is a tetrahydrofolate-dependent methyltransferase system.
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PMID:Coexistence of two different O demethylation systems in lignin metabolism by Sphingomonas paucimobilis SYK-6: cloning and sequencing of the lignin biphenyl-specific O-demethylase (LigX) gene. 1078 91

Ingression of primary mesenchyme cells (PMC) is associated with the encounter of basal lamina including pamlin. It was found that sea urchin embryos have a protein that binds antihuman focal adhesion kinase (FAK) antibodies, yet it has a 62 kDa homo-dimeric structure. Thus, this protein was distinctive from known FAK, and was named SUp62. In mesenchyme blastulae, one of the subunits increased its apparent molecular mass slightly but distinctively, then restored the original molecular mass in early gastrulae. This temporal and stage-specific shifting of the molecular mass was associated with the occurrence of tyrosine phosphorylation of a subunit that did not increase the apparent molecular mass. Herbimycin A induced the hyperphosphorylation of tyrosine residues of SUp62, and inhibited the occurrence of molecular mass shifting. Immunohistochemistry showed a strong positive signal of SUp62 and phosphotyrosine in PMC. Herbimycin A also severely but reversibly inhibited PMC dissociation, migration and gastrulation. Tyrosine phosphorylation of SUp62 was induced when PMC were incubated with pamlin in vitro, and it was initiated within 10 min after onset of the incubation. It reached its peak in 1 h, and declined gradually in the next 1 h, indicating that pamlin-induced tyrosine phosphorylation of SUp62 occurs closely associated with acquiring PMC migration activity.
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PMID:Pamlin-induced tyrosine phosphorylation of SUp62 protein in primary mesenchyme cells during early embryogenesis in the sea urchin, Hemicentrotus pulcherrimus. 1104 93

Sphingomonas paucimobilis SYK-6 is able to grow on various dimeric lignin compounds, which are converted to vanillate and syringate by the actions of unique lignin degradation enzymes in this strain. Vanillate and syringate are degraded by the O-demethylase and converted into protocatechuate (PCA) and 3-O-methylgallate (3MGA), respectively. PCA is further degraded via the PCA 4,5-cleavage pathway, while the results suggested that 3MGA is degraded through another pathway in which PCA 4,5-dioxygenase is not involved. In a 10.5-kb EcoRI fragment carrying the genes for PCA 4,5-dioxygenase (ligAB), 2-pyrone-4,6-dicarboxylate hydrolase (ligI), and a portion of 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase (ligC), we found the ligJ gene encoding 4-oxalomesaconate (OMA) hydratase, which catalyzes the conversion of OMA into 4-carboxy-4-hydroxy-2-oxoadipate. The ligJ gene is transcribed in the same direction as ligABC genes and consists of an 1,023-bp open reading frame encoding a polypeptide with a molecular mass of 38,008 Da, which is located 73-bp upstream from ligA. The ligJ gene product (LigJ), expressed in Escherichia coli, was purified to near homogeneity and was estimated to be a homodimer (69.5 kDa) by gel filtration chromatography. The isoelectric point was determined to be 4.9, and the optimal temperature is 30 degrees C. The K(m) for OMA and the V(max) were determined to be 138 microM and 440 U/mg, respectively. LigJ activity was inhibited by the addition of thiol reagents, suggesting that some cysteine residue is part of the catalytic site. The ligJ gene disruption in SYK-6 caused the growth defect on and the accumulation of common metabolites from both vanillate and syringate, indicating that the ligJ gene is essential to the degradation of these two compounds. These results indicated that syringate is converted into OMA via 3MGA, and it enters the PCA 4,5-cleavage pathway.
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PMID:The 4-oxalomesaconate hydratase gene, involved in the protocatechuate 4,5-cleavage pathway, is essential to vanillate and syringate degradation in Sphingomonas paucimobilis SYK-6. 1109 55

Sphingomonas paucimobilis SYK-6 is able to grow on a wide variety of dimeric lignin compounds. These compounds are degraded via vanillate and syringate by a unique enzymatic system, composed of etherases, O demethylases, ring cleavage oxygenases and side chain cleaving enzymes. These unique and specific lignin modification enzymes are thought to be powerful tools for utilization of the most abundant aromatic biomass, lignin. Here, we focus on the genes and enzymes involved in beta-aryl ether cleavage and biphenyl degradation. Two unique etherases are involved in the reductive cleavage of beta-aryl ether. These two etherases have amino acid sequence similarity with the glutathione S-transferases, and use glutathione as a hydrogen donor. It was found that 5,5'-dehydrodivanillate, which is a typical lignin-related biphenyl structure, was transformed into 5-carboxyvanillate by the reaction sequence of O-demethylation, meta-ring cleavage, and hydrolysis, and the genes involved in the latter two reactions have been characterized. Vanillate and syringate are the most common intermediate metabolites in lignin catabolism. These compounds are initially O-demethylated and the resulting diol compounds, protocatechuate (PCA) and 3-O-methylgallate, respectively, are subjected to ring cleavage catalyzed by PCA 4,5-dioxygenase. The ring cleavage products generated are further degraded through the PCA 4,5-cleavage pathway. We have isolated and characterized genes for enzymes involved in this pathway. Disruption of a gene for 2-pyrone-4,6-dicarboxylate hydrolase (ligI) in this pathway suggested that an alternative route for 3-O-methylgallate degradation, in which ligI is not involved, would play a role in syringate catabolism. In this article, we describe the genetic and biochemical features of the S. paucimobilis SYK-6 genes involved in degradation of lignin-related compounds. A possible application of the SYK-6 lignin degradation system to produce a valuable chemical material is also described.
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PMID:Characterization of Sphingomonas paucimobilis SYK-6 genes involved in degradation of lignin-related compounds. 1142 57

The granulocyte-derived hemoregulatory peptide pyroGlu-Glu-Asp-Cys-Lys = pEEDCK is known to keep hematopoietic cells quiescent. When oxidized to its dimeric form (pEEDCK)2, it activates growth of hematopoietic progenitors in association with stroma-derived cytokines. (pEEDCK)2 has a Cys-Cys motif which is also a typical feature of the macrophage inflammatory protein (MIP-1alpha). The present study was designed to analyze differences between the response of normal and leukemic progenitor cells to (pEEDCK)2 or MIP-1alpha. When long-term bone marrow cultures (LTBMCs) were incubated with (pEEDCK)2 or MIP-1alpha and/or cytokines, the stimulatory effect on colony-forming units-granulocyte/erythroid/macrophage/megakaryocyte of LTBMC from chronic myeloid leukemia (CML) patients was less than 50% compared to LTBMC from healthy humans. No difference in oncogene expression could be observed in LTBMC from CML patients regarding reduction of Philadelphia chromosome-associated transcription of the BCR-ABL gene. With respect to the expression of growth and differentiation-associated genes (Galpha16, 5-lipoxygenase, phospholipaseA2, c-kit, and CD34), which were analyzed from LTBMC by semiquantitative reverse transcriptase-polymerase chain reaction, the same transcription rate was observed in CML patients and healthy donors. However, two isoforms of a key enzyme of oxidative metabolism, carnitine palmitoyltransferase (CPT1A and CPT1B), showed 50-fold higher expression rates in LTBMC cells of healthy donors compared to CML patients. It is known that a decrease in oxidative metabolism is associated with an increase in redox equivalents in malignancy. This might result in a reduction of disulphide bonds in (pEEDCK)2 or MIP-1alpha, thus inducing a downregulation of these factors in bone marrow from CML patients.
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PMID:Effect of the hemoregulatory peptide (pEEDCK)2 (pyroGlu-Glu-Asp-Cys-Lys)2 and MIP-1alpha is reduced in bone marrow cultures from patients with chronic myeloid leukemia (CML). 1146 52

For the treatment of chronic myelogenous leukemia (CML), attempts have been made to design various ribozyme motifs that can specifically recognize and cleave BCR-ABL fusion mRNAs. In the case of L6 BCR-ABL b2a2 mRNA, it is difficult to cleave the abnormal mRNA specifically because the mRNA includes no sequences that can be cleaved efficiently by conventional hammerhead ribozymes near the BCR-ABL junction. We recently succeeded in designing a novel maxizyme, which specifically cleaves BCR-ABL fusion mRNA, as a result of the formation of a dimeric structure [Kuwabara, T.; et al. Mol. Cell 1998, 2, 617-627; Tanabe, T.; et al. Nature 2000, 406, 473-474]. Specifically, we tailored the maxizyme with molecular switching function: the maxizyme splices a cleavable GUC site, but only when it appears within a strand of mRNA that possesses the abnormal splice junction. We demonstrated that this approach is generalizable [Tanabe, T.; et al. Biomacromolecules 2000, 1, 108-117]. All the maxizymes designed in the past functioned as a result of the formation of a dimeric structure. Questions have been asked whether a similar molecular switching might be possible within a single molecule when two monomer units of the maxizyme were connected via a linker sequence. We found that an analogous conformational change could not be induced within a single molecule when two maxizyme units were simply connected via a nonregulatable linker sequence. However, an active conformation was achieved by the introduction of an antisense modulator within the linker sequence that adjusted the overall structure to the correct form. Results of studies in cultured cells suggested that the desired conformational change could indeed be induced within the modified single-chained maxizyme and such a construct caused apoptosis only in leukemic cells with the Philadelphia chromosome.
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PMID:Allosterically controlled single-chained maxizymes with extremely high and specific activity. 1171 33

Galectin-1, a beta-galactoside-binding dimeric lectin, is involved in adhesion, migration, and proliferation of vascular smooth muscle cells (SMC), the key steps in the development of atherosclerosis and restenosis. Here we investigated the molecular basis of the interactions between galectin-1 and SMCs. Galectin-1 modulated SMC attachment in a dose- and beta-galactoside-dependent manner. Direct binding of galectin-1 to beta1 integrin was detected by the immune precipitation of beta1 integrin after chemical cross-linking of 125I-labelled galectin-1 to the cell surface proteins. Galectin-1 transiently increased availability of beta1 integrins on the cell surface to antibodies against beta1 integrin. Incubation of SMCs with galectin-1 transiently increased the amount of the active form of beta1 integrin and tyrosine phosphorylation of two cytoskeleton-associated proteins; one of them coincided with focal adhesion kinase (FAK). Galectin-1 is likely to affect SMC adhesion by interacting with beta1 integrin on the cell surface of SMCs and inducing outside-in signalling.
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PMID:Galectin-1 interacts with beta-1 subunit of integrin. 1455 Mar 5

Binding of erythropoietin to the erythropoietin receptor (EpoR) extracellular domain orients the transmembrane (TM) and cytosolic regions of the receptor dimer into an unknown activated conformation. By replacing the EpoR extracellular domain with a dimeric coiled coil, we engineered TM EpoR fusion proteins where the helical TM domains were constrained into seven possible relative orientations. We identify one dimeric TM conformation that imparts full activity to the cytosolic domain of the receptor and signals via JAK2, STAT proteins, and MAP kinase, one partially active orientation that preferentially activates MAP kinase, and one conformation corresponding to the inactive receptor. The active and inactive conformations were independently identified by computational searches for low-energy TM dimeric structures. We propose a specific EpoR-activated interface and suggest its use for structural and signaling studies.
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PMID:Active and inactive orientations of the transmembrane and cytosolic domains of the erythropoietin receptor dimer. 1463 81


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