RESUMO
The compound U0126 (1,4-diamino-2,3-dicyano-1, 4-bis[2-aminophenylthio]butadiene) was identified as an inhibitor of AP-1 transactivation in a cell-based reporter assay. U0126 was also shown to inhibit endogenous promoters containing AP-1 response elements but did not affect genes lacking an AP-1 response element in their promoters. These effects of U0126 result from direct inhibition of the mitogen-activated protein kinase kinase family members, MEK-1 and MEK-2. Inhibition is selective for MEK-1 and -2, as U0126 shows little, if any, effect on the kinase activities of protein kinase C, Abl, Raf, MEKK, ERK, JNK, MKK-3, MKK-4/SEK, MKK-6, Cdk2, or Cdk4. Comparative kinetic analysis of U0126 and the MEK inhibitor PD098059 (Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci U. S. A. 92, 7686-7689) demonstrates that U0126 and PD098059 are noncompetitive inhibitors with respect to both MEK substrates, ATP and ERK. We further demonstrate that the two compounds bind to deltaN3-S218E/S222D MEK in a mutually exclusive fashion, suggesting that they may share a common or overlapping binding site(s). Quantitative evaluation of the steady state kinetics of MEK inhibition by these compounds reveals that U0126 has approximately 100-fold higher affinity for deltaN3-S218E/S222D MEK than does PD098059. We further tested the effects of these compounds on the activity of wild type MEK isolated after activation from stimulated cells. Surprisingly, we observe a significant diminution in affinity of both compounds for wild type MEK as compared with the deltaN3-S218E/S222D mutant enzyme. These results suggest that the affinity of both compounds is mediated by subtle conformational differences between the two activated MEK forms. The MEK affinity of U0126, its selectivity for MEK over other kinases, and its cellular efficacy suggest that this compound will serve as a powerful tool for in vitro and cellular investigations of mitogen-activated protein kinase-mediated signal transduction.
Assuntos
Butadienos/farmacologia , Inibidores Enzimáticos/farmacologia , Nitrilas/farmacologia , Inibidores de Proteínas Quinases , Animais , Butadienos/química , Células COS , DNA/metabolismo , Inibidores Enzimáticos/química , Flavonoides/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Cinética , MAP Quinase Quinase 1 , Quinases de Proteína Quinase Ativadas por Mitógeno , Nitrilas/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-fos/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-jun/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-raf/metabolismo , Transdução de Sinais/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Fator de Transcrição AP-1/metabolismoRESUMO
The inducible isoform of human prostaglandin G/H synthase (human cyclooxygenase; hCOX2) has been produced in Sf21 insect cells using the baculovirus expression system. The full-length gene for hCOX2 was placed under the control of the hybrid pCap/PolH promoter and recombinant virus generated by homologous recombination. Insect cells infected with recombinant virus synthesized active hCOX2 at levels exceeding 5% of total cellular protein 72 h postinfection. Optimal production on a preparative scale and high activity yields were attained in 8-liter spinner flasks using a supplemented Grace's medium containing 10% FCS. The apo-enzyme was purified to homogeneity by detergent extraction and ion exchange chromatography and functionally reconstituted with heme to form the holo-enzyme. The purified enzyme from insect cells was identified as hCOX2 by enzymatic activity, Western immunoassay, and N-terminal sequence analysis; the latter also indicated correct processing of the hCOX2 signal sequence. Insect recombinant hCOX2 displays high specific activity for both cyclooxygenase and peroxidase activities at levels indistinguishable from mammalian derived enzyme. Spectroscopic analysis suggests that the recombinant enzyme adopts native-like secondary and tertiary structure. The data presented here demonstrate that this system is capable of providing high yields of active enzyme for biochemical, biophysical, and pharmacological investigations.
Assuntos
Prostaglandina-Endoperóxido Sintases/genética , Prostaglandina-Endoperóxido Sintases/metabolismo , Animais , Ácido Araquidônico/farmacologia , Baculoviridae/genética , Western Blotting , Linhagem Celular , Dicroísmo Circular , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Expressão Gênica , Vetores Genéticos , Humanos , Cinética , Peroxidases/metabolismo , Prostaglandina-Endoperóxido Sintases/química , Prostaglandina-Endoperóxido Sintases/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Espectrofotometria , Spodoptera , Tetrametilfenilenodiamina/metabolismoRESUMO
The binding interactions for the three primary reactants of the fibroblast growth factor (FGF) system, basic FGF (bFGF), an FGF receptor, FGFR1, and the cofactor heparin/heparan sulfate (HS), were explored by isothermal titrating calorimetry, ultracentrifugation, and molecular modeling. The binding reactions were first dissected into three binary reactions: (1) FGFR1 + bFGF<==>FGFR1/bFGF, K1 = 41 (+/- 12) nM; (2) FGFR1 + HS<==>FGFR1/HS, K2 = 104 (+/- 17) microM; and (3) bFGF + HS<==>bFGF/HS, K3 = 470 (+/- 20) nM, where HS = low MW heparin, approximately 3 kDa. The first, binding of bFGF to FGFR1 in the absence of HS, was found to be a simple binary binding reaction that is enthalpy dominated and characterized by a single equilibrium constant, K1. The conditional reactions of bFGF and FGFR1 in the presence of heparin were then examined under conditions that saturate only the bFGF heparin site (1.5 equiv of HS/bFGF) or saturate the HS binding sites of both bFGF and FGFR1 (1.0 mM HS). Both 3-and 5-kDa low MW heparins increased the affinity for FGFR1 binding to bFGF by approximately 10-fold (Kd = 4.9 +/- 2.0 nM), relative to the reaction with no HS. In addition, HS, at a minimum of 1.5 equiv/bFGF, induced a second FGFR1 molecule to bind to another lower affinity secondary site on bFGF (K4 = 1.9 +/- 0.7 microM) in an entropy-dominated reaction to yield a quaternary complex containing two FGFR1, one bFGF, and at least one HS. Molecular weight estimates by analytical ultracentrifugation of such fully bound complexes were consistent with this proposed composition. To understand these binding reactions in terms of structural components of FGFR1, a three-dimensional model of FGFR1 was constructed using segment match modeling. Electrostatic potential calculations confirmed that an elongated cluster, approximately 15 x 35 A, of nine cationic residues focused positive potential (+2kBT) to the solvent-exposed beta-sheet A, B, E, C' surface of the D(II) domain model, strongly implicating this locus as the HS binding region of FGFR1. Structural models for HS binding to FGFR1, and HS binding to bFGF, were built individually and then assembled to juxtapose adjacent binding sites for receptor and HS on bFGF, against matching proposed growth factor and HS binding sites on FGFR1. The calorimetric binding results and the molecular modeling exercises suggest that bFGF and HS participate in a concerted bridge mechanism for the dimerization of FGFR1 in vitro and presumably for mitogenic signal transduction in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Heparina/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Cromatografia de Afinidade , Clonagem Molecular , DNA Complementar/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Glicosilação , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Poliéster Sulfúrico de Pentosana/metabolismo , Ligação Proteica , Conformação Proteica , Receptores de Fatores de Crescimento de Fibroblastos/química , Receptores de Fatores de Crescimento de Fibroblastos/genética , TermodinâmicaRESUMO
A refolding and purification method for economically producing large quantities of H-ras isolated from Escherichia coli inclusion bodies is described. Experiments were performed to optimize the yield of refolded H-ras for structural analysis by NMR spectroscopy. Protein concentration, temperature, and the presence of 10% glycerol during refolding were varied. The yield of H-ras was highest when the protein was refolded at concentrations less than or equal to 0.1 mg/ml and was independent of the presence of 10% glycerol. The yield was slightly higher at 4 degrees C than at 25 degrees C. The refolded H-ras was purified by anion exchange chromatography to yield protein with a purity of > 90%, as determined by C4 reverse-phase HPLC, and a GDP-binding stoichiometry greater than 0.9. NMR structural analysis of refolded and soluble H-ras was conducted using [15N]glycine- and [15N]serine-enriched protein. The NMR data indicate that the refolded ras protein is structurally similar to ras isolated from the soluble fraction.
Assuntos
Escherichia coli/química , Dobramento de Proteína , Proteínas Proto-Oncogênicas p21(ras)/química , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Glicerol/farmacologia , Glicina/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Espectroscopia de Ressonância Magnética , Proteínas Proto-Oncogênicas p21(ras)/isolamento & purificação , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Serina/química , Solubilidade , TemperaturaAssuntos
Acetolactato Sintase/isolamento & purificação , Isoenzimas/isolamento & purificação , Oxo-Ácido-Liases/isolamento & purificação , Salmonella typhimurium/isolamento & purificação , Acetolactato Sintase/análise , Acetolactato Sintase/metabolismo , Cromatografia em Gel/métodos , Cromatografia por Troca Iônica/métodos , Estabilidade Enzimática , Indicadores e Reagentes , Isoenzimas/análise , Isoenzimas/metabolismo , Cinética , Salmonella typhimurium/análise , Salmonella typhimurium/metabolismo , Espectrofotometria Ultravioleta/métodosRESUMO
A facile purification has been devised for recombinantly produced Salmonella typhimurium acetolactate synthase isozyme II. Purification of the enzyme was made possible by determining the complex set of factors that lead to loss of enzymic activity with this rather labile enzyme. When complexed with thiamin pyrophosphate, FAD, and magnesium, acetolactate synthase is subject to oxygen-dependent inactivation, a property not shared by the enzyme-FAD complex. When divorced from all of its tightly bound cofactors, losses of the enzymic activity are encountered at low ionic strength, especially at low protein concentrations. If purified and stored as the enzyme-FAD complex, acetolactate synthase is quite stable. The enzyme is composed of two types of subunits, a result that was not anticipated from previous studies of ilvG (the gene that codes for the large subunit of acetolactate synthase). These subunits were determined to be in equal molar ratio in the purified enzyme from the distribution of radioactivity between the two subunits after carboxymethylation with iodo[14C]acetate and their respective amino acid compositions. Besides the expected ilvG gene product (59.3 kDa), purified acetolactate synthase contained a smaller subunit (9.7 kDa; designated here as the ilvM gene product). On the basis of sequence homology of the small subunit with that coded for by the corresponding Escherichia coli gene sequence [Lawther, R. P., Calhoun, D. H., Adams, C. W., Hauser, C. A., Gray, J., & Hatfield, G. W. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 922-925], it is encoded by the region between ilvG and ilvE, beginning at base-pair (bp) 1914 (relative to the point of transcription initiation).(ABSTRACT TRUNCATED AT 250 WORDS)