RESUMO
OBJECTIVE: The aim of this study was to investigate the expression of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in juvenile rats with nephrotic syndrome, and to explore its effects on inflammatory changes and renal injury. MATERIALS AND METHODS: 24 Sprague-Dawley (SD) rats were randomly divided into the normal group (n=12) and model group (n=12). Rats in the normal group were intraperitoneally injected with normal saline. Meanwhile, rats in the model group were given azithromycin hydrochloride injection to establish the model of nephrotic syndrome. After 24 h of modeling, the samples were collected. The expression of NF-κB was detected via immunohistochemistry. Moreover, the protein expression of NF-κB was determined through Western blotting. Quantitative Polymerase Chain Reaction (qPCR) was used to measure the messenger ribonucleic acid (mRNA) expression levels of interleukin-1 (IL-1) and IL-6. Meanwhile, the content of IL-1 and IL-6 was detected by enzyme-linked immunosorbent assay (ELISA). The serum levels of urea nitrogen and serum creatinine were measured by an automatic biochemical analyzer. Furthermore, the correlation between NF-κB protein with IL-1 and IL-6 were studied via Pearson analysis. RESULTS: Compared with the normal group, rats in the model group exhibited significantly increased expression and protein expression of NF-κB (p<0.05). Meanwhile, the mRNA expression levels and content of IL-1 and IL-6 (p<0.05), as well as the serum levels of urea nitrogen and creatinine (p<0.05) of the model group were markedly higher than those of the normal group. Furthermore, NF-κB protein was positively correlated with IL-1 and IL-6 contents. CONCLUSIONS: NF-κB is highly expressed in juvenile rats with nephrotic syndrome, which promotes the expressions of inflammatory factors (IL-1 and IL-6) and aggravates the renal injury.
Assuntos
Rim/patologia , NF-kappa B/metabolismo , Síndrome Nefrótica/patologia , Animais , Azitromicina/toxicidade , Nitrogênio da Ureia Sanguínea , Creatinina/sangue , Feminino , Interleucina-1/análise , Interleucina-1/genética , Interleucina-1/metabolismo , Interleucina-6/análise , Interleucina-6/genética , Interleucina-6/metabolismo , Rim/metabolismo , Masculino , Síndrome Nefrótica/induzido quimicamente , Síndrome Nefrótica/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
One of the challenges in gene therapy is to ensure stable transgene expression at the site of disease with a high degree of accuracy and safety. In this paper, we examine both viral and cellular elements that may affect the level of transgene expression mediated by herpes simplex virus type 1 (HSV-1) adeno-associated virus (AAV) amplicon vectors. These elements include the AAV inverted terminal repeats (ITRs), the AAV Rep proteins, and the allelic status of 19q in human glioma cell lines. The latter is of particular interest because the AAV integration site (AAVS1) is located on the long arm of chromosome 19 and 30-40% of human glioblastoma tumors are reported to have loss of heterozygosity in this region of chromosome 19q. Fluorescence-activated cell-sorting analysis results indicate that inclusion of minimal or full-length AAV ITRs in HSV-1 amplicon vectors markedly increases the efficiency of transgene expression. On the other hand, insertion of the AAV rep gene decreases the level of transgene expression, apparently because of the cytotoxic effects of Rep proteins. Further, the levels of transgene expression appear to be independent of 19q allelic status or the number of endogenous AAVS1 sequences in the various glioma cell lines studied. Taken together, these data support employing AAV ITRs, in the context of HSV-1 amplicon vectors, to enhance short-term levels of transgene expression.
Assuntos
Dependovirus , Técnicas de Transferência de Genes , Vetores Genéticos , Glioblastoma/genética , Simplexvirus , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Genes Reporter , Glioblastoma/metabolismo , Humanos , Hibridização in Situ Fluorescente , Transgenes/fisiologia , Células Tumorais CultivadasRESUMO
Cyclooxygenase-2 (COX-2) gene overexpression is suggested to play important roles in colorectal tumorigenesis. Epidemiological studies revealed that nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin and sulindac, which inhibit COX activity, reduce colorectal cancer mortality. Current investigations have focused on delineating the molecular mechanisms that regulate COX-2 gene expression and the roles of NSAIDs in cancer chemoprevention. COX-2 catalyzes the production of prostaglandins (PGs) from arachidonic acid (AA), generated by phospholipases A2 (PLA2s), a family of acyl esterases that cause the release of AA from cellular phospholipids. Pancreatic secretory PLA2 (sPLA2), via its receptor (sPLA2R), transcriptionally activates COX-2 gene expression in several cell types, although a specific transcription factor mediating COX-2 expression has not yet been identified. Here, we report that a transcription factor, CCAAT/enhancer-binding protein beta(C/EBPbeta), plays a critical role in sPLA2IB-induced, receptor-mediated COX-2 gene expression in MC3T3E1 and NIH3T3 cells. Furthermore, treatment of these cells with NSAIDs in the presence of sPLA2IB appears to potentiate the stimulatory effects on COX-2 mRNA and COX-2 protein expression and a concomitant elevation in PG production. Most significantly, NSAID treatment appears to drastically suppress the production of cytosolic PLA2 (cPLA2) mRNA. The lack of sPLA2IB, sPLA2IIA, and sPLA2V mRNA expression in both NIH3T3 and MC3T3E1 cells suggests that cPLA2 is the most likely enzyme that catalyzes the release of AA, the rate-limiting substrate of COX for the production of PGs. Our results suggest that: (a) sPLA2IB receptor-mediated COX-2 expression is mediated via C/EBPbeta; (b) NSAIDs in the presence of sPLA2IB potentiate the stimulatory effects of sPLA2IB on COX-2 mRNA expression; and (c) despite the apparent stimulation of COX-2 expression by NSAIDs, they strikingly deprive COX-2 of its substrate, AA, by suppressing cPLA2 mRNA expression. Both AA and PGs regulate many vital biological functions (e.g., motility and invasiveness) that are dysregulated in most cancer cells, and they have profound effects on cellular differentiation. Our results raise the possibility that deprivation of COX-2 of its substrate by the suppression of cPLA2 mRNA expression is an additional mechanism used by NSAIDs to inhibit tumorigenesis.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Isoenzimas/genética , Prostaglandina-Endoperóxido Sintases/genética , Células 3T3 , Animais , Proteínas Estimuladoras de Ligação a CCAAT , Ciclo-Oxigenase 2 , Proteínas de Ligação a DNA/fisiologia , Camundongos , Proteínas Nucleares/fisiologia , Fosfolipases A/genética , Fosfolipases A/farmacologia , Fosfolipases A2 , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
To determine the physiological function(s) of uteroglobin (UG), a steroid-inducible, homodimeric, secreted protein, we have generated transgenic mice that either are completely UG-deficient due to UG gene-knockout (UG-KO) or are partially UG-deficient due to the expression of UG antisense RNA (UG-AS). Both the UG-KO and UG-AS mice develop immunoglobulin A (IgA) nephropathy (IgAN), characterized by microhematuria, albuminuria, and renal glomerular deposition of IgA, fibronectin (Fn), collagen, and C3 complement. This phenotype of both UG-KO and UG-AS mice is virtually identical to that of human IgAN, the most common primary glomerulopathy worldwide. The molecular mechanism by which UG prevents this disease in mice appears to center around UG's interaction with Fn. Since Fn, IgA, and UG are present in circulation and high plasma levels of IgA-Fn complex have been reported in human IgAN, we sought to determine whether UG interacts with Fn and prevents Fn-Fn and/or IgA-Fn interactions, essential for abnormal tissue deposition of Fn and IgA. Our coimmunoprecipitation studies uncovered the formation of Fn-UG heteromers in vitro and these heteromers are detectable in the plasma of normal mice, but not UG-KO mice. Further, high plasma levels of IgA-Fn complex, a characteristic of human IgAN patients, were also found in UG-KO mice. Finally, coadministration of UG + Fn or UG + IgA to UG-KO mice prevented glomerular deposition of Fn and IgA, respectively. Our results define a possible molecular mechanism of IgAN and provide insight into at least one important physiological function of UG in maintaining normal renal function in mice.
Assuntos
Glomerulonefrite por IGA/genética , Rim/metabolismo , Camundongos Knockout/metabolismo , Camundongos Transgênicos/metabolismo , RNA Antissenso , Uteroglobina/deficiência , Uteroglobina/genética , Animais , Colágeno/genética , Complemento C3/metabolismo , Fibronectinas/sangue , Fibronectinas/genética , Mesângio Glomerular/imunologia , Mesângio Glomerular/metabolismo , Mesângio Glomerular/patologia , Glomerulonefrite por IGA/imunologia , Glomerulonefrite por IGA/patologia , Hematúria/imunologia , Hematúria/patologia , Hematúria/fisiopatologia , Humanos , Imunoglobulina A/sangue , Rim/patologia , Rim/fisiopatologia , Camundongos , Camundongos Knockout/genética , Camundongos Knockout/imunologia , Camundongos Transgênicos/genética , Camundongos Transgênicos/imunologia , Fenótipo , Fator de Crescimento Derivado de Plaquetas/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , RNA Antissenso/genética , RNA Mensageiro/metabolismo , Uteroglobina/biossínteseRESUMO
Uteroglobin (UG) is a multifunctional, secreted protein with anti-inflammatory and antichemotactic properties. While its anti-inflammatory effects, in part, stem from the inhibition of soluble phospholipase A2 (sPLA2) activity, the mechanism(s) of its antichemotactic effects is not clearly understood. Although specific binding of UG on microsomal and plasma membranes has been reported recently, how this binding affects cellular function is not clear. Here, we report that recombinant human UG (hUG) binds to both normal and cancer cells with high affinity (20-35 nM, respectively) and specificity. Affinity cross-linking studies revealed that 125I-hUG binds to the NIH 3T3 cell surface with two proteins of apparent molecular masses of 190 and 49 kDa, respectively. UG affinity chromatography yielded similar results. While both the 190- and 49-kDa proteins were expressed in the heart, liver, and spleen, the lung and trachea expressed only the 190-kDa protein. Some cancer cells (e.g., mastocytoma, sarcoma, and lymphoma) expressed both the 190- and 49-kDa proteins. Further, using functional assays, we found that UG dramatically suppressed the motility and extracellular matrix invasion of both NIH 3T3 and some cancer cells. In order to further characterize the anti-ECM-invasive properties of UG, we induced expression of hUG into cancer cell lines derived from organs that, under physiological circumstances, secrete UG at a high level. Interestingly, it has been reported that a high percentage of the adenocarcinomas arising from the same organs fail to express UG. Our results on induced hUG expression in these cells show that inhibition of motility and ECM invasion requires the expression of both UG and its binding proteins. Taken together, our data define receptor-mediated functions of UG in which this protein regulates vital cellular functions by both autocrine and paracrine pathways.
Assuntos
Proteínas de Transporte/metabolismo , Movimento Celular/genética , Invasividade Neoplásica/genética , Ligação Proteica/genética , Receptores de Superfície Celular/metabolismo , Células Tumorais Cultivadas/metabolismo , Uteroglobina/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/farmacologia , Movimento Celular/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Humanos , Camundongos , Invasividade Neoplásica/patologia , Ligação Proteica/efeitos dos fármacos , Ensaio Radioligante/estatística & dados numéricos , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/patologia , Uteroglobina/genética , Uteroglobina/farmacologiaRESUMO
Blastokinin or uteroglobin (UG) is a steroid-inducible, evolutionarily conserved, multifunctional protein secreted by the mucosal epithelial of virtually all mammals. It is present in the blood and in other body fluids including urine. An antigen immunoreactive to UG antibody is also detectable in the mucosal epithelia of all vertebrates. UG-binding proteins (putative receptor), expressed on several normal and cancer cell types, have been characterized. The human UG gene is mapped to chromosome 11q12.2 13.1, a region that is frequently rearranged or deleted in many cancers. The generation of UG knockout mice revealed that disruption of this gene causes: (i) severe renal disease due to an abnormal deposition of fibronectin and collagen in the glomeruli; (ii) predisposition to a high incidence of malignancies; and (iii) a lack of polychlorinated biphenyl binding and increased oxygen toxicity in the lungs. The mechanism(s) of UG action is likely to be even more complex as it also functions via a putative receptor-mediated pathway that has not yet been clearly defined. Molecular characterization of the UG receptor and signal transduction via this receptor pathway may show that this protein belongs to a novel cytokine/chemokine family.
Assuntos
Citocinas/fisiologia , Uteroglobina/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Citocinas/química , Citocinas/genética , DNA Complementar/genética , Regulação da Expressão Gênica , Genes Supressores de Tumor , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Receptores de Citocinas/fisiologia , Homologia de Sequência de Aminoácidos , Uteroglobina/química , Uteroglobina/genéticaRESUMO
Uteroglobin (UG) is a multifunctional, secreted protein that has receptor-mediated functions. The human UG (hUG) gene is mapped to chromosome 11q12.2-13.1, a region frequently rearranged or deleted in many cancers. Although high levels of hUG expression are characteristic of the mucosal epithelia of many organs, hUG expression is either drastically reduced or totally absent in adenocarcinomas and in viral-transformed epithelial cells derived from the same organs. In agreement with these findings, in an ongoing study to evaluate the effects of aging on UG-knockout mice, 16/16 animals developed malignant tumors, whereas the wild-type littermates (n = 25) remained apparently healthy even after 11/2 years. In the present investigation, we sought to determine the effects of induced-expression of hUG in human cancer cells by transfecting several cell lines derived from adenocarcinomas of various organs with an hUG-cDNA construct. We demonstrate that induced hUG expression reverses at least two of the most important characteristics of the transformed phenotype (i.e., anchorage-independent growth on soft agar and extracellular matrix invasion) of only those cancer cells that also express the hUG receptor. Similarly, treatment of the nontransfected, receptor-positive adenocarcinoma cells with purified recombinant hUG yielded identical results. Taken together, these data define receptor-mediated, autocrine and paracrine pathways through which hUG reverses the transformed phenotype of cancer cells and consequently, may have tumor suppressor-like effects.
Assuntos
Adenocarcinoma/genética , Adenocarcinoma/patologia , Transformação Celular Neoplásica , Cromossomos Humanos Par 11 , Uteroglobina/genética , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Mapeamento Cromossômico , Feminino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Knockout , Invasividade Neoplásica , Neoplasias Experimentais/genética , Fenótipo , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Transfecção , Células Tumorais Cultivadas , Neoplasias Uterinas/genética , Neoplasias Uterinas/patologia , Uteroglobina/deficiência , Uteroglobina/fisiologiaRESUMO
Blastokinin or uteroglobin (UG) is an evolutionarilly conserved, steroid-inducible, homodimeric, multifunctional, secreted protein with potent Immunomodulatory/antiinflammatory properties. Recently, a UG-receptor expressed on several malignant and normal cell types has been characterized. Although the biochemistry, structural, and molecular biology of UG have been extensively studied, its physiological function(s), until recently, remained unknown. By generating UG-null (UG-/-) mice, we determined that an essential role of UG is to prevent severe renal disease caused by an abnormal deposition of predominantly multimeric fibronectin (Fn) and collagen in the glomerulus. The molecular mechanisms by which UG prevents this disease in control (UG+/+) mice, at least in part, is attributable to its high-affinity binding to Fn and the formation of Fn-UG heteromers, which counteract both Fn-Fn and Fn-collagen interactions, required for abnormal tissue deposition. In addition, by inhibiting secretory phospholipase A2 (sPLA2) activity and decreasing the level of lysophosphatidic acid (LPA), UG may indirectly prevent the activation of integrins (eg, alpha5beta1) that enhance abnormal tissue deposition of Fn. The mechanism(s) of UG action is likely to be even more complex, because it also functions through a receptor-mediated pathway that has not yet been clearly defined. Nevertheless, the UG gene-knockout mice provide a valuable animal model for investigation of human glomerulopathies in general and familial Fn-deposit glomerulopathy in particular.
Assuntos
Genes/genética , Glomérulos Renais/fisiologia , Uteroglobina/fisiologia , Animais , Clonagem Molecular , Regulação da Expressão Gênica/fisiologia , Humanos , Camundongos , Camundongos Knockout , Relação Estrutura-Atividade , Uteroglobina/química , Uteroglobina/deficiência , Uteroglobina/genéticaRESUMO
Despite myriads of biological activities ascribed to uteroglobin (UG), a steroid-inducible secreted protein, its physiological functions are unknown. Mice in which the uteroglobin gene was disrupted had severe renal disease that was associated with massive glomerular deposition of predominantly multimeric fibronectin (Fn). The molecular mechanism that normally prevents Fn deposition appears to involve high-affinity binding of UG with Fn to form Fn-UG heteromers that counteract Fn self-aggregation, which is required for abnormal tissue deposition. Thus, UG is essential for maintaining normal renal function in mice, which raises the possibility that an analogous pathogenic mechanism may underlie genetic Fn-deposit human glomerular disease.
Assuntos
Fibronectinas/metabolismo , Glomérulos Renais , Uteroglobina/fisiologia , Animais , Células Cultivadas , Cruzamentos Genéticos , Marcação de Genes , Humanos , Nefropatias/embriologia , Nefropatias/genética , Nefropatias/patologia , Glomérulos Renais/embriologia , Glomérulos Renais/metabolismo , Glomérulos Renais/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Uteroglobina/deficiência , Uteroglobina/genéticaRESUMO
Using site-directed mutagenesis, we proposed that an autoinhibitory domain(s) is located at the C-terminal region (301-386) of the phosphorylase kinase gamma-subunit (Huang, C.-Y.F., Yuan C.-J., Livanova, N.B., and Graves, D.J. (1993) Mol. Cell. Biochem. 127/128, 7-18). Removal of the putative inhibitory domain(s) by truncation results in the generation of a constitutively active and calmodulin-independent form, gamma 1-300. To probe the structural basis of autoinhibition of gamma-subunit activity, two synthetic peptides, PhK13 (gamma 303-327) and PhK5 (gamma 343-367), corresponding to the two calmodulin-binding regions, were assayed for their ability to inhibit gamma 1-300. Competitive inhibition of gamma 1-300 by PhK13 was found versus phosphorylase b (Ki = 1.8 microM) and noncompetitive inhibition versus ATP. PhK5 showed noncompetitive inhibition with respect to both phosphorylase b and ATP. Calmodulin released the inhibition caused by both peptides. These results indicate that there are two distinct auto-inhibitory domains within the C terminus of the gamma-subunit and that these two domains overlap with the calmodulin-binding regions. Two mutant forms of gamma 1-300, E111K and E154R, were used to probe the enzyme-substrate-binding region using peptide substrate analogs corresponding to residues 9-18 of phosphorylase b (KRK11Q12ISVRGL). The data suggest that Glu111 interacts with the P-3 position of the substrate (Lys11) and Glu154 interacts with the P-2 site (Gln12). Both E111K and E154R were competitively inhibited with respect to phosphorylase b by PhK13, with 14- and 8-fold higher Ki values, respectively, than that observed with the wild-type enzyme. These data are consistent with a model for the regulation of the gamma-subunit of phosphorylase kinase in which PhK13 acts as a competitive pseudosubstrate that directly binds the substrate binding site of the gamma-subunit (Glu111 and Glu154).
Assuntos
Calmodulina/metabolismo , Fosforilase Quinase/química , Fosforilase Quinase/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ligação Competitiva , Cinética , Substâncias Macromoleculares , Dados de Sequência Molecular , Músculo Esquelético/enzimologia , Mutagênese Sítio-Dirigida , Peptídeos/síntese química , Peptídeos/química , Fosforilase Quinase/isolamento & purificação , Fosforilase b/metabolismo , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
A truncated form of the gamma subunit of phosphorylase kinase is inactivated by Cu2+ with the formation of two intra-molecular disulfide bonds. The formation of a disulfide bond between Cys-36 and Cys-172 (semioxidized form) results in approximately 50% loss of specific activity because the Km for MgATP is about 10-fold higher. The second disulfide bond is between Cys-184 and Cys-197 and causes further loss of activity. Eight Cys mutants, i.e. C36S, C36A, C42S, C138S, C172S, C184S, C184A, and C197S, were expressed and purified. Kinetic studies suggest that Cys-36 is important for interaction at the nucleotide site because of its hydrophobicity. With Cys-184 mutants, C184S and C184A, tyrosyl phosphorylation of angiotensin II is affected much more than serine kinase activity. The loss of tyrosine kinase activity is related to a lowered activity with Mn2+. With Mn2+, angiotensin II is a competitive inhibitor with respect to seryl kinase activity of C184S. With Mg2+, however, angiotensin II is a noncompetitive inhibitor. We suggest that metal ions influence the conformation of truncated gamma and that the protein substrate binding region containing Cys-184 is important for the dual specificity of this kinase.
Assuntos
Fosforilase Quinase/metabolismo , Compostos de Sulfidrila/metabolismo , Sequência de Aminoácidos , Angiotensina II/metabolismo , Catálise , Cobre/farmacologia , Cisteína/metabolismo , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oxirredução , Fosforilase Quinase/antagonistas & inibidores , Fosforilase Quinase/genética , FosforilaçãoRESUMO
Phosphorylase kinase (PhK) and truncated gamma subunit, denoted gamma 1-300, can phosphorylate seryl and tyrosyl residues dependent on the metal ion [Yuan, C.-J., Huang, C. F., & Graves, D. J. (1993) J. Biol. Chem. 268, 17683-17686]. Recombinant gamma 1-300 was used to explore its dual specificity and the location of the metal ion binding sites by using site-directed mutagenesis. Two approaches were taken to generate 26 mutants. First, on the basis of the crystal structure of cAMP-dependent protein kinase (cAPK), the invariant Asn155 and highly conserved Asp168-Phe169-Gly170 residues were mutated. Changes included production of N155H, D168E, D168N, F169R, G170V, G170I, G170L (less than 1% of enzymatic activities were found in these mutants), F169W, and G170A mutants. Second, charge to alanine and charge reversal scanning mutations were used to probe the metal ion binding sites. Two mutants, E111K and E154R, showed very different metal ion response compared to wild-type gamma and were further characterized. The mutants F169W, G170A, E111K, and E154R had 15%, 5%, 8%, and 25% specific activity relative to wild-type gamma, respectively. The folding pattern of wild-type and mutated enzyme forms of gamma was determined by photoacoustic infrared spectroscopy. Conformational disruptions were found in G170V, G170I, and G170L mutants, but the conformation of the rest of the mutants was similar to that of wild-type gamma, suggesting that the loss of enzymatic activities of these mutants was not because of incorrect refolding.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Metais/metabolismo , Fragmentos de Peptídeos/metabolismo , Fosforilase Quinase/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Análise Mutacional de DNA , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fosforilase Quinase/química , Fosforilase Quinase/genética , Proteínas Tirosina Quinases/metabolismo , Espectrofotometria Infravermelho , Especificidade por SubstratoRESUMO
A catalytic fragment, gamma 1-298, derived from limited chymotryptic digestion of phosphorylase b kinase (Harris, W.R. et al., J. Biol. Chem., 265: 11740-11745, 1990), is reported to have about six-fold greater specific activity than does the gamma subunit-calmodulin complex. To test whether there is an inhibitory domain located outside the catalytic core of the gamma subunit, full-length wild-type and seven truncated forms of gamma were expressed in E. coli. Recombinant proteins accumulate in the inclusion bodies and can be isolated, solubilized, renatured, and purified further by ammonium sulfate precipitation and Q-Sepharose column. Four out of seven truncated mutants show similar (gamma 1-353 and gamma 1-341) or less (gamma 1-331 and gamma 1-276) specific activity than does the full-length wild-type gamma, gamma 1-386. Three truncated forms, gamma 1-316, gamma 1-300, and gamma 1-290 have molar specific activities approximately twice as great as those of the full-length wild-type gamma and the nonactivated holoenzyme. All recombinant gamma s exhibit similar Km values for both substrates, i.e., about 18 microM for phosphorylase b and about 75 microM for MgATP. Three truncated gamma s, gamma 1-316, gamma 1-300, and gamma 1-290, have a 1.9- to 2.5-fold greater catalytic efficiency (Vmax/Km) than that of the full-length wild-type gamma and a 3.5- to 4.5-fold greater efficiency than that of the truncated gamma 1-331. This evidence suggests that there is at least one inhibitory domain in the C-terminal region of gamma, which is located at gamma 301-331. gamma 1-290, but not gamma 1-276, which contains the highly conserved kinase domain, is the minimum sequence required for the gamma subunit to exhibit phosphotransferase activity. Both gamma 1-290 and gamma 1-300 have several properties similar to full-length wild-type gamma, including metal ion responses (activation by free Mg2+ and inhibition by free Mn2+), pH dependency, and substrate specificities.
Assuntos
Fosforilase Quinase/biossíntese , Sequência de Aminoácidos , Animais , Sítios de Ligação , Clonagem Molecular , Escherichia coli , Cinética , Substâncias Macromoleculares , Dados de Sequência Molecular , Peso Molecular , Músculos/enzimologia , Mutagênese , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/metabolismo , Fosforilase Quinase/isolamento & purificação , Fosforilase Quinase/metabolismo , Fosforilase b/metabolismo , Plasmídeos , Coelhos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Deleção de SequênciaRESUMO
Phosphorylase kinase is shown to be a dual specificity kinase. The specificity of phosphorylation is determined by divalent cation. Mg2+ causes seryl phosphorylation of phosphorylase b, but Mn2+ activates tyrosine phosphorylation of angiotensin II. In contrast to seryl phosphorylation, the tyrosine kinase activity of holoenzyme is not regulated by Ca2+. Preincubation of the holoenzyme with Ca2+, Mg2+ and ATP that causes autophosphorylation activates tyrosine kinase activity. The tyrosyl kinase activity is a property of the gamma subunit. Addition of varying amounts of Mn2+ to a truncated form of the gamma subunit of phosphorylase kinase containing MgATP inhibits serine kinase but activates tyrosine kinase activity. This result along with an oxidative reaction caused by Cu2+ and site-directed mutagenesis of the putative catalytic base inhibiting both serine and tyrosine kinase activity suggest that one active site is involved in both activities. Kinetic studies with Mn2+ and ATP show that Km for nucleotide is not changed with a seryl or tyrosyl substrate. The Vm values are different, and the value for tyrosyl phosphorylation is similar to other tyrosyl kinases. We propose two conformations for the active site; one favors seryl phosphorylation, and the second tyrosyl phosphorylation is caused by the binding of divalent cation at a second metal ion binding site.
Assuntos
Cálcio/farmacologia , Magnésio/farmacologia , Manganês/farmacologia , Músculos/enzimologia , Fosforilase Quinase/metabolismo , Proteínas Tirosina Quinases/metabolismo , Trifosfato de Adenosina/metabolismo , Angiotensina II , Animais , Cinética , Fosforilase Quinase/isolamento & purificação , Fosforilase b/metabolismo , Fosforilação , Coelhos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoAssuntos
Músculos/enzimologia , Fosforilase Quinase/isolamento & purificação , Proteínas Quinases/isolamento & purificação , Animais , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Ativação Enzimática , Escherichia coli/genética , Cinética , Substâncias Macromoleculares , Fosforilase Quinase/genética , Fosforilase Quinase/metabolismo , Proteínas Quinases/metabolismo , Coelhos , Proteínas Recombinantes/metabolismo , TermodinâmicaRESUMO
Two gastrin analogs containing a D- and a L-tetrafluorinated tyrosyl residue (Arg-Arg-Leu-Glu-Glu-Glu-Glu-Glu-Ala-(F4)Tyr-Gly) were synthesized and tested as substrates and inhibitors of the insulin receptor kinase. No phosphorylation of these peptides was observed, but both gastrin analogs were effective inhibitors in the microM range. Although the D- and L-tetrafluorotyrosine-gastrin analogs differ in the sequence by only 1 amino acid residue, a different inhibitory pattern was obtained with the insulin receptor. The inhibition of all-L-isomer is competitive with respect to both the protein substrate, reduced, S-carboxymethylated, and maleylated lysozyme (RCMM-lysozyme), and ATP with a Ki value of 4 microM. This result corroborates a previous finding (Walker, D. H., Kuppuswamy, D., Visvanathan, A., and Pike, L. J. (1987) Biochemistry 26, 1428-1433) that the kinetic mechanism for insulin receptor is a random Bi Bi mechanism. Different from the L-isomer, the D-analog is competitive to RCMM-lysozyme and noncompetitive toward ATP and gives an apparent inhibition constant of 20 microM. A free tetrafluorotyrosine also shows a competitive inhibition to protein substrate, RCMM-lysozyme (Ki = 18 mM) whereas free tyrosine shows no effect on the activity of insulin receptor. These results show the importance of the charge state and nucleophilicity of the phenolic component in substrate recognition and catalysis and provide a rationale for the design of inhibitors of tyrosyl phosphorylation.
Assuntos
Gastrinas/síntese química , Oligopeptídeos/síntese química , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Desenho de Fármacos , Gastrinas/metabolismo , Gastrinas/farmacologia , Cinética , Modelos Estruturais , Dados de Sequência Molecular , Oligopeptídeos/farmacologia , Fosforilação , Receptor de InsulinaRESUMO
A strong Ca2+-independent interaction between the isolated, active gamma subunit of phosphorylase kinase and dansyl-calmodulin (dansyl-CaM) was observed by monitoring changes in fluorescence intensity in the absence of calcium ion. The pure, active gamma subunit of phosphorylase kinase was simply prepared by dialyzing the HPLC-purified, inactive gamma subunit against 8 M urea, containing 0.1 mM DTT, 0.1 M Hepes at pH 6.8 or 0.1 M Tris at pH 8.2, followed by dilution of urea with pH 6.8 or 8.2 buffer. The dissociation constants determined by fluorescence spectroscopy for the gamma subunit to dansyl-CaM are 25.7 +/- 0.6 and 104 +/- 12 nM at pH 6.8 in the presence and absence of CaCl2. At pH 8.2, these values are 4.9 +/- 0.3 and 29 +/- 8 nM in the presence and absence of CaCl2. As the free Ca2+ decreases to as low as 10(-9) M, the fluorescence intensity and the fluorescence polarization of the gamma subunit and dansyl-CaM complex do not decrease in parallel, indicating that the complex does not come apart at low Ca2+ concentration. The presence of Mg2+ affects the interaction between dansyl-CaM and the gamma subunit, as indicated by the increase in the polarization of fluorescence of dansyl-CaM. Mn2+ interferes with the interaction of the gamma subunit and dansyl-CaM. Free ATP has little effect.
Assuntos
Cálcio/fisiologia , Fosforilase Quinase/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Sítios de Ligação/efeitos dos fármacos , Calmodulina/isolamento & purificação , Calmodulina/metabolismo , Bovinos , Cromatografia Líquida de Alta Pressão , Ácido Egtázico , Ativação Enzimática/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Cinética , Magnésio/farmacologia , Fosforilase Quinase/isolamento & purificação , Espectrometria de FluorescênciaRESUMO
Quantitative structure activity relationship (QSAR) analyses have been performed to analyze the basis for the antigelling activity of a series of aryl-substituted alanine analogs, including both phenylalanine and tryptophan analogs. We find that these two groups of compounds should be treated separately, probably due to the difference in shape between the phenyl and indole rings in the respective side chains. For the phenylalanine analogs, the hydrophobicity of the side chain, as measured by the pi constants of the aromatic ring and their substituents, explains about 50% of the variance in antigelling activity. The square of the aromatic dipole moment and the steric overlap volume, as obtained from three dimensional molecular shape analysis, account for an additional 20% and 10% of the variance, respectively. For the tryptophan analogs, the majority of the variance is explained either by the square of the aromatic dipole moment (70%) or by the steric overlap volume (60%), with the two descriptors being highly correlated. This result suggests that the tryptophan analogs exhibit a relatively tight steric fit within their binding sites. The separate QSAR's of phenylalanine and tryptophan analogs also suggest that these two groups of compounds may bind to hemoglobin through different mechanisms or at different sites. For phenylalanine analogs, the hydrophobic interaction is dominant, and the dipole-dipole interaction and steric effects contribute to lesser extents. For the tryptophan analogs, the dipole-dipole interaction prevails with a high degree of steric shape complimentarity, but without any significant contribution from hydrophobic interactions. This information should assist in the future development of more specific and effective agents.
Assuntos
Alanina , Alanina/análogos & derivados , Géis , Hemoglobinas , Modelos Biológicos , Conformação Molecular , Estrutura Molecular , Fenilalanina , Solubilidade , Relação Estrutura-Atividade , TriptofanoRESUMO
A cDNA encoding the entire tau subunit of rabbit skeletal muscle phosphorylase kinase was reconstructed and inserted into a plasmid containing the Escherichia coli ptac promoter and a constructed plasmid containing the ptac promoter and bacterial chloramphenicol acetyl transferase (CAT) gene, respectively. A significant phosphorylase kinase activity was found, in the first case. In the second case, a fused protein containing 73 amino acids from the CAT protein was obtained. After renaturation, the CAT-tau subunit protein shows enzymatic activity similar to the HPLC-purified and renatured tau subunit.