RESUMO
BACKGROUND: Lung adenocarcinoma (LADCA) patients with epidermal growth factor receptor (EGFR) mutations are in general associated with relatively high clinical response rate to EGFR-tyrosine kinase inhibitors (TKIs) but not all responded to TKI. It has therefore become important to identify the additional surrogate markers regarding EGFR-TKI sensitivity. METHODS: We first examined the effects of EGFR-TKIs, gefitinib and erlotinib, upon cell proliferation of lung adenocarcinoma cell lines. We then evaluated the gene profiles related to EGFR-TKI sensitivity using a microarray analysis. Results of microarray analysis led us to focus on carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family, CEACAM 3, 5, 6, 7, and 19, as potential further surrogate markers of EGFR-TKI sensitivity. We then examined the correlation between the status of CEACAM 3, 5, 6, 7, and 19 immunoreactivity in LADCA and clinicopathological parameters of individual cases. RESULTS: In the cases with EGFR mutations, the status of all CEACAMs examined was significantly higher than that in EGFR wild-type patients, but there were no significant differences in the status of CEACAMs between TKI responder and nonresponder among 22 patients who received gefitinib therapy. However, among 115 EGFR mutation-negative LADCA patients, both CEACAM6 and CEACAM3 were significantly associated with adverse clinical outcome (CEACAM6) and better clinical outcome (CEACAM3). CONCLUSION: CEACAMs examined in this study could be related to the presence of EGFR mutation in adenocarcinoma cells but not represent the effective surrogate marker of EGFR-TKI in LADCA patients. However, immunohistochemical evaluation of CEACAM3/6 in LADCA patients could provide important information on their clinical outcome.
Assuntos
Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/metabolismo , Biomarcadores Tumorais/metabolismo , Antígeno Carcinoembrionário/metabolismo , Moléculas de Adesão Celular/metabolismo , Receptores ErbB/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Adenocarcinoma/enzimologia , Adenocarcinoma/genética , Adenocarcinoma de Pulmão , Antígeno Carcinoembrionário/genética , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/genética , Receptores ErbB/metabolismo , Cloridrato de Erlotinib , Gefitinibe , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Mutação/efeitos dos fármacos , Quinazolinas/farmacologiaRESUMO
In Saccharomyces cerevisiae, phosphoacetylglucosamine mutase is encoded by an essential gene called AGM1. The human AGM1 cDNA (HsAGM1) and the Candida albicans AGM1 gene (CaAGM1) were functionally cloned and characterized by using an S. cerevisiae strain in which the endogenous phosphoacetylglucosamine mutase was depleted. When expressed in Escherichia coli as fusion proteins with glutathione S-transferase, both HsAgm1 and CaAgm1 proteins displayed phosphoacetylglucosamine mutase activities, demonstrating that they indeed specify phosphoacetylglucosamine mutase. Sequence comparison of HsAgm1p with several hexose-phosphate mutases yielded three domains that are highly conserved among phosphoacetylglucosamine mutases and phosphoglucomutases of divergent organisms. Mutations of the conserved amino acids found in these domains, which were designated region I, II, and III, respectively, demonstrated that alanine substitutions for Ser(64) and His(65) in region I, and for Asp(276), Asp(278), and Arg(281) in region II of HsAgm1p severely diminished the enzyme activity and the ability to rescue the S. cerevisiae agm1Delta null mutant. Conservative mutations of His(65) and Asp(276) restored detectable activities, whereas those of Ser(64), Asp(278), and Arg(281) did not. These results indicate that Ser(64), Asp(278), and Arg(281) of HsAgm1p are residues essential for the catalysis. Because Ser(64) corresponds to the phosphorylating serine in the E. coli phosphoglucosamine mutase, it is likely that the activation of HsAgm1p also requires phosphorylation on Ser(64). Furthermore, alanine substitution for Arg(496) in region III significantly increased the K(m) value for N-acetylglucosamine-6-phosphate, demonstrating that Arg(496) serves as a binding site for N-acetylglucosamine-6-phosphate.
Assuntos
Fosfotransferases (Fosfomutases)/genética , Fosfotransferases (Fosfomutases)/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Candida albicans/enzimologia , Candida albicans/genética , Catálise , Clonagem Molecular , Análise Mutacional de DNA , DNA Complementar/análise , Biblioteca Genômica , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Homologia de Sequência de AminoácidosRESUMO
The amino acid sequence of the Saccharomyces cerevisiae mRNA 5'-triphosphatase (TPase) diverges from those of higher eukaryotes. In order to confirm the sequence divergence of TPases in lower and higher eukaryotes, the Candida albicans gene for TPase was identified and characterized. This gene designated CaCET1 (C. albicans mRNA 5'-capping enzyme triphosphatase 1) has an open reading frame of 1.5 kb, which can encode a 59-kDa protein. Although the N-terminal one-fifth of S. cerevisiae TPase (ScCet1p) is missing in CaCet1p, CaCet1p shares significant sequence similarity with ScCet1p over the entire region of the protein; the recombinant CaCet1p, which was expressed as a fusion protein with glutathione S-transferase (GST), displayed TPase activity in vitro. CaCET1 rescued CET1-deficient S. cerevisiae cells when expressed under the control of the ADH1 promoter, whereas the human capping enzyme derivatives that are active for TPase activity but defective in mRNA 5'-guanylyltransferase (GTase) activity did not. Yeast two-hybrid analysis revealed that C. albicans Cet1p can bind to the S. cerevisiae GTase in addition to its own partner, the C. albicans GTase. In contrast, neither the full-length human capping enzyme nor its TPase domain interacted with the yeast GTase. These results indicate that the failure of the human TPase activity to complement an S. cerevisiae cet1delta null mutation is attributable, at least in part, to the inability of the human capping enzyme to associate with the yeast GTase, and that the physical association of GTase and TPase is essential for the function of the capping enzyme in vivo.
Assuntos
Hidrolases Anidrido Ácido/genética , Hidrolases Anidrido Ácido/isolamento & purificação , Candida albicans/enzimologia , Genes Fúngicos , Capuzes de RNA/metabolismo , Hidrolases Anidrido Ácido/metabolismo , Sequência de Aminoácidos , Candida albicans/genética , Clonagem Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Teste de Complementação Genética , Humanos , Dados de Sequência Molecular , Mutagênese , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Capuzes de RNA/genética , Saccharomyces cerevisiae/genéticaRESUMO
When chitin synthase 2 of Saccharomyces cerevisiae was overexpressed in yeast cells using GAL1 promoter, deletion of the N-terminal 193 amino acids significantly increased the level of the protein without affecting its characteristics. We partially purified N-terminally truncated chitin synthase 2 by product entrapment and ion exchange column chromatography, and found that it was active even without trypsin treatment when appropriate divalent cations were present in the reaction mixture. This chitin synthase activity was independent of the N-terminal 193 amino acid truncation, because partially purified full length enzyme also exhibited the activity without trypsin treatment in the presence of appropriate cations. Furthermore, the molecular weights of these two forms of chitin synthase 2 were coincident with those estimated from the deduced amino acid sequence, and most of the chitin synthase 2 in the yeast membrane was present as an unprocessed form, as judged from its molecular weight. Treatment of either full length or truncated enzyme with trypsin, however, further increased the enzyme activity by four to fivefold, and produced a 35 kDa polypeptide that specifically reacted with monoclonal antibody raised against the region containing the putative active site of chitin synthase 2. Thus, it appears that predominant native (unprocessed) chitin synthase 2 is active, but the 35 kDa region encompassing the active site is sufficient for the catalytic activity.
Assuntos
Quitina Sintase/metabolismo , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/enzimologia , Anticorpos Antifúngicos , Anticorpos Monoclonais , Sequência de Bases , Sítios de Ligação , Cátions Bivalentes , Membrana Celular/enzimologia , Quitina/biossíntese , Quitina Sintase/biossíntese , Quitina Sintase/química , Quitina Sintase/genética , Quitina Sintase/isolamento & purificação , Quimotripsina , Ativação Enzimática , Precursores Enzimáticos/metabolismo , Expressão Gênica , Dados de Sequência Molecular , Peso Molecular , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/imunologia , Deleção de Sequência , TripsinaRESUMO
A complementary DNA of the Aspergillus nidulans chsB gene encoding chitin synthase, an essential gene for hyphal growth, was obtained by RT-PCR and expressed in Saccharomyces cerevisiae by using the GAL1 promoter in a multicopy plasmid. The biochemical characteristics of chitin synthase B (ChsB) expressed in S. cerevisiae were examined. The chitin synthase B produced in galactose medium showed zymogenicity due to activation by trypsin treatment and required Mg2+ ion to exert maximal activity. It was competitively inhibited by polyoxin D. The Ki value of the inhibitor was 10 microM, and the K(m) for the substrate was 1.6 mM. The activity was enhanced by the addition of N-acetylglucosamine. The optimal pH is 7.5 when Mg2+ is used. These characteristics are the same as those of other chitin synthases.
Assuntos
Aspergillus nidulans/genética , Quitina Sintase/genética , Saccharomyces cerevisiae/genética , Aspergillus nidulans/enzimologia , Aspergillus nidulans/crescimento & desenvolvimento , DNA Complementar , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação Fúngica da Expressão Gênica/fisiologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/fisiologia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
Cancer stem cells have been proposed to be responsible for tumorigenesis and recurrence in various neoplastic diseases, including multiple myeloma (MM). We have previously reported that MM cells specifically express HLA class I at high levels and that single-chain Fv diabody against this molecule markedly induces MM cell death. Here we investigated the effect of a new diabody (C3B3) on cancer stem cell-like side population (SP) cells. SP fraction of MM cells highly expressed ABCG2 and exhibited resistance to chemotherapeutic agents; however, C3B3 induced cytotoxicity in both SP cells and main population (MP) cells to a similar extent. Moreover, C3B3 suppressed colony formation and tumorigenesis of SP cells in vitro and in vivo. Crosslinking of HLA class I by C3B3 mediated disruption of lipid rafts and actin aggregation, which led to inhibition of gene expression of ß-catenin and pluripotency-associated transcription factors such as Sox2, Oct3/4 and Nanog. Conversely, knockdown of Sox2 and Oct3/4 mRNA reduced the proportion of SP cells, suggesting that these factors are essential in maintenance of SP fraction in MM cells. Thus, our findings reveal that immunotherapeutic approach by engineered antibodies can overcome drug resistance, and provide a new basis for development of cancer stem cell-targeted therapy.
Assuntos
Antígenos HLA/imunologia , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/terapia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Células da Side Population/metabolismo , Anticorpos de Cadeia Única/uso terapêutico , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Antineoplásicos/uso terapêutico , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos SCID , Mieloma Múltiplo/patologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/imunologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células da Side Population/imunologia , Células da Side Population/patologia , Anticorpos de Cadeia Única/imunologia , beta Catenina/metabolismoRESUMO
Point mutation of the c-H-ras gene significantly increases cellular transforming activities of Ras. Since posttranslational modification and subsequent membrane localization are essential for the biological activities of Ras, we examined whether or not the mutation also affects these two factors. The normal (Gly(12)) or the transforming (Val(12)) c-H-ras gene was expressed in NIH3T3 cells using a metallothionein promoter. Expression of either type of Ras was efficiently induced by the cadmium treatment of these cells, and immunoprecipitation of metabolically labeled cell extracts revealed that both normal and transforming Ras were expressed as four differently migrating forms on SDS-polyacrylamide gels, two of which were slower migrating cytosolic precursors and the other two were faster migrating membrane-bound forms. There was no significant difference in half lives between normal and transforming Ras; however, posttranslational modification was quite different between the two types of Ras. Transforming Ras was processed and became membrane-bound forms much more efficiently than normal Ras. Interestingly, posttranslational modification and membrane localization of Ras was significantly inhibited when the c-myc oncogene was co-expressed with Ras. In contrast to the c-myc oncogene, expression of either wild type or mutant p53 did not affect the posttranslational modification of Ras, suggesting that the c-myc oncogene specifically impairs the posttranslational modification of Ras.
Assuntos
Genes ras , Mutação Puntual , Processamento de Proteína Pós-Traducional , Transformação Genética , Células 3T3 , Animais , Genes myc , Genes p53 , CamundongosRESUMO
A search of the yeast data base for a protein homologous to Escherichia coli UDP-N-acetylglucosamine pyrophosphorylase yielded UAP1 (UDP-N-acetylglucosamine pyrophosphorylase), the Saccharomyces cerevisiae gene for UDP-N-acetylglucosamine pyrophosphorylase. The Candida albicans and human homologs were also cloned by screening a C. albicans genomic library and a human testis cDNA library, respectively. Sequence analysis revealed that the human UAP1 cDNA was identical to previously reported AGX1. A null mutation of the S. cerevisiae UAP1 (ScUAP1) gene was lethal, and when expressed under the control of ScUAP1 promoter, both C. albicans and Homo sapiens UAP1 (CaUAP1 and HsUAP1) rescued the ScUAP1-deficient S. cerevisiae cells. All the recombinant ScUap1p, CaUap1p, and HsUap1p possessed UDP-N-acetylglucosamine pyrophosphorylase activities in vitro. The yeast Uap1p utilized N-acetylglucosamine-1-phosphate as the substrate, and together with Agm1p, it produced UDP-N-acetylglucosamine from N-acetylglucosamine-6-phosphate. These results demonstrate that the UAP1 genes indeed specify eukaryotic UDP-GlcNAc pyrophosphorylase and that phosphomutase reaction precedes uridyltransfer. Sequence comparison with other UDP-sugar pyrophosphorylases revealed that amino acid residues, Gly112, Gly114, Thr115, Arg116, Pro122, and Lys123 of ScUap1p are highly conserved in UDP-sugar pyrophosphorylases reported to date. Among these amino acids, alanine substitution for Gly112, Arg116, or Lys123 severely diminished the activity, suggesting that Gly112, Arg116, or Lys123 are possible catalytic residues of the enzyme.
Assuntos
Candida albicans/enzimologia , Nucleotidiltransferases/química , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Clonagem Molecular , Proteínas Fúngicas/química , Teste de Complementação Genética , Humanos , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida/genética , Proteínas Recombinantes/química , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Like bacteria and many fungi, the pathogenic fungus Candida albicans can utilize GlcNAc as a carbon source for growth. A cluster of six genes was identified in the C. albicans genome. One of the genes in the cluster was CaNAG1, which is responsible for GlcN6P deaminase and is therefore essential for GlcNAc-dependent growth. The other five genes were designated CaNAG2, CaNAG3, CaNAG4, CaNAG5 and CaNAG6. The mRNA levels of CaNAG1, CaNAG2 and CaNAG5 were significantly induced by GlcNAc, whereas those of CaNAG3, CaNAG4 and CaNAG6 were not. Neither CaNAG2 nor CaNAG5 was essential for growth, but disruption of CaNAG2 or CaNAG5 greatly retarded the growth of cells using GlcNAc as the sole carbon source. Although no homolog of CaNAG2 or CaNAG5 was found in the Saccharomyces cerevisiae genome, CaNag2p displayed sequence similarities to Escherichia coli nagA, and CaNag5p is homologous to a wide variety of hexose kinases. When expressed as a fusion protein with glutathione S-transferase (GST), CaNag5p produced GlcNAc-P from GlcNAc in the presence of ATP, whereas GST alone did not. Furthermore, the recombinant GST-CaNag2p fusion protein converted GlcNAcP, which was produced by CaNag5p, into GlcNP. These results clearly demonstrate that CaNAG2 and CaNAG5 encode GlcNAcP deacetylase and GlcNAc kinase, respectively. CaNag5p recognized glucose and mannose as substrates, whereas the recently identified human GlcNAc kinase was specific to GlcNAc. Deletion of CaNAG2 or CaNAG5 markedly, and that of CaNAG1 moderately, attenuated the virulence of C. albicans in a mouse systemic infection model. Thus, it appears that GlcNAc metabolism of C. albicans is closely associated with its virulence.
Assuntos
Amidoidrolases/genética , Candida albicans/enzimologia , Candida albicans/genética , Proteínas Fúngicas , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Animais , Candida albicans/patogenicidade , Carbono/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Deleção de Genes , Glutationa Transferase/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Família Multigênica , Mutagênese Sítio-Dirigida , Plasmídeos , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Especificidade por Substrato , Fatores de TempoRESUMO
The mRNA-capping enzyme (mRNA 5'-guanylyltransferase) gene was cloned from a Candida albicans genomic DNA library by functional complementation of a Saccharomyces cerevisiae ceg1 delta null mutation. This gene, referred to as CGT1 (C. albicans guanylyltransferase 1), can encode a 52 kDa protein that is highly homologous to S. cerevisiae Ceg1p. CGT1 in a single-copy plasmid complemented the lethality of the S. cerevisiae ceg1 delta null mutation and, like S. cerevisiae Ceg1p, bacterially expressed Cgt1p was able to form a stable complex with the GMP moiety of GTP and to synthesize the cap structure in vitro, demonstrating that CGT1 is the C. albicans mRNA 5'-guanylyltransferase gene. CGT1 seemed to exist as a single copy in the C. albicans genome and was actively transcribed into mRNA. Another ORF was found in an opposite strand very close to the CGT1 locus. This gene shared significant sequence homology with S. cerevisiae FRE1, the gene encoding ferric reductase, and therefore was designated CFL1 (C. albicans ferric-reductase-like gene 1). Despite its sequence homology with S. cerevisiae FRE1, CFL1 mRNA was not induced by iron deprivation, and CFL1 did not complement the slow growth of a S. cerevisiae fre1 delta null mutant in the absence of iron, suggesting that CFL1 is functionally distinct from S. cerevisiae FRE1.
Assuntos
Candida albicans/genética , FMN Redutase , Proteínas Fúngicas/genética , NADH NADPH Oxirredutases/genética , Nucleotidiltransferases/genética , Oxirredutases , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Southern Blotting , Clonagem Molecular , Regulação Fúngica da Expressão Gênica , Biblioteca Gênica , Teste de Complementação Genética , Dados de Sequência Molecular , Mutagênese Insercional , Mapeamento por Restrição , Saccharomyces cerevisiae/genética , Homologia de Sequência de AminoácidosRESUMO
Cell wall beta-glucan in a pathogenic fungus, Candida albicans, is highly branched with beta-1,3 and beta-1,6 linkages. We have isolated the C. albicans cDNAs for KRE6 and SKN1, the genes required for beta-1,6-glucan synthesis in Saccharomyces cerevisiae. The results of Northern blot analysis revealed that C. albicans KRE6 was expressed at a higher level than SKN1 in the yeast phase, while SKN1 expression was strongly induced upon induction of hyphal formation. In addition, the C. albicans KRE6 and SKN1 mRNAs but not the actin mRNA were shortened during the yeast-hypha transition. Unlike S. cerevisiae, more than 50% of cell wall glucan was beta-1,6 linked in C. albicans. Neither beta-1,3-glucan nor beta-1,6-glucan was affected by the homozygous C. albicans skn1 delta null mutation. Although we never succeeded in generating the homozygous C. albicans kre6 delta null mutant, the hemizygous kre6 delta mutation decreased the KRE6 mRNA level by about 60% and also caused a more than 80% reduction of beta-1,6-glucan without affecting beta-1,3-glucan. The physiological function of KRE6 was further examined by studying gene regulation in C. albicans. When KRE6 transcription was suppressed by using the HEX1 promoter, C. albicans cells exhibited the partial defect in cell separation and increased susceptibility to Calcofluor White. These results demonstrate that KRE6 plays important roles in beta-1,6-glucan synthesis and budding in C. albicans.
Assuntos
Candida albicans/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Proteínas de Membrana/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , beta-Glucanas , Sequência de Aminoácidos , Sequência de Bases , Parede Celular/metabolismo , Clonagem Molecular , Regulação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Glucanos/biossíntese , Dados de Sequência Molecular , Mutagênese Insercional , RNA Mensageiro/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Solubilidade , Especificidade da EspécieRESUMO
Transformation of NIH3T3 cells with the ras, the sis, or the neu oncogene rendered cells less susceptible to cis-diamminedichloroplatinum(II). Since resistance to cis-diamminedichloroplatinum(II) is reported to be associated with increased levels of metallothionein, we examined effects of these oncogenes on metallothionein gene expression. NIH3T3 cells were first transfected with the lacZ gene whose transcription is under the control of mouse metallothionein I promoter and then with the ras, the sis, or the neu oncogene. The ras and the sis oncogenes increased beta-galactosidase activities which were induced either by metal (cadmium and zinc) or by glucocorticoid (dexamethasone), whereas the neu oncogene repressed its activity. When SV40 early promoter was used instead of metallothionein I promoter for the lacZ gene transcription, the beta-galactosidase activities were not affected by metal, dexamethasone, or any of these oncogenes. This result was coincident with that of reverse transcription polymerase chain reaction that metal-induced MT I mRNA was only detected in the sis- or the ras-transformed cells, whereas any of these oncogenes did not affect the metal-induced transcription of the MT II gene. These results demonstrate that the ras and the sis oncogenes upregulate the metal- or glucocorticoid-induced transcription from metallothionein I promoter, but the neu oncogene negatively regulates it. Thus, resistance to the chemotherapeutic agent by oncogenic transformation is partly associated with the metallothionein gene expression, and MT I and MT II gene expressions are differently controlled by different oncogenes.
Assuntos
Cisplatino/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Metalotioneína/genética , Proteínas Oncogênicas/toxicidade , Oncogenes/fisiologia , beta-Galactosidase/genética , Células 3T3/efeitos dos fármacos , Animais , Sequência de Bases , Northern Blotting , Cádmio/toxicidade , Primers do DNA/química , Dexametasona/toxicidade , Resistência a Medicamentos/genética , Regulação da Expressão Gênica/genética , Genes erbB-2/efeitos dos fármacos , Genes erbB-2/fisiologia , Genes ras/efeitos dos fármacos , Genes ras/fisiologia , Óperon Lac/efeitos dos fármacos , Óperon Lac/fisiologia , Camundongos , Dados de Sequência Molecular , Oncogenes/efeitos dos fármacos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética , Transfecção , Zinco/toxicidade , beta-Galactosidase/metabolismoRESUMO
The human mRNA 5'-capping enzyme cDNA was identified. Three highly related cDNAs, HCE1 (human mRNAcappingenzyme1), HCE1A and HCE1B , were isolated from a HeLa cDNA library. The HCE1 cDNA has the longest ORF, which can encode a 69 kDa protein. A short region of 69 bp in the 3'-half of the HCE1 ORF was missing in HCE1A and HCE1B , and, additionally, HCE1B has an early translation termination signal, which suggests that the latter two cDNAs represent alternatively spliced product. When expressed in Escherichia coli as a fusion protein with glutathione S -transferase, Hce1p displayed both mRNA 5'-triphosphatase (TPase) and mRNA 5'-guanylyltransferase (GTase) activities, and it formed a cap structure at the 5'-triphosphate end of RNA, demonstrating that it indeed specifies an active mRNA 5'-capping enzyme. The recombinant proteins derived from HCE1A and HCE1B possessed only TPase activity. When expressed from ADH1 promoter, HCE1 but not HCE1A and HCE1B complemented Saccharomyces cerevisiae CEG1 and CET1 , the genes for GTase and TPase, respectively. These results demonstrate that the N-terminal part of Hce1p is responsible for TPase activity and the C-terminal part is essential for GTase activity. In addition, the human TPase domain cannot functionally substitute for the yeast enzyme in vivo.
Assuntos
Nucleotidiltransferases/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , DNA Complementar/isolamento & purificação , DNA Complementar/metabolismo , Biblioteca Gênica , Teste de Complementação Genética , Células HeLa , Humanos , Dados de Sequência Molecular , Nucleotidiltransferases/química , Nucleotidiltransferases/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Saccharomyces cerevisiae harbors three chitin synthases termed Chs1p, Chs2p and Chs3p. Previously, we demonstrated that con1, a region that is highly conserved among all chitin synthases, contains amino acids essential for the catalytic activity of the enzyme and that Asp562, Gln601, Arg604, and Trp605 found in con1 together with Asp441 were probable catalytic sites of the enzyme. Here we report that another region, con2, in the C-terminal half of Chs2p is also conserved exclusively in chitin synthases that resemble S. cerevisiae Chs1p and Chs2p. Alanine substitutions for the conserved amino acids in con2 identified five amino acids, Asn797, His799, Asp800, Trp803, and Thr805, the mutation of which severely diminished enzymatic activity and the enzyme's ability to rescue the yeast chs2 delta chs3 delta null mutant strain. Although the activities of some of the mutant enzymes were too low to measure enzyme kinetics, most of the alanine mutations in con2 affected the kcat values rather than the K(m) values. Whereas a conservative mutation of Asn797 restored the activity, those of His799, Asp800, Trp803, and Thr805 did not. A fine alignment of the amino acid sequences of con2 and Chs3p revealed that Asp800, Trp803 and Thr805 are completely conserved near the C-terminal ends of Chs3p and its homologs in other fungi. On the basis of these findings, we propose that Asp800, Trp803, and Thr805 in con2 are additional residues involved in catalysis, and hypothesise that Asp800 together with the previously identified Asp441 and Asp562 serve as polar residues necessary for the acid-based catalytic reaction of chitin synthase.
Assuntos
Aminoácidos/química , Quitina Sintase/química , Quitina Sintase/genética , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Aminoácidos/metabolismo , Aminoácidos/fisiologia , Catálise , Quitina Sintase/metabolismo , Sequência Conservada , Análise Mutacional de DNA , Teste de Complementação Genética , Dados de Sequência Molecular , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Alinhamento de SequênciaRESUMO
The Saccharomyces cerevisiae gene, YFL017C, for a putative acetyltransferase was characterized. Disruption of YFL017C was lethal, leading to a morphology similar to those caused by the depletion of AGM1 or UAP1, the genes encoding phospho-N-acetylglucosamine mutase and UDP-N-acetylglucosamine pyrophosphorylase, respectively. This implies the involvement of YFL017C in UDP-N-acetylglucosamine synthesis. The recombinant protein for YFL017C displayed phosphoglucosamine acetyltransferase activities in vitro and utilized glucosamine 6-phosphate as the substrate. When incubated with Agm1p and Uap1p, the Yfl017c protein produced UDP-N-acetylglucosamine from glucosamine 6-phosphate. These results indicate that YFL017C specifies glucosamine-6-phosphate acetyltransferase; therefore, the gene was designated GNA1 (glucosamine-6-phosphate acetyltransferase). In addition, whereas bacterial phosphoglucosamine acetyltransferase and UDP-N-acetylglucosamine pyrophosphorylase activities are intrinsic in a single polypeptide, they are encoded by distinct essential genes in yeast. When the sequence of ScGna1p was compared with those of other acetyltransferases, Ile97, Glu98, Val102, Gly112, Leu115, Ile116, Phe142, Tyr143, and Gly147 were found to be highly conserved. When alanine was substituted for these amino acids, the enzyme activity for the substituted Phe142 or Tyr143 enzymes was severely diminished. Although the activity of Y143A was too low to perform kinetics, F142A displayed a significantly increased Km value for acetyl-CoA, suggesting that the Phe142 and Tyr143 residues are essential for the catalysis.
Assuntos
Acetiltransferases/genética , Saccharomyces cerevisiae/genética , Uridina Difosfato N-Acetilglicosamina/biossíntese , Acetiltransferases/química , Acetiltransferases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Catálise , Glucosamina 6-Fosfato N-Acetiltransferase , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae , Homologia de Sequência de AminoácidosRESUMO
As in Saccharomyces cerevisiae, the pathogenic fungus Candida albicans harbors three chitin synthases called CaChs1p, CaChs2p, and CaChs3p, which are structurally and functionally analogous to the S. cerevisiae ScChs2p, ScChs1p, and ScChs3p, respectively. In S. cerevisiae, ScCHS1, ScCHS2, and ScCHS3 are all non-essential genes; only the simultaneous disruption of ScCHS2 and ScCHS3 is lethal. The fact that a null mutation of the CaCHS1 is impossible, however, implies that CaCHS1 is required for the viability of C. albicans. To gain more insight into the physiological importance of CaCHS1, we identified and characterized a novel inhibitor that was highly specific to CaChs1p. RO-09-3143 inhibited CaChs1p with a K(i) value of 0.55 nm in a manner that was non-competitive to the substrate UDP-N-acetylglucosamine. RO-09-3143 also hampered the growth of the C. albicans cells with an MIC(50) value of 0.27 microm. In the presence of RO-09-3143, the C. albicans cells failed to form septa and displayed an aberrant morphology, confirming the involvement of the C. albicans Chs1p in septum formation. Although the effect of RO-09-3143 on the wild-type C. albicans was fungistatic, it caused cell death in the cachs2Delta null mutants but not in the cachs3Delta null mutants. Thus, it appears that in C. albicans, inhibition of CaChs1p causes cell growth arrest, but simultaneous inhibition of CaChs1p and CaChs2p is lethal.
Assuntos
Aminoglicosídeos , Antifúngicos/farmacologia , Candida albicans/fisiologia , Quitina Sintase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Oxazinas/farmacologia , Antibacterianos/farmacologia , Candida albicans/efeitos dos fármacos , Candida albicans/enzimologia , Divisão Celular/efeitos dos fármacos , Quitina Sintase/genética , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/genética , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Cinética , Proteínas Recombinantes/antagonistas & inibidores , Saccharomyces cerevisiae/enzimologiaRESUMO
A canCHS1A gene encoding the chitin synthase of Candida albicans was cloned. DNA sequencing and comparison with another canCHS1 gene described elsewhere indicated that the canCHS1A gene encoded a polypeptide with 775 amino acid residues, a protein with one less amino acid than that encoded by the canCHS1 gene. A six-base alteration was observed between the two genes, suggesting that the canCHS1A gene is a variant gene of the canCHS1. The pH profile/activity relationship of canChs1A in permeabilized cells was identical to that of canChs1. The canChs1A enzyme was competitively inhibited by polyoxin D (5.2 microM) and nikkomycin Z (12 microM). When the cloned gene was expressed in a Saccharomyces cerevisiae chs2 mutant that exhibited aberrant morphology, the normal structure was restored. We conclude that the function of the canCHS1A gene is similar to that of sacCHS2 in S. cerevisiae.
Assuntos
Aminoglicosídeos , Candida albicans/genética , Quitina Sintase/genética , Genes Fúngicos , Variação Genética , Antibacterianos/farmacologia , Sequência de Bases , Candida albicans/enzimologia , Clonagem Molecular , Relação Dose-Resposta a Droga , Inibidores Enzimáticos , Teste de Complementação Genética , Dados de Sequência Molecular , Nucleosídeos de Pirimidina/farmacologia , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Especificidade da EspécieRESUMO
The CHS2 and CHS3 genes of Candida albicans were disrupted. The double disruptant was still viable. Assessment of chitin and of calcofluor white resistance shows that CHS1 is responsible for septum formation and CHS3 is responsible for overall chitin synthesis otherwise. There were only small differences in virulence to immunocompromised mice of homozygous chs2 delta amd chs3 delta null mutants.
Assuntos
Candida albicans/crescimento & desenvolvimento , Quitina Sintase/genética , Quitina/biossíntese , Genes Bacterianos , Animais , Candida albicans/genética , Candida albicans/patogenicidade , Candidíase/microbiologia , Parede Celular/fisiologia , Proteínas Fúngicas/genética , Hospedeiro Imunocomprometido , Camundongos , MutagêneseRESUMO
We previously isolated the Saccharomyces cerevisiae HKR1 gene that confers on S. cerevisiae cells resistance to HM-1 killer toxin secreted by Hansenula mrakii (S. Kasahara, H. Yamada, T. Mio, Y. Shiratori, C. Miyamoto, T. Yabe, T. Nakajima, E. Ichishima, and Y. Furuichi, J. Bacteriol. 176:1488-1499, 1994). HKR1 encodes a type 1 membrane protein that contains a calcium-binding consensus sequence (EF hand motif) in the cytoplasmic domain. Although the null mutation of HKR1 is lethal, disruption of the 3' part of the coding region, which would result in deletion of the cytoplasmic domain of Hkr1p, did not affect the viability of yeast cells. This partial disruption of HKR1 significantly reduced beta-1,3-glucan synthase activity and the amount of beta-1,3-glucan in the cell wall and altered the axial budding pattern of haploid cells. Neither chitin synthase activity nor chitin content was significantly affected in the cells harboring the partially disrupted HKR1 allele. Immunofluorescence microscopy with an antibody raised against Hkr1p expressed in Escherichia coli revealed that Hkr1p was predominantly localized on the cell surface. The cell surface localization of Hkr1p required the N-terminal signal sequence because the C-terminal half of Hkr1p was detected uniformly in the cells. These results demonstrate that HKR1 encodes a cell surface protein that regulates both cell wall beta-glucan synthesis and budding pattern and suggest that bud site assembly is somehow related to beta-glucan synthesis in S. cerevisiae.