RESUMO
The human serine protease serine 2 TMPRSS2 is involved in the priming of proteins of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and represents a possible target for COVID-19 therapy. The TMPRSS2 gene may be co-expressed with SARS-CoV-2 cell receptor genes angiotensin-converting enzyme 2 (ACE2) and Basigin (BSG), but only TMPRSS2 demonstrates tissue-specific expression in alveolar cells according to single-cell RNA sequencing data. Our analysis of the structural variability of the TMPRSS2 gene based on genome-wide data from 76 human populations demonstrates that a functionally significant missense mutation in exon 6/7 in the TMPRSS2 gene is found in many human populations at relatively high frequencies, with region-specific distribution patterns. The frequency of the missense mutation encoded by rs12329760, which has previously been found to be associated with prostate cancer, ranged between 10% and 63% and was significantly higher in populations of Asian origin compared with European populations. In addition to single-nucleotide polymorphisms, two copy number variants were detected in the TMPRSS2 gene. A number of microRNAs have been predicted to regulate TMPRSS2 and BSG expression levels, but none of them is enriched in lung or respiratory tract cells. Several well-studied drugs can downregulate the expression of TMPRSS2 in human cells, including acetaminophen (paracetamol) and curcumin. Thus, the interactions of TMPRSS2 with SARS-CoV-2, together with its structural variability, gene-gene interactions, expression regulation profiles, and pharmacogenomic properties, characterize this gene as a potential target for COVID-19 therapy.
Assuntos
Tratamento Farmacológico da COVID-19 , COVID-19/terapia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Terapia de Alvo Molecular , SARS-CoV-2/fisiologia , Serina Endopeptidases/genética , Acetaminofen/farmacologia , Acetaminofen/uso terapêutico , Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Enzima de Conversão de Angiotensina 2/biossíntese , Enzima de Conversão de Angiotensina 2/genética , Ásia/epidemiologia , Basigina/biossíntese , Basigina/genética , Basigina/fisiologia , COVID-19/etnologia , COVID-19/genética , Curcumina/farmacologia , Curcumina/uso terapêutico , Europa (Continente)/epidemiologia , Éxons/genética , Frequência do Gene , Predisposição Genética para Doença , Variação Genética , Humanos , MicroRNAs/genética , Mutação de Sentido Incorreto , Testes Farmacogenômicos , Mapeamento de Interação de Proteínas , Receptores Virais/antagonistas & inibidores , Receptores Virais/biossíntese , Receptores Virais/genética , Serina Endopeptidases/biossíntese , Serina Endopeptidases/fisiologia , Análise de Célula Única , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
After the first case of Coronavirus disease 2019 (COVID-19) was reported in Wuhan, COVID-19 has rapidly spread to almost all parts of world. Angiotensin converting enzyme 2 (ACE2) receptor can bind to spike protein of SARS-CoV-2. Then, the spike protein of SARS-CoV-2 can be cleaved and activated by transmembrane protease, serine 2 (TMPRSS2) of the host cells for SARS-CoV-2 infection. Therefore, ACE2 and TMPRSS2 are potential antiviral targets for treatment of prevention of SARS-CoV-2 infection. In this study, we discovered that 10-250 µg/mL of GB-2, from Tian Shang Sheng Mu of Chiayi Puzi Peitian Temple, can inhibit ACE2 mRNA expression and ACE2 and TMPRSS2 protein expression in HepG2 and 293 T cells without cytotoxicity. GB-2 treatment could decrease ACE2 and TMPRSS2 expression level of lung tissue and kidney tissue without adverse effects, including nephrotoxicity and hepatotoxicity, in animal model. In the compositions of GB-2, we discovered that 50 µg/mL of theaflavin could inhibit protein expression of ACE2 and TMPRSS2. Theaflavin could inhibit the mRNA expression of ACE2. In conclusion, our results suggest that GB-2 and theaflavin could act as potential compounds for ACE2 and TMPRSS2 inhibitors in the further clinical study.
Assuntos
Enzima de Conversão de Angiotensina 2/biossíntese , Medicamentos de Ervas Chinesas/farmacologia , Serina Endopeptidases/biossíntese , Enzima de Conversão de Angiotensina 2/genética , Inibidores da Enzima Conversora de Angiotensina/isolamento & purificação , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Animais , COVID-19/epidemiologia , Medicamentos de Ervas Chinesas/isolamento & purificação , Medicamentos de Ervas Chinesas/uso terapêutico , Expressão Gênica/efeitos dos fármacos , Células HEK293 , Células Hep G2 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Inibidores de Proteases/isolamento & purificação , Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêutico , SARS-CoV-2 , Serina Endopeptidases/genética , Tratamento Farmacológico da COVID-19RESUMO
Reelin is an extracellular glycoprotein which contributes to synaptic plasticity and function of memory in the adult brain. It has been indicated that the Reelin signaling cascade participates in Alzheimer's disease (AD). Besides the neurons, glial cells such as astrocytes also express Reelin protein. While functional loss of astrocytes has been reported to be associated with AD, dysfunction of astrocytic Reelin signaling pathway has not received much attention. Therefore, we investigated the effects of α-boswellic acid (ABA) as one of the major component of Boswellia serrata resin on primary fetal human astrocytes under a stress paradigm as a possible model for AD through study on Reelin cascade. For this aim, we used streptozotocin (STZ), in which from an outlook generates Alzheimer's hallmarks in astrocytes, and assayed Reelin expression, Tau and Akt phosphorylation as well as reactive oxygen species (ROS) generation and apoptosis in the presences of ABA. Our results indicated that while STZ (100 µM) down-regulated the expression of Reelin, ABA (25 µM) up-regulated its expression (p < 0.01) for 24 h. ABA efficiently reduced hyperphosphorylated Tau (Ser404) in STZ-treated astrocytes (p < 0.01). Furthermore, STZ-induced apoptosis by increasing cleaved caspase three (p < 0.01) and ROS generation (p < 0.01), a further pathological hallmark of Tauopathy. On the other hand, ABA decreased ROS generation and promoted proliferation of astrocytes through elevating Survivin expression (p < 0.01). These results showed that ABA could be considered as a potent therapeutic agent for prevention and decreasing the progression of Alzheimer's hallmarks in astrocytes; however, more in vivo studies would be needed.
Assuntos
Astrócitos/efeitos dos fármacos , Moléculas de Adesão Celular Neuronais/biossíntese , Proteínas da Matriz Extracelular/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Triterpenos Pentacíclicos/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Serina Endopeptidases/biossíntese , Proteínas tau/metabolismo , Apoptose/efeitos dos fármacos , Astrócitos/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Proteínas da Matriz Extracelular/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hipotálamo/citologia , Hipotálamo/embriologia , Proteínas Inibidoras de Apoptose/biossíntese , Proteínas Inibidoras de Apoptose/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Estresse Oxidativo , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína Reelina , Serina Endopeptidases/genética , Estreptozocina/farmacologia , SurvivinaRESUMO
BACKGROUND: Proprotein convertase subtilisin/kexin type 9 (PCSK9) post-transcriptionally downregulates the low-density lipoprotein receptor (LDLR) by binding to the receptor's epidermal growth factor repeat A on the cell surface and shuttling the LDLR to the lysosomes for degradation. Mutations in the PCSK9 gene have been shown to cause either hypo- or hypercholesterolemia. Here we investigated the effect of berberine, a natural plant extract, on PCSK9 expression in HepG2 cells. RESULTS: Berberine decreases PCSK9 mRNA and protein levels in a time- and dose-dependent manner. This was not due to increased degradation of PCSK9 mRNA but most likely due to a decreased transcription of the PCSK9 gene. We also show that a combination of berberine and mevastatin increases LDLR mRNA and protein levels, while suppressing the increase in PCSK9 mRNA levels caused by mevastatin alone. CONCLUSION: Berberine may be a useful supplement to statin treatment due to its effects on PCSK9 mRNA and protein levels.
Assuntos
Berberina/farmacologia , Regulação da Expressão Gênica , Serina Endopeptidases/biossíntese , Anticolesterolemiantes/farmacologia , Linhagem Celular , Dactinomicina/farmacologia , Relação Dose-Resposta a Droga , Humanos , Lovastatina/análogos & derivados , Lovastatina/farmacologia , Extratos Vegetais/metabolismo , Pró-Proteína Convertase 9 , Pró-Proteína Convertases , RNA Mensageiro/metabolismo , Receptores de LDL/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Transcrição GênicaRESUMO
Celiac disease is caused by an immune response to the dietary protein gluten. The only available treatment is the strict exclusion of gluten from the diet; however, this is marred by the virtual omnipresence of this protein. The enzymatic degradation of gluten might become an alternative to the gluten-free diet, and recent work indicates that such approaches are getting close to being tested in clinical trials.
Assuntos
Doença Celíaca/dietoterapia , Suplementos Nutricionais , Glutens/metabolismo , Serina Endopeptidases/farmacologia , Biotransformação/fisiologia , Doença Celíaca/fisiopatologia , Cisteína Endopeptidases/administração & dosagem , Cisteína Endopeptidases/biossíntese , Cisteína Endopeptidases/farmacocinética , Cisteína Endopeptidases/farmacologia , Humanos , Prolil Oligopeptidases , Serina Endopeptidases/administração & dosagem , Serina Endopeptidases/biossíntese , Serina Endopeptidases/farmacocinéticaRESUMO
The purpose of our investigations was to evaluate the supposed beneficial effects of gamma-linolenic (GLA) and docosahexaenoic acid (DHA) in a canine mastocytoma cell line (C2) as a model for canine atopic dermatitis. Cells were cultured in a basic medium (DEH) and in DEH supplemented with 14.3 microM GLA (DEH-GLA) or 14.3 microM DHA (DEH-DHA) for 8 days. Chymase and tryptase activity, as well as histamine and prostaglandin (PG)E(2) release were measured. To stimulate histamine and PGE(2) release, cells were incubated with the wasp venom peptide mastoparan (50 microM) for 30 min. GLA increased tryptase activity and decreased histamine release after C2 stimulation. DHA diminished PGE(2) production in activated C2. These results support the prescription of GLA- and DHA-enriched diets to reduce inflammatory signs in canine atopic dermatitis.
Assuntos
Dermatite Atópica/veterinária , Ácidos Docosa-Hexaenoicos/farmacologia , Doenças do Cão/tratamento farmacológico , Mediadores da Inflamação/metabolismo , Mastócitos/efeitos dos fármacos , Ácido gama-Linolênico/farmacologia , Animais , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Quimases , Dermatite Atópica/tratamento farmacológico , Suplementos Nutricionais , Dinoprostona/biossíntese , Ácidos Docosa-Hexaenoicos/administração & dosagem , Ácidos Docosa-Hexaenoicos/uso terapêutico , Cães , Histamina/biossíntese , Mastócitos/metabolismo , Serina Endopeptidases/biossíntese , Triptases , Ácido gama-Linolênico/administração & dosagem , Ácido gama-Linolênico/uso terapêuticoRESUMO
OBJECTIVE: To establish a cell model of secreted alkaline phosphatase (SEAP) co-controlled by HCV 5'NCR and NS3 serine protease in an effort to develop new antiviral agents. METHODS: The fragments of HCV 5'NCR and NS3/4A-SEAP were amplified by PCR. They were fused into pBluescript SK+ to generate 5'NCR-NS3/4A-SEAP chimeric plasmid. The resulting chimeric gene was subcloned into HindIII/Bsu36 I site of pSEAP2-Control (a SEAP eukaryotic expression plasmid), to generate pNCR-NS3/4A-SEAP, in which the SEAP was fused in-frame to the downstream of NS4A/4B cleavage site. The SEAP activity in the culture media of transiently transfected cells was monitored quantitatively. The regulatory effect of HCV 5'NCR and NS3 serine protease on SEAP expression was measured by treatment of transfected cells with antisense oligodeoxynucleotide (ASODN) against HCV 5'NCR and TPCK, a irreversible serine protease inhibitor. RESULTS: The SEAP activity in the culture media reached 80801+/-4794 RLU, and was significantly inhibited by 5 micromol/L, 10 micromol/L of ASODN (t=4.315, p<0.01; t=6.985, p<0.001) and 100 micromol/L of TPCK (t=6.949, P<0.001). CONCLUSION: A cell model of SEAP co-controlled by HCV 5'NCR and NS3 serine protease has been successfully established. This might promote the screening of anti-viral drugs
Assuntos
Fosfatase Alcalina/metabolismo , Hepacivirus/genética , Hepatócitos/virologia , Serina Endopeptidases/biossíntese , Proteínas não Estruturais Virais/genética , Antivirais , Avaliação Pré-Clínica de Medicamentos , Hepatócitos/enzimologia , Humanos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Serina Endopeptidases/genética , Proteínas não Estruturais Virais/biossínteseRESUMO
In the present work, three zebrafish cDNA clones encoding transferrin, intestinal fatty acid binding protein (IFABP), and elastaseB were cloned and their expression patterns in early zebrafish development were characterized as differentiation markers for the three major endoderm organs: liver, intestine, and exocrine pancreas. transferrin and ifabp mRNAs exhibit a biphasic expression pattern during early development. transferrin mRNAs were first expressed at approximately 7 hours postfertilization (hpf) in the yolk syncytial layer (YSL) and later in the liver rudiment (from approximately 48 hpf) and in the esophagus transiently (72-96 hpf). Ifabp mRNAs were initially expressed in the YSL at the ventral side during late epiboly (8-9 hpf), spread throughout the YSL of later stage embryos, and appeared in the intestine rudiment at approximately 36 hpf. In contrast to the transferrin and ifabp mRNAs, elastaseB mRNAs were not expressed in the yolk sac or YSL, and these transcripts were detected exclusively in the exocrine pancreas after approximately 56 hpf.
Assuntos
Proteínas de Transporte/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Serina Endopeptidases/biossíntese , Transferrina/biossíntese , Sequência de Aminoácidos , Animais , Northern Blotting , Carpas , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Diferenciação Celular , Núcleo Celular/metabolismo , DNA Complementar/metabolismo , Proteínas de Ligação a Ácido Graxo , Hibridização In Situ , Mucosa Intestinal/metabolismo , Ferro/metabolismo , Fígado/metabolismo , Dados de Sequência Molecular , Pâncreas/metabolismo , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/genética , Fatores de Tempo , Transferrina/genética , Saco Vitelino/metabolismo , Peixe-ZebraRESUMO
AIM: To study the regulatory effects of Ganoderma lucidum polysaccharides (Gl-PS) on cytotoxicity and mechanism of specific cytotoxic T-lymphocytes (CTL) induced by dendritic cells (DC) in vitro during the stage of antigen presentation. METHODS: Cultured murine bone marrow-derived DC were pulsed with P815 tumor cell lysates and co-incubated with or without various concentrations of Gl-PS (0.8, 3.2, or 12.8 mg/L) at the same time. P815 specific CTL were induced by spleen lymphocytes stimulated with mature DC. Non-adherent cells and culture supernatants were harvested on d 5 for analysis of specific cytotoxicity with lactate dehydrogenase (LDH) activity assay, mRNA expression of IFNgamma, granzyme B with RT-PCR assay, and protein expression of IFNgamma, granzyme B with ELISA or Western blot assay, respectively. RESULTS: Three concentrations of Gl-PS promoted LDH activities released into culture supernatants (P<0.01). It also increased mRNA expression of IFNgamma in CTL (Gl-PS 12.8 mg/L vs RPMI medium 1640, P<0.05) and granzyme B in CTL (P<0.01). Protein production of IFNgamma in culture supernatants (P<0.05) and protein expression of granzyme B in CTL (Gl-PS 12.8 mg/L vs RPMI medium 1640, P<0.05) were also augmented by Gl-PS. CONCLUSION: Gl-PS is shown to promote the cytotoxicity of specific CTL induced by DC which were pulsed with P815 tumor antigen during the stage of antigen presentation, and the mechanism of cytotoxicity is believed to be going through IFNgamma and granzyme B pathways.
Assuntos
Células Dendríticas/imunologia , Interferon gama/biossíntese , Polissacarídeos/farmacologia , Reishi , Linfócitos T Citotóxicos/metabolismo , Animais , Apresentação de Antígeno , Células da Medula Óssea/citologia , Células Dendríticas/citologia , Feminino , Granzimas , Interferon gama/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plantas Medicinais/química , Polissacarídeos/isolamento & purificação , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reishi/química , Serina Endopeptidases/biossíntese , Serina Endopeptidases/genética , Linfócitos T Citotóxicos/enzimologiaRESUMO
Anther development in angiosperms culminates in the programmed cell death of specific tissues to facilitate the release of pollen. Despite a wealth of morphological descriptions of this process, there have been few reports on the regulation of dehiscence or the coordination of events between tissues. We have cloned an anther-specific tomato gene encoding a serine proteinase that is expressed during meiosis and late microsporogenesis. The conceptualized tomato meiotic proteinase (TMP) is a member of a family of genes that exhibit characteristics of mammalian proprotein convertases. To examine the role of TMP in microsporogenesis, we generated transgenic plants harboring an antisense construct of the gene. Some of these plants produced little or no detectable TMP, yet no phenotypic abnormalities were observed. Zymogram analyses revealed that multiple proteinases are present in mature anthers and that proteinase activity increases as development proceeds. Taken together, these data indicate that the role of TMP during microsporogenesis, if any, may be compensated for by other proteinases.
Assuntos
Genes de Plantas , Oligonucleotídeos Antissenso/farmacologia , Pólen/fisiologia , Serina Endopeptidases/genética , Solanum lycopersicum/genética , Sequência de Aminoácidos , Clonagem Molecular , DNA/metabolismo , DNA Complementar/metabolismo , Immunoblotting , Dados de Sequência Molecular , Plantas Geneticamente Modificadas , Pólen/genética , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/biossíntese , Fatores de Tempo , TransgenesRESUMO
Anthracycline antibiotics are effective anticancer agents but their use is limited due to unwanted adverse side effects. The toxic effects of doxorubicin (adriamycin) include the development of defined cardiac lesions leading to cardiomyopathy in some patients. This has been reported to be due to reductions in cardiac protein synthesis. However, virtually all of these previous studies have failed to consider the specific radioactivity of the precursor pool in their measurements or have carried out their studies in vitro. To further resolve the above we measured fractional rates of cardiac protein synthesis using the "flooding dose" method in rats treated with adriamycin (5 mg/kg body wt). Controls were identically treated and injected with saline. At 2.5 or 24 h after adriamycin injection, rates of protein synthesis were measured with a flooding dose of l-[4-(3)H]phenylalanine. Measurements included free (S(i)) and protein-bound (S(b)) phenylalanine-specific radioactivities, the protein synthetic capacity (RNA/protein ratio; C(s)), the fractional rates of protein synthesis calculated from the ratio S(b)/S(i), and the protein synthetic efficiency calculated from the ratio k(s)/C(s). Complementary analyses included assays of lysosomal (cathepsins B, D, H, and L and diaminopeptidases I and II) and cytoplasmic proteases (alanyl aminopeptidase, arginyl aminopeptidase, leucyl aminopeptidase, diaminopeptidase IV, tripeptidyl aminopeptidase, and proline endopeptidase). These enzymes constitute the most active proteases in this tissue and represent an index of protein degradation capacity in cardiac muscle. The results showed that in 2.5-h dosed rats, adriamycin had no effect on S(i), S(b), C(s), k(s), or k(RNA) (P > 0.05, not significant (NS) in all instances). In 2.5-h dosed rats, levels of diaminopeptidase I activity were reduced (P < 0.05), whereas the activities of other proteases were not significantly altered (NS in all instances). In 24-h dosed rats, adriamycin reduced cardiac S(b) (P < 0.001), which would normally be interpreted as a reduction in protein synthesis. However, S(i) was also decreased in 24-h adriamycin-injected rats (P < 0.025%). C(s) was not changed (NS). Consequently, the calculated k(s) and k(RNA) values were not significantly affected in 24-h adriamycin-dosed rats (NS). There were also significant reductions in proline endopeptidase activities in rats exposed for 24 h to adriamycin. The activities of other proteases were not significantly affected at this time point (NS in all instances). In conclusion, adriamycin reduces amino acid labeling of cardiac proteins, an effect that is a consequence of altered free phenylalanine-specific radioactivities. There was some evidence of limited altered intracellular proteolysis.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Catepsina C/biossíntese , Doxorrubicina/farmacologia , Miocárdio/metabolismo , Biossíntese de Proteínas , Serina Endopeptidases/biossíntese , Animais , Catepsina C/genética , Indução Enzimática , Coração/efeitos dos fármacos , Masculino , Miocárdio/enzimologia , Fenilalanina/metabolismo , Prolil Oligopeptidases , Ratos , Ratos Wistar , Serina Endopeptidases/genéticaRESUMO
Proteases have been implicated in the extracellular modulation required for tumor growth and invasion. In an effort to categorize those proteases contributing to ovarian carcinoma progression, we have utilized redundant primers to conserved amino acid (AA) domains surrounding the catalytic triad of His, Asp and Ser to amplify serine proteases that are differentially expressed in carcinomas. Using this method, we have identified and cloned a serine protease named TADG-15 (tumor-associated differentially expressed gene 15) that is overexpressed in ovarian tumors. TADG-15 is a transmembrane multidomain serine protease which includes ligand binding domains and a serine protease in the extracellular space.
Assuntos
Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/enzimologia , Serina Endopeptidases/biossíntese , Sequência de Aminoácidos , Animais , Ácido Aspártico/química , Sequência de Bases , Northern Blotting , Western Blotting , Domínio Catalítico , Linhagem Celular , Membrana Celular/metabolismo , Clonagem Molecular , Primers do DNA/metabolismo , DNA Complementar/metabolismo , Feminino , Histidina/química , Humanos , Imuno-Histoquímica , Ligantes , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Ovário/metabolismo , Ovário/patologia , Reação em Cadeia da Polimerase , Ligação Proteica , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Serina/química , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
Retinoic acid-treated mesenchyme cells of the budding ascidian Polyandrocarpa misakiensis acquire an organizer activity to induce a secondary body axis when implanted into developing buds. We identified several different mRNAs that were upregulated in the mesenchyme cells after retinoic acid treatment. We isolated a cDNA clone corresponding to one of these mRNAs. The C-terminal region of the predicted protein product is homologous to the catalytic domain of serine proteases that belong to the trypsin family. The N-terminal region contains several types of protein-protein interaction domains. We therefore named this protein tunicate retinoic acid-inducible modular protease (TRAMP). Expression of the TRAMP mRNA in mesenchyme cells during budding and its upregulation by retinoic acid were demonstrated by reverse transcription-PCR and in situ hybridization. A glutathione S-transferase-TRAMP fusion protein showed a protease activity with trypsin-like substrate specificity and stimulated proliferation of the cell line established in this species.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Serina Endopeptidases/genética , Tretinoína/farmacologia , Urocordados/enzimologia , Urocordados/crescimento & desenvolvimento , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA Complementar , Indução Enzimática , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Dados de Sequência Molecular , Especificidade de Órgãos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/biossíntese , Serina Endopeptidases/química , Transcrição Gênica/efeitos dos fármacos , Urocordados/genéticaRESUMO
The plant pathogenic fungus Verticillium dahliae produced extracellular alkaline protease activity when grown in liquid medium supplemented with a protein source. A serine protease was purified 80-fold in a single step, using cation-exchange chromatography, from the filtrate of cultures grown with skim milk as a protein source. N-terminal amino acid sequence analysis of the 30-kDa protein (VDP30) that copurified with the serine protease activity suggested that VDP30 is a trypsin-like protein. The purified enzyme hydrolyzed the synthetic substrate N alpha-benzoyl-DL-arginine p-nitroanilide hydrochloride (BAPNA), and the activity on BAPNA was inhibited by leupeptin, further verifying the trypsin-like nature of the enzyme.
Assuntos
Fungos Mitospóricos/enzimologia , Fungos Mitospóricos/metabolismo , Serina Endopeptidases/biossíntese , Serina Endopeptidases/isolamento & purificação , Sequência de Aminoácidos , Animais , Benzoilarginina Nitroanilida/metabolismo , Biomassa , Cromatografia por Troca Iônica , Meios de Cultura/metabolismo , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Leupeptinas/farmacologia , Leite/metabolismo , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/farmacologia , Fatores de TempoRESUMO
Preadipocyte factor-1 (Pref-1), a novel gene product isolated from murine preadipocyte 3T3-L1 cells, is thought to function as a negative regulator of adipocyte differentiation. We investigated the regulation of Pref-1 expression in 3T3-L1 preadipocytes during proliferation, growth arrest, and early differentiation in the presence and absence of three well described differentiation antagonists: interleukin-11 (IL-11), transforming growth factor-beta, and tumor necrosis factor-alpha. Northern blot analysis was used to determine messenger RNA (mRNA) steady state expression of Pref-1 and two differentiation-specific genes, adipsin and glycerol-3-phosphate dehydrogenase. We confirmed that Pref-1 mRNA is abundant in proliferating preadipocytes and that its expression is dramatically reduced early in differentiation. However, proliferating and growth-arrested cells treated with the differentiation inhibitor IL-11 demonstrated a modest decrease in Pref-1 mRNA abundance. Transforming growth factor-beta and tumor necrosis factor-alpha had little effect. The reduction of Pref-1 mRNA was most dramatic in differentiating preadipocytes treated with IL-11, occurring despite inhibition of adipogenesis, as judged by cell morphology and adipocyte-specific gene expression (adipsin and glycerol-3-phosphate dehydrogenase). This effect of IL-11 on Pref-1 suggests that different mechanisms are responsible for the IL-11-induced and the differentiation- associated down-regulation of Pref-1, thus dissociating Pref-1 regulation from differentiation. We conclude that Pref-1 expression is not a reliable marker of preadipocytes, and that decreased Pref-1 abundance does not function as a trigger for adipocyte differentiation.
Assuntos
Adipócitos/citologia , Diferenciação Celular , Regulação da Expressão Gênica , Proteínas de Membrana/biossíntese , Proteínas Repressoras/biossíntese , 1-Metil-3-Isobutilxantina/farmacologia , Células 3T3 , Adipócitos/metabolismo , Animais , Sequência de Bases , Northern Blotting , Proteínas de Ligação ao Cálcio , Divisão Celular/efeitos dos fármacos , Fator D do Complemento , Primers do DNA , DNA Complementar , Dexametasona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glicerolfosfato Desidrogenase/biossíntese , Insulina/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular , Interleucina-1/farmacologia , Cinética , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Serina Endopeptidases/biossíntese , Transcrição Gênica/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Our objective was to determine the coordination of transcript and/or protein abundances of stromal enzymes during leaf senescence. First trifolioliate leaves of Phaseolus vulgaris L. plants were sampled beginning at the time of full leaf expansion; at this same time, half of the plants were switched to a nutrient solution lacking N. Total RNA and soluble protein abundances decreased after full leaf expansion whereas chlorophyll abundance remained constant; N stress enhanced the decline in these traits. Abundances of ribulose-1,5-bisposphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39), Rubisco activase and phosphoribulokinase (Ru5P kinase; EC 2.7.1.19) decreased after full leaf expansion in a coordinated manner for both treatments. In contrast, adenosine diphosphate glucose (ADPGlc) pyrophosphorylase (EC 2.7.7.27) abundance was relatively constant during natural senescence but did decline similar to the other enzymes under N stress. Northern analyses indicated that transcript abundances for all enzymes declined markedly on a fresh-weight basis just after full leaf expansion. This rapid decline was particularly strong for the Rubisco small subunit (rbcS) transcript. The decline was enhanced by N stress for rbcS and Rubisco activase (rca), but not for Ru5P kinase (prk) and ADPGlc pyrophosphorylase (agp). Transcripts of the Clp protease subunits clpC and clpP declined in abundance just after full leaf expansion, similar to the other mRNA species. When Northern blots were analyzed using equal RNA loads, rbcS transcripts still declined markedly just after full leaf expansion whereas rca and clpC transcripts increased over time. The results indicated that senescence was initiated near the time of full leaf expansion, was accelerated by N stress, and was characterized by large decline in transcripts of stromal enzymes. The decreased mRNA abundances were in general associated with steadily declining stromal protein abundances, with ADPGlc pyrophosphorylase being the notable exception. Transcript analyses for the Clp subunits supported a recent report (Shanklin et al., 1995, Plant Cell 7: 1713-1722) indicating that the Clp protease subunits were constitutive throughout development and suggested that ClpC and ClpP do not function as a senescence-specific proteolytic system in Phaseolus.
Assuntos
Fabaceae/enzimologia , Regulação da Expressão Gênica de Plantas , Plantas Medicinais , Serina Endopeptidases/biossíntese , Adenosina Trifosfatases/biossíntese , Sequência de Bases , Primers do DNA , Endopeptidase Clp , Fabaceae/crescimento & desenvolvimento , Regulação Enzimológica da Expressão Gênica , Glucose-1-Fosfato Adenililtransferase , Nucleotidiltransferases/biossíntese , Folhas de Planta , Reação em Cadeia da Polimerase , Biossíntese de Proteínas , RNA Mensageiro/biossíntese , Serina Endopeptidases/química , Transcrição GênicaRESUMO
We previously reported on the purification of a serum calcium-decreasing factor, referred to as caldecrin, from porcine pancreas, that is thought to be a serine protease (Tomomura, A., Fukushige, T., Noda, T., Noikura, T., and Saheki, T. (1992) FEBS Lett. 301, 277-281). In the present study, we purified caldecrin from rat pancreas and determined its primary structure by cDNA cloning. The predicted caldecrin protein is presumed to be synthesized as a preproenzyme of 268 amino acids with a signal peptide of 16 amino acids and an activation peptide of 13 amino acids, and is, with the exception of a central region, almost identical to the reported rat pancreatic elastase IV sequence. The caldecrin gene is selectively expressed in the pancreas, as judged by Northern blot analysis. After expression in BMT-10 cells, immunoreactive caldecrin was found in the culture supernatant, and it inhibited the parathyroid hormone-stimulated 45Ca release from cultured fetal long bones. Catalytic site mutants were synthesized in a baculovirus system, and recombinant mutants also decreased the serum calcium level of mice. These data implicate caldecrin, a protease closely related to elastase IV, in the regulation of blood calcium levels.
Assuntos
Reabsorção Óssea , Antagonistas de Hormônios/farmacologia , Pâncreas/metabolismo , Serina Endopeptidases/biossíntese , Serina Endopeptidases/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Bioensaio , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Cálcio/metabolismo , Clonagem Molecular , Primers do DNA , DNA Complementar , Feto , Expressão Gênica , Antagonistas de Hormônios/isolamento & purificação , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Técnicas de Cultura de Órgãos , Especificidade de Órgãos , Pâncreas/química , Elastase Pancreática/biossíntese , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Mutação Puntual , Reação em Cadeia da Polimerase , Sinais Direcionadores de Proteínas/biossíntese , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/isolamento & purificação , SuínosRESUMO
OBJECTIVE: Swainsonine (SW), an inhibitor of mammalian Golgi alpha-mannosidase II, blocks the processing of high mannose to complex type oligosaccharides. In this study, the effect of SW on the cytotoxicity of lymphokine-activated killer (LAK) cells against autologous thyroid cancer was investigated. DESIGN: Peripheral blood lymphocytes from patients with thyroid cancer were incubated with recombinant interleukin 2 (100 U/mL) and 0.5 mg/L of SW for 7 days, and thyroid cancer cells obtained from surgical specimens were pretreated with SW (0.5 mg/L) for 18 hours. The cytotoxicity of SW-treated LAK cells against tumor cells tested in a standard 4-hour radioactive chromium Cr 51 release assay. RESULTS: The cytotoxicity of SW-treated LAK cells against autologous thyroid cancer cells was found to be significantly greater than that of standard LAK cells incubated with interleukin 2 alone. The N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester esterase activity of LAK cells, this activity being a cytotoxic factor that is necessary for the lethal hit stage, was also increased by SW treatment. Further, thyroid cancer cells incubated with SW, as compared with nontreated tumor cells, showed much higher susceptibility to LAK killing. CONCLUSIONS: Our results suggest that SW might have potential immunomodulatory properties in the treatment of thyroid cancer.
Assuntos
Adenocarcinoma Papilar/imunologia , Adjuvantes Imunológicos/farmacologia , Citotoxicidade Imunológica/efeitos dos fármacos , Células Matadoras Ativadas por Linfocina/imunologia , Swainsonina/farmacologia , Neoplasias da Glândula Tireoide/imunologia , Imunofluorescência , Granzimas , Humanos , Interleucina-2/farmacologia , Células Matadoras Ativadas por Linfocina/efeitos dos fármacos , Células Matadoras Ativadas por Linfocina/enzimologia , Serina Endopeptidases/biossíntese , Células Tumorais Cultivadas/imunologiaRESUMO
The proteolytic activity produced by a Bacillus subtilis isolated from a hot spring was investigated. Maximum protease production was obtained after 38 h of fermentation. Effects of various carbon and nitrogen sources indicate the requirement of starch and bacteriological peptone to be the best inducers for maximum protease production. Requirement for phosphorus was very evident, and the protease was secreted over a wide range of pH 5-11. The partially purified enzyme was stable at 60 degrees C for 30 min. Calcium ions were effective in stabilizing the enzyme, especially at higher temperature. The enzyme was extremely salt tolerant and retained 100% activity in 5M NaCl over 96 h. The molecular weight of the purified enzymes as determined by SDS-PAGE was 28,000. The enzyme was completely inactivated by PMSF, but little affected by urea, sodium dodecyl sulfate, and sodium tripoly phosphate.