RESUMO
Herpes simplex virus 1 (HSV-1) causes a number of clinical manifestations including cold sores, keratitis, meningitis and encephalitis. Although current drugs are available to treat HSV-1 infection, they can cause side effects such as nephrotoxicity. Moreover, owing to the emergence of drug-resistant HSV-1 strains, new anti-HSV-1 compounds are needed. Because many viruses exploit cellular host proteases and encode their own viral proteases for survival, we investigated the inhibitory effects of a panel of protease inhibitors (TLCK, TPCK, E64, bortezomib, or MG132) on HSV-1 replication and several host cell signaling pathways. We found that HSV-1 infection suppressed c-Raf-MEK1/2-ERK1/2-p90RSK signaling in host cells, which facilitated viral replication. The mechanism by which HSV-1 inhibited ERK signaling was mediated through the polyubiquitination and proteasomal degradation of Ras-guanine nucleotide-releasing factor 2 (Ras-GRF2). Importantly, the proteasome inhibitor MG132 inhibited HSV-1 replication by reversing ERK suppression in infected cells, inhibiting lytic genes (ICP5, ICP27 and UL42) expression, and overcoming the downregulation of Ras-GRF2. These results indicate that the suppression of ERK signaling via proteasomal degradation of Ras-GRF2 is necessary for HSV-1 infection and replication. Given that ERK activation by MG132 exhibits anti-HSV-1 activity, these results suggest that the proteasome inhibitor could serve as a novel therapeutic agent against HSV-1 infection.
Assuntos
Antivirais/farmacologia , Herpesvirus Humano 1/efeitos dos fármacos , Leupeptinas/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Animais , Bortezomib/farmacologia , Caspases/metabolismo , Chlorocebus aethiops , Replicação do DNA/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Leucina/análogos & derivados , Leucina/farmacologia , Modelos Biológicos , Inibidor de NF-kappaB alfa/metabolismo , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Poliubiquitina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Estabilidade Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Tosilina Clorometil Cetona/farmacologia , Tosilfenilalanil Clorometil Cetona/farmacologia , Células Vero , Replicação Viral/efeitos dos fármacosRESUMO
Among the many immunomodulatory and anti-tumor activities, IFN-γ up-regulates tumor cell death mediated by Fas receptor (FasR). Our and several other studies have demonstrated the involvement of trypsin-like proteases (TLPs) in the mode of action of IFN-γ. In the present study, we tried to unravel the role of serine proteases in IFN-γ induced Fas-mediated cell death. Our present results show that both tosyl-l-Lysine chloromethylketone (TLCK), a trypsin like protease inhibitor and tosyl-l-phenylalanine chloromethylketone (TPCK) - a chymotrypsin like protease (CLP) inhibitor, sensitize HeLa cells to Fas-mediated cell death. The combined effect of these protease inhibitors with anti-Fas was stronger than additive. In contrast, elastase inhibitor III (EI), which also contains the chloromethyl ketone moiety, was not active. Furthermore, co-addition of TLCK or TPCK with IFN-γ markedly enhanced Fas-induced cell death. IFN-γ led to up-regulation of FasR on its own, which was further enhanced by the co-addition of TLCK or TPCK. This was evident both by increased expression of Fas receptor on cell surface and by elevated Fas mRNA level. This study may provide the basis for the design of a novel combinatory therapeutic strategy that could enhance the eradication of tumors.
Assuntos
Apoptose/efeitos dos fármacos , Interferon gama/farmacologia , Neoplasias/tratamento farmacológico , Inibidores de Serina Proteinase/farmacologia , Receptor fas/biossíntese , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Proteína Ligante Fas/metabolismo , Células HT29 , Células HeLa , Humanos , Neoplasias/patologia , RNA Mensageiro/biossíntese , Serina Endopeptidases/metabolismo , Tosilina Clorometil Cetona/farmacologia , Tosilfenilalanil Clorometil Cetona/farmacologia , Regulação para Cima , Receptor fas/genéticaRESUMO
OBJECTIVE: To establish osteoblast apoptosis model induced by gingipains, and to examine the expression of pro-apoptotic protein Bcl-2 interacting mediator (Bim), Bcl-2 associated X protein (Bax) and Bcl-2 antagonist/killer (Bak). METHODS: Gingipain and gingipain acticity were extracted and measured. Mouse osteoblast cell line MC3T3-E1 cells were cultured in the presence of 0.453, 0.906, 1.812 U/L gingipains for 0, 16, 24 and 48 h. Apoptosis was examined by 4',6-diamidino-2-phenylindole (DAPI) staining or annexin V/propidine iodide (PI) staining.Protein expression of Bim, Bax and Bak was determined by Western blotting after osteoblasts were cultured with 1.812 U/L gingipain for 0, 4, 8, 16, 24 and 48 h. Osteoblasts were cultured with 1.812 U/L gingipain which had been inhibited with N-alpha-tosyl L-lysyl-chlorom ethylketone (TLCK). Western blotting was used to detect Bim expression and DAPI staining to measure apoptosis. RESULTS: Arginine-specific proteinases (Rgp) activity was (18.11 ± 2.11) U/L and specific proteinases (Kgp) was (1.02 ± 0.25) U/L. Percentage of osteoblast apoptosis induced by 1.812 U/L gingipain rose to (6.31 ± 0.37)% after 16 h, and reached (11.20 ± 0.35)% at 24 h and (10.80 ± 0.46)% after 48 h with DAPI staining. Annexin V/PI staining supported the result from DAPI staining.Bim protein level increased during osteoblast apoptosis, the relative fold rose to (0.31 ± 0.03) after 4 h (about 2 fold compared to control), peaking at 24 h (0.57 ± 0.05, 3-4 fold compared to control). Proteinase inhibitor TLCK effectively blocked the activity of gingipain and inhibited up-regulation of Bim induced by gingipains from (0.58 ± 0.04) to (0.14 ± 0.03). The percentage of osteoblast apoptosis decreased from (11.20 ± 0.35)% to (4.31 ± 0.38)% in the presence of TLCK. Expression of Bax remained unchanged when cells were cultured with or without gingipains. Bak was under the detectable level in MC3T3-E1. CONCLUSIONS: 1.812 U/L gingipains induced osteoblast apoptosis. Protein expression of Bim was up-regulated during cell apoptosis and was down-regulated when gingipain inhibited with TLCK, suggesting that Bim was involved in osteoblast apoptosis induced by gingipain. Inhibition of Bim protein expression protected osteoblast from apoptosis.
Assuntos
Adesinas Bacterianas/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/efeitos dos fármacos , Cisteína Endopeptidases/farmacologia , Proteínas de Membrana/metabolismo , Osteoblastos , Proteínas Proto-Oncogênicas/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Proteína 11 Semelhante a Bcl-2 , Linhagem Celular , Cisteína Endopeptidases Gingipaínas , Humanos , Células MCF-7 , Camundongos , Osteoblastos/citologia , Osteoblastos/metabolismo , Tosilina Clorometil Cetona/farmacologiaRESUMO
AIM: To determine the levels of LL-37 in and its susceptibility to degradation by components of gingival crevicular fluid (GCF) in periodontal health and disease. MATERIALS AND METHODS: Levels of LL-37 in GCF from periodontitis patients and periodontally healthy subjects were determined by ELISA. In addition, degradation of synthetic/exogenous LL-37 by components of GCF in the presence and absence of inhibitors was determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry. RESULTS: The concentration of native LL-37 in GCF from Porphyromonas gingivalis positive (Pg+) and P. gingivalis negative (Pg-) sites in periodontitis patients was significantly higher than in GCF from healthy subjects. When synthetic LL-37 was added to healthy GCF, the peptide was not degraded. Conversely, GCF from Pg+ sites rapidly degraded synthetic LL-37 which was prevented in the presence of Arg- and Lys- gingipain inhibitors. Synthetic LL-37 was degraded more slowly by GCF from Pg- sites. CONCLUSIONS: LL-37 is detectable in GCF in periodontal health and disease. The rapid degradation of synthetic LL-37 in periodontitis GCF, particularly in Pg+ sites, limits its role as a potential therapeutic in the gingival crevice. These results highlight the need to design stable peptide mimetics of LL-37 as future therapeutics in periodontitis.
Assuntos
Antibacterianos/análise , Catelicidinas/análise , Cisteína Proteases/metabolismo , Líquido do Sulco Gengival/enzimologia , Periodontite/metabolismo , Periodonto/metabolismo , Adesinas Bacterianas/análise , Adesinas Bacterianas/efeitos dos fármacos , Adulto , Idoso , Peptídeos Catiônicos Antimicrobianos , Cisteína Endopeptidases/análise , Cisteína Endopeptidases/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , Placa Dentária/microbiologia , Ensaio de Imunoadsorção Enzimática , Cisteína Endopeptidases Gingipaínas , Líquido do Sulco Gengival/microbiologia , Humanos , Leupeptinas/farmacologia , Pessoa de Meia-Idade , Fragmentos de Peptídeos/análise , Periodontite/enzimologia , Periodontite/microbiologia , Periodonto/enzimologia , Porphyromonas gingivalis/enzimologia , Porphyromonas gingivalis/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Tosilina Clorometil Cetona/farmacologiaRESUMO
The aim of the present study was to determine the correlation between angiogenesis and the differential expression of growth factors and their receptors when myocardial microvascular endothelial cells (MMVECs) were co-cultured with mast cell granules (MCGs) under hyperglycemic conditions. MMVECs and mast cells (MCs) were isolated from Wistar rats. An in vitro angiogenesis assay was used to observe any differences when MMVECs were co-cultured with MCGs in normal or hyperglycemic medium. The mRNA and protein expression of growth factors and their receptors were analyzed by real-time reverse transcription (RT)-PCR and western blot analysis. Real-time RT-PCR analysis demonstrated the upregulated mRNA and protein expression of vascular endothelial growth factor (VEGF) in the MMVECs; however, the expression of its receptor, fms-like tyrosine kinase-1 (Flt-1) and fetal liver kinase-1 (Flk1), decreased significantly, and the angiogenic ability of the MMVECs decreased under hyperglycemic conditions. The angiogenic ability of the MMVECs cultured under hyperglycemic conditions (even after the addition of MCGs) was inferior to that of the MMVECs cultured under normal glucose conditions. The specific inhibitor of tryptase, N-tosyl-L-lysine chloromethyl ketone (TLCK), suppressed angiogenesis regardless of the glucose concentration, and the specific inhibitor of chymase, N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK), was not as effective as TLCK, which was mainly detected under hyperglycemic conditions. High glucose levels have a profound effect on angiogenesis; this effect may be more pronounced than the effects of MCGs on angiogenesis.
Assuntos
Células Endoteliais/metabolismo , Glucose/farmacologia , Hiperglicemia/patologia , Mastócitos/metabolismo , Microvasos/citologia , Miocárdio/citologia , Neovascularização Fisiológica/efeitos dos fármacos , Animais , Técnicas de Cocultura , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Hiperglicemia/metabolismo , Masculino , Mastócitos/citologia , Mastócitos/efeitos dos fármacos , Neovascularização Fisiológica/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tosilina Clorometil Cetona/farmacologia , Tosilfenilalanil Clorometil Cetona/farmacologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Cicatrização/efeitos dos fármacosRESUMO
OBJECTIVE: To investigate the regulatory mechanisms of integrin α5 and ß1 in osteoblast in the process of gingipains-induced apoptosis. METHODS: Gingipains were isolated and purified from supernatants of Porphyromonas gingivalis W83 which was cultured under standard anaerobic conditions. MC3T3-E1 was challenged with or without 8.3480 U/L gingipains for 48 h and apoptosis was examined by transferase-mediated deoxyuridine triphosphate-biotin nick end labeling-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (TUNEL-DAPI) staining. The expression of integrin α5 and ß1 was analyzed by Western blotting after MC3T3-E1 was treated under different conditions. RESULTS: Arginine-specific proteinases(Rgp) activity was (41.74 ± 2.11) U/L and lysine-specific proteinase(Kgp) was (1.02 ± 0.25) U/L.Gingipains induced MC3T3-E1 cells apoptosis after 48 h. Compared with control group, expression of integrin α5 and ß1 was down-regulated by gingipains in a time-dependent manner within short periods ( ≤ 72 h), integrin α5 and ß1 relative expression was (0.485 ± 0.039),(0.504 ± 0.002) at 48 h,(0.398 ± 0.058),(0.179 ± 0.001) at 72 h respectively (P < 0.05). After 72 h, integrin α5 expression in MC3T3-E1 cells was stable compared with control group while integrin ß1 was still lower(control group:1.000 ± 0.000, 96 h:0.604 ± 0.003, 120 h: 0.357 ± 0.002) (P < 0.05). Proteinase inhibitor tosyl- L- lysine-chloromethyl-ketone(TLCK) effectively blocked the activity of gingipain and inhibited down-regulation of integrin α5 and ß1 induced by gingipains from (0.398 ± 0.058,0.179 ± 0.001 ) to (0.781 ± 0.012, 0.857 ± 0.060) (P < 0.05). TLCK alone did not have any effect on integrin α5 and ß1(P > 0.05). Gingipains also decreased integrin α5 and ß1 in a dose-dependent manner.When cells were treated with 20.8700 U/L gingipains, integrin α5 and ß1 relative expression reached to the lowest(0.105 ± 0.004,0.020 ± 0.000) (P < 0.05). CONCLUSIONS: Gingipains inhibited the expression of integrin α5 and ß1 in a time- and dose- dependent manner in osteoblasts in the process of apoptosis, which may not be mediated by direct proteolytic effect.
Assuntos
Adesinas Bacterianas/farmacologia , Apoptose/efeitos dos fármacos , Cisteína Endopeptidases/farmacologia , Integrina alfa5/metabolismo , Integrina beta1/metabolismo , Osteoblastos , Adesinas Bacterianas/administração & dosagem , Adesinas Bacterianas/isolamento & purificação , Animais , Cisteína Endopeptidases/administração & dosagem , Cisteína Endopeptidases/isolamento & purificação , Relação Dose-Resposta a Droga , Regulação para Baixo , Cisteína Endopeptidases Gingipaínas , Camundongos , Osteoblastos/citologia , Osteoblastos/metabolismo , Porphyromonas gingivalis/química , Inibidores de Serina Proteinase/farmacologia , Fatores de Tempo , Tosilina Clorometil Cetona/farmacologiaRESUMO
Midgut trypsins are associated with Bt protoxin activation and toxin degradation. Proteinase inhibitors have potential insecticidal toxicity against a wide range of insect species. This study was conducted to evaluate the interaction of proteinase inhibitors with Bt toxin and to examine midgut trypsin gene profile of Heliothis virescens. A sublethal dose (15 ppb) of Cry1Ac, 0.75% soybean trypsin inhibitor, and 0.1% and 0.2% N-α-tosyl-L-lysine chloromethyl ketone significantly suppressed midgut proteinase activities, and resulted in reductions in larval and pupal size and mass. The treatment with inhibitor+Bt suppressed approximately 65% more larval body mass and 21% more enzymatic activities than the inhibitor-only or Bt-only. Eleven trypsin-like cDNAs were sequenced from the midgut of H. virescens. All trypsins contained three catalytic center residues (H(73), D(153), and S(231)), substrate specificity determinant residues (D(225), G(250), and G(261)), and six cysteines for disulfide bridges. These putative trypsins were separated into three distinct groups, indicating the diverse proteinases evolved in this polyphagous insect. These results indicated that the insecticidal activity of proteinase inhibitors may be used to enhance Bt toxicity and delay resistance development.
Assuntos
Proteínas de Bactérias/farmacologia , Endotoxinas/farmacologia , Proteínas Hemolisinas/farmacologia , Mariposas/efeitos dos fármacos , Proteínas de Soja/farmacologia , Tosilina Clorometil Cetona/farmacologia , Inibidores da Tripsina/farmacologia , Tripsina/genética , Sequência de Aminoácidos , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Clonagem Molecular , Endotoxinas/genética , Proteínas Hemolisinas/genética , Resistência a Inseticidas , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Dados de Sequência Molecular , Mariposas/crescimento & desenvolvimento , Alinhamento de Sequência , Especificidade por Substrato , Tripsina/químicaRESUMO
In this study we investigated the peptidase activity in Leishmania (L.) amazonensis live amastigote by confocal microscopy using peptidyl-MCA as substrates, the hydrolysis of which releases the MCA fluorophore inside the cells. Cell pre-treatment with peptidase inhibitors indicated the presence of cysteine and serine peptidases. It was noteworthy that Leishmania amastigotes incorporate only substrates (Z-FR-MCA, Z-RR-MCA) or inhibitors (E64, TLCK) containing positively charged groups. The peptidase activities in the supernatants of amastigotes and promastigotes lysates were also evaluated with the same peptidyl-MCA substrates and inhibitors in the pH range 4.5-9.0. The effects of temperature and different salts were also included in this study. The hydrolytic activities of supernatants on Z-FR-MCA clearly indicate the presence of different cysteine peptidases that adapted to work in different environment conditions. Intact Leishmania cells incorporated Z-RR-MCA, the hydrolysis of which was inhibited only by TLCK indicating the presence of at least one serine peptidase. The pH profile of Z-RR-MCA hydrolysis by amastigotes and promastigotes lysate supernatants, and the hydrolysis time course of the FRET peptide Abz-AGRRRAQ-EDDnp at RA bond, followed by removal of the two C-termini R to yield Abz-AGR-OH that is a unique characteristic of oligopeptidase B, indicate its presence in the parasite.
Assuntos
Leishmania/enzimologia , Peptídeo Hidrolases/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Citratos/química , Cricetinae , Inibidores de Cisteína Proteinase/farmacologia , Concentração de Íons de Hidrogênio , Leishmania/citologia , Leucina/análogos & derivados , Leucina/farmacologia , Mesocricetus , Oligopeptídeos/química , Pepstatinas/farmacologia , Peptídeo Hidrolases/química , Proteólise , Proteínas de Protozoários/química , Salinidade , Inibidores de Serina Proteinase/farmacologia , Citrato de Sódio , Sulfatos/química , Temperatura , Tosilina Clorometil Cetona/farmacologiaRESUMO
OBJECTIVE: The purpose of the present study was to characterize the collagenolytic activity in a sonicated extract of Tannerella forsythia and to investigate the activation of proMMMP-2 and -9 by the T. forsythia extract. METHODS: The T. forsythia extract was incubated with type I collagen. The cleaved products were then analyzed by SDS-PAGE using the method of Laemmli. We studied the effects of cysteine, DTT, CaCl(2), and various proteinase inhibitors on collagenolytic activity. A HT1080 cell culture supernatant containing proMMP-2 and -9 was incubated with the T. forsythia extract and analyzed for the activation of proMMP-2 and -9 by gelatin zymography. RESULTS: The T. forsythia extract degraded type I collagen. Cysteine increased the collagenolytic activity of the extract, and 5mM CaCl(2) was required for this activity. The collagenolytic activity of T. forsythia was inhibited by N-ethylmaleimide, iodoacetamide, iodoacetic acid, EDTA, and leupeptin, but not by PMSF, E-64, TLCK, or TPCK. When proMMP-2 and -9 were incubated with the T. forsythia extract, gelatinases with the relative molecular masses of MMP-2 and -9 were produced. CONCLUSION: The present study suggests that T. forsythia extract is able to degrade type I collagen and activate proMMP-2 and -9.
Assuntos
Bacteroides/metabolismo , Colágeno Tipo I/metabolismo , Cloreto de Cálcio/farmacologia , Catepsinas/antagonistas & inibidores , Linhagem Celular Tumoral , Quelantes/farmacologia , Cisteína/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Ácido Edético/farmacologia , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Precursores Enzimáticos/metabolismo , Etilmaleimida/farmacologia , Gelatinases/metabolismo , Humanos , Iodoacetamida/farmacologia , Ácido Iodoacético/farmacologia , Leucina/análogos & derivados , Leucina/farmacologia , Leupeptinas/farmacologia , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Metaloproteinase 9 da Matriz/metabolismo , Fluoreto de Fenilmetilsulfonil/farmacologia , Inibidores de Proteases/farmacologia , Inibidores de Serina Proteinase/farmacologia , Tosilina Clorometil Cetona/farmacologia , Tosilfenilalanil Clorometil Cetona/farmacologiaRESUMO
DNA fragmentation is a hallmark of apoptosis that occurs in a variety of cell types; however, it remains unclear whether caspase-3 is required for its induction. To investigate this, we produced caspase-3 knockout Chinese hamster ovary (CHO)-K1 cells and examined the effects of gene knockout and treatment with caspase-3 inhibitors. Okadaic acid (OA) is a potent inhibitor of the serine/threonine protein phosphatases (PPs) PP1 and PP2A, which induce apoptotic cellular reactions. Treatment of caspase-3(-/-) cells with OA induced DNA fragmentation, indicating that caspase-3 is not an essential requirement. However, in the presence of benzyloxycarbonyl-Asp-Glu-Val-Asp (OMe) fluoromethylketone (z-DEVD-fmk), DNA fragmentation occurred in CHO-K1 cells but not in caspase-3(-/-) cells, suggesting that caspase-3 is involved in OA-induced DNA fragmentation that does not utilize DEVDase activity. In the absence of caspase-3, DEVDase activity may play an important role. In addition, OA-induced DNA fragmentation was reduced but not blocked in CHO-K1 cells, suggesting that caspase-3 is involved in caspase-independent OA-induced DNA fragmentation. Furthermore, OA-induced cleavage of caspase-3 and DNA fragmentation were blocked by pretreatment with the wide-ranging serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride. These results suggest that serine proteases regulate DNA fragmentation upstream of caspase-3.
Assuntos
Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Caspase 3/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Ácido Okadáico/farmacologia , Animais , Sequência de Bases , Células CHO , Caspase 3/deficiência , Caspase 3/genética , Inibidores de Caspase , Cricetinae , Cricetulus , Inibidores de Cisteína Proteinase/farmacologia , Primers do DNA/genética , DNA Complementar/genética , Dados de Sequência Molecular , Oligopeptídeos/farmacologia , Peptídeo Hidrolases/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Inibidores de Serina Proteinase/farmacologia , Tosilina Clorometil Cetona/farmacologia , Tosilfenilalanil Clorometil Cetona/farmacologiaRESUMO
The purpose of this work was to obtain information about conformational changes of the plasma membrane Ca(2+)-pump (PMCA) in the membrane region upon interaction with Ca(2+), calmodulin (CaM) and acidic phospholipids. To this end, we have quantified labeling of PMCA with the photoactivatable phosphatidylcholine analog [(125)I]TID-PC/16, measuring the shift of conformation E(2) to the auto-inhibited conformation E(1)I and to the activated E(1)A state, titrating the effect of Ca(2+) under different conditions. Using a similar approach, we also determined the CaM-PMCA dissociation constant. The results indicate that the PMCA possesses a high affinity site for Ca(2+) regardless of the presence or absence of activators. Modulation of pump activity is exerted through the C-terminal domain, which induces an apparent auto-inhibited conformation for Ca(2+) transport but does not modify the affinity for Ca(2+) at the transmembrane domain. The C-terminal domain is affected by CaM and CaM-like treatments driving the auto-inhibited conformation E(1)I to the activated E(1)A conformation and thus modulating the transport of Ca(2+). This is reflected in the different apparent constants for Ca(2+) in the absence of CaM (calculated by Ca(2+)-ATPase activity) that sharply contrast with the lack of variation of the affinity for the Ca(2+) site at equilibrium. This is the first time that equilibrium constants for the dissociation of Ca(2+) and CaM ligands from PMCA complexes are measured through the change of transmembrane conformations of the pump. The data further suggest that the transmembrane domain of the PMCA undergoes major rearrangements resulting in altered lipid accessibility upon Ca(2+) binding and activation.
Assuntos
Azirinas/metabolismo , Cálcio/metabolismo , Calmodulina/metabolismo , Membrana Celular/enzimologia , Sondas Moleculares/metabolismo , Fosfatidilcolinas/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Membrana Celular/efeitos dos fármacos , Quimotripsina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Cinética , Ácido Oleico/farmacologia , Ácidos Fosfatídicos/farmacologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Estrutura Terciária de Proteína , Deleção de Sequência/efeitos dos fármacos , Titulometria , Tosilina Clorometil Cetona/farmacologiaRESUMO
DrTI was effective against trypsin-like enzymes from A. kuehniella and C. cephalonica, however an artificial diet was insufficient to affect the survival and body weight of either insect. The inhibitor stimulated chymotrypsin-like enzymes and probably induced the synthesis of enzymes insensitive to TLCK in neonate larvae.
Assuntos
Fabaceae/química , Lepidópteros/efeitos dos fármacos , Peptídeo Hidrolases/metabolismo , Peptídeos/farmacologia , Proteínas de Plantas/farmacologia , Sementes/química , Animais , Larva/efeitos dos fármacos , Larva/enzimologia , Lepidópteros/enzimologia , Peptídeos/química , Proteínas de Plantas/química , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologia , Tosilina Clorometil Cetona/farmacologiaRESUMO
Analogues of the irreversible protease inhibitors TPCK and TLCK have been synthesized and tested as inhibitors of the bacterial cysteine protease IdeS excreted by Streptococcuspyogenes. Eight compounds were identified as inhibitors of IdeS in an in vitro assay. The most potent compounds contained an aldehyde function, thus acting as efficient reversible inhibitors, nitrile and azide derivatives showed moderate activity.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/síntese química , Inibidores de Cisteína Proteinase/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cisteína Endopeptidases/química , Cisteína Proteases/metabolismo , Inibidores de Cisteína Proteinase/química , Cinética , Ligação Proteica , Streptococcus pyogenes/enzimologia , Streptococcus pyogenes/patogenicidade , Relação Estrutura-Atividade , Tosilina Clorometil Cetona/química , Tosilina Clorometil Cetona/farmacologia , Tosilfenilalanil Clorometil Cetona/química , Tosilfenilalanil Clorometil Cetona/farmacologia , VirulênciaRESUMO
INTRODUCTION: Tumour necrosis factor-alpha converting enzyme (TACE), also known as ADAM17, is a membrane-bound metalloprotease and disintegrin. It is produced by a number of host cells and is known to shed and release cell-bound cytokines, particularly members of the tumour necrosis factor family. The aim of this study was to investigate the effect of Porphyromonas gingivalis on TACE production by a human T-cell line, to identify putative virulence factors involved in this process, and to investigate the effect of doxycycline. METHODS: P. gingivalis 6-day culture supernatants were used to challenge Jurkat T cells for 6 h. Secreted and cell-associated TACE levels were measured by enzyme-linked immunosorbent assay, whereas messenger RNA expression was investigated by quantitative real-time polymerase chain reaction. To investigate the involvement of cysteine proteases or proteinaceous components in general, P. gingivalis culture supernatants were treated with the specific chemical inhibitor TLCK or heat-inactivated, respectively. The effect of doxycycline on the regulation of TACE secretion by P. gingivalis was also investigated. RESULTS: P. gingivalis challenge resulted in a concentration-dependent enhancement of TACE messenger RNA expression and protein release by Jurkat cells. TLCK treatment or heat treatment of P. gingivalis culture supernatants decreased TACE release to control levels. Doxycycline inhibited TACE secretion dose dependently. CONCLUSION: The induction of TACE by T cells in response to P. gingivalis may in turn favour the shedding of host cell-bound cytokines into the local microenvironment, potentially amplifying the inflammatory response. In the present experimental system, P. gingivalis cysteine proteases are involved in TACE release by T cells.
Assuntos
Proteínas ADAM/biossíntese , Cisteína Endopeptidases/metabolismo , Células Jurkat/enzimologia , Porphyromonas gingivalis/fisiologia , Proteína ADAM17 , Adesinas Bacterianas/metabolismo , Antibacterianos/farmacologia , Meios de Cultivo Condicionados/farmacologia , Doxiciclina/farmacologia , Expressão Gênica , Cisteína Endopeptidases Gingipaínas , Humanos , Células Jurkat/efeitos dos fármacos , Células Jurkat/microbiologia , Lipopolissacarídeos/fisiologia , Inibidores de Proteases/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/biossíntese , Tosilina Clorometil Cetona/farmacologia , Fatores de VirulênciaRESUMO
Porphyromonas gingivalis strains W50 and ATCC 33277 were shown to bind to cultured human fibroblast (MRC-5) cells using flow cytometry. As the concentration of P. gingivalis strain W50 cells was increased relative to the concentration of MRC-5 cells, the number of W50 cells bound per MRC-5 cell increased, as did the percentage of MRC-5 cells with bacteria bound. However, this relationship was only seen for P. gingivalis strain ATCC 33277 at low cell concentrations: at high bacterial cell concentrations strain ATCC 33277 auto-aggregated and binding to the MRC-5 cells decreased. Strain W50 was therefore chosen to study the role of the surface proteinase-adhesin complexes (RgpA-Kgp complexes) in binding to MRC-5 cells. P. gingivalis W50 cells treated with an inhibitor of the RgpA-Kgp complexes exhibited reduced binding to MRC-5 cells. The purified active and proteinase-inactive RgpA-Kgp complexes competitively inhibited binding of W50 to MRC-5 cells, and isogenic mutants of W50 lacking RgpA/B and Kgp displayed reduced binding. P. gingivalis W50 mutant cells lacking Kgp exhibited the lowest binding to MRC-5 cells, suggesting an important role for this proteinase and its associated adhesins in binding to fibroblasts.
Assuntos
Adesinas Bacterianas/metabolismo , Aderência Bacteriana , Cisteína Endopeptidases/metabolismo , Porphyromonas gingivalis/metabolismo , Adesinas Bacterianas/efeitos dos fármacos , Linhagem Celular , Cisteína Endopeptidases/efeitos dos fármacos , Escherichia coli/metabolismo , Fibroblastos/microbiologia , Citometria de Fluxo , Fluoresceína-5-Isotiocianato , Cisteína Endopeptidases Gingipaínas , Humanos , Microscopia de Fluorescência , Tosilina Clorometil Cetona/farmacologiaRESUMO
In this work, we re-examine the previously reported phenomenon of the creation of a superactive glutamate dehydrogenase by proteolytic modification by chymotrypsin and explore the various discrepancies that came to light during those studies. We find that superactivation is caused by cleavage at the N terminus of the protein and not the C-terminal allosteric site, as has previously been suggested. N-terminal sequencing reveals that TLCK-treated chymotrypsin cleaves bovine glutamate dehydrogenase at phenylalanine 10. We suggest that trypsin contamination in nontreated chymotrypsin may have led to the production of the larger 4-5 kDa digestion product, previously misinterpreted as having caused the activation. In line with some previous studies, we can confirm that GTP inhibition is attenuated to some extent by the proteolysis, while ADP activation is almost abolished. Utilizing the recently solved structures of bovine glutamate dehydrogenase, we illustrate the cleavage points.
Assuntos
Glutamato Desidrogenase/química , Glutamato Desidrogenase/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Regulação Alostérica/efeitos dos fármacos , Animais , Bovinos , Quimotripsina/metabolismo , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática/efeitos dos fármacos , Guanosina Trifosfato/metabolismo , Modelos Moleculares , Estrutura Secundária de Proteína , Inibidores de Serina Proteinase/farmacologia , Soroalbumina Bovina/metabolismo , Tosilina Clorometil Cetona/farmacologia , Tripsina/metabolismoRESUMO
Enzymes involved in sexual differentiation and fertilization of the human malaria parasite Plasmodium falciparum represent potential targets for transmission blocking strategies. Parasite proteases are putatively involved in several steps during fertilization, but the types of proteases, their targets and modes of action remain hitherto unknown. We investigated the involvement of proteases in gametogenesis via exflagellation and immunofluorescence assays, using a variety of commercially available as well as newly designed protease inhibitors. The assays revealed a blockade of microgamete formation by the cysteine/serine protease inhibitors TLCK and TPCK. The serine protease inhibitor PMSF, the falcipain-targeting inhibitor RV112D, and the aspartic protease inhibitor EPNP also significantly decreased formation of microgametes. The metalloprotease inhibitor 1,10-phenanthroline, on the other hand, inhibited exflagellation by interfering with microgamete motility. Furthermore, EPNP reduced the activation of male and female gametocytes. Our data point to a major involvement of serine proteases and a non-thermolysin-like zinc metalloprotease in microgametocyte exflagellation.
Assuntos
Flagelos/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/fisiologia , Inibidores de Proteases/classificação , Inibidores de Proteases/farmacologia , Serina Endopeptidases/efeitos dos fármacos , Animais , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Inibidores de Cisteína Proteinase , Metaloproteases/antagonistas & inibidores , Testes de Sensibilidade Parasitária , Plasmodium falciparum/enzimologia , Reprodução/efeitos dos fármacos , Reprodução/fisiologia , Inibidores de Serina Proteinase/farmacologia , Tosilina Clorometil Cetona/farmacologia , Tosilfenilalanil Clorometil Cetona/farmacologiaRESUMO
Serine protease inhibitors N-alpha-tosyl-L-lysinyl-chloromethylketone (TLCK) and N-tosyl-L-phenylalaninyl-chloromethylketone (TPCK) exhibit multiple effects on cell death pathways in mammalian cells. Thus, they are able to induce apoptosis by itself or promote cell death induced by other cytotoxic stimuli [King et al., 2004; Murn et al., 2004]. On the other hand, TLCK and TPCK were reported to prevent apoptosis by inhibiting the processing of caspases in response to some cell death inducing stimuli [Stefanis et al., 1997; Jones et al., 1998]. We observed that the pretreatment of HL-60 cells with TLCK or TPCK diminished caspases 3 and -7 (DEVDase) and caspase-6 (VEIDase) activity in response to various cell death inducing stimuli such as staurosporine (STS), etoposide (ETP), or N6-(2-isopentenyl)adenosine. In addition, TLCK but not TPCK inhibited collapse of mitochondrial transmembrane potential Delta Psi m (delta psi) in dying HL-60 cells. Such effects used to be considered as protective, however, the protection was only presumable since neither TLCK nor TPCK actually prevented cells from death. Our results further indicated that serine protease inhibitors TLCK and particularly TPCK acted as efficient direct inhibitors of mature caspases. Indeed, experiments with human recombinant caspases provided unequivocal evidence that TLCK and TPCK are very potent but non-specific inhibitors of activated caspases, namely caspases 3, -6, and -7. Interestingly, TPCK exhibited similar efficiency towards human recombinant caspases to that found for panspecific caspase inhibitor Boc-D-CMK. Such properties of TLCK and TPCK, previously considered as specific inhibitors of serine proteases, might offer novel consistent explanation for several protective or protective-like effects on apoptotic cells.
Assuntos
Apoptose/efeitos dos fármacos , Inibidores de Caspase , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Inibidores de Serina Proteinase/farmacologia , Tosilina Clorometil Cetona/farmacologia , Tosilfenilalanil Clorometil Cetona/farmacologia , Caspases/química , Caspases/metabolismo , Células HL-60 , Humanos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Decapod crustaceans synthesize highly active proteolytic enzymes in the midgut gland and release at least a part of them into the stomach where they facilitate the first step in peptide hydrolysis. The most common proteinases in the gastric fluid characterized so far are serine proteinases, that is, trypsin and chymotrypsin. These enzymes show highest activities at neutral or slightly alkaline conditions. The presence of acid proteinases, as they prevail in vertebrates, has been discussed contradictorily yet in invertebrates. In this study, we show that acid aspartic proteinases appear in the gastric fluid of several decapods. Lobsters Homarus gammarus showed the highest activity with a maximum at pH 3. These activities were almost entirely inhibited by pepstatin A, which indicates a high share of aspartic proteinases. In other species (Panulirus interruptus, Cancer pagurus, Callinectes arcuatus and Callinectes bellicosus), proteolytic activities were present at acid conditions but were distinctly lower than in H. gammarus. Zymograms at pH 3 showed in each of the studied species at least one, but mostly two-four bands of activity. The apparent molecular weight of the enzymes ranged from 17.8 to 38.6 kDa. Two distinct bands were identified which were inhibited by pepstatin A. Acid aspartic proteinases may play an important role in the process of extracellular digestion in decapod crustaceans. Activities were significantly higher in clawed lobster than in spiny lobster and three species of brachyurans. Therefore, it may be suggested that the expression of acid proteinases is favored in certain groups and reduced in others.
Assuntos
Ácido Aspártico Endopeptidases/análise , Ácido Aspártico Endopeptidases/metabolismo , Decápodes/enzimologia , Animais , Inibidores de Cisteína Proteinase/farmacologia , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática/efeitos dos fármacos , Trato Gastrointestinal/enzimologia , Concentração de Íons de Hidrogênio , Leucina/análogos & derivados , Leucina/farmacologia , Pepstatinas/farmacologia , Inibidores de Proteases/farmacologia , Inibidores de Serina Proteinase/farmacologia , Tosilina Clorometil Cetona/farmacologia , Tripsina/farmacologiaRESUMO
In this article we investigated the platelet aggregating activity of whole crotoxin and its subunits isolated from Crotalus durissus cascavella venom. During the purification protocols of the venom, using HPLC molecular exclusion, we detected the presence of two different serine protease activities in the gyroxin fraction, and another in the crotoxin fraction, which induced strong and irreversible platelet aggregation, in addition to blood coagulation. From crotoxin, we isolated PLA2, crotapotin (both fractions corresponding approximately 85% of whole crotoxin) and another minor fraction (F20) that exhibited serine protease activity. After a new fractionation on reverse phase HPLC chromatography, we obtained three other fractions named as F201, F202 and F203. F202 was obtained with high degree of molecular homogeneity with molecular mass of approximately 28 kDa and a high content of acidic amino residues, such as aspartic acid and glutamic acid. Other important amino acids were histidine, cysteine and lysine. This protein exhibited a high specificity for BApNA, a Michaelis-Menten behavior with Vmax estimated in 5.64 microM/min and a Km value of 0.58 mM for this substrate. In this work, we investigated the ability of F202 to degrade fibrinogen and observed alpha and beta chain cleavage. Enzymatic as well as the platelet aggregation activities were strongly inhibited when incubated with TLCK and PMSF, specific inhibitors of serine protease. Also, F202 induced platelet aggregation in washed and platelet-rich plasma, and in both cases, TLCK inhibited its activity. The N-terminal amino acid sequence of F202 presented a high amino acid sequence homology with other thrombin-like proteins, but it was significantly different from gyroxin. These results showed that crotoxin is a highly heterogeneous protein composed of PLA2, thrombin-like and other fractions that might explain the diversity of physiological and pharmacological activities of this protein.