RESUMO
The Ananas comosus stem extract is a complex mixture containing various cysteine ââproteases of the C1A subfamily, such as bromelain and ananain. This mixture used for centuries in Chinese medicine, has several potential therapeutic applications as anti-cancer, anti-inflammatory and ecchymosis degradation agent. In the present work we determined the structures of bromelain and ananain, both in their free forms and in complex with the inhibitors E64 and TLCK. These structures combined with protease-substrate complexes modeling clearly identified the Glu68 as responsible for the high discrimination of bromelain in favor of substrates with positively charged residues at P2, and unveil the reasons for its weak inhibition by cystatins and E64. Our results with purified and fully active bromelain, ananain and papain show a strong reduction of cell proliferation with MDA-MB231 and A2058 cancer cell lines at a concentration of about 1 µM, control experiments clearly emphasizing the need for proteolytic activity. In contrast, while bromelain and ananain had a strong effect on the proliferation of the OCI-LY19 and HL-60 non-adherent cell lines, papain, the archetypal member of the C1A subfamily, had none. This indicates that, in this case, sequence/structure identity beyond the active site of bromelain and ananain is more important than substrate specificity.
Assuntos
Ananas/química , Bromelaínas/química , Cisteína Endopeptidases/química , Inibidores de Cisteína Proteinase/química , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Bromelaínas/antagonistas & inibidores , Bromelaínas/metabolismo , Bromelaínas/farmacologia , Domínio Catalítico , Linhagem Celular Tumoral , Cisteína/química , Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases/farmacologia , Inibidores de Cisteína Proteinase/metabolismo , Dissulfetos/química , Humanos , Leucina/análogos & derivados , Leucina/química , Leucina/metabolismo , Modelos Moleculares , Caules de Planta/química , Conformação Proteica , Espectrometria de Massas por Ionização por Electrospray , Especificidade por Substrato , Tosilina Clorometil Cetona/química , Tosilina Clorometil Cetona/metabolismoRESUMO
Glia cells are involved in upper motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Protease activated receptor 1 (PAR1) pathway is related to brain pathologies. Brain PAR1 is located on peri-synaptic astrocytes, adjacent to pyramidal motor neurons, suggesting possible involvement in ALS. Brain thrombin activity in superoxide dismutase 1 (SOD1) mice was measured using a fluorometric assay, and PAR1 levels by western blot. PAR1 was localized using immunohistochemistry staining. Treatment targeted PAR1 pathway on three levels; thrombin inhibitor TLCK (N-Tosyl-Lys-chloromethylketone), PAR1 antagonist SCH-79797 and the Ras intracellular inhibitor FTS (S-trans-trans-farnesylthiosalicylic acid). Mice were weighed and assessed for motor function and survival. SOD1 brain thrombin activity was increased (p < 0.001) particularly in the posterior frontal lobe (p = 0.027) and hindbrain (p < 0.01). PAR1 levels were decreased (p < 0.001, brain, spinal cord, p < 0.05). PAR1 and glial fibrillary acidic protein (GFAP) staining decreased in the cerebellum and cortex. SOD1 mice lost weight (≥17 weeks, p = 0.047), and showed shorter rotarod time (≥14 weeks, p < 0.01). FTS 40mg/kg significantly improved rotarod scores (p < 0.001). Survival improved with all treatments (p < 0.01 for all treatments). PAR1 antagonism was the most efficient, with a median survival improvement of 10 days (p < 0.0001). Our results support PAR1 pathway involvement in ALS.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Encéfalo/metabolismo , Modelos Animais de Doenças , Receptor PAR-1/metabolismo , Superóxido Dismutase-1/metabolismo , Esclerose Lateral Amiotrófica/genética , Animais , Astrócitos/metabolismo , Peso Corporal/efeitos dos fármacos , Farneseno Álcool/análogos & derivados , Farneseno Álcool/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Neurônios Motores/metabolismo , Mutação , Pirróis/farmacologia , Quinazolinas/farmacologia , Salicilatos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Superóxido Dismutase-1/genética , Análise de Sobrevida , Tosilina Clorometil Cetona/farmacologiaRESUMO
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
Crystalline (Cry) proteins from Bacillus thuringiensis (Bt) are used extensively for insect control in sprays and transgenic plants, but their efficacy is reduced by evolution of resistance in pests. Here we evaluated reduced activation of Cry1Ac protoxin as a potential mechanism of resistance in the invasive pest Helicoverpa armigera. Based on the concentration killing 50% of larvae (LC50) for a laboratory-selected resistant strain (LF120) divided by the LC50 for its susceptible parent strain (LF), the resistance ratio was 1600 for Cry1Ac protoxin and 1200 for trypsin-activated Cry1Ac toxin. The high level of resistance to activated toxin as well as to protoxin indicates reduced activation of protoxin is not a major mechanism of resistance to Cry1Ac in LF120. For both insect strains, treatment with either the trypsin inhibitor N-a-tosyl-L-lysine chloromethyl ketone (TLCK) or the chymotrypsin inhibitor N-a-tosyl-L-phenylalanine chloromethyl ketone (TPCK) did not significantly affect the LC50 of Cry1Ac protoxin. Enzyme activity was higher for LF than LF120 for trypsin-like proteases, but did not differ between strains for chymotrypsin-like proteases. The results here are consistent with previous reports indicating that reduced activation of protoxin is generally not a major mechanism of resistance to Bt proteins.
Assuntos
Toxinas Bacterianas/farmacologia , Mariposas/efeitos dos fármacos , Precursores de Proteínas/farmacologia , Animais , Controle de Insetos , Resistência a Inseticidas , Larva/efeitos dos fármacos , Tosilina Clorometil Cetona/farmacologiaRESUMO
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
A metal-free decarboxylative amination of 4-(ethoxycarbonyl)-2,3-allenols by TsNCO via base-induced aza-Michael addition/elimination has been developed. A variety of substituted N-tosyl 1,3-dien-2-yl amines were obtained in good yields and excellent regio- and stereoselectivity. Moreover, this transformation could be applied in preparation of 2-amino-trienes.
Assuntos
Alcadienos/síntese química , Aminas/química , Aminas/síntese química , Tosilina Clorometil Cetona/análogos & derivados , Tosilina Clorometil Cetona/química , Alcadienos/química , Aminação , Catálise , Estrutura Molecular , EstereoisomerismoRESUMO
It is well-established that activation of proteases, such as caspases, calpains and cathepsins are essential components in signaling pathways of programmed cell death (PCD). Although these proteases have also been linked to mechanisms of neuronal cell death, they are dispensable in paradigms of intrinsic death pathways, e.g. induced by oxidative stress. However, emerging evidence implicated a particular role for serine proteases in mechanisms of PCD in neurons. Here, we investigated the role of trypsin-like serine proteases in a model of glutamate toxicity in HT-22 cells. In these cells glutamate induces oxytosis, a form of caspase-independent cell death that involves activation of the pro-apoptotic protein BH3 interacting-domain death agonist (Bid), leading to mitochondrial demise and ensuing cell death. In this model system, the trypsin-like serine protease inhibitor Nα-tosyl-l-lysine chloromethyl ketone hydrochloride (TLCK) inhibited mitochondrial damage and cell death. Mitochondrial morphology alterations, the impairment of the mitochondrial membrane potential and ATP depletion were prevented and, moreover, lipid peroxidation induced by glutamate was completely abolished. Strikingly, truncated Bid-induced cell death was not affected by TLCK, suggesting a detrimental activity of serine proteases upstream of Bid activation and mitochondrial demise. In summary, this study demonstrates the protective effect of serine protease inhibition by TLCK against oxytosis-induced mitochondrial damage and cell death. These findings indicate that TLCK-sensitive serine proteases play a crucial role in cell death mechanisms upstream of mitochondrial demise and thus, may serve as therapeutic targets in diseases, where oxidative stress and intrinsic pathways of PCD mediate neuronal cell death.
Assuntos
Mitocôndrias/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Inibidores de Serina Proteinase/farmacologia , Tosilina Clorometil Cetona/farmacologia , Animais , Apoptose/efeitos dos fármacos , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ácido Glutâmico/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Neurônios/metabolismo , Estresse Oxidativo , Transdução de Sinais , Tosilfenilalanil Clorometil Cetona/farmacologiaRESUMO
The serine protease inhibitor (SERPIN) family member corticosteroid-binding globulin (CBG) is the main carrier of glucocorticoids in plasma. Human CBG mediates the targeted release of cortisol at sites of inflammation through cleavage of its reactive center loop (RCL) by neutrophil elastase. The RCLs of SERPIN family members are targeted by diverse endogenous and exogenous proteases, including several bacterial proteases. We tested different bacteria for their ability to secrete proteases that disrupt CBG cortisol-binding activity, and characterized the responsible protease and site of CBG cleavage. Serum CBG integrity was assessed by Western blotting and cortisol-binding capacity assay. Effects of time, pH, temperature, and protease inhibitors were tested. Proteolytically active proteins from bacterial media were purified by fast protein liquid chromatography, and the active protease and CBG cleavage sites were identified by mass spectrometry. Among the bacteria tested, medium from Pseudomonas aeruginosa actively disrupted the cortisol-binding activity of CBG. This proteolytic activity was inhibited by zinc chelators and occurred most efficiently at pH 7 and elevated physiological temperature (ie, 41°C). Mass spectrometric analysis of a semi-purified fraction of P. aeruginosa media identified the virulence factor LasB as the responsible protease, and this was confirmed by assaying media from LasB-deficient P. aeruginosa. This metalloprotease cleaves the CBG RCL at a major site, distinct from that targeted by neutrophil elastase. Our results suggest that humoral responses to P. aeruginosa infection are influenced by this pathogen's ability to secrete a protease that promotes the release of the anti-inflammatory steroid, cortisol, from its plasma transport protein.
Assuntos
Proteínas de Bactérias/toxicidade , Hidrocortisona/metabolismo , Metaloendopeptidases/toxicidade , Pseudomonas aeruginosa/enzimologia , Transcortina/metabolismo , Proteínas de Bactérias/fisiologia , Meios de Cultivo Condicionados , Humanos , Hidrocortisona/antagonistas & inibidores , Concentração de Íons de Hidrogênio , Elastase de Leucócito/fisiologia , Metaloendopeptidases/fisiologia , Pseudomonas aeruginosa/patogenicidade , Temperatura , Tosilina Clorometil Cetona , Transcortina/antagonistas & inibidores , Fatores de Virulência/toxicidade , ZincoRESUMO
In mammals, proteases are present in sperm acrosome and play key role in fertilization. Sturgeon sperm has an acrosome, but its physiology, biochemistry, and potential role in fertilization are unknown. In the present study, we have observed high protease activity in acidic extract of intact sperm compared to that of seminal plasma in sterlet (Acipenser ruthenus). The protease activity was decreased and increased in acidic extract of motility-activated sperm and in the activation medium, respectively. Molecular analysis revealed total protease and serine (acrosin) protease activities in sperm acidic extract which was accumulated in a protein band with relative molecular mass of 35 kDa. Immunoelectron microscopy using an affinity-purified polyclonal antibody for boar acrosin localized the protease at the acrosome region. Moreover, initiation of sperm motility was inhibited after activation in the presence of inhibitors for both trypsin-like and chymotrypsin-like proteases, while the effects of protease inhibitors on sperm velocity were uncertain. Our results indicate similarities in physiology and biochemistry of acrosome between sturgeon and mammals and suggest potential role of protease in the initiation of sperm motility in sturgeon.
Assuntos
Peixes/fisiologia , Peptídeo Hidrolases/farmacologia , Inibidores de Proteases/farmacologia , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/enzimologia , Espermatozoides/fisiologia , Acrosina/metabolismo , Acrossomo/enzimologia , Análise de Variância , Animais , Técnicas Histológicas/veterinária , Masculino , Microscopia Imunoeletrônica/veterinária , Corantes de Rosanilina , Sêmen/enzimologia , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/efeitos dos fármacos , Estatísticas não Paramétricas , Tosilina Clorometil Cetona/farmacologia , Tosilfenilalanil Clorometil Cetona/farmacologiaRESUMO
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
Trichomonas gallinae, the aetiological agent of avian trichomonosis, was shown to secrete soluble factors involved in cytopathogenic effect on a permanent chicken liver (LMH) cell culture. The present study focused on the characterization of these molecules. The addition of specific peptidase inhibitors to the cell-free filtrate partially inhibited the monolayer destruction, which implied the presence of peptidases in the filtrate and their involvement in the cytopathogenic effect. One-dimensional substrate (gelatin) SDS-PAGE confirmed the proteolytic character of the filtrate by demonstrating the proteolytic activity within the molecular weight range from 38 to 110 kDa. In addition, the proteolytic activity was specifically inhibited by addition of TLCK and E-64 cysteine peptidase inhibitors implying their cysteine peptidase nature. Furthermore, variations in the intensity and the number of proteolytic bands were observed between cell-free filtrates of low and high passages of the same T. gallinae clonal culture. Two-dimensional substrate gel electrophoresis of concentrated T. gallinae cell-free filtrate identified at least six proteolytic spots. The mass spectrometric analysis of spots from 2-D gels identified the presence of at least two different Clan CA, family C1, cathepsin L-like cysteine peptidases in the cell-free filtrate of T. gallinae. In parallel, a PCR approach using degenerated primers based on the conserved amino acid sequence region of cysteine peptidases from Trichomonas vaginalis identified the coding sequences for four different Clan CA, family C1, cathepsin L-like cysteine peptidases. Finally, this is the first report analyzing molecules secreted by T. gallinae and demonstrating the ubiquity of peptidases secreted by this protozoon.
Assuntos
Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Hepatócitos/metabolismo , Trichomonas/enzimologia , Animais , Células Cultivadas , Galinhas , Cisteína Endopeptidases/genética , Primers do DNA/genética , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Leucina/análogos & derivados , Espectrometria de Massas , Tosilina Clorometil CetonaRESUMO
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
Entre os alvos mais promissores para o desenvolvimento de novos agentes antiparasitários, destacam-se as proteases que nos protozoários participam de processos metabólicos e fisiológicos, atuando como importantes fatores de virulência. Como a atividade dessas moléculas pode ser controlada por inibidores específicos, essas substâncias têm sido avaliadas quanto ao potencial terapêutico em diferentes infecções parasitárias, inclusive por Giardia. O presente estudo foi desenvolvido com o objetivo de avaliar o efeito in vitro dos inibidores de cisteína (IAA e E-64) e serina-proteases (antipaina, leupeptina e TLCK) sobre o crescimento, aderência, viabilidade e ultraestrutura de trofozoítos de cepa de Giardia isolada e axenizada em Botucatu. Para isso, trofozoítos foram incubados em meio contendo os inibidores a diferentes concentrações durante 24, 48 e 72 horas. Nos ensaios de crescimento e aderência, o número de trofozoítos foi estimado a partir de contagens em hemocitômetro, enquanto que a viabilidade celular e as alterações ultraestruturais foram avaliadas, respectivamente, pelo método de redução do MTT e por microscopia eletrônica de transmissão. De acordo com as observações feitas no presente estudo, todos os inibidores de proteases apresentaram efeito sobre o crescimento, aderência e viabilidade dos trofozoítos. Entretanto, melhor desempenho quanto à capacidade de reduzir os parâmetros avaliados foi demonstrado nos ensaios com os inibidores de cisteína-proteases, especialmente a IAA. As maiores porcentagens de inibição do crescimento e aderência e as menores taxas de viabilidade foram observadas após o tratamento com IAA...
The quest for new antiparasitic alternatives has led researchers to base their studies on insights into biology, host-parasite interactions and pathogenesis. In light of this, the proteolytic enzymes or proteases have excited the researchers interest, once they have been identified as important virulence factors as well as potential chemotherapeutic targets in parasites. Considering that proteases are naturally regulated by specific inhibitors, these substances have been evaluated for their therapeutic potential in parasitic infections including Giardia. In this way, we proposed to evaluate the in vitro effect of inhibitors of cysteine (IAA and E-64) and serine proteases (antipain, leupeptin and TLCK) on growth, adherence, viability and ultrastructure of Giardia trophozoites of a strain isolated and axenized in Botucatu. For this, trophozoites were incubated in medium containing the inhibitors at various concentrations for 24, 48 and 72 hours. In growth and adherence assays, the number of trophozoites was estimated microscopically in a haemocytometer, whereas cell viability and ultrastructural changes were evaluated, respectively, by the method of MTT and transmission electron microscopy. In this study, all protease inhibitors showed effect on growth, adherence and viability of trophozoites. However, better performance in their ability to reduce the parameters assessed was demonstrated in experiments with cysteine proteases inhibitors, especially IAA...
Assuntos
Humanos , Antipaína/antagonistas & inibidores , Cisteína/antagonistas & inibidores , Giardia lamblia/isolamento & purificação , Técnicas In Vitro , Leupeptinas/antagonistas & inibidores , Inibidores de Proteases , Serina Endopeptidases , Tosilina Clorometil Cetona/antagonistas & inibidores , TrofozoítosRESUMO
A series of novel ethyl 5-(4-aminophenyl)-1H-pyrazole-3-carboxylate derivatives were designed and synthesized and their in vitro acrosin inhibitory activities were evaluated. Most of the compounds exhibited acrosin inhibitory activities. Among them, three compounds (5l, 5n, and 5v) were more potent than that of the control TLCK. These provide a new structural type for the development of novel contraceptive acrosin inhibitory agents.
Assuntos
Acrosina/antagonistas & inibidores , Ácidos Carboxílicos/síntese química , Anticoncepcionais/síntese química , Pirazóis/síntese química , Inibidores de Serina Proteinase/síntese química , Software , Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacologia , Anticoncepcionais/química , Anticoncepcionais/farmacologia , Desenho de Fármacos , Fertilização/fisiologia , Humanos , Masculino , Terapia de Alvo Molecular , Pirazóis/química , Pirazóis/farmacologia , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologia , Relação Estrutura-Atividade , Tosilina Clorometil Cetona/química , Tosilina Clorometil Cetona/metabolismo , Tosilina Clorometil Cetona/farmacologiaRESUMO
Malaria parasites reside in human erythrocytes within a parasitophorous vacuole. The parasites are transmitted from the human to the mosquito by the uptake of intraerythrocytic gametocytes during a blood meal, which in the midgut become activated by external stimuli and subsequently egress from the enveloping erythrocyte. Gametocyte egress is a crucial step for the parasite to prepare for fertilization, but the molecular mechanisms of egress are not well understood. Via electron microscopy, we show that Plasmodium falciparum gametocytes exit the erythrocyte by an inside-out type of egress. The parasitophorous vacuole membrane (PVM) ruptures at multiple sites within less than a minute following activation, a process that requires a temperature drop and parasite contact with xanthurenic acid. PVM rupture can also be triggered by the ionophore nigericin and is sensitive to the cysteine protease inhibitor E-64d. Following PVM rupture the subpellicular membrane begins to disintegrate. This membrane is specific to malaria gametocytes, and disintegration is impaired by the aspartic protease inhibitor EPNP and the cysteine/serine protease inhibitor TLCK. Approximately 15 min post activation, the erythrocyte membrane ruptures at a single breaking point, which can be inhibited by inhibitors TLCK and TPCK. In all cases inhibitor treatment results in interrupted gametogenesis.
Assuntos
Culicidae/parasitologia , Eritrócitos/parasitologia , Plasmodium falciparum/crescimento & desenvolvimento , Animais , Compostos de Epóxi/metabolismo , Eritrócitos/ultraestrutura , Humanos , Membranas Intracelulares/ultraestrutura , Leucina/análogos & derivados , Leucina/metabolismo , Microscopia Eletrônica , Nigericina/metabolismo , Nitrofenóis/metabolismo , Plasmodium falciparum/ultraestrutura , Temperatura , Tosilina Clorometil Cetona/metabolismo , Vacúolos/parasitologia , Vacúolos/ultraestrutura , Xanturenatos/metabolismoRESUMO
This study reports the biochemical characterization and comparative analyses of highly active serine proteases in the larval and pupal developmental stages of Aedes aegypti (Linnaeus) using substrate-SDS-PAGE. Zymographic analysis of larval stadia detected proteolytic activity in 6-8 bands with apparent molecular masses ranging from 20 to 250 kDa, with activity observed from pH 5.5 to 10.0. The pupal stage showed a complex proteolytic activity in at least 11 bands with apparent Mr ranging from 25 to 250 kDa, and pH optimum at 10.0. The proteolytic activities of both larval and pupal stages were strongly inhibited by phenyl-methyl sulfonyl-fluoride and N-α-Tosyl-L-lysine chloromethyl ketone hydrochloride, indicating that the main proteases expressed by these developmental stages are trypsin-like serine proteases. The enzymes were active at temperatures ranging from 4 to 85°C, with optimal activity between 37 and 60°C, and low activity at 85°C. Comparative analysis between the proteolytic enzymes expressed by larvae and pupae showed that substantial changes in the expression of active trypsin-like serine proteases occur during the developmental cycle of A. aegypti.