RESUMO
Prosthetic infections are associated with high morbidity, mortality, and relapse rates, making them still a serious problem for implantology. Staphylococcus aureus is one of the most common bacterial pathogens causing prosthetic infections. In response to the increasing rate of bacterial resistance to commonly used antibiotics, this work proposes a method for combating pathogenic microorganisms by modifying the surfaces of synthetic polymeric biomaterials using proteolytic enzyme inhibitors (serine protease inhibitors-4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride and puromycin). While using techniques based on the immobilization of biologically active molecules, it is important to monitor the changes occurring on the surface of the modified biomaterial, where spectroscopic techniques (e.g., FTIR) are ideal. ATR-FTIR measurements demonstrated that the immobilization of both inhibitors caused large structural changes on the surface of the tested vascular prostheses (polyester or polytetrafluoroethylene) and showed that they were covalently bonded to the surfaces of the biomaterials. Next, the bactericidal and antibiofilm activities of the tested serine protease inhibitors were determined using the CLSM microscopic technique with fluorescent staining. During LIVE/DEAD analyses, a significant decrease in the formation of Staphylococcus aureus biofilm after exposure to selected concentrations of native inhibitors (0.02-0.06 mg/mL for puromycin and 0.2-1 mg/mL for 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride) was demonstrated.
Assuntos
Anti-Infecciosos , Infecções Estafilocócicas , Sulfonas , Humanos , Prótese Vascular , Antibacterianos/farmacologia , Biofilmes , Inibidores de Serina Proteinase/farmacologia , Staphylococcus aureus , Materiais Biocompatíveis , Puromicina , Peptídeo HidrolasesRESUMO
Abstract: Viruses invade the host cells and maneuver the cellular translation machinery to translate the viral proteins in substantial amounts, which may disturb Endoplasmic Reticulum homeostasis leading to induction of Unfolded Protein Response (UPR), a host response pathway involved in viral pathogenesis. Here, we investigated the effect of UPR pathways on the pathogenesis of chikungunya virus infection. We observed that chikungunya virus mediated the modulation of UPR. A positive modulation was observed in the activation of IRE1 and ATF6 branch while the PERK branch of UPR observed suppressed upon virus infection. We further investigated the effect of the inhibition of UPR pathways on chikungunya virus replication using inhibitors for each branch. Cells treated with 3-ethoxy-5,6-dibromosalicylaldehyde (IRE1 inhibitor) and AEBSF (ATF6 inhibitor) significantly inhibits the viral replication process. This study has provided a novel perspective in designing antivirals against chikungunya virus. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-022-01046-5.
RESUMO
Application of a protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), during the cell culture process was demonstrated to effectively reduce proteolytic activity at a specific amino acid site during the production of an HIV-1 broadly neutralizing antibody (bNAb). However, the addition of AEBSF could potentially introduce some modifications to the bNAb protein. Experimental design from sample preparation to LC-MS characterization was performed using middle-up and bottom-up approaches to identify AEBSF-modified species for the bNAb using an AEBSF supplementation in the cell culture media. Modified species along with the unmodified control sample were also subjected to binding activity assessment. The results showed that two amino acids (Tyr177 and Lys250) were susceptible to AEBSF modification in the bNAb test articles but at a negligible level and not in the CDR regions, which therefore did not reduce the in vitro binding activity of the bNAb.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Imunoconjugados/imunologia , Inibidores de Proteases/imunologia , Sulfonas/imunologia , Sequência de Aminoácidos , Anticorpos Neutralizantes/química , Anticorpos Anti-HIV/química , Infecções por HIV/virologia , Humanos , Imunoconjugados/química , Inibidores de Proteases/química , Sulfonas/química , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Host proteases have been shown to play important roles in many viral activities such as entry, uncoating, viral protein production and disease induction. Therefore, these cellular proteases are putative targets for the development of antivirals that inhibit their activity. Host proteases have been described to play essential roles in Ebola, HCV, HIV and influenza, such that specific protease inhibitors are able to reduce infection. RSV utilizes a host protease in its replication cycle but its potential as antiviral target is unknown. Therefore, we evaluated the effect of protease inhibitors on RSV infection. METHODS: To measure the sensitivity of RSV infection to protease inhibitors, cells were infected with RSV and incubated for 18 h in the presence or absence of the inhibitors. Cells were fixed, stained and studied using fluorescence microscopy. RESULTS: Several protease inhibitors, representing different classes of proteases (AEBSF, Pepstatin A, E-64, TPCK, PMSF and aprotinin), were tested for inhibitory effects on an RSV A2 infection of HEp-2 cells. Different treatment durations, ranging from 1 h prior to inoculation and continuing for 18 h during the assay, were evaluated. Of all the inhibitors tested, AEBSF and TPCK significantly decreased RSV infection. To ascertain that the observed effect of AEBSF was not a specific feature related to HEp-2 cells, A549 and BEAS-2B cells were also used. Similar to HEp-2, an almost complete block in the number of RSV infected cells after 18 h of incubation was observed and the effect was dose-dependent. To gain insight into the mechanism of this inhibition, AEBSF treatment was applied during different phases of an infection cycle (pre-, peri- and post-inoculation treatment). The results from these experiments indicate that AEBSF is mainly active during the early entry phase of RSV. The inhibitory effect was also observed with other RSV isolates A1998/3-2 and A2000/3-4, suggesting that this is a general feature of RSV. CONCLUSION: RSV infection can be inhibited by broad serine protease inhibitors, AEBSF and TPCK. We confirmed that AEBSF inhibition is independent of the cell line used or RSV strain. The time point at which treatment with the inhibitor was most potent, was found to coincide with the expected moment of entry of the virion with the host cell.
Assuntos
Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Vírus Sincicial Respiratório Humano/patogenicidade , Inibidores de Serina Proteinase/farmacologia , Sulfonas/antagonistas & inibidores , Internalização do Vírus/efeitos dos fármacos , Células A549 , Aprotinina/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Endopeptidases/efeitos dos fármacos , Humanos , Cinética , Leucina/análogos & derivados , Leucina/antagonistas & inibidores , Pepstatinas/antagonistas & inibidores , Fatores de Tempo , Tosilfenilalanil Clorometil Cetona/antagonistas & inibidores , Proteínas Virais/metabolismoRESUMO
Tuberin is a negative regulator of mTOR pathway. To investigate the function of tuberin during liver regeneration, we performed 70% hepatectomy on wild-type and Tsc2+/- mice. We found the tuberin phosphorylation correlated with mTOR activation during early liver regeneration in wild-type mice. However, liver regeneration in the Tsc2+/- mice was not enhanced. Instead, the Tsc2+/- livers failed to accumulate lipid bodies, and this was accompanied by increased mortality. These findings suggest that tuberin plays a critical role in liver energy balance by regulating hepatocellular lipid accumulation during early liver regeneration. These effects may influence the role of mTORC1 on cell growth and proliferation.
Assuntos
Heterozigoto , Metabolismo dos Lipídeos , Regeneração Hepática , Fígado/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Peso Corporal , Ativação Enzimática , Hepatectomia , Fígado/patologia , Fígado/cirurgia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Tamanho do Órgão , Fosforilação , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Fatores de Tempo , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/deficiênciaRESUMO
Trypanosoma congolense is a haemoprotozoan parasite that causes African animal trypanosomosis, a wasting disease of cattle and small ruminants. Current control methods are unsatisfactory and no conventional vaccine exists due to antigenic variation. An anti-disease vaccine approach to control T. congolense has been proposed requiring the identification of parasitic factors that cause disease. Immunoprecipitation of T. congolense antigens using sera from infected trypanotolerant cattle allowed the identification of several immunogenic antigens including two M1 type aminopeptidases (APs). The two APs were cloned and expressed in Escherichia coli. As the APs were expressed as insoluble inclusion bodies it was necessary to develop a method for solubilisation and subsequent refolding to restore conformation and activity. The refolded APs both showed a distinct substrate preference for H-Ala-AMC, an optimum pH of 8.0, puromycin-sensitivity, inhibition by bestatin and amastatin, and cytoplasmic localisation. The two APs are expressed in procyclic metacyclic and bloodstream form parasites. Down-regulation of both APs by RNAi resulted in a slightly reduced growth rate in procyclic parasites in vitro.
Assuntos
Antígenos CD13/metabolismo , Trypanosoma congolense/enzimologia , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/química , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/isolamento & purificação , Western Blotting , Antígenos CD13/química , Antígenos CD13/genética , Cátions Bivalentes/farmacologia , Bovinos , Cromatografia Líquida , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Regulação Enzimológica da Expressão Gênica , Concentração de Íons de Hidrogênio , Imunoprecipitação , Metais/farmacologia , Camundongos , Redobramento de Proteína , RNA de Protozoário/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Massas em Tandem , Trypanosoma congolense/genética , Trypanosoma congolense/imunologiaRESUMO
von Hippel-Lindau protein (pVHL) is the tumor suppressor responsible for ubiquitylating the hypoxia-inducible factor (HIF) family of transcription factors for degradation under normoxic conditions. There are two major pVHL isoforms with the shorter isoform (pVHL19 ) lacking the acidic domain present in the N-terminus of the longer isoform (pVHL30 ). Although both isoforms can degrade HIF and suppress tumor formation in experimental systems, previous research suggests that pVHL30 can undergo posttranslational modifications (PTM) and interact with unique proteins. Indeed, pVHL30 has long been observed to migrate as two species on a reducing polyacrylamide gel, indicating the presence of an uncharacterized PTM on the slower-migrating pVHL30 without an identifiable biological consequence. Thus, there has been considerable effort to elucidate the exclusive biological activity of pVHL30 , if any, by first defining the unique features of the slower-migrating species. We show here that the migration of pVHL30 , but not pVHL19 , is retarded by 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), an irreversible serine protease inhibitor commonly found in protease inhibitor cocktails.
Assuntos
Sulfonas/química , Proteína Supressora de Tumor Von Hippel-Lindau/química , Humanos , Isoenzimas/química , Domínios ProteicosRESUMO
Dengue virus (DENV) infection has evolved into a major global health menace and economic burden due to its intensity and geographic distribution. DENV infection in humans can cause a wide range of symptoms including dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). An antiviral agent that is effective against all four serotypes of DENV is urgently needed to prevent and to manage this condition. Reducing the viral load during the early phase of infection may minimize the chance of patients progressing to more severe DHF or DSS. In this study, we set forth to investigate the anti-viral effect of five commercially available protease inhibitors on DENV infection since both viral and host proteases can contribute to effective viral replication. Previously, the serine protease inhibitor AEBSF [4-(2-aminoethyl) benzene sulfonyl fluoride] has been shown to inhibit DENV NS3 protease activity. The results of the present study revealed that DENV genome replication and protein synthesis were significantly inhibited by AEBSF in a dose-dependent manner. AEBSF inhibited the expression of genes such as 3-hydroxy 3-methyl-glutaryl-CoA synthase (HMGCS), 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR), and low-density lipoprotein receptor (LDLR). Moreover, AEBSF significantly inhibited HMGCR activity and intracellular cholesterol synthesis after DENV infection. The anti-DENV effect of AEBSF was confirmed in all four DENV serotypes and in three different cell lines. These results indicate that AEBSF reduces DENV infection via both viral and host protease activities.
Assuntos
Colesterol/biossíntese , Vírus da Dengue/efeitos dos fármacos , Dengue/metabolismo , Dengue/virologia , Inibidores de Serina Proteinase/farmacologia , Sulfonas/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Vírus da Dengue/classificação , Vírus da Dengue/genética , Genoma Viral , Humanos , Replicação ViralRESUMO
During research of a broadly neutralizing antibody (bNAb) for HIV-1 infection, site-specific clipping was observed during cell culture incubation. Protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), was supplemented to the cell culture feeding to mitigate clipping as one of the control strategies. It led to the need and development of a new assay to monitor the free AEBSF-related impurities during the purification process. In this work, a reversed-phase liquid chromatography (RPLC-UV) method was developed to measure the total concentration of AEBSF and its major degradant product, 4-(aminoethyl) benzenesulfonic acid (AEBS-OH). This quantitative approach involved hydrolysis pre-treatment to drive all AEBSF to AEBS-OH, a filtration step to remove large molecules, followed by RPLC-UV analysis. The method was qualified and shown to be capable of measuring AEBS-OH down to 0.5⯵M with good accuracy and precision, which was then applied for process clearance studies. The results demonstrated that a Protein A purification step in conjunction with a mock ultrafiltration/diafiltration (UF/DF) step could remove AEBSF-related impurities below the detection level. Overall, this study is the first to provide a unique approach for monitoring the clearance of free AEBSF and its related degradant, AEBS-OH, in support of the bNAb research.
Assuntos
Cromatografia de Fase Reversa/métodos , Contaminação de Medicamentos , Sulfonas/análise , Fármacos Anti-HIV/química , Fármacos Anti-HIV/normas , Anticorpos Neutralizantes/química , Anticorpos Anti-HIV/química , Humanos , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos Testes , Tecnologia FarmacêuticaRESUMO
Background & Aims: Antibiotic (ABx) therapy is associated with increased risk for Crohn's disease but underlying mechanisms are unknown. We observed high fecal serine protease activity (PA) to be a frequent side effect of ABx therapy. The aim of the present study was to unravel whether this rise in large intestinal PA may promote colitis development via detrimental effects on the large intestinal barrier. Methods: Transwell experiments were used to assess the impact of high PA in ABx-treated patients or vancomycin/metronidazole-treated mice on the epithelial barrier. Serine protease profiling was performed using liquid chromatography-mass spectrometry/mass spectrometry analysis. The impact of high large intestinal PA on the intestinal barrier in wild-type and interleukin (IL)10-/- mice and on colitis development in IL10-/- mice was investigated using vancomycin/metronidazole with or without oral serine protease inhibitor (AEBSF) treatment. Results: The ABx-induced, high large intestinal PA was caused by significantly increased levels of pancreatic proteases and impaired epithelial barrier integrity. In wild-type mice, the rise in PA caused a transient increase in intestinal permeability but did not affect susceptibility to chemically induced acute colitis. In IL10-/- mice, increased PA caused a consistent impairment of the intestinal barrier associated with inflammatory activation in the large intestinal tissue. In the long term, the vancomycin/metronidazole-induced lasting increase in PA aggravated colitis development in IL10-/- mice. Conclusions: High large intestinal PA is a frequent adverse effect of ABx therapy, which is detrimental to the large intestinal barrier and may contribute to the development of chronic intestinal inflammation in susceptible individuals.
Assuntos
Antibacterianos/efeitos adversos , Colite/metabolismo , Intestino Grosso/enzimologia , Serina Proteases/metabolismo , Animais , Colite/induzido quimicamente , Sulfato de Dextrana/farmacologia , Modelos Animais de Doenças , Fezes/enzimologia , Fezes/microbiologia , Humanos , Intestino Grosso/microbiologia , Metronidazol/efeitos adversos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Risco , Sulfonas/farmacologia , Vancomicina/efeitos adversosRESUMO
The Rho guanine nucleotide exchange factor protoDbl is involved in different biochemical pathways affecting cell proliferation and migration. The N-terminal sequence of protoDbl contains negative regulatory elements that restrict the catalytic activity of the DH-PH module. Here, we report the identification of a new mouse protoDbl splice variant lacking exon 3. We found that the splice variant mRNA is expressed in the spleen and bone marrow lymphocytes, adrenal gland, gonads and brain. The protoDbl variant protein was detectable in the brain. The newly identified variant displays the disruption of the SEC14 domain, positioned on exons 2 and 3 in the protoDbl N-terminal region. We show here that an altered SEC14 sequence leads to enhanced Dbl translocation to the plasma membrane and to augmented transforming and exchange activity.
Assuntos
Processamento Alternativo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proto-Oncogenes , Sequência de Aminoácidos , Animais , Linfócitos B/metabolismo , Encéfalo/metabolismo , Células COS , Membrana Celular/metabolismo , Éxons , Feminino , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Proteínas de Transferência de Fosfolipídeos/metabolismo , Estrutura Terciária de Proteína , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro , Proteínas de Saccharomyces cerevisiae/metabolismo , Baço/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
Senescence-related proteases play important roles in leaf senescence by regulating protein degradation and nutrient recycling. A 98.9kDa senescence-related protease EP3 in wheat leaves was purified by ammonium sulfate precipitation, Q-Sepharose fast flow anion exchange chromatography and gel slicing after gel electrophoresis. Due to its relatively high thermal stability, its protease activity did not decrease after incubation at 40°C for 1-h. EP3 protease was suggested to be a metal-dependent serine protease, because its activity was inhibited by serine protease inhibitors PMSF and AEBSF and metal related protease inhibitor EGTA. It was identified as a subtilisin-like serine protease of the S8A family based on data from both mass spectrometry and the cloned cDNA sequence. Therefore, these data suggest that a serine protease of the S8A subfamily with specific biochemical properties is involved in senescence-associated protein degradation.
Assuntos
Senescência Celular , Escuridão , Folhas de Planta/fisiologia , Proteínas de Plantas/metabolismo , Serina Endopeptidases/metabolismo , Triticum/enzimologia , Sequência de Aminoácidos , Clonagem Molecular , DNA Complementar , Ácido Egtázico/farmacologia , Temperatura Alta , Dados de Sequência Molecular , Folhas de Planta/enzimologia , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Proteólise , Serina Endopeptidases/química , Serina Endopeptidases/isolamento & purificação , Inibidores de Serina Proteinase/farmacologia , Estresse Fisiológico , Subtilisina/metabolismo , Sulfonas/farmacologia , Triticum/química , Triticum/fisiologiaRESUMO
The objective of this study was to determine the neuroprotective role of tropisetron on retinal ganglion cells (RGCs) as well as to explore the possible mechanisms associated with alpha7 nAChR-induced neuroprotection. Adult pig RGCs were isolated from all other retinal tissue using a two-step panning technique. Once isolated, RGCs were cultured for 3 days under control untreated conditions, in the presence of 500 µM glutamate to induce excitotoxicity, and when tropisetron was applied before glutamate to induce neuroprotection. 500 µM glutamate decreased RGC survival by an average of 62% compared to control conditions. However, RGCs pretreated with 100 nM tropisetron before glutamate increased cell survival to an average of 105% compared to controls. Inhibition studies using the alpha7 nAChR antagonist, MLA (10 nM), support the hypothesis that tropisetron is an effective neuroprotective agent against glutamate-induced excitotoxicity; mediated by α7 nAChR activation. ELISA studies were performed to determine if signaling cascades normally associated with excitotoxicity and neuroprotection were up- or down-regulated after tropisetron treatment. Tropisetron had no discernible effects on pAkt levels but significantly decreased p38 MAPK levels associated with excitotoxicity from an average of 15 ng/ml to 6 ng/ml. Another mechanism shown to be associated with neuroprotection involves internalization of NMDA receptors. Double-labeled immunocytochemistry and electrophysiology studies provided further evidence that tropisetron caused internalization of NMDA receptor subunits. The findings of this study suggest that tropisetron could be an effective therapeutic agent for the treatment of degenerative disorders of the central nervous system that involves excitotoxicity.
Assuntos
Ácido Glutâmico/toxicidade , Indóis/farmacologia , Fármacos Neuroprotetores/farmacologia , Agonistas Nicotínicos/farmacologia , Receptor Nicotínico de Acetilcolina alfa7/agonistas , Aconitina/análogos & derivados , Aconitina/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Indóis/antagonistas & inibidores , Antagonistas Nicotínicos/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Suínos , Tropizetrona , Receptor Nicotínico de Acetilcolina alfa7/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Human cysteine cathepsins (Cats) are implicated in lung injuries and tissue remodeling and have recently emerged as important players in pulmonary inflammations. The proteolytic activities of Cat B, L, K, S and H are dramatically increased in the sputum of patients with cystic fibrosis (CF), suggesting a possible involvement in the CF pathophysiology. We found that pulmonary surfactant protein A (SP-A) that participates to innate host defense is extensively degraded in CF expectorations. Breakdown of SP-A was markedly decreased in CF sputum by E-64 and Mu-Leu-Hph-VSPh, a Cat S inhibitor. Cat S cleaved efficiently and specifically SP-A within critical residues of the solvent-exposed loop of its carbohydrate recognition (C-type lectin) domain that allows binding to pathogens. Cat S decreased aggregation properties of SP-A (self-aggregation, aggregation of phospholipid vesicles and rough LPS). Moreover cleavage of SP-A by Cat S reduced binding to yeast mannan and impaired agglutination of Escherichia coli and Pseudomonas aeruginosa, a foremost detrimental pathogen colonizing the lungs of CF patients. Besides human neutrophil serine proteases and bacterial proteases, we propose that Cat S may participate in the pathophysiology of CF by weakening the antibacterial activity of SP-A. More broadly, present results provide further indication that Cat S, along with Cats B and L, could display immuno-modulatory functions by inactivating key proteins involved in the innate immunity defense.