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1.
Nat Commun ; 11(1): 4046, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792488

RESUMO

2-oxoglutarate (2-OG or α-ketoglutarate) relates mitochondrial metabolism to cell function by modulating the activity of 2-OG dependent dioxygenases involved in the hypoxia response and DNA/histone modifications. However, metabolic pathways that regulate these oxygen and 2-OG sensitive enzymes remain poorly understood. Here, using CRISPR Cas9 genome-wide mutagenesis to screen for genetic determinants of 2-OG levels, we uncover a redox sensitive mitochondrial lipoylation pathway, dependent on the mitochondrial hydrolase ABHD11, that signals changes in mitochondrial 2-OG metabolism to 2-OG dependent dioxygenase function. ABHD11 loss or inhibition drives a rapid increase in 2-OG levels by impairing lipoylation of the 2-OG dehydrogenase complex (OGDHc)-the rate limiting step for mitochondrial 2-OG metabolism. Rather than facilitating lipoate conjugation, ABHD11 associates with the OGDHc and maintains catalytic activity of lipoyl domain by preventing the formation of lipoyl adducts, highlighting ABHD11 as a regulator of functional lipoylation and 2-OG metabolism.


Assuntos
Complexo Cetoglutarato Desidrogenase/metabolismo , Ácidos Cetoglutáricos/metabolismo , Mitocôndrias/metabolismo , Mutagênese/fisiologia , Serina Proteases/metabolismo , Metabolismo Energético/genética , Metabolismo Energético/fisiologia , Células HeLa , Humanos , Complexo Cetoglutarato Desidrogenase/genética , Modelos Biológicos , Mutagênese/genética , Serina Proteases/genética
2.
J Biol Chem ; 295(36): 12686-12696, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32675285

RESUMO

Type II transmembrane serine proteases (TTSPs) are a group of enzymes participating in diverse biological processes. Some members of the TTSP family are implicated in viral infection. TMPRSS11A is a TTSP expressed on the surface of airway epithelial cells, which has been shown to cleave and activate spike proteins of the severe acute respiratory syndrome (SARS) and the Middle East respiratory syndrome coronaviruses (CoVs). In this study, we examined the mechanism underlying the activation cleavage of TMPRSS11A that converts the one-chain zymogen to a two-chain enzyme. By expression in human embryonic kidney 293, esophageal EC9706, and lung epithelial A549 and 16HBE cells, Western blotting, and site-directed mutagenesis, we found that the activation cleavage of human TMPRSS11A was mediated by autocatalysis. Moreover, we found that TMPRSS11A activation cleavage occurred before the protein reached the cell surface, as indicated by studies with trypsin digestion to remove cell surface proteins, treatment with cell organelle-disturbing agents to block intracellular protein trafficking, and analysis of a soluble form of TMPRSS11A without the transmembrane domain. We also showed that TMPRSS11A was able to cleave the SARS-CoV-2 spike protein. These results reveal an intracellular autocleavage mechanism in TMPRSS11A zymogen activation, which differs from the extracellular zymogen activation reported in other TTSPs. These findings provide new insights into the diverse mechanisms in regulating TTSP activation.


Assuntos
Células Epiteliais/metabolismo , Proteínas de Membrana/metabolismo , Proteólise , Serina Proteases/metabolismo , Células A549 , Células Cultivadas , Células HEK293 , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mutação , Domínios Proteicos , Transporte Proteico , Mucosa Respiratória/citologia , Serina Proteases/química , Serina Proteases/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Tripsina/metabolismo
3.
Eur J Pharm Sci ; 153: 105495, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32730844

RESUMO

In December 2019, a new coronavirus was identified in the Hubei province of central china and named SARS-CoV-2. This new virus induces COVID-19, a severe respiratory disease with high death rate. A putative target to interfere with the virus is the host transmembrane serine protease family member II (TMPRSS2). This enzyme is critical for the entry of coronaviruses into human cells by cleaving and activating the spike protein (S) of SARS-CoV-2. Repositioning approved, investigational and experimental drugs on the serine protease domain of TMPRSS2 could thus be valuable. There is no experimental structure for TMPRSS2 but it is possible to develop quality structural models for the serine protease domain using comparative modeling strategies as such domains are highly structurally conserved. Beside the TMPRSS2 catalytic site, we predicted on our structural models a main exosite that could be important for the binding of protein partners and/or substrates. To block the catalytic site or the exosite of TMPRSS2 we used structure-based virtual screening computations and two different collections of approved, investigational and experimental drugs. We propose a list of 156 molecules that could bind to the catalytic site and 100 compounds that may interact with the exosite. These small molecules should now be tested in vitro to gain novel insights over the roles of TMPRSS2 or as starting point for the development of second generation analogs.


Assuntos
Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Serina Endopeptidases/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/efeitos dos fármacos , Catálise , Biologia Computacional , Simulação por Computador , Reposicionamento de Medicamentos , Humanos , Modelos Moleculares , Pandemias , Serina Proteases/química , Relação Estrutura-Atividade
4.
Toxicon ; 184: 19-27, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32479836

RESUMO

Bothrops brazili is a pitviper from Amazonian region, responsible for many accidents in Peru. Despite its relevance, its venom has not been extensively characterized. In the present work, Bothrops brazili venom (BbV) components were analyzed by RP-HPLC, SDS-PAGE and MALDI-TOF/TOF. Approximately 37 proteins were identified, belonging to 7 families. Snake venom metalloproteinases (SVMPs) were the most abundant proteins of the venom (33.05%), followed by snake venom serine proteinases (SVSPs, 26.11%), phospholipases A2 (PLA2, 25.57%), snake C-type lectins (CTLs, 9.61%), L-aminoacid oxidase (LAAO, 3.80%), cystein-rich secretory proteins (CRISP, 1.67%) and Bradykinin-potentiating peptide (BPP, 0.20%). In vitro enzymatic activities of BbV showed high levels of SVMP activity and reduced Hyal activity in comparison with other bothropic venoms. Furthermore, BbV reduced VERO cells viability. ELISA and Western Blotting showed that both Peruvian and Brazilian bothropic antivenoms were able to recognize BbV components. This work provides an overview of BbV venom content and indicates a potential efficiency of Peruvian and Brazilian antivenoms to treat accidents with this species.


Assuntos
Bothrops , Venenos de Crotalídeos/toxicidade , Animais , Antivenenos , Western Blotting , Brasil , Chlorocebus aethiops , Cromatografia Líquida de Alta Pressão , Venenos de Crotalídeos/metabolismo , Eletroforese em Gel de Poliacrilamida , L-Aminoácido Oxidase/metabolismo , Peru , Fosfolipases A2/química , Proteômica , Serina Proteases/metabolismo , Células Vero
5.
Front Immunol ; 11: 1229, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32574272

RESUMO

COVID-19 is caused by the Severe Acute Respiratory Syndrome (SARS) coronavirus (Cov)-2, an enveloped virus with a positive-polarity, single-stranded RNA genome. The initial outbreak of the pandemic began in December 2019, and it is affecting the human health of the global community. In common with previous pandemics (Influenza H1N1 and SARS-CoV) and the epidemics of Middle east respiratory syndrome (MERS)-CoV, CoVs target bronchial and alveolar epithelial cells. Virus protein ligands (e.g., haemagglutinin or trimeric spike glycoprotein for Influenza and CoV, respectively) interact with cellular receptors, such as (depending on the virus) either sialic acids, Dipeptidyl peptidase 4 (DPP4), or angiotensin-converting enzyme 2 (ACE2). Host proteases, e.g., cathepsins, furin, or members of the type II transmembrane serine proteases (TTSP) family, such as Transmembrane protease serine 2 (TMPRSS2), are involved in virus entry by proteolytically activating virus ligands. Also involved are Toll Like Receptor (TLR) family members, which upregulate anti-viral and pro-inflammatory mediators [interleukin (IL)-6 and IL-8 and type I and type III Interferons among others], through the activation of Nuclear Factor (NF)-kB. When these events (virus cellular entry and innate immune responses) are uncontrolled, a deleterious systemic response is sometimes encountered in infected patients, leading to the well-described "cytokine storm" and an ensuing multiple organ failure promoted by a downregulation of dendritic cell, macrophage, and T-cell function. We aim to describe how the lung and systemic host innate immune responses affect survival either positively, through downregulating initial viral load, or negatively, by triggering uncontrolled inflammation. An emphasis will be put on host cellular signaling pathways and proteases involved with a view on tackling these therapeutically.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/imunologia , Imunidade Inata , Pulmão/imunologia , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/imunologia , Transdução de Sinais , Animais , Antivirais/uso terapêutico , Infecções por Coronavirus/metabolismo , Sistemas de Liberação de Medicamentos , Células Epiteliais/virologia , Humanos , Pulmão/virologia , Camundongos , Células Mieloides/virologia , Pandemias , Pneumonia Viral/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Virais/metabolismo , Serina Proteases/metabolismo , Internalização do Vírus
6.
Food Chem ; 330: 127324, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32569938

RESUMO

Enzymes currently used in cheesemaking have various drawbacks, and there is a continual need to find new coagulants. This study describes the extraction and biochemical characterization of two proteases from the red alga Gracilaria edulis. The proteases were extracted with phosphate buffer and partially purified by ammonium sulphate precipitation and dialysis. The enzymes exhibited optimum caseinolytic activity at 60 °C and a pH range of 6-8. They showed a high ratio of milk-clotting over caseinolytic activity, indicating they had an excellent milk-clotting ability. The proteases were confirmed to be serine protease and metalloprotease with molecular weight (MW) of 44 and 108 kDa. They exhibited high hydrolytic activity on κ-caseins, cleaving κ-casein at four main sites, one of which being the same as that of calf rennet, which is the first reported for an algal protease. The findings demonstrated that the proteases could potentially be used as a milk coagulant in cheesemaking.


Assuntos
Caseínas/metabolismo , Gracilaria/enzimologia , Peptídeo Hidrolases/isolamento & purificação , Peptídeo Hidrolases/metabolismo , Alga Marinha/enzimologia , Sulfato de Amônio , Animais , Caseínas/química , Fracionamento Químico , Quimosina/metabolismo , Eletroforese em Gel de Poliacrilamida , Gracilaria/química , Concentração de Íons de Hidrogênio , Hidrólise , Leite/química , Leite/metabolismo , Peso Molecular , Peptídeo Hidrolases/química , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/metabolismo , Alga Marinha/química , Serina Proteases/química , Serina Proteases/isolamento & purificação , Serina Proteases/metabolismo , Espectrometria de Massas em Tandem , Temperatura
7.
PLoS One ; 15(6): e0234780, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32579589

RESUMO

Obesity epidemic continues to spread and obesity rates are increasing in the world. In addition to public health effort to reduce obesity, there is a need to better understand the underlying biology to enable more effective treatment and the discovery of new pharmacological agents. Abhydrolase domain-containing protein 11 (ABHD11) is a serine hydrolase enzyme, localized in mitochondria, that can synthesize the endocannabinoid 2-arachidonoyl glycerol (2AG) in vitro. In vivo preclinical studies demonstrated that knock-out ABHD11 mice have a similar 2AG level as WT mice and exhibit a lean metabolic phenotype. Such mice resist to weight gain in Diet Induced Obesity studies (DIO) and display normal biochemical plasma parameters. Metabolic and transcriptomic analyses on serum and tissues of ABHD11 KO mice from DIO studies show a modulation in bile salts associated with reduced fat intestinal absorption. These data suggest that modulating ABHD11 signaling pathway could be of therapeutic value for the treatment of metabolic disorders.


Assuntos
Serina Proteases/metabolismo , Ganho de Peso , Animais , Fezes/enzimologia , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Células MCF-7 , Camundongos , Mitocôndrias/metabolismo , Serina Proteases/deficiência , Serina Proteases/genética , Transdução de Sinais
8.
Toxicon ; 184: 180-191, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32585218

RESUMO

In Colombia, Lachesis acrochorda causes 2-3% of all snake envenomations. The accidents promote a high mortality rate (90%) due to blood and cardiovascular complications. Here, the effects of the snake venom of L. acrochorda (SVLa) were analyzed on human blood cells and on cardiovascular parameters of rats. SVLa induced blood coagulation, as measured by the prothrombin time test, but did not reduce the cell viability of neutrophils and platelets evaluated by the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay and by the lactate dehydrogenase (LDH) enzyme assay. In fact, SVLa increased the absorbance in tests made with platelets subjected to the MTT assay. SVLa induced platelet aggregation whose magnitude was comparable to that of the positive control adenosine diphosphate (ADP), and occurred earlier with increasing SVLa concentration. Acetylsalicylic acid (ASA, a cyclooxygenase inhibitor) or clopidogrel (an ADP receptor blocker) inhibited the aggregating effect of SVLa. Inhibition of SVLa-elicited platelet aggregation also resulted from the treatment with disodium ethylenediaminetetraacetate (Na2-EDTA; metalloproteinase inhibitor) and with 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF, serine protease inhibitor). In isolated right atrium of rats, SVLa increased slightly, but significantly, the magnitude of the spontaneous contractions and, in isolated rat aorta, SVLa relaxed KCl- or phenylephrine-induced contractions. In vivo, SVLa induced hypotension and bradycardia in rats, with detection of hemorrhage in pulmonary and renal tissues. Altogether, under experimental conditions, SVLa induced blood coagulation, platelet aggregation, hypotension and bradycardia. Part of the effects presented here may be explained by the presence of snake venom metalloproteinases (SVMPs) and snake venom serine proteases (SVSPs), constituents of SVLa.


Assuntos
Sistema Cardiovascular/efeitos dos fármacos , Venenos de Víboras/toxicidade , Viperidae , Animais , Células Sanguíneas , Coagulação Sanguínea , Plaquetas , Colômbia , Fibrinogênio , Hemorragia , Humanos , Hipotensão , Metaloproteases , Agregação Plaquetária , Tempo de Protrombina , Ratos , Serina Endopeptidases , Serina Proteases , Inibidores de Serino Proteinase , Mordeduras de Serpentes
9.
Am J Vet Res ; 81(7): 572-580, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32584177

RESUMO

OBJECTIVE: To investigate the activities of gelatinases (matrix metalloproteinase [MMP]-2 and MMP-9) and serine proteases in the colorectal mucosa of Miniature Dachshunds (MDs) with inflammatory colorectal polyps (ICRPs). ANIMALS: 15 MDs with ICRPs and 5 dogs with non-ICRP-related large bowel diarrhea (controls). PROCEDURES: Zymographic methods were used to evaluate the activities of MMP-2, MMP-9, latent forms of MMP-2 and MMP-9 (pro-MMP-2 and pro-MMP-9), and serine proteases in inflamed and noninflamed tissue samples from MDs with ICRPs and in noninflamed tissue samples from control dogs. The associations of serine protease activities with MMP-2 or MMP-9 activity were also analyzed. RESULTS: Activities of pro-MMP-2 and pro-MMP-9 were detected in most tissue samples, regardless of the tissue type, whereas activities of MMP-2 and MMP-9 were not detected in control tissue samples. In the inflamed tissue samples from MDs with ICRPs, the activities of MMP-2, pro-MMP-9, and MMP-9 were significantly higher than those in the noninflamed tissue samples from those dogs. Serine protease activities were significantly higher in the inflamed and noninflamed tissue samples from MDs with ICRP, compared with findings for control tissue samples. A weak correlation was detected between serine protease activities and MMP-9 activity. CONCLUSIONS AND CLINICAL RELEVANCE: Study results suggested that gelatinase and serine protease activities are upregulated in the colorectal mucosa of MDs with ICRPs, possibly contributing to the pathogenesis of this disease through the functions of these enzymes in degradation of extracellular matrix and promotion of inflammatory cell migration and inflammatory responses.


Assuntos
Pólipos do Colo/veterinária , Neoplasias Colorretais/veterinária , Doenças do Cão , Animais , Cães , Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz , Serina Proteases
10.
PLoS Negl Trop Dis ; 14(6): e0008299, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32511239

RESUMO

Snake venoms are complex mixtures of proteins with toxic activities, with many distinct isoforms, affecting different physiological targets, comprised in a few protein families. It is currently accepted that this diversity in venom composition is an adaptive advantage for venom efficacy on a wide range of prey. However, on the other side, variability on isoforms expression has implications in the clinics of human victims of snakebites and in the efficacy of antivenoms. B. atrox snakes are responsible for most of the human accidents in Brazilian Amazon and the type and abundance of protein families on their venoms present individual variability. Thus, in this study we attempted to correlate the individual venom proteome of the snake brought to the hospital by the patient seeking for medical assistance with the clinical signs observed in the same patient. Individual variability was confirmed in venoms of the 14 snakes selected for the study. The abundance of each protein family was quite similar among the venom samples, while the isoforms composition was highly variable. Considering the protein families, the SVMP group presented the best correlation with bleeding disorders and edema. Considering individual isoforms, some isoforms of venom metalloproteinase (SVMP), C-type lectin-like toxins (CTL) and snake venom serine proteinases (SVSP) presented expression levels that with statistically significant positive correlation to signs and symptoms presented by the patients as bleeding disorders, edema, ecchymosis and blister formation. However, some unexpected data were also observed as the correlation between a CTL, CRISP or LAAO isoforms with blister formation, still to be confirmed with a larger number of samples. Although this is still a small number of patient samples, we were able to indicate that venom composition modulates clinical manifestations of snakebites, to confirm at the bedside the prominent role of SVMPs and to include new possible toxin candidates for the development of toxin inhibitors or to improve antivenom selectiveness, important actions for the next generation treatments of snakebites.


Assuntos
Bothrops , Venenos de Crotalídeos/análise , Proteoma/análise , Serina Proteases/análise , Animais , Antivenenos , Brasil , Metaloproteases/análise , Isoformas de Proteínas/análise , Proteômica , Mordeduras de Serpentes/terapia
12.
Molecules ; 25(10)2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32455942

RESUMO

Processing of certain viral proteins and bacterial toxins by host serine proteases is a frequent and critical step in virulence. The coronavirus spike glycoprotein contains three (S1, S2, and S2') cleavage sites that are processed by human host proteases. The exact nature of these cleavage sites, and their respective processing proteases, can determine whether the virus can cross species and the level of pathogenicity. Recent comparisons of the genomes of the highly pathogenic SARS-CoV2 and MERS-CoV, with less pathogenic strains (e.g., Bat-RaTG13, the bat homologue of SARS-CoV2) identified possible mutations in the receptor binding domain and in the S1 and S2' cleavage sites of their spike glycoprotein. However, there remains some confusion on the relative roles of the possible serine proteases involved for priming. Using anthrax toxin as a model system, we show that in vivo inhibition of priming by pan-active serine protease inhibitors can be effective at suppressing toxicity. Hence, our studies should encourage further efforts in developing either pan-serine protease inhibitors or inhibitor cocktails to target SARS-CoV2 and potentially ward off future pandemics that could develop because of additional mutations in the S-protein priming sequence in coronaviruses.


Assuntos
Antivirais/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Serina Proteases/metabolismo , Inibidores de Serino Proteinase/farmacologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Animais , Antígenos de Bactérias/toxicidade , Antivirais/uso terapêutico , Toxinas Bacterianas/toxicidade , Betacoronavirus/patogenicidade , Sítios de Ligação , Sistemas de Liberação de Medicamentos , Feminino , Furina/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Pandemias , Células RAW 264.7 , Inibidores de Serino Proteinase/uso terapêutico , Glicoproteína da Espícula de Coronavírus/química
14.
PLoS One ; 15(5): e0233866, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32470085

RESUMO

Several candidate HIV subunit vaccines based on recombinant envelope (Env) glycoproteins have been advanced into human clinical trials. To facilitate biopharmaceutical production, it is necessary to produce these in CHO (Chinese Hamster Ovary) cells, the cellular substrate used for the manufacturing of most recombinant protein therapeutics. However, previous studies have shown that when recombinant Env proteins from clade B viruses, the major subtype represented in North America, Europe, and other parts of the world, are expressed in CHO cells, they are proteolyzed and lack important glycan-dependent epitopes present on virions. Previously, we identified C1s, a serine protease in the complement pathway, as the endogenous CHO protease responsible for the cleavage of clade B laboratory isolates of -recombinant gp120s (rgp120s) expressed in stable CHO-S cell lines. In this paper, we describe the development of two novel CHOK1 cell lines with the C1s gene inactivated by gene editing, that are suitable for the production of any protein susceptible to C1s proteolysis. One cell line, C1s-/- CHOK1 2.E7, contains a deletion in the C1s gene. The other cell line, C1s-/- MGAT1- CHOK1 1.A1, contains a deletion in both the C1s gene and the MGAT1 gene, which limits glycosylation to mannose-5 or earlier intermediates in the N-linked glycosylation pathway. In addition, we compare the substrate specificity of C1s with thrombin on the cleavage of both rgp120 and human Factor VIII, two recombinant proteins known to undergo unintended proteolysis (clipping) when expressed in CHO cells. Finally, we demonstrate the utility and practicality of the C1s-/- MGAT1- CHOK1 1.A1 cell line for the expression of clinical isolates of clade B Envs from rare individuals that possess broadly neutralizing antibodies and are able to control virus replication without anti-retroviral drugs (elite neutralizer/controller phenotypes). The Envs represent unique HIV vaccine immunogens suitable for further immunogenicity and efficacy studies.


Assuntos
Vacinas contra a AIDS/imunologia , Edição de Genes , Proteólise , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Alelos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Sítios de Ligação , Células CHO , Sequência Consenso , Cricetinae , Cricetulus , Fator VIII/metabolismo , Glicosilação , Humanos , Polissacarídeos/metabolismo , Domínios Proteicos , Proteínas Recombinantes/metabolismo , Serina Proteases/química , Serina Proteases/metabolismo , Homologia Estrutural de Proteína , Especificidade por Substrato , Trombina/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/química
15.
Toxicon ; 183: 1-10, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32445841

RESUMO

Echis carinatus (EC) envenomation causes severe immune response by the accumulation of tissue debris in the form of DAMPs resulting in chronic inflammation and progressive tissue necrosis at the bitten site. Clearing of tissue debris is a prerequisite to enhance the healing of venom-induced necrotic wounds. Tricosanthus tricuspidata is a medicinal plant used extensively for the treatment of snake bite-induced toxicities. The active component responsible for the observed pharmacological action is a serine protease, tricuspidin. The topical application of tricuspidin was able to neutralize ECV-induced mouse footpad tissue necrosis and open wound in rabbits. Tricuspidin exerted its healing action via proteolytic activity as a consequence of upregulation of MMP-8 and down regulation of MMP-9. Further, tricuspidin reduced ECV-induced inflammation by decreasing the expression of TNF-α, IL-6 and MPO, and by increasing the level of VEGF-A and TGF-ß1. The modulation of ECV induced immune/inflammatory mediators by tricuspidin was found to be more effective than trypsin. Moreover, tricuspidin and trypsin activated MAPKs via protease activated receptors-2 (PAR-2). These data indicate that the proteolytic activity of tricuspidin directly involved in the healing of ECV-induced chronic wound.


Assuntos
Necrose/tratamento farmacológico , Extratos Vegetais/uso terapêutico , Serina Proteases/uso terapêutico , Trichosanthes , Venenos de Víboras/toxicidade , Animais , Serina Proteases/metabolismo , Viperidae , Cicatrização/efeitos dos fármacos
16.
Toxicon ; 183: 29-35, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32445842

RESUMO

The ant-like bethylid ectoparasitoid Scleroderma guani (Hymenoptera: Bethylidae) envenomates host to suppress immune response. Yet, the roles of its venom in inhibiting melanization of the host hemolymph have not been fully characterized. Here, we demonstrated that S. guani envenomation induced strong inhibition of melanization of the hemolymph from Tenebrio molitor (Coleoptera: Tenebrionidae), permitting the successful development of parasitoid offspring. To reveal venom component associated with such function, a serine proteinase homolog (SguaSPH) rich in the venom of S. guani was characterized. It was found that one of the catalytic triad residues for serine proteinase is absent in the amino acid sequence of SguaSPH. This venom component was abundantly expressed in venom apparatus and adult stages. By enzymatic assays, SguaSPH displayed low trypsin and no chymotrypsin activity, and was able to inhibit phenoloxidase activity in the hemolymph of Ostrinia furnacalis (Lepidoptera: Crambidae). The findings suggest that SguaSPH is essential for interfering with hemolymph melanization of S. guani envenomated host via phenoloxidase cascade disruption.


Assuntos
Monofenol Mono-Oxigenase/metabolismo , Serina Proteases/metabolismo , Animais , Hemolinfa/metabolismo , Proteínas de Insetos/metabolismo , Larva , Tenebrio/metabolismo
17.
Toxicon ; 178: 61-68, 2020 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-32112787

RESUMO

Snakebites cause upwards of 1.8 million envenomings, 138,000 deaths and 500,000 cases of long term morbidity each year. Viper snake venoms (family Viperidae) generally contain a high proportion of proteases which can cause devastating effects such as hemorrhage, coagulopathy, edema, necrosis, and severe pain, in envenomed victims. In this study, analytical techniques were combined with enzymatic assays to develop a novel method for the detection of snake venom protease activity by using rhodamine-110-peptide substrate. In the so called at-line nanofractionation set up, crude venoms were first separated with reversed phase liquid chromatography, after which fractions were collected onto 384-well plates. Protease activity assays were then performed in the 384-well plates and bioassay chromatograms were constructed revealing protease activity. Parallel obtained UV absorbance, MS and proteomics data from a previous study facilitated toxin identification. The application of the rhodamine-110-peptide substrate assay showed significantly greater sensitivity compared to prior assays using casein-FITC as the substrate. Moreover, cross referencing UV and MS data and resulted in the detection of a number of tentative proteases suspected to exhibit protease activity, including snake venom serine proteases from Calloselasma rhodostoma and Daboia russelli venom and a snake venom metalloproteinase from the venom of Echis ocellatus. Our data demonstrate that his methodology can be a useful tool for selectively identifying snake venom proteases, and can be applied to provide a better understanding of protease-induced pathologies and the development of novel therapeutics for treating snakebite.


Assuntos
Venenos de Víboras/química , Animais , Fracionamento Químico , Cromatografia de Fase Reversa , Ensaios de Triagem em Larga Escala , Metaloproteases , Peptídeos , Rodaminas , Serina Proteases/química , Mordeduras de Serpentes , Viperidae
18.
Microbiol Res ; 236: 126468, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32208189

RESUMO

Extracellular proteases from haloarchaea (halolysins) can resist high salt conditions. In this study, the gene encoding a halolysin from Halococcus salifodinae was identified. The hlyA gene encoded an active halolysin with the classical Asp-His-Ser catalytic triad of serine proteases. Site-directed mutagenesis showed that the three cysteine residues in the catalytic domain were important for the extracellular proteolytic activity and displayed an additive effect on the activity. Truncation mutants of the C-terminal extension (CTE) domain displayed very low or almost no extracellular protease activity towards milk and small peptide substrates, indicating its importance for the function of HlyA. CTE can be functionally interchangeable among halolysins. Additionally, the HlyA expressing strain as a starter culture for fish sauce fermentation significantly increased the peptide release and total free amino acid content in fish sauce. This study enriches our knowledge of the key amino acid residues and domains of halolysins, and provides an opportunity for applications of halolysins in fish sauce fermentation.


Assuntos
Halococcus/genética , Serina Endopeptidases , Serina Proteases/biossíntese , Sequência de Aminoácidos/genética , Proteínas de Bactérias/análise , Biotecnologia , Domínio Catalítico/genética , Fermentação , Produtos Pesqueiros , Serina Endopeptidases/biossíntese , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Proteases/química , Serina Proteases/genética
19.
Mol Immunol ; 121: 47-58, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32163758

RESUMO

Shigellosis is a diarrheal disease that causes high mortality every year, especially in children, elderly and immunocompromised patients. Recently, resistance strains to antibiotic therapy are in the rise and the World Health Organization prioritizes the development of a safe vaccine against the most common causal agent of shigellosis, Shigella flexneri. This pathogen uses autotransporter proteins such as SigA, Pic and Sap to increase virulence and some of them have been described as highly immunogenic proteins. In this study, we used immune-informatics analysis to identify the most antigenic epitope as a vaccine candidate on three passenger domains of auto-transporter proteins encoded on the pathogenic island SHI-1, to induce immunity against S. flexneri. Epitope identification was done using various servers such as Bepipred, Bcepred, nHLAPRED, NetMHCII, Rankpep and IEDB and the final selection was done based on its antigenicity using the VaxiJen server. Moreover, to enhance immunity, the GroEL adjuvant was added to the final construct as a Toll-like receptor 2 (TLR2) agonist. On the other hand, to predict the tertiary structure, the I-TASSER server was used, and the best model was structurally validated using the ProSA-web software and the Ramachandran plot. Subsequently, the model was refined and used for docking and molecular dynamics analyses with TLR2, which demonstrated an appropriate and stable interaction. In summary, a potential subunit vaccine candidate, that contains B and T cell epitopes with proper physicochemical properties was designed. This multiepitope vaccine is expected to elicit robust humoral and cellular immune responses and vest protective immunity against S. flexneri.


Assuntos
Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Disenteria Bacilar/terapia , Serina Proteases/imunologia , Shigella flexneri/imunologia , Sistemas de Secreção Tipo V/imunologia , Adjuvantes Imunológicos/farmacologia , Antígenos de Bactérias/imunologia , Vacinas Bacterianas/uso terapêutico , Chaperonina 60/imunologia , Chaperonina 60/farmacologia , Biologia Computacional , Simulação por Computador , Disenteria Bacilar/microbiologia , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Humanos , Imunidade Celular , Imunidade Humoral , Imunogenicidade da Vacina , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Domínios Proteicos/imunologia , Receptor 2 Toll-Like/agonistas , Vacinas de Subunidades/imunologia , Vacinas de Subunidades/uso terapêutico
20.
PLoS One ; 15(3): e0230166, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32182256

RESUMO

Over 100 metabolic serine hydrolases are present in humans with confirmed functions in metabolism, immune response, and neurotransmission. Among potentially clinically-relevant but uncharacterized human serine hydrolases is OVCA2, a serine hydrolase that has been linked with a variety of cancer-related processes. Herein, we developed a heterologous expression system for OVCA2 and determined the comprehensive substrate specificity of OVCA2 against two ester substrate libraries. Based on this analysis, OVCA2 was confirmed as a serine hydrolase with a strong preference for long-chain alkyl ester substrates (>10-carbons) and high selectivity against a variety of short, branched, and substituted esters. Substitutional analysis was used to identify the catalytic residues of OVCA2 with a Ser117-His206-Asp179 classic catalytic triad. Comparison of the substrate specificity of OVCA2 to the model homologue FSH1 from Saccharomyces cerevisiae illustrated the tighter substrate selectivity of OVCA2, but their overlapping substrate preference for extended straight-chain alkyl esters. Conformation of the overlapping biochemical properties of OVCA2 and FSH1 was used to model structural information about OVCA2. Together our analysis provides detailed substrate specificity information about a previously, uncharacterized human serine hydrolase and begins to define the biological properties of OVCA2.


Assuntos
Proteínas/química , Proteínas de Saccharomyces cerevisiae/química , Serina Proteases/química , Sequência de Aminoácidos , Ésteres/metabolismo , Humanos , Modelos Moleculares , Conformação Proteica , Proteínas/metabolismo , Saccharomyces cerevisiae , Homologia de Sequência de Aminoácidos , Serina Proteases/metabolismo , Homologia Estrutural de Proteína , Especificidade por Substrato
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