RESUMEN

The high content and quality of protein in Andean legumes make them valuable for producing protein hydrolysates using proteases from bacteria isolated from extreme environments. This study aimed to carry out a single-step purification of a haloprotease from Micrococcus sp. PC7 isolated from Peru salterns. In addition, characterize and apply the enzyme for the production of bioactive protein hydrolysates from underutilized Andean legumes. The PC7 protease was fully purified using only tangential flow filtration (TFF) and exhibited maximum activity at pH 7.5 and 40 °C. It was characterized as a serine protease with an estimated molecular weight of 130 kDa. PC7 activity was enhanced by Cu2+ (1.7-fold) and remained active in the presence of most surfactants and acetonitrile. Furthermore, it stayed completely active up to 6% NaCl and kept Ì´ 60% of its activity up to 8%. The protease maintained over 50% of its activity at 25 °C and 40 °C and over 70% at pH from 6 to 10 for up to 24 h. The determined Km and Vmax were 0.1098 mg mL-1 and 273.7 U mL-1, respectively. PC7 protease hydrolyzed 43%, 22% and 11% of the Lupinus mutabilis, Phaseolus lunatus and Erythrina edulis protein concentrates, respectively. Likewise, the hydrolysates from Lupinus mutabilis and Erythrina edulis presented the maximum antioxidant and antihypertensive activities, respectively. Our results demonstrated the feasibility of a simple purification step for the PC7 protease and its potential to be applied in industrial and biotechnological processes. Bioactive protein hydrolysates produced from Andean legumes may lead to the development of nutraceuticals and functional foods contributing to address some United Nations Sustainable Development Goals (SDGs).

Asunto(s)

Fabaceae , Micrococcus , Hidrolisados de Proteína , Micrococcus/metabolismo , Micrococcus/enzimología , Concentración de Iones de Hidrógeno , Hidrolisados de Proteína/química , Hidrolisados de Proteína/metabolismo , Peso Molecular , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/aislamiento & purificación , Perú , Temperatura , Serina Proteasas/metabolismo , Serina Proteasas/aislamiento & purificación , Serina Proteasas/química , Estabilidad de Enzimas , Cloruro de Sodio/metabolismo , Cloruro de Sodio/farmacología , Hidrólisis , Cinética

RESUMEN

Serine peptidases and metallopeptidases are the primary toxins found in Bothrops snakes venoms, which act on proteins in the tissues of victims or prey, and release of peptides formed through proteolytic activity. Various studies have indicated that these peptides, released by the proteolytic activity of heterologous enzymes, generate molecules with unidentified functions, referred to as cryptids. To address this, we purified serine peptidases from Bothrops jararaca venom using molecular exclusion chromatography and then incubated them with the endogenous substrate myoglobin. As a control, we also incubated the substrate with trypsin. The resulting proteolytic fragments were analyzed, separated, and collected via HPLC. These fractions were then tested on cell cultures, the active fractions were sequenced (ALELFR and TGHPETLEK) and synthesized. After confirming their activity, the peptides underwent sequencing and synthesis for additional cell tests, including the increase of cell viability, cycle phases, proliferation, signaling, growth kinetics, angiogenesis, and migration. The results revealed that the synthesized peptides exhibited cellular repair properties, suggesting a potential role in tissue repair in the range of 0.05-5 µ M. Additionally, the effects of fragments resulting from myoglobin degradation isolated (ALELFR and TGHPETLEK) revealed a regenerative action on tissue.

Asunto(s)

Bothrops , Venenos de Crotálidos , Mioglobina , Serina Proteasas , Animales , Venenos de Crotálidos/química , Serina Proteasas/metabolismo , Serina Proteasas/química , Mioglobina/metabolismo , Péptidos/farmacología , Péptidos/química , Humanos , Supervivencia Celular/efectos de los fármacos , Bothrops jararaca

RESUMEN

Snakebite accidents, neglected tropical diseases per the WHO, pose a significant public health threat due to their severity and frequency. Envenomation by Bothrops genus snakes leads to severe manifestations due to proteolytic enzymes. While the antibothropic serum produced by the Butantan Institute saves lives, its efficacy is limited as it fails to neutralize certain serine proteases. Hence, developing new-generation antivenoms, like monoclonal antibodies, is crucial. This study aimed to explore the inhibitory potential of synthetic peptides homologous to the CDR3 regions of a monoclonal antibody targeting a snake venom thrombin-like enzyme (SVTLE) from B. atrox venom. Five synthetic peptides were studied, all stable against hydrolysis by venoms and serine proteases. Impressively, four peptides demonstrated uncompetitive SVTLE inhibition, with Ki values ranging from 10-6 to 10-7 M. These findings underscore the potential of short peptides homologous to CDR3 regions in blocking snake venom toxins, suggesting their promise as the basis for new-generation antivenoms. Thus, this study offers potential advancements in combatting snakebites, addressing a critical public health challenge in tropical and subtropical regions.

Asunto(s)

Anticuerpos Monoclonales , Bothrops , Péptidos , Serina Proteasas , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacología , Péptidos/química , Péptidos/farmacología , Serina Proteasas/química , Serina Proteasas/metabolismo , Antivenenos/química , Antivenenos/inmunología , Antivenenos/farmacología , Regiones Determinantes de Complementariedad/química , Venenos de Crotálidos/antagonistas & inhibidores , Venenos de Crotálidos/inmunología , Venenos de Crotálidos/enzimología , Venenos de Crotálidos/química , Secuencia de Aminoácidos , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/farmacología

RESUMEN

BACKGROUND: Neuronal ceroid lipofuscinoses (NCL) are a group of autosomal recessive, inherited, lysosomal, and neurodegenerative diseases that causes progressive dementia, seizures, movement disorders, language delay/regression, progressive visual failure, and early death. Neuronal ceroid lipofuscinosis type 2 (CLN2), caused by biallelic pathogenic variants of the TPP1 gene, is the only NCL with an approved targeted therapy. The laboratory diagnosis of CLN2 is established through highly specific tests, leading to diagnostic delays and eventually hampering the provision of specific treatment for patients with CLN2. Epilepsy is a common and clinically-identifiable feature among NCLs, and seizure onset is the main driver for families to seek medical care. OBJECTIVE: To evaluate the results of the Latin America Epilepsy and Genetics Program, an epilepsy gene panel, as a comprehensive tool for the investigation of CLN2 among other genetic causes of epilepsy. METHODS: A total of 1,284 patients with epilepsy without a specific cause who had at least 1 symptom associated with CLN2 were screened for variants in 160 genes associated with epilepsy or metabolic disorders presenting with epilepsy through an epilepsy gene panel. RESULTS: Variants of the TPP1 gene were identified in 25 individuals (1.9%), 21 of them with 2 variants. The 2 most frequently reported variants were p.Arg208* and p.Asp276Val, and 2 novel variants were detected in the present study: p.Leu308Pro and c.89 + 3G > C Intron 2. CONCLUSION: The results suggest that these genetic panels can be very useful tools to confirm or exclude CLN2 diagnosis and, if confirmed, provide disease-specific treatment for the patients.

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ANTECEDENTES: As lipofuscinoses ceroides neuronais (neuronal ceroid lipofuscinoses, NCLs, em inglês) são um grupo de doenças autossômicas recessivas, hereditárias, lisossomais e neurodegenerativas que causam demência progressiva, crises epiléticas, distúrbios de movimento, atraso/regressão da linguagem, deficiência visual progressiva e morte precoce. A lipofuscinose ceroide neuronal tipo 2 (neuronal ceroid lipofuscinosis type 2, CLN2, em inglês), causada por variantes patogênicas bialélicas do gene TPP1, é a única com terapia-alvo aprovada. O diagnóstico laboratorial é realizado por testes específicos, o que leva a atrasos diagnósticos e, consequentemente, prejudica a disponibilização de tratamento. A epilepsia é uma característica comum e clinicamente identificável entre as NCLs, e o início das convulsões é o principal motivo para as famílias buscarem atendimento médico. OBJETIVO: Avaliar os resultados do Programa de Epilepsia e Genética da América Latina, um painel genético, como uma ferramenta abrangente para a investigação de CLN2 entre outras causas genéticas de epilepsia. MéTODOS: Um total de 1.284 pacientes com epilepsia sem uma causa específica e que tinham pelo menos 1 sintoma associado à CLN2 foram rastreados em busca de variantes em 160 genes associados à epilepsia ou a distúrbios metabólicos que apresentam epilepsia, por meio de um painel genético. RESULTADOS: Variantes do gene TPP1 foram identificadas em 25 indivíduos (1,9%), sendo que ; 21 apresentavam duas variantes. As duas variantes mais frequentes foram p.Arg208* e p.Asp276Val, e duas variantes novas foram detectadas neste: p.Leu308Pro e c.89 + 3G > C Intron 2. CONCLUSãO: Os resultados sugerem que os painéis genéticos de epilepsia podem ser uma ferramenta útil para confirmar ou excluir o diagnóstico de CLN2 e, se confirmado, fornecer tratamento específico para os pacientes.

Asunto(s)

Aminopeptidasas , Epilepsia , Lipofuscinosis Ceroideas Neuronales , Serina Proteasas , Tripeptidil Peptidasa 1 , Humanos , Lipofuscinosis Ceroideas Neuronales/genética , Femenino , Masculino , Epilepsia/genética , Aminopeptidasas/genética , Serina Proteasas/genética , Niño , Adolescente , Adulto , Adulto Joven , Preescolar , Proteínas de Unión a Telómeros/genética , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/genética , Mutación , Pruebas Genéticas/métodos , Persona de Mediana Edad , Lactante

RESUMEN

Mannheimiahaemolytica is an opportunistic agent of the respiratory tract of bovines, a member of the Pasteurellaceae family, and the causal agent of fibrinous pleuropneumonia. This bacterium possesses different virulence factors, allowing it to colonize and infect its host. The present work describes the isolation and characterization of a serine protease secreted by M. haemolytica serotype 1. This protease was isolated from M. haemolytica cultured media by precipitation with 50 % methanol and ion exchange chromatography on DEAE-cellulose. It is a 70-kDa protease able to degrade sheep and bovine fibrinogen or porcine gelatin but not bovine IgG, hemoglobin, or casein. Mass spectrometric analysis indicates its identity with protease IV of M. haemolytica. The proteolytic activity was active between pH 5 and 9, with an optimal pH of 8. It was stable at 50 °C for 10 min but inactivated at 60 °C. The sera of bovines with chronic or acute pneumonia recognized this protease. Still, it showed no cross-reactivity with rabbit hyperimmune serum against the secreted metalloprotease from Actinobacilluspleuropneumoniae, another member of the Pasteurellaceae family. M. haemolytica secreted proteases could contribute to the pathogenesis of this bacterium through fibrinogen degradation, a characteristic of this fibrinous pleuropneumonia.

Asunto(s)

Fibrinógeno , Mannheimia haemolytica , Serina Proteasas , Animales , Mannheimia haemolytica/enzimología , Ovinos , Bovinos , Fibrinógeno/metabolismo , Concentración de Iones de Hidrógeno , Serina Proteasas/metabolismo , Serina Proteasas/aislamiento & purificación , Temperatura , Proteolisis , Peso Molecular , Gelatina/metabolismo , Estabilidad de Enzimas , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/aislamiento & purificación , Espectrometría de Masas , Cromatografía por Intercambio Iónico , Porcinos , Factores de Virulencia/metabolismo , Factores de Virulencia/aislamiento & purificación

Asunto(s)

Enfermedades Desatendidas , Humanos , Enfermedades Desatendidas/tratamiento farmacológico , Proteasas de Cisteína/metabolismo , Serina Proteasas/metabolismo , Descubrimiento de Drogas , Inhibidores de Serina Proteinasa/farmacología , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/uso terapéutico

RESUMEN

Nematophagous fungi have been widely evaluated in the biological control of parasitic helminths in animals, both through their direct use and the use of their derived products. Fungal bioproducts can include extracellular enzymes, silver nanoparticles (AgNPs), as well as secondary metabolites. The aim of this study was to conduct a systematic review covering the evaluation of products derived from nematophagous fungi in the biological control of parasitic helminths in animals. In total, 33 studies met the inclusion criteria and were included in this review. The majority of the studies were conducted in Brazil (72.7%, 24/33), and bioproducts derived from the fungus Duddingtonia flagrans were the most commonly evaluated (36.3%, 12/33). The studies involved the production of extracellular enzymes (48.4%, 16/33), followed by crude enzymatic extract (27.2%, 9/33), secondary metabolites (15.1%, 5/33) and biosynthesis of AgNPs (9.1%, 3/33). The most researched extracellular enzymes were serine proteases (37.5%, 6/16), with efficacies ranging from 23.9 to 85%; proteases (31.2%, 5/16), with efficacies from 41.4 to 95.4%; proteases + chitinases (18.7%, 3/16), with efficacies from 20.5 to 43.4%; and chitinases (12.5%, 2/16), with efficacies ranging from 12 to 100%. In conclusion, extracellular enzymes are the most investigated derivatives of nematophagous fungi, with proteases being promising strategies in the biological control of animal helminths. Further studies under in vivo and field conditions are needed to explore the applicability of these bioproducts as tools for biological control.

Asunto(s)

Helmintos , Animales , Agentes de Control Biológico/metabolismo , Brasil , Duddingtonia/metabolismo , Hongos/metabolismo , Nanopartículas del Metal/química , Control Biológico de Vectores/métodos , Serina Proteasas/metabolismo , Plata/metabolismo

RESUMEN

Introduction: Serine proteases play a critical role during SARS-CoV-2 infection. Therefore, polymorphisms of transmembrane protease serine 2 (TMPRSS2) and serpine family E member 1 (SERPINE1) could help to elucidate the contribution of variability to COVID-19 outcomes. Methods: To evaluate the genetic variants of the genes previously associated with COVID-19 outcomes, we performed a cross-sectional study in which 1536 SARS-CoV-2-positive participants were enrolled. TMPRSS2 (rs2070788, rs75603675, rs12329760) and SERPINE1 (rs2227631, rs2227667, rs2070682, rs2227692) were genotyped using the Open Array Platform. The association of polymorphisms with disease outcomes was determined by logistic regression analysis adjusted for covariates (age, sex, hypertension, type 2 diabetes, and obesity). Results: According to our codominant model, the GA genotype of rs2227667 (OR=0.55; 95% CI = 0.36-0.84; p=0.006) and the AG genotype of rs2227667 (OR=0.59; 95% CI = 0.38-0.91; p=0.02) of SERPINE1 played a protective role against disease. However, the rs2227692 T allele and TT genotype SERPINE1 (OR=1.45; 95% CI = 1.11-1.91; p=0.006; OR=2.08; 95% CI = 1.22-3.57; p=0.007; respectively) were associated with a decreased risk of death. Similarly, the rs75603675 AA genotype TMPRSS2 had an OR of 1.97 (95% CI = 1.07-3.6; p=0.03) for deceased patients. Finally, the rs2227692 T allele SERPINE1 was associated with increased D-dimer levels (OR=1.24; 95% CI = 1.03-1.48; p=0.02). Discussion: Our data suggest that the rs75603675 TMPRSS2 and rs2227692 SERPINE1 polymorphisms are associated with a poor outcome. Additionally, rs2227692 SERPINE1 could participate in hypercoagulable conditions in critical COVID-19 patients, and this genetic variant could contribute to the identification of new pharmacological targets and treatment strategies to block the inhibition of TMPRSS2 entry into SARS-CoV-2.

Asunto(s)

COVID-19 , Diabetes Mellitus Tipo 2 , Humanos , COVID-19/genética , Serina Proteasas , SARS-CoV-2 , Estudios Transversales

RESUMEN

A novel trypsin inhibitor from Cajanus cajan (TIC) fresh leaves was partially purified by affinity chromatography. SDS-PAGE revealed one band with about 15 kDa with expressive trypsin inhibitor activity by zymography. TIC showed high affinity for trypsin (Ki = 1.617 µM) and was a competitive inhibitor for this serine protease. TIC activity was maintained after 24 h of treatment at 70 °C, after 1 h treatments with different pH values, and ß-mercaptoethanol increasing concentrations, and demonstrated expressive structural stability. However, the activity of TIC was affected in the presence of oxidizing agents. In order to study the effect of TIC on secreted serine proteases, as well as on the cell culture growth curve, SK-MEL-28 metastatic human melanoma cell line and CaCo-2 colon adenocarcinoma was grown in supplemented DMEM, and the extracellular fractions were submitted salting out and affinity chromatography to obtain new secreted serine proteases. TIC inhibited almost completely, 96 to 89%, the activity of these serine proteases and reduced the melanoma and colon adenocarcinoma cells growth of 48 and 77% respectively. Besides, it is the first time that a trypsin inhibitor was isolated and characterized from C. cajan leaves and cancer serine proteases were isolated and partial characterized from SK-MEL-28 and CaCo-2 cancer cell lines. Furthermore, TIC shown to be potent inhibitor of tumor protease affecting cell growth, and can be one potential drug candidate to be employed in chemotherapy of melanoma and colon adenocarcinoma.

Asunto(s)

Cajanus , Hojas de la Planta , Humanos , Cajanus/química , Hojas de la Planta/química , Células CACO-2 , Proliferación Celular/efectos de los fármacos , Línea Celular Tumoral , Inhibidores de Tripsina/farmacología , Inhibidores de Tripsina/química , Inhibidores de Tripsina/aislamiento & purificación , Proteínas de Plantas/farmacología , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Serina Proteasas/química , Serina Proteasas/aislamiento & purificación , Serina Proteasas/metabolismo

RESUMEN

Plasmid-encoded toxin (Pet) is an autotransporter protein of the serine protease autotransporters of Enterobacteriaceae (SPATE) family, important in the pathogenicity of Escherichia coli. The pet gene was initially found in the enteroaggregative E. coli (EAEC) virulence plasmid, pAA2. Although this virulence factor was initially described in EAEC, an intestinal E. coli pathotype, pet may also be present in other pathotypes, including extraintestinal pathogenic strains (ExPEC). The complement system is an important defense mechanism of the immune system that can be activated by invading pathogens. Proteases produced by pathogenic bacteria, such as SPATEs, have proteolytic activity and can cleave components of the complement system, promoting bacterial resistance to human serum. Considering these factors, the proteolytic activity of Pet and its role in evading the complement system were investigated. Proteolytic assays were performed by incubating purified components of the complement system with Pet and Pet S260I (a catalytic site mutant) proteins. Pet, but not Pet S260I, could cleave C3, C5 and C9 components, and also inhibited the natural formation of C9 polymers. Furthermore, a dose-dependent inhibition of ZnCl2-induced C9 polymerization in vitro was observed. E. coli DH5α survived incubation with human serum pre-treated with Pet. Therefore, Pet can potentially interfere with the alternative and the terminal pathways of the complement system. In addition, by cleaving C9, Pet may inhibit membrane attack complex (MAC) formation on the bacterial outer membrane. Thus, our data are suggestive of a role of Pet in resistance of E. coli to human serum.

Asunto(s)

Toxinas Bacterianas , Infecciones por Escherichia coli , Proteínas de Escherichia coli , Humanos , Escherichia coli/metabolismo , Toxinas Bacterianas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas del Sistema Complemento/metabolismo , Serina Proteasas/metabolismo , Infecciones por Escherichia coli/microbiología , Plásmidos/genética

RESUMEN

Accidents with snakes are responsible for about 32,000 deaths annually in sub-Saharan Africa, caused mostly by snakes from the genus Bitis, in particular Bitis arietans. B. arietans venom is composed of a complex mixture of toxins, mainly metalloproteases, serine proteases, phospholipases, lectins, and disintegrins. In this work, we compared two approaches to anti-B. arietans antivenom production: immunization with crude snake venom ("traditional approach") and immunization with selected key toxins isolated from the snake venom ("toxin oriented" approach). Fractions from B. arietans venom were isolated by size exclusion chromatography. Crude venom and samples containing serine proteases or metalloproteases were selected for the immunization of BALB/c mice. Anti-B. arietans and anti-serine proteases plasmas showed a similar recognition profile and higher titers and affinity than the anti-metalloproteases plasma. Cross-recognition of other Bitis venoms was observed, but with low intensity. Although the plasma of all experimental groups inhibited the enzymatic activity of B. arietans venom in vitro, in vivo protection was not achieved. Our results have shown limitations in both approaches considered. Based on this, we proposed a model of polyclonal, species-specific, monovalent antivenoms that could be used as a base to produce customizable polyvalent sera for use in sub-Saharan Africa.

Asunto(s)

Antivenenos , Toxinas Biológicas , Animales , Ratones , Antivenenos/farmacología , Venenos de Serpiente , Serina Endopeptidasas , Serina Proteasas , Ratones Endogámicos BALB C

RESUMEN

Extracellular proteases from halophilic archaea displays increased enzymatic activities in hypersaline environment. In this study, an extracellular protease-coding gene, hly34, from the haloarchaeal strain Halococcus salifodinae PRR34, was obtained through homologous search. The protease activity produced by this strain at 20% NaCl, 42 °C, and pH 7.0 was 32.5 ± 0.5 (U·mL-1). The codon-optimized hly34 which is specific for Escherichia coli can be expressed in E. coli instead of native hly34. It exhibits proteolytic activity under a wide range of low- or high-salt concentrations, slightly acidic or alkaline conditions, and slightly higher temperatures. The Hly34 presented the highest proteolytic activity at 50 °C, pH 9.0, and 0-1 M NaCl. It was found that the Hly34 showed a higher enzyme activity under low-salt conditions. Hly34 has good stability at different NaCl concentrations (1-4 M) and pH (6.0-10.0), as well as good tolerance to some metal ions. However, at 60 °C, the stability is reduced. It has a good tolerance to some metal ions. The proteolytic activity was completely inhibited by phenylmethanesulfonyl fluoride, suggesting that the Hly34 is a serine protease. This study further deepens our understanding of haloarchaeal extracellular protease, most of which found in halophilic archaea are classified as serine proteases. These proteases exhibit a certain level of alkaline resistance and moderate heat resistance, and they may emerge with higher activity under low-salt conditions than high-salt conditions. The protease Hly34 is capable of degrading a number of proteins, including substrate proteins, such as azocasein, whey protein and casein. It has promising applications in industrial production.

Asunto(s)

Halococcus , Halococcus/genética , Halococcus/metabolismo , Cloruro de Sodio/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Serina Proteasas , Serina Endopeptidasas , Metales , Iones , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Temperatura

RESUMEN

Serine proteases play crucial biological roles and have their activity controlled by inhibitors, such as the EcTI, a serine protease inhibitor purified from Enterolobium contortisiliquum seeds, which has anticancer activity. This study aimed to conjugate EcTI with quantum dots (QDs), fluorophores with outstanding optical properties, and investigate the interaction of QDs-EcTI nanoprobe with cancer cells. The conjugation was evaluated by fluorescence correlation spectroscopy (FCS) and fluorescence microplate assay (FMA). EcTI inhibitory activity after interaction with QDs was also analyzed. From FCS, the conjugate presented a hydrodynamic diameter about 4× greater than bare QDs, suggesting a successful conjugation. This was supported by FMA, which showed a relative fluorescence intensity of ca. 3815% for the nanosystem, concerning bare QDs or EcTI alone. The EcTI inhibitory activity remained intact after its interaction with QDs. From flow cytometry analyses, approximately 62% of MDA-MB-231 and 90% of HeLa cells were labeled with the QD-EcTI conjugate, suggesting that their membranes have different protease levels to which EcTI exhibits an affinity. Concluding, the QD-EcTI represents a valuable nanotool to study the interaction of this inhibitor with cancer cells using fluorescence-based techniques with the potential to unravel the intricate dynamics of interplays between proteases and inhibitors in cancer biology.

Asunto(s)

Fabaceae , Neoplasias , Puntos Cuánticos , Humanos , Inhibidores de Tripsina/farmacología , Células HeLa , Fabaceae/química , Serina Proteasas , Colorantes

RESUMEN

Serine protease autotransporters of Enterobacteriaceae (SPATE) constitute a superfamily of virulence factors, resembling the trypsin-like superfamily of serine proteases. SPATEs accomplish multiple functions associated to disease development of their hosts, which could be the consequence of SPATE cleavage of host cell components. SPATEs have been divided into class-1 and class-2 based on structural differences and biological effects, including similar substrate specificity, cytotoxic effects on cultured cells, and enterotoxin activity on intestinal tissues for class-1 SPATEs, whereas most class-2 SPATEs exhibit a lectin-like activity with a predilection to degrade a variety of mucins, including leukocyte surface O-glycoproteins and soluble host proteins, resulting in mucosal colonization and immune modulation. In this review, the structure of class-1 and class-2 are analyzed, making emphasis on their putative functional subdomains as well as a description of their function is provided, including prototypical mechanism of action.

Asunto(s)

Proteínas de Escherichia coli , Serina Proteasas , Serina Proteasas/metabolismo , Enterobacteriaceae/genética , Enterobacteriaceae/metabolismo , Sistemas de Secreción Tipo V , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Células Cultivadas , Glicoproteínas de Membrana

RESUMEN

The use of force spectroscopy approaches performed with optical tweezers can be very useful in determining the binding modes and the physical chemistry of DNA interactions with ligands, from small drugs to proteins. Helminthophagous fungi, on the other hand, have important enzyme secretion mechanisms for various purposes, and the interactions between such enzymes and nucleic acids are very poorly studied. Therefore, the main goal of the present work was to investigate, at the molecular level, the mechanisms of interaction between fungal serine proteases and the double-stranded (ds) DNA molecule. Experimental assays performed with this single molecule technique consist in exposing different concentrations of the protease of this fungus to dsDNA until saturation while monitoring the changes on the mechanical properties of the macromolecular complexes formed, from where the physical chemistry of the interaction can be deduced. It was found that the protease binds strongly to the double-helix, forming aggregates and changing the persistence length of the DNA molecule. The present work thus allowed us to infer information at the molecular level on the pathogenicity of these proteins, an important class of biological macromolecules, when applied to a target specimen.

Asunto(s)

Ascomicetos , Serina Proteasas , Serina Proteasas/genética , Ascomicetos/genética , Serina Endopeptidasas , ADN

RESUMEN

Given the importance of serine proteases for biochemical processes, we have studied the peptide bond rupture mechanism using three sequential scale models as representations of the KLK5 enzyme (a protein overexpressed in ovarian cancer). The first model contains the basic functional groups of the residues that conform to the catalytic triad present in serine proteases; the second model contains some additional residues and, finally, the last representation includes all atoms of the KLK5 protein together with 10.000 explicit water molecules. This separation into three scale models allows us to separate the intrinsic reactivity of the catalytic triad from the process taking place in the enzyme. The methodologies employed in this work include full DFT calculations with a dielectric continuum in the first two models and a multi-level setup with a Quantum Mechanics/Molecular Mechanics (QM/MM) partition in the whole protein system. Our results show that the peptide-bond rupture mechanism is a stepwise process involving two proton transfer reactions. The rate-determining step is the second proton transfer from the imidazole group to the amidic nitrogen of the substrate. In addition, we find that the simplest model does not provide accurate results compared to the full protein system. This can be attributed to the electronic stabilization conferred by the residues around the reaction site. Interestingly, the energy profile obtained with the second scale model with additional residues shows the same trends as the full system and could therefore be considered an appropriate model system. It could be used for studying the peptide bond rupture mechanism in case full QM/MM calculations cannot be performed, or as a rapid tool for screening purposes.

Asunto(s)

Protones , Serina Proteasas , Serina Proteasas/metabolismo , Serina Endopeptidasas/metabolismo , Proteínas , Simulación de Dinámica Molecular , Péptidos , Teoría Cuántica

RESUMEN

The Kunitz-Soybean Trypsin Inhibitor (Kunitz-STI) family is a large family of proteins with most of its members being protease inhibitors. The versatility of the inhibitory profile and the structural plasticity of these proteins, make this family a promising scaffold for designing new multifunctional proteins. Historically, Kunitz-STI inhibitors have been classified as canonical serine protease inhibitors, but new inhibitors with novel inhibition mechanisms have been described in recent years. Different inhibition mechanisms could be the result of different evolutionary pathways. In the present work, we performed a structural analysis of all the crystallographic structures available for Kunitz-STI inhibitors to characterize serine protease-binding loop structural features and locations. Our study suggests a relationship between the conformation of serine protease-binding loops and the inhibition mechanism, their location in the ß-trefoil fold, and the plant source of the inhibitors. The classical canonical inhibitors of this family are restricted to plants from the Fabales order and bind their targets via the ß4-ß5 loop, whereas serine protease-binding loops in inhibitors from other plants lie mainly in the ß5-ß6 and ß9-ß10 loops. In addition, we found that the ß5-ß6 loop is used to inhibit two different families of serine proteases through a steric blockade inhibition mechanism. This work will help to change the general perception that all Kunitz-STI inhibitors are canonical inhibitors and proteins with protease-binding loops adopting noncanonical conformations are exceptions. Additionally, our results will help in the identification of protease-binding loops in uncharacterized or newly discovered inhibitors, and in the design of multifunctional proteins.

Asunto(s)

Serina Proteasas , Inhibidor de la Tripsina de Soja de Kunitz , Inhibidor de la Tripsina de Soja de Kunitz/química , Serina , Secuencia de Aminoácidos , Serina Endopeptidasas , Inhibidores de Serina Proteinasa/farmacología , Inhibidores de Serina Proteinasa/química

RESUMEN

A high diversity of rattlesnake species can be found in the Baja California peninsula and the island of the Gulf of California, nevertheless, their venom has been poorly evaluated. The aim of this work was to present the first characterization of endemic Crotalus mitchellii, micro endemic C. polisi and C. thalassoporus venoms. All samples provoke human plasma coagulation showing doses in the rank of 2.3-41.0 µg and also produce rapid hydrolysis of the alpha chain of bovine fibrinogen while the beta chain is attacked at larger incubation periods by C. polisi and especially by C. thalassoporus. Phospholipase activity ranging from 23.2 to 173.8 U/mg. The venoms of C. thalassoporus and C. polisi show very high hemorrhagic activity (from 0.03 to 0.31 µg). A total of 130 toxin-related proteins were identified and classified into ten families. Crotalus mitchellii venom was characterized by high abundance of crotoxin-like and other phospholipase proteins (34.5%) and serine proteinases (29.8%). Crotalus polisi showed a similar proportion of metalloproteinases (34%) and serine proteinases (22.8%) components with important contribution of C-type lectins (14.3%) and CRiSP (14.0%) proteins. Venom of C. thalassoporus is dominated by metalloproteases that amount to more than 66% of total toxin proteins. These results provide a foundation for comprehending the biological, ecological and evolutionary significance of venom composition of speckled rattlesnake from the Baja California peninsula.

Asunto(s)

Venenos de Crotálidos , Crotalus , Animales , Venenos de Crotálidos/metabolismo , Crotalus/metabolismo , Metaloproteasas/metabolismo , México , Fosfolipasas/metabolismo , Proteínas/metabolismo , Serina Proteasas/metabolismo

RESUMEN

BACKGROUND: The invasive gastropod Pomacea canaliculata has received great attention in the last decades as a result of its negative impact on crops agriculture, yet knowledge of their digestive physiology remains incomplete, particularly the enzymatic breakdown of macromolecules such as proteins and lipids. RESULTS: Discovery proteomics revealed aspartic peptidases, cysteine peptidases, serine peptidases, metallopeptidases and threonine peptidases, as well as acid and neutral lipases and phospholipases along the digestive tract of P. canaliculata. Peptides specific to peptidases (139) and lipases (14) were quantified by targeted mass spectrometry. Digestion begins in the mouth via diverse salivary peptidases (nine serine peptidases; seven cysteine peptidases, one aspartic peptidase and 22 metallopeptidases) and then continues in the oesophagus (crop) via three luminal metallopeptidases (Family M12) and six serine peptidases (Family S1). Downstream, the digestive gland provides a battery of enzymes composed of aspartic peptidase (one), cysteine peptidases (nine), serine peptidases (12) and metallopeptidases (24), including aminopeptidases, carboxypeptidases and dipeptidases). The coiled gut has M1 metallopeptidases that complete the digestion of small peptides. Lipid extracellular digestion is completed by triglyceride lipases. CONCLUSION: From an integrative physiological and anatomical perspective, P. canaliculata shows an unexpected abundance and diversity of peptidases, which participate mainly in extracellular digestion. Moreover, the previously unknown occurrence of luminal lipases from the digestive gland is reported for the first time. Salivary and digestive glands were the main tissues involved in the synthesis and secretion of these enzymes, but plausibly the few luminally exclusive peptidases are secreted by ventrolateral pouches or epithelial unicellular glands. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Asunto(s)

Gastrópodos , Animales , Proteómica , Cisteína , Tracto Gastrointestinal , Péptidos , Metaloproteasas , Serina Proteasas , Serina Endopeptidasas , Serina

RESUMEN

Plant fungal diseases cause major problems for the global economy. Antimicrobial peptides have aroused great interest in the control of phytopathogens, as they are natural molecules and have a broad spectrum of inhibitory activity. Herein, we have tried to identify and characterize antimicrobial peptides present in fruits of Capsicum chinense and to evaluate their enzymatic and antifungal activities. The retained fraction obtained in the anion exchange chromatography with strong antifungal activity was subjected to molecular exclusion chromatography and obtained four fractions named G1, G2, G3, and G4. The 6.0-kDa protein band of G2 showed similarity with protease inhibitors type II, and it was able to inhibit 100% of trypsin and α-amylase activities. The protein band with approximately 6.5 kDa of G3 showed similarity with sequences of protease inhibitors from genus Capsicum and showed growth inhibition of 48% for Colletotrichum lindemuthianum, 49% for Fusarium lateritium, and 51% for F. solani and F. oxysporum. Additionally, G3 causes morphological changes, membrane permeabilization, and ROS increase in F. oxysporum cells. The 9-kDa protein band of G4 fraction was similar to a nsLTP type 1, and a protein band of 6.5 kDa was similar to a nsLTP type 2. The G4 fraction was able to inhibit 100% of the activities of glycosidases tested and showed growth inhibition of 35 and 50% of F. oxysporum and C. lindemuthianum, respectively. C. chinense fruits have peptides with antifungal activity and enzyme inhibition with biotechnological potential.

Asunto(s)

Antifúngicos , Capsicum , Antifúngicos/farmacología , Antifúngicos/química , Frutas/microbiología , Capsicum/microbiología , Serina Proteasas/análisis , Péptidos Antimicrobianos , alfa-Amilasas , Hongos , Inhibidores de Proteasas/análisis