Structures of BlEst2 from Bacillus licheniformis in its propeptide and mature forms reveal autoinhibitory effects of the C-terminal domain.

Carboxylesterases comprise a major class of α/ß-fold hydrolases responsible for the cleavage and formation of ester bonds. Found ubiquitously in nature, these enzymes are crucial for the metabolism of both endogenous and exogenous carboxyl esters in animals, plants and microorganisms. Beyond their essential physiological roles, carboxylesterases stand out as one of the important classes of biocatalysts for biotechnology. BlEst2, an enzyme previously classified as Bacillus licheniformis esterase, remains largely uncharacterized. In the present study, we elucidate the structural biology, molecular dynamics and biochemical features of BlEst2. Our findings reveal a canonical α/ß-hydrolase fold similar to the ESTHER block L of lipases, further augmented by two additional accessory C-terminal domains. Notably, the catalytic domain demonstrates two insertions, which occupy conserved locations in α/ß-hydrolase proteins and commonly form the lid domain in lipase structures. Intriguingly, our in vitro cleavage of C-terminal domains revealed the structure of the active form of BlEst2. Upon activation, BlEst2 showed a markedly elevated hydrolytic activity. This observation implies that the intramolecular C-terminal domain serves as a regulatory intramolecular inhibitor. Interestingly, despite exhibiting esterase-like activity, BlEst2 structural characteristics align more closely with lipases. This suggests that BlEst2 could potentially represent a previously unrecognized subgroup within the realm of carboxyl ester hydrolases.

Molecular characterization of EcCLP1, a new putative cathepsin L protease from Echinococcus canadensis.

Echinococcus granulosus sensu lato is a platyhelminth parasite and the etiological cause of cystic echinococcosis (CE), a zoonotic and neglected disease that infects animals and humans worldwide. As a part of the biological arsenal of the parasite, cathepsin L proteases are a group of proteins that are believed to be essential for parasite penetration, immune evasion, and establishment in the tissues of the host. In this work, we have cloned and sequenced a new putative cathepsin L protease from Echinococcus canadensis (EcCLP1). The bioinformatic analysis suggests that EcCLP1 could be synthesized as a zymogen and activated after proteolytic cleavage. The multiple sequence alignment with other cathepsin proteases reveals important functional conserved features like a conserved active site, an N-linked glycosylation residue, a catalytic triad, an oxyanion hole, and three putative disulfide bonds. The phylogenetic analysis suggests that EcCLP1 could indeed be a cathepsin L cysteine protease from clade 1 as it grouped with cathepsins from other species in this clade. Modeling studies suggest that EcCLP1 has two domains forming a cleft where the active site is located and an occluding role for the propeptide. The transcriptomic analysis reveals different levels of cathepsin transcript expression along the different stages of the parasite life cycle. The whole-mount immunohistochemistry shows an interesting superficial punctate pattern of staining which suggests a secretory pattern of expression. The putative cathepsin L protease characterized here may represent an interesting tool for diagnostic purposes, vaccine design, or a new pharmacological target for antiparasitic intervention.

Title: Caractérisation moléculaire d'EcCLP1, une nouvelle protéase putative de type cathepsine L d'Echinococcus canadensis. Abstract: Echinococcus granulosus sensu lato est un Plathelminthe parasite et la cause étiologique de l'échinococcose kystique (EK), une maladie zoonotique et négligée qui infecte les animaux et les humains dans le monde entier. En tant que partie de l'arsenal biologique du parasite, les protéases de type cathepsine L sont un groupe de protéines considérées comme essentielles à la pénétration du parasite, l'évasion immunitaire et son établissement dans les tissus de l'hôte. Dans ce travail, nous avons cloné et séquencé une nouvelle protéase putative de type cathepsine L d'Echinococcus canadensis (EcCLP1). L'analyse bioinformatique suggère qu'EcCLP1 pourrait être synthétisée sous forme de zymogène et activée après clivage protéolytique. L'alignement de séquences multiples avec d'autres protéases de type cathepsine révèle d'importantes caractéristiques fonctionnelles conservées telles qu'un site actif conservé, un résidu de glycosylation lié à N, une triade catalytique, un trou oxyanion et trois liaisons disulfure putatives. L'analyse phylogénétique suggère qu'EcCLP1 pourrait en effet être une protéase de type cathepsine L du clade 1 car elle se regroupe avec les cathepsines d'autres espèces de ce clade. Les études de modélisation suggèrent qu'EcCLP1 possède deux domaines formant une fente où se trouve le site actif et un rôle d'occlusion pour le propeptide. L'analyse transcriptomique révèle différents niveaux d'expression du transcrit de la cathepsine au cours des différentes étapes du cycle de vie du parasite. L'immunohistochimie de montages entiers montre un intéressant motif de coloration ponctuée superficielle qui suggère un modèle d'expression sécrétoire. La protéase putative de type cathepsine L caractérisée ici peut représenter un outil intéressant à des fins de diagnostic, de conception de vaccins ou une nouvelle cible pharmacologique pour une intervention antiparasitaire.

Regulation of the Fasciola hepatica newly excysted juvenile cathepsin L3 (FhCL3) by its propeptide: a proposed 'clamp-like' mechanism of binding and inhibition.

BACKGROUND: The zoonotic worm parasite Fasciola hepatica secretes an abundance of cathepsin L peptidases that are associated with virulence, invasiveness, feeding and migration. The peptidases are produced as inactive zymogens that activate at low pH by autocatalytic removal of their N-terminal pro-domain or propeptide. Propeptides bind to their cognate enzyme with high specificity. Little is known, however, about the mechanism by which the propeptide of FhCL3, a cathepsin L peptidase secreted by the infective newly excysted juveniles (NEJs), regulates the inhibition and activation of the mature enzyme before it is secreted into host tissues. RESULTS: Immunolocalisation/immunoblotting studies show that the FhCL3 zymogen is produced and secreted by gastrodermal cells of the NEJs gut. A recombinant propeptide of FhCL3 (ppFhCL3) was shown to be a highly potent and selective inhibitor of native and recombinant F. hepatica FhCL3 peptidase, and other members of the cathepsin L family; inhibition constant (Ki) values obtained for FhCL1, FhCL2 and FhCL3 were 0.04 nM, 0.004 nM and < 0.002 nM, respectively. These values are at least 1000-fold lower than those Ki obtained for human cathepsin L (HsCL) and human cathepsin K (HsCK) demonstrating the selectivity of the ppFhCL3 for parasite cathepsins L. By exploiting 3-D structural data we identified key molecular interactions in the specific binding between the ppFhCL3 and FhCL3 mature domain. Using recombinant variants of ppFhCL3 we demonstrated the critical importance of a pair of propeptide residues (Tyr46Lys47) for the interaction with the propeptide binding loop (PBL) of the mature enzyme and other residues (Leu66 and Glu68) that allow the propeptide to block the active site. CONCLUSIONS: The FhCL3 peptidase involved in host invasion by F. hepatica is produced as a zymogen in the NEJs gut. Regulation of its activation involves specific binding sites within the propeptide that are interdependent and act as a "clamp-like" mechanism of inhibition. These interactions are disrupted by the low pH of the NEJs gut to initiate autocatalytic activation. Our enzyme kinetics data demonstrates high potency and selectivity of the ppFhCL3 for its cognate FhCL3 enzyme, information that could be utilised to design inhibitors of parasite cathepsin L peptidases.

Uso de NT-proBNP como predictor de evolución en el posoperatorio de cirugía cardíaca / Use of NT-proBNP to predict postoperative evolution in cardiac surgery / Uso do NT-proBNP como preditor de evolução no pós-operatório de cirurgia cardíaca

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Summary: Introduction: the amino-terminal pro brain-type natriuretic peptide (NT-proBNP) is a diagnostic and prognostic biomarker in heart failure. Its use as a prognosis predictor of postoperative evolution in cardiac surgery has not been established. Objective: to determine if the value of preoperative NT in cardiac surgery is associated with postoperative evolution parameters. Primary objective: to evaluate its association with the length of stay in intensive care unit. Secondary objectives: to evaluate its association with the time of mechanical ventilation and inotropic agents requirements. To determine the evolution of NT-proBNP concentration after cardiopulmonary bypass. Methods: multicentric retrospective study, endorsed by the Research Committee of the National Institute of Cardiac Surgery and the Ethic Research Committee of the Clinic Hospital. It included patients who underwent cardiac surgery between March and August 2018. NT-proBNP was measured during anesthesia induction and after cardiopulmonary bypass. A possible association of preoperative NT-proBNP with risk factors and type of procedure performed was studied. By analysing the ROC curve, the area under curve (AUC) was calculated and then, the best cut-off value of NT-proBNP to predict prolonged intensive care unit stay was determined. Intensive care unit stays, mechanical ventilation and inotropic requirements were defined as prolonged when they exceeded 2 days, 6 and 24 hours respectively. Through the use of multivaried logistics, the predicting value of NT-proBNP was determined for each one of the aforementioned variables. A value of alfa 0.05 was considered significant. Results: a total of 155 patients were included in the study. Age, creatininemia, and left ventricular ejection fraction were 65.8±11.4 years, 1.15±1.10 mg/dl and 52.8±11.9% respectively. Female prevalence was 30.3%, arterial hypertension 77.4%, diabetes mellitus 25.2% and dyslipidemia 50.3%. In 42.6% isolated myocardial revascularization was performed, in 12.9% myocardial revascularization plus one or more valve procedures, and in 44.5% isolated valve procedures. In all cases there was a significant reduction between preoperative (443 pg/ml, interquartile range 143-1.193) and postoperative NT-proBNP (362 pg/ml, interquartile range 138-939) (p<0.001). Age, creatininemia, left ventricular ejection fraction, functional classification IV of the New York Heart Association and dyslipidemia turned out to be predictors of preoperative NT-proBNP. Preoperative NT-proBNP was higher in patients with prolonged intensive care unit stay, mechanical ventilation and inotropic requirements. However, it turned out to be an independent predictor only for prolonged intensive care unit stay. (OR=1.62; IC95%:1.11-2.35. p=0.012). The best cut-off value for prolonged intensive care unit stay was 409 pg/ml (AUC=0.68). Conclusion: preoperative determination of NT-proBNP is an efficient tool to predict postoperative evolution. Cardiopulmonary bypass is associated to a significant drop in that marker.

Resumo: Introdução: a porção terminal amino do peptído natriurético tipo B é um biomarcador diagnóstico e prognóstico na insuficiência cardíaca. Seu uso como preditor prognóstico no pós-operatório de cirurgia cardíaca não está estabelecido. Objetivo: determinar se o valor de NT-proBNP no período pré-operatório de cirurgia cardíaca está associado a parâmetros de evolução pós-operatória. Objetivo primário: avaliar sua associação com tempo prolongado de internação em unidade de terapia intensiva. Objetivos secundários: associação com tempo de ventilação mecânica e necessidade inotrópica. Determinar a evolução da concentração de NT-proBNP após circulação extracorpórea. Método: estudo multicêntrico retrospectivo endossado pelo Comitê de Pesquisa do Instituto Nacional de Cirurgia Cardíaco e pelo Comitê de Ética da Pesquisa do Hospital da Clínica. Foram incluídos pacientes operados de março a agosto de 2018. O NT-proBNP foi dosado durante a indução anestésica e após a circulação extracorpórea. O NT-proBNP foi comparado de acordo com os fatores de risco e procedimentos realizados. A área da curva (AUC) foi determinada pela análise da curva ROC e o melhor ponto de corte NT-proBNP foi estabelecido na previsão do tempo prolongado de internação em unidade de terapia intensiva. O tempo de internação em unidade de terapia intensiva, tempo de ventilação mecânica e necessidade inotrópica prolongada foram definidos como maiores que 2 dias, 6 e 24 horas, respectivamente. Um alfa de 0,05 foi considerado significativo. Resultados: 155 pacientes foram incluídos. Idade, creatininemia e fração de ejeção do ventrículo esquerdo foram 65,8± 11,4 anos, 1,15 ± 1,10 mg/dl e 52,8 ± 11,9 %, respectivamente. A prevalência do sexo feminino foi 30,3%, hipertensão arterial 77,4%, diabetes mellitus 25,2% e dislipidemia 50,3%. Em 42,6%, foi realizada revascularização do miocárdio isolada, em 12,9% revascularização do miocárdio associada a um ou mais procedimentos valvares, e em 44,5% procedimentos puros da válvula. Em todos os casos, houve uma diminuição significativa entre o pré-operatório (443 pg/ml, rango interquartílico 143-1.193) e o pós-operatório de NT-proBNP (362 pg/ml, rango interquartilico 138-939) (p <0,001). Idade, cretininemia, fração de ejeção do ventrículo esquerdo, classe funcional IV da New York Heart Association e dislipidemia foram preditores de NT-proBNP pré-operatório. NT-proBNP pré-operatório foi elevado em pacientes com tempo prolongado de internação em unidade de terapia intensiva, tempo de ventilação mecânica e necessidade inotrópica prolongada, mas apenas acabou por ser preditor independente em internação prolongada em unidade de terapia intensiva (OR=1,62; IC95%:1,11-2,35. p=0,012). O melhor valor de corte para internação prolongada foi de 409 pg/ml (AUC = 0,68). Conclusão: a determinação pré-operatória de NT-proBNP é útil como ferramenta na previsão da evolução pós-operatória. A circulação extracorpórea está associada a uma diminuição significativa desse marcador.

Procollagen Type I and III Aminoterminal Propeptide Levels and Severity of Interstitial Lung Disease in Mexican Women With Progressive Systemic Sclerosis.

BACKGROUND: Interstitial lung disease (ILD) is a frequent complication in progressive systemic sclerosis (SSc), being present in 25% to 90% of cases. OBJECTIVES: To evaluate whether serum levels of procollagen typei and iii aminoterminal propeptide (PINP and PIIINP) correlate with severity and patterns of ILD in Mexican women with SSc. METHODS: Thirty three SSc patients were assessed for disease characteristics and anti-topoisomerase antibodies (topoi), and also underwent pulmonary function tests and high-resolution computed tomography (HRCT). Nineteen patients had ILD+SSc, and 14 had no lung involvement (no ILD-SSc); data were compared with those from 45 healthy controls. PINP and PIIINP were assessed in all 3 groups. RESULTS: Patients with SSc had higher PINP and PIIINP vs controls (P=.001, P<.001, respectively). Compared to no ILD-SSc patients, those with ILD+SSc had longer disease duration in years (P=.005), higher modified Rodnan skin score (P<.001), higher Health Assessment Questionnaire-Disability-Index scores (P<.001), higher topoi U/mL (P<.001), PINP (49.28±28.63 vs. 32.12±18.58µg/L, P=.05), and PIIINP (4.33±1.03 vs. 2.67±1.26µg/L, P<.001) levels. ILD severity based on total HRCT correlated with PINP (r=.388, P=.03) and PIIINP (P=.594, P<.001). On adjusted analysis, ILD severity was associated with disease duration (P=.037), PIIINP (P=.038), and topoi (P=.045). CONCLUSIONS: PINP and PIIINP are useful markers for severe ILD+SSc, suggesting they could play a role in the follow-up of this complication in SSc.