RESUMEN

Lowe Syndrome (LS) is a rare X-linked disorder characterized by renal dysfunction, cataracts, and several central nervous system (CNS) anomalies. The mechanisms underlying the neurological dysfunction in LS remain unclear, albeit they share some phenotypic characteristics similar to the deficiency or dysfunction of the Reelin signaling, a relevant pathway with roles in CNS development and neuronal functions. In this study, we investigated the role of OCRL1, an inositol polyphosphate 5-phosphatase encoded by the OCRL gene, mutated in LS, focusing on its impact on endosomal trafficking and receptor recycling in human neuronal cells. Specifically, we tested the effects of OCRL1 deficiency in the trafficking and signaling of ApoER2/LRP8, a receptor for the ligand Reelin. We found that loss of OCRL1 impairs ApoER2 intracellular trafficking, leading to reduced receptor expression and decreased levels at the plasma membrane. Additionally, human neurons deficient in OCRL1 showed impairments in ApoER2/Reelin-induced responses. Our findings highlight the critical role of OCRL1 in regulating ApoER2 endosomal recycling and its impact on the ApoER2/Reelin signaling pathway, providing insights into potential mechanisms underlying the neurological manifestations of LS.

Asunto(s)

Moléculas de Adhesión Celular Neuronal , Endosomas , Proteínas de la Matriz Extracelular , Proteínas Relacionadas con Receptor de LDL , Proteínas del Tejido Nervioso , Neuronas , Monoéster Fosfórico Hidrolasas , Transporte de Proteínas , Proteína Reelina , Serina Endopeptidasas , Humanos , Monoéster Fosfórico Hidrolasas/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/deficiencia , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/deficiencia , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/deficiencia , Moléculas de Adhesión Celular Neuronal/metabolismo , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/deficiencia , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/deficiencia , Endosomas/metabolismo , Neuronas/metabolismo , Proteínas Relacionadas con Receptor de LDL/metabolismo , Proteínas Relacionadas con Receptor de LDL/genética , Transducción de Señal , Síndrome Oculocerebrorrenal/genética , Síndrome Oculocerebrorrenal/metabolismo

RESUMEN

Dengue virus (DENV) infection is known to affect host cell metabolism, but the molecular players involved are still poorly known. Using a proteomics approach, we identified six DENV proteins associated with mitochondria isolated from infected hepatocytes, and most of the peptides identified were from NS3. We also found an at least twofold decrease of several electron transport system (ETS) host proteins. Thus, we investigated whether NS3 could modulate the ETS function by incubating recombinant DENV NS3 constructs in mitochondria isolated from mouse liver. We found that NS3pro (NS3 protease domain), but not the correspondent catalytically inactive mutant (NS3proS135A), impairs complex I (CI)-dependent NADH:ubiquinone oxidoreductase activity, but not the activities of complexes II, III, IV, or V. Accordingly, using high-resolution respirometry, we found that both NS3pro and full-length NS3 decrease the respiratory rates associated with malate/pyruvate oxidation in mitochondria. The NS3-induced impairment in mitochondrial respiration occurs without altering either leak respiration or mitochondria's capacity to maintain membrane potential, suggesting that NS3 does not deeply affect mitochondrial integrity. Remarkably, CI activity is also inhibited in DENV-infected cells, supporting that the NS3 effects observed in isolated mitochondria may be relevant in the context of the infection. Finally, in silico analyses revealed the presence of potential NS3 cleavage sites in 17 subunits of mouse CI and 16 subunits of human CI, most of them located on the CI surface, suggesting that CI is prone to undergo proteolysis by NS3. Our findings suggest that DENV NS3 can modulate mitochondrial bioenergetics by directly affecting CI function. IMPORTANCE: Dengue virus (DENV) infection is a major public health problem worldwide, affecting about 400 million people yearly. Despite its importance, many molecular aspects of dengue pathogenesis remain poorly known. For several years, our group has been investigating DENV-induced metabolic alterations in the host cells, focusing on the bioenergetics of mitochondrial respiration. The results of the present study reveal that the DENV non-structural protein 3 (NS3) is found in the mitochondria of infected cells, impairing mitochondrial respiration by directly targeting one of the components of the electron transport system, the respiratory complex I (CI). NS3 acts as the viral protease during the DENV replication cycle, and its proteolytic activity seems necessary for inhibiting CI function. Our findings uncover new nuances of DENV-induced metabolic alterations, highlighting NS3 as an important player in the modulation of mitochondria function during infection.

Asunto(s)

Virus del Dengue , Complejo I de Transporte de Electrón , Mitocondrias , Proteínas no Estructurales Virales , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Animales , Virus del Dengue/fisiología , Virus del Dengue/genética , Ratones , Complejo I de Transporte de Electrón/metabolismo , Complejo I de Transporte de Electrón/genética , Humanos , Mitocondrias/metabolismo , Hepatocitos/virología , Hepatocitos/metabolismo , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , Dengue/virología , Dengue/metabolismo , Respiración de la Célula , Proteómica , Proteasas Virales

RESUMEN

BACKGROUND: Peptide drugs are advantageous because they are subject to rational design and exhibit highly diverse structures and broad biological activities. The NS2B-NS3 protein is a particularly promising flavivirus therapeutic target, with extensive research on the development of inhibitors as therapeutic candidates, and was used as a model in this work to determine the mechanism by which GA-Hecate inhibits ZIKV replication. OBJECTIVE: The present study aimed to evaluate the potential of GA-Hecate, a new antiviral developed by our group, against the Brazilian Zika virus and to evaluate the mechanism of action of this compound on the flavivirus NS2B-NS3 protein. METHODS: Solid-phase peptide Synthesis, High-Performance Liquid Chromatography, and Mass Spectrometry were used to obtain, purify, and characterize the synthesized compound. Real-time and enzymatic assays were used to determine the antiviral potential of GA-Hecate against ZIKV. RESULTS: The RT-qPCR results showed that GA-Hecate decreased the number of ZIKV RNA copies in the virucidal, pre-treatment, and post-entry assays, with 5- to 6-fold fewer RNA copies at the higher nontoxic concentration in Vero cells (HNTC: 10 µM) than in the control cells. Enzymatic and kinetic assays indicated that GA-Hecate acts as a competitive ZIKV NS2B-NS3 protease inhibitor with an IC50 of 32 nM and has activity against the yellow fever virus protease. CONCLUSION: The results highlight the antiviral potential of the GA-Hecate bioconjugate and open the door for the development of new antivirals.

Asunto(s)

Antivirales , Proteínas no Estructurales Virales , Replicación Viral , Virus Zika , Virus Zika/efectos de los fármacos , Antivirales/farmacología , Antivirales/química , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Chlorocebus aethiops , Células Vero , Replicación Viral/efectos de los fármacos , Serina Endopeptidasas/metabolismo , Péptidos/farmacología , Péptidos/química , ARN Helicasas/metabolismo , ARN Helicasas/antagonistas & inhibidores , Infección por el Virus Zika/tratamiento farmacológico , Infección por el Virus Zika/virología , Humanos , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/química , Proteasas Virales , Nucleósido-Trifosfatasa , ARN Helicasas DEAD-box

RESUMEN

COVID-19, caused by SARS-CoV-2, affects neuronal cells, causing several symptoms such as memory loss, anosmia and brain inflammation. Curcuminoids (Me08 e Me23) and curcumin (CUR) are derived from Curcuma Longa extract (EXT). Many therapeutic actions have been linked to these compounds, including antiviral action. Given the severe implications of COVID-19, especially within the central nervous system, our study aims to shed light on the therapeutic potential of curcuminoids against SARS-CoV-2 infection, particularly in neuronal cells. Here, we investigated the effects of CUR, EXT, Me08 and Me23 in human neuroblastoma SH-SY5Y. We observed that Me23 significantly decreased the expression of plasma membrane-associated transmembrane protease serine 2 (TMPRSS2) and TMPRSS11D, consequently mitigating the elevated ROS levels induced by SARS-CoV-2. Furthermore, Me23 exhibited antioxidative properties by increasing NRF2 gene expression and restoring NQO1 activity following SARS-CoV-2 infection. Both Me08 and Me23 effectively reduced SARS-CoV-2 replication in SH-SY5Y cells overexpressing ACE2 (SH-ACE2). Additionally, all of these compounds demonstrated the ability to decrease proinflammatory cytokines such as IL-6, TNF-α, and IL-17, while Me08 specifically reduced INF-γ levels. Our findings suggest that curcuminoid Me23 could serve as a potential agent for mitigating the impact of COVID-19, particularly within the context of central nervous system involvement.

Asunto(s)

Antiinflamatorios , Antioxidantes , Antivirales , Tratamiento Farmacológico de COVID-19 , Curcumina , SARS-CoV-2 , Humanos , Curcumina/farmacología , Curcumina/análogos & derivados , Antioxidantes/farmacología , Antivirales/farmacología , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/fisiología , Antiinflamatorios/farmacología , Línea Celular Tumoral , Curcuma/química , Serina Endopeptidasas/metabolismo , COVID-19/virología , COVID-19/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Extractos Vegetales/farmacología , Citocinas/metabolismo , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/virología

RESUMEN

Transmembrane serine protease 2 (TMPRSS2) is a membrane-bound protease belonging to the type II transmembrane serine protease (TTSP) family. It is a multidomain protein, including a serine protease domain responsible for its self-activation. The protein has been implicated as an oncogenic transcription factor and for its ability to cleave (prime) the SARS-CoV-2 spike protein. In order to characterize the TMPRSS2 biochemical properties, we expressed the serine protease domain (rTMPRSS2_SP) in Komagataella phaffii using the pPICZαA vector and purified it using immobilized metal affinity (Ni Sepharose™ excel) and size exclusion (Superdex 75) chromatography. We explored operational fluorescence resonance energy transfer FRET peptides as substrates. We chose the peptide Abz-QARK-(Dnp)-NH2 (Abz = ortho-aminobenzoic acid, the fluorescence donor, and Dnp = 2,4-dinitrophenyl, the quencher group) as a substrate to find the optimal conditions for maximum enzymatic activity. We found that metallic ions such as Ca2+ and Na+ increased enzymatic activity, but ionic surfactants and reducing agents decreased catalytic capacity. Finally, we determined the rTMPRSS2_SP stability for long-term storage. Altogether, our results represent the first comprehensive characterization of TMPRSS2's biochemical properties, providing valuable insights into its serine protease domain.

Asunto(s)

Transferencia Resonante de Energía de Fluorescencia , Proteínas Recombinantes , Serina Endopeptidasas , Cinética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidad por Sustrato , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/química , Serina Endopeptidasas/genética , Humanos , Estabilidad de Enzimas , Dominios Proteicos , Saccharomycetales/enzimología , Saccharomycetales/metabolismo , Saccharomycetales/genética , SARS-CoV-2/enzimología , Péptidos/metabolismo , Péptidos/química , Glicoproteína de la Espiga del Coronavirus

RESUMEN

Beer is a beverage that contains gluten and cannot be consumed by people with celiac disease. In this context, the enzyme prolyl endoprotease (PEP) can be used to reduce the gluten content in beer. The present study aimed to produce the PEP from Aspergillus sp. FSDE 16 using solid-state fermentation with 5 conditions and comparing with a similar commercial enzyme produced from Aspergillus niger in the production of a gluten-free beer. The results of the performed cultures showed that during the culture, the most increased protease activity (54.46 U/mL) occurred on the 4th day. In contrast, for PEP, the highest activity (0.0356 U/mL) was obtained on the 3rd day of culture in condition. Regarding beer production, cell growth, pH, and total soluble solids showed similar behavior over the 7 days for beers produced without enzyme addition or with the addition of commercial enzyme and with the addition of the enzyme extract produced. The addition of the enzyme and the enzyme extract did not promote changes, and all the beers produced showed similar and satisfactory results, with acid pH between 4 and 5, total soluble solids ranging from 4.80 to 5.05, alcohol content ranging from 2.83% to 3.08%, and all beers having a dark character with deep amber and light copper color. Gluten removal was effectively using the commercial enzyme and the enzyme produced according to condition (v) reaching gluten concentrations equal to 17 ± 5.31 and 21.19 ± 11.28 ppm, respectively. In this way, the production of the enzyme by SSF and its application in the removal of gluten in beer was efficient.

Asunto(s)

Cerveza , Serina Endopeptidasas , Humanos , Cerveza/análisis , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Prolil Oligopeptidasas , Fermentación , Glútenes/análisis , Glútenes/metabolismo , Aspergillus niger , Extractos Vegetales

RESUMEN

Fe-deficiency anaemia is a major public health concern in children under 5 years of age. TMPRSS6 gene, encoding matriptase-2 protein, is implicated in Fe homoeostasis and has been associated with anaemia and Fe status in various populations. The aim of this cross-sectional study was to investigate the associations between the single nucleotide polymorphism (SNP) TMPRSS6 rs855791 and biomarkers of anaemia and Fe deficiency in Brazilian children attending day care centres. A total of 163 children aged 6-42 months were evaluated. Socio-economic, demographic, biochemical, haematological, immunological and genotype data were collected. Multiple logistic and linear regressions with hierarchical selection were used to assess the effects of independent variables on categorised outcomes and blood marker concentrations. Minor allele (T) frequency of rs855791 was 0·399. Each copy of the T allele was associated with a 4·49-fold increased risk of developing anaemia (P = 0·005) and a 4·23-fold increased risk of Fe deficiency assessed by serum soluble transferrin receptor (sTfR) (P < 0·001). The dose of the T allele was associated with an increase of 0·18 mg/l in sTfR concentrations and reductions of 1·41 fl and 0·52 pg in mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH), respectively. In conclusion, the T allele of SNP TMPRSS6 rs855791 was significantly associated with anaemia and Fe deficiency assessed by sTfR in Brazilian children attending day care centres. The effect was dose dependent, with each copy of the T allele being associated with lower MCV and MCH and higher concentrations of sTfR.

Asunto(s)

Anemia Ferropénica , Anemia , Deficiencias de Hierro , Preescolar , Humanos , Anemia/epidemiología , Anemia/genética , Anemia Ferropénica/epidemiología , Anemia Ferropénica/genética , Brasil/epidemiología , Estudios Transversales , Centros de Día , Proteínas de la Membrana/genética , Polimorfismo de Nucleótido Simple , Receptores de Transferrina , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo

RESUMEN

The aims of this study are to characterize the antiplatelet activity of StSBTc-3, a potato serine protease with fibrino (geno) lytic activity, and to provide information on its mechanism of action. The results obtained show that StSBTc-3 inhibits clot retraction and prevents platelet aggregation induced by thrombin, convulxin, and A23187. Platelet aggregation inhibition occurs in a dose-dependent manner and is not affected by inactivation of StSBTc-3 with the inhibitor of serine proteases phenylmethylsulfonyl fluoride (PMSF). In addition, StSBTc-3 reduces fibrinogen binding onto platelets. In-silico calculations show a high binding affinity between StSBTc-3 and human α2bß3 integrin suggesting that the antiplatelet activity of StSBTc-3 could be associated with the fibronectin type III domain present in its amino acid sequence. Binding experiments show that StSBTc-3 binds to α2bß3 preventing the interaction between α2bß3 and fibrinogen and, consequently, inhibiting platelet aggregation. StSBTc-3 represents a promising compound to be considered as an alternative to commercially available drugs used in cardiovascular therapies.

Asunto(s)

Solanum tuberosum , Humanos , Serina/metabolismo , Plaquetas/metabolismo , Agregación Plaquetaria , Serina Endopeptidasas/metabolismo , Fibrinógeno/metabolismo , Subtilisinas/metabolismo

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

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

Structural variability is a feature of snake venom proteins, and glycosylation is a post-translational modification that contributes to the diversification of venom proteomes. Studies by our group have shown that Bothrops venoms are distinctly defined by their glycoprotein content, and that most hybrid/complex N-glycans identified in these venoms contain sialic acid. Considering that metalloproteases and serine proteases are abundant components of Bothrops venoms and essential in the envenomation process, and that these enzymes contain several glycosylation sites, the role of sialic acid in venom proteolytic activity was evaluated. Here we show that removal of sialic acid by treatment of nine Bothrops venoms with neuraminidase (i) altered the pattern of gelatinolysis in zymography of most venoms and reduced the gelatinolytic activity of all venoms, (ii) decreased the proteolytic activity of some venoms on fibrinogen and the clotting activity of human plasma of all venoms, and (iii) altered the proteolysis profile of plasma proteins by B. jararaca venom, suggesting that sialic acid may play a role in the interaction of proteases with their protein substrates. In contrast, the profile of venom amidolytic activity on Bz-Arg-pNA did not change after removal of sialic acid, indicating that this monosaccharide is not essential in N-glycans of serine proteases acting on small substrates. In summary, these results expand the knowledge about the variability of the subproteomes of Bothrops venom proteases, and for the first time point to the importance of carbohydrate chains containing sialic acid in the enzymatic activities of venom proteases relevant in human envenomation.

Asunto(s)

Bothrops , Venenos de Crotálidos , Animales , Humanos , Ácido N-Acetilneuramínico/metabolismo , Venenos de Serpiente , Serina Proteasas/metabolismo , Venenos de Crotálidos/química , Glicoproteínas/metabolismo , Serina Endopeptidasas/metabolismo , Polisacáridos/metabolismo , Bothrops/metabolismo

RESUMEN

The Zika virus protease NS2B-NS3 has a binding site formed with the participation of a H51-D75-S135 triad presenting two forms, active and inactive. Studies suggest that the inactive conformation is a good target for the design of inhibitors. In this paper, we evaluated the co-crystallized structures of the protease with the inhibitors benzoic acid (5YOD) and benzimidazole-1-ylmethanol (5H4I). We applied a protocol consisting of two steps: first, classical molecular mechanics energy minimization followed by classical molecular dynamics were performed, obtaining stabilized molecular geometries; second, the optimized/relaxed geometries were used in quantum biochemistry and molecular mechanics/Poisson-Boltzmann surface area (MM-PBSA) calculations to estimate the ligand interactions with each amino acid residue of the binding pocket. We show that the quantum-level results identified essential residues for the stabilization of the 5YOD and 5H4I complexes after classical energy minimization, matching previously published experimental data. The same success, however, was not observed for the MM-PBSA simulations. The application of quantum biochemistry methods seems to be more promising for the design of novel inhibitors acting on NS2B-NS3.

Asunto(s)

Infección por el Virus Zika , Virus Zika , Simulación de Dinámica Molecular , Péptido Hidrolasas/metabolismo , Inhibidores de Proteasas/química , Serina Endopeptidasas/metabolismo , Succinatos , Proteínas no Estructurales Virales/metabolismo , Virus Zika/metabolismo

RESUMEN

BACKGROUND: Drosophila melanogaster lipophorin receptors (LpRs), LpR1 and LpR2, are members of the LDLR family known to mediate lipid uptake in a range of organisms from Drosophila to humans. The vertebrate orthologs of LpRs, ApoER2 and VLDL-R, function as receptors of a glycoprotein involved in development of the central nervous system, Reelin, which is not present in flies. ApoER2 and VLDL-R are associated with the development and function of the hippocampus and cerebral cortex, important association areas in the mammalian brain, as well as with neurodevelopmental and neurodegenerative disorders linked to those regions. It is currently unknown whether LpRs play similar roles in the Drosophila brain. RESULTS: We report that LpR-deficient flies exhibit impaired olfactory memory and sleep patterns, which seem to reflect anatomical defects found in a critical brain association area, the mushroom bodies (MB). Moreover, cultured MB neurons respond to mammalian Reelin by increasing the complexity of their neurite arborization. This effect depends on LpRs and Dab, the Drosophila ortholog of the Reelin signaling adaptor protein Dab1. In vitro, two of the long isoforms of LpRs allow the internalization of Reelin, suggesting that Drosophila LpRs interact with human Reelin to induce downstream cellular events. CONCLUSIONS: These findings demonstrate that LpRs contribute to MB development and function, supporting the existence of a LpR-dependent signaling in Drosophila, and advance our understanding of the molecular factors functioning in neural systems to generate complex behaviors in this model. Our results further emphasize the importance of Drosophila as a model to investigate the alterations in specific genes contributing to neural disorders.

Asunto(s)

Proteínas de Drosophila , Drosophila melanogaster , Cuerpos Pedunculados , Receptores Citoplasmáticos y Nucleares , Animales , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/farmacología , Cuerpos Pedunculados/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteína Reelina , Serina Endopeptidasas/metabolismo

RESUMEN

DNA damage triggers a widely conserved stress response in bacteria called the SOS response, which involves two key regulators, the activator RecA and the transcriptional repressor LexA. Despite the wide conservation of the SOS response, the number of genes controlled by LexA varies considerably between different organisms. The filamentous soil-dwelling bacteria of the genus Streptomyces contain LexA and RecA homologs, but their roles in Streptomyces have not been systematically studied. Here, we demonstrate that RecA and LexA are required for the survival of Streptomyces venezuelae during DNA-damaging conditions and for normal development during unperturbed growth. Monitoring the activity of a fluorescent recA promoter fusion and LexA protein levels revealed that the activation of the SOS response is delayed in S. venezuelae. By combining global transcriptional profiling and chromatin immunoprecipitation sequencing (ChIP-seq) analysis, we determined the LexA regulon and defined the core set of DNA damage repair genes that are expressed in response to treatment with the DNA-alkylating agent mitomycin C. Our results show that DNA damage-induced degradation of LexA results in the differential regulation of LexA target genes. Using surface plasmon resonance, we further confirmed the LexA DNA binding motif (SOS box) and demonstrated that LexA displays tight but distinct binding affinities to its target promoters, indicating a graded response to DNA damage. IMPORTANCE The transcriptional regulator LexA functions as a repressor of the bacterial SOS response, which is induced under DNA-damaging conditions. This results in the expression of genes important for survival and adaptation. Here, we report the regulatory network controlled by LexA in the filamentous antibiotic-producing Streptomyces bacteria and establish the existence of the SOS response in Streptomyces. Collectively, our work reveals significant insights into the DNA damage response in Streptomyces that will promote further studies to understand how these important bacteria adapt to their environment.

Asunto(s)

Proteínas Bacterianas , Streptomyces , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Daño del ADN , Regulación Bacteriana de la Expresión Génica , Rec A Recombinasas/genética , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Streptomyces/genética , Streptomyces/metabolismo

RESUMEN

Proteases are virulence factors with a recognized impact on the Leishmania spp. life cycle. This study considers a set of analyses measuring phenotypic factors of L. (V.) braziliensis clinical isolates as promastigotes growth curves, murine peritoneal macrophages infection, inflammatory mediators production, and serine proteases gene expression (subtilisin 13: S13, subtilisin 28: S28, oligopeptidase B: OPB) assessing these isolates' fitness on in vitro conditions. Parasites had different behavior during the early growth phase from day zero to day three, and all isolates reached the stationary growth phase between days four and seven. Macrophages infection showed two tendencies, one of decreased infection rate and number of parasites per macrophage (Infection Index <1000) and another with a constant infection index (≥1400). TNF-α (≥10 pg/mL) detected in infections by 75% of isolates, IL-6 (≥80 pg/mL) by 30% of isolates and low levels of NO (≥0.01µM) in almost all infections. Gene expression showed higher values of S13 (≥2RQ) in the intracellular amastigotes of all the isolates evaluated. On the contrary, S28 expression was low (≤1RQ) in all isolates. OPB expression was different between promastigotes and intracellular amastigotes, being significantly higher (≥2RQ) in the latter form of 58% of the isolates. Predictive structural assays of S13 and OPB were performed to explore temperature influence on gene expression and the encoded proteases. Gene expression data is discussed based on in silico predictions of regulatory regions that show plasticity in the linearity index of secondary structures of S13 and OPB 3'-untranslated regions of mRNA, dependent on temperature changes. While hairpin structures suggest an active region of mRNA for both genes above 26°C, pseudoknot structure found in S13 is an indication of a particular profile of this gene at mammalian host temperatures (37°C). Furthermore, the predicted 3D structures are in accordance with the influence of these temperatures on the catalytic site stability of both enzymes, favoring their action over peptide substrates. Data gathered here suggest that L. (V.) braziliensis serine proteases can be influenced by the temperature conditions affecting parasite fitness throughout its life cycle.

Asunto(s)

Leishmania braziliensis , Serina Endopeptidasas , Subtilisina , Temperatura , Animales , Leishmania braziliensis/enzimología , Estadios del Ciclo de Vida , Ratones , ARN Mensajero , Serina Endopeptidasas/metabolismo

RESUMEN

Several strategies are used by Escherichia coli to evade the host innate immune system in the blood, such as the cleavage of complement system proteins by secreted proteases. Members of the Serine Proteases Autotransporters of Enterobacteriaceae (SPATE) family have been described as presenting proteolytic effects against complement proteins. Among the SPATE-encoding genes sat (secreted autotransporter toxin) has been detected in high frequencies among strains of E. coli isolated from bacteremia. Sat has been characterized for its cytotoxic action, but the possible immunomodulatory effects of Sat have not been investigated. Therefore, this study aimed to evaluate the proteolytic effects of Sat on complement proteins and the role in pathogenesis of BSI caused by extraintestinal E. coli (ExPEC). E. coli EC071 was selected as a Sat-producing ExPEC strain. Whole-genome sequencing showed that sat sequences of EC071 and uropathogenic E. coli CFT073 present 99% identity. EC071 was shown to be resistant to the bactericidal activity of normal human serum (NHS). Purified native Sat was used in proteolytic assays with proteins of the complement system and, except for C1q, all tested substrates were cleaved by Sat in a dose and time-dependent manner. Moreover, E. coli DH5α survived in NHS pre-incubated with Sat. EC071-derivative strains harboring sat knockout and in trans complementations producing either active or non-active Sat were tested in a murine sepsis model. Lethality was reduced by 50% when mice were inoculated with the sat mutant strain. The complemented strain producing active Sat partially restored the effect caused by the wild-type strain. The results presented in this study show that Sat presents immunomodulatory effects by cleaving several proteins of the three complement system pathways. Therefore, Sat plays an important role in the establishment of bloodstream infections and sepsis.

Asunto(s)

Bacteriemia , Toxinas Bacterianas , Proteínas de Escherichia coli , Escherichia coli Uropatógena , Animales , Toxinas Bacterianas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ratones , Serina Endopeptidasas/metabolismo , Serina Proteasas/genética , Sistemas de Secreción Tipo V/genética , Sistemas de Secreción Tipo V/metabolismo

RESUMEN

AIMS: Angiotensin-converting enzyme (ACE) 2 is the receptor for severe acute respiratory syndrome coronavirus 2 which causes coronavirus disease 2019 (COVID-19). Viral cellular entry requires ACE2 and transmembrane protease serine 2 (TMPRSS2). ACE inhibitors (ACEIs) or angiotensin (Ang) receptor blockers (ARBs) influence ACE2 in animals, though evidence in human lungs is lacking. We investigated ACE2 and TMPRSS2 in type II pneumocytes, the key cells that maintain lung homeostasis, in lung parenchymal of ACEI/ARB-treated subjects compared to untreated control subjects. MAIN METHODS: Ang II and Ang-(1-7) levels and ACE2 and TMPRSS2 protein expression were measured by radioimmunoassay and immunohistochemistry, respectively. KEY FINDINGS: We found that the ratio Ang-(1-7)/Ang II, a surrogate marker of ACE2 activity, as well as the amount of ACE2-expressing type II pneumocytes were not different between ACEI/ARB-treated and untreated subjects. ACE2 protein content correlated positively with smoking habit and age. The percentage of TMPRSS2-expressing type II pneumocytes was higher in males than females and in subjects under 60 years of age but it was not different between ACEI/ARB-treated and untreated subjects. However, there was a positive association of TMPRSS2 protein content with age and smoking in ACEI/ARB-treated subjects, with high TMPRSS2 protein levels most evident in ACEI/ARB-treated older adults and smokers. SIGNIFICANCE: ACEI/ARB treatment influences human lung TMPRSS2 but not ACE2 protein content and this effect is dependent on age and smoking habit. This finding may help explain the increased susceptibility to COVID-19 seen in smokers and older patients with treated cardiovascular-related pathologies.

Asunto(s)

Células Epiteliales Alveolares/metabolismo , Antagonistas de Receptores de Angiotensina/farmacología , Enzima Convertidora de Angiotensina 2/metabolismo , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Sistema Renina-Angiotensina/fisiología , Serina Endopeptidasas/metabolismo , Adulto , Factores de Edad , Anciano , Células Epiteliales Alveolares/química , Células Epiteliales Alveolares/efectos de los fármacos , Angiotensina I/metabolismo , Angiotensina II/metabolismo , Enzima Convertidora de Angiotensina 2/análisis , Enzima Convertidora de Angiotensina 2/antagonistas & inhibidores , Femenino , Humanos , Pulmón/química , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Masculino , Persona de Mediana Edad , Fragmentos de Péptidos/metabolismo , Sistema Renina-Angiotensina/efectos de los fármacos , Estudios Retrospectivos , Serina Endopeptidasas/análisis , Fumar/metabolismo , Fumar/patología

Asunto(s)

COVID-19/complicaciones , Neoplasias de la Próstata/epidemiología , SARS-CoV-2/aislamiento & purificación , Serina Endopeptidasas/metabolismo , COVID-19/metabolismo , COVID-19/virología , Humanos , Masculino , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/virología , Serina Endopeptidasas/genética

RESUMEN

Although dengue virus (DENV) replication occurs in the cytoplasm, the nucleus plays an essential role during infection. Both the capsid protein (C) and non-structural protein 5 (NS5) are translocated into the infected cell nucleus to favor viral replication. Previously, our group reported the nuclear localization of the NS3 protein during DENV infection of mosquito cells; however, the nuclear localization of the DENV NS3 protein in human host cells has not been described. Here, we demonstrated that NS3 is present in the nucleus of Huh7 cells at early infection times, and later, it is mainly located in the cytoplasm.

Asunto(s)

Núcleo Celular/metabolismo , Virus del Dengue/metabolismo , Serina Endopeptidasas/metabolismo , Línea Celular Tumoral , Citoplasma/metabolismo , Humanos

RESUMEN

The COVID-19 pandemic is an unexpected worldwide situation, and all countries have implemented their own policies to curb the spread of the virus. The pathophysiology of COVID-19 has opened numerous hypotheses of functional alterations in different physiological aspects. The direct impact of SARS-CoV-2 on the urogenital organs of males and females is still to be assessed. Nevertheless, based on biological similarities between SARS-CoV and SARS-CoV-2, several hypotheses have been proposed. In this study, we will discuss the possible mechanism of action, and potential effects on the male/female reproductive system and fertility.

Asunto(s)

COVID-19 , Fertilidad , Reproducción , SARS-CoV-2 , Enzima Convertidora de Angiotensina 2/inmunología , Enzima Convertidora de Angiotensina 2/metabolismo , Genitales/inmunología , Genitales/metabolismo , Genitales/virología , Humanos , Serina Endopeptidasas/inmunología , Serina Endopeptidasas/metabolismo