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1.
Int J Mol Sci ; 22(21)2021 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-34768820

RESUMO

Disseminated intravascular coagulation (DIC) is a severe condition characterized by the systemic formation of microthrombi complicated with bleeding tendency and organ dysfunction. In the last years, it represents one of the most frequent consequences of coronavirus disease 2019 (COVID-19). The pathogenesis of DIC is complex, with cross-talk between the coagulant and inflammatory pathways. The objective of this study is to investigate the anti-inflammatory action of ultramicronized palmitoylethanolamide (um-PEA) in a lipopolysaccharide (LPS)-induced DIC model in rats. Experimental DIC was induced by continual infusion of LPS (30 mg/kg) for 4 h through the tail vein. Um-PEA (30 mg/kg) was given orally 30 min before and 1 h after the start of intravenous infusion of LPS. Results showed that um-PEA reduced alteration of coagulation markers, as well as proinflammatory cytokine release in plasma and lung samples, induced by LPS infusion. Furthermore, um-PEA also has the effect of preventing the formation of fibrin deposition and lung damage. Moreover, um-PEA was able to reduce the number of mast cells (MCs) and the release of its serine proteases, which are also necessary for SARS-CoV-2 infection. These results suggest that um-PEA could be considered as a potential therapeutic approach in the management of DIC and in clinical implications associated to coagulopathy and lung dysfunction, such as COVID-19.


Assuntos
Amidas/uso terapêutico , Transtornos da Coagulação Sanguínea/tratamento farmacológico , Coagulação Intravascular Disseminada/tratamento farmacológico , Etanolaminas/uso terapêutico , Ácidos Palmíticos/uso terapêutico , Sepse/complicações , Amidas/química , Amidas/farmacologia , Animais , Transtornos da Coagulação Sanguínea/etiologia , COVID-19/patologia , COVID-19/virologia , Citocinas/sangue , Citocinas/metabolismo , Modelos Animais de Doenças , Coagulação Intravascular Disseminada/etiologia , Etanolaminas/química , Etanolaminas/farmacologia , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Lipopolissacarídeos/toxicidade , Pulmão/metabolismo , Pulmão/patologia , Masculino , Mastócitos/citologia , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Ácidos Palmíticos/química , Ácidos Palmíticos/farmacologia , Tempo de Tromboplastina Parcial , Tempo de Protrombina , Ratos , Ratos Sprague-Dawley , SARS-CoV-2/isolamento & purificação , Sepse/patologia , Serina Proteases/metabolismo
3.
Appl Microbiol Biotechnol ; 105(21-22): 8129-8138, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34605969

RESUMO

The non-spore forming Gram-positive actinomycetes Amycolatopsis keratiniphila subsp. keratiniphila D2T (DSM 44,409) has a high potential for keratin valorization as demonstrated by a novel biotechnological microbial conversion process consisting of a bacterial growth phase and a keratinolytic phase, respectively. Compared to the most gifted keratinolytic Bacillus species, a very large number of 621 putative proteases are encoded by the genome of Amycolatopsis keratiniphila subsp. keratiniphila D2T, as predicted by using Peptide Pattern Recognition (PPR) analysis. Proteome analysis by using LC-MS/MS on aliquots of the supernatant of A. keratiniphila subsp. keratiniphila D2T culture on slaughterhouse pig bristle meal, removed at 24, 48, 96 and 120 h of growth, identified 43 proteases. This was supplemented by proteome analysis of specific fractions after enrichment of the supernatant by anion exchange chromatography leading to identification of 50 proteases. Overall 57 different proteases were identified corresponding to 30% of the 186 proteins identified from the culture supernatant and distributed as 17 metalloproteases from 11 families, including an M36 protease, 38 serine proteases from 4 families, and 13 proteolytic enzymes from other families. Notably, M36 keratinolytic proteases are prominent in fungi, but seem not to have been discovered in bacteria previously. Two S01 family peptidases, named T- and C-like proteases, prominent in the culture supernatant, were purified and shown to possess a high azo-keratin/azo-casein hydrolytic activity ratio. The C-like protease revealed excellent thermostability, giving promise for successful applications in biorefinery processes. Notably, the bacterium seems not to secrete enzymes for cleavage of disulfides in the keratinous substrates. KEY POINTS: • A. keratiniphila subsp. keratiniphila D2T is predicted to encode 621 proteases. • This actinomycete efficiently converts bristle meal to a protein hydrolysate. • Proteome analysis identified 57 proteases in its secretome.


Assuntos
Actinobacteria , Actinomyces , Amycolatopsis , Animais , Cromatografia Líquida , Queratinas , Peptídeo Hidrolases , Serina Proteases , Suínos , Espectrometria de Massas em Tandem
4.
J Food Biochem ; 45(10): e13919, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34486135

RESUMO

Marine pharaoh cuttlefish Sepia pharaonis (family Sepiidae) is regarded as an economically important class of cephalopod in the coastal Mediterranean and Asian regions. Bioassay-guided chromatographic purification of solvent extract of S. pharaonis led to the identification of a trans-decalin based spirolactone, spiropharanone, which was characterized as 1-hydroxy-7-(4'-methoxy-3-methylbut-2-enyl)-3,9,15-trimethyl-8-oxo-octahydro-5H-spiro[furan-8,9-naphtho]-8-yl-acetate by spectroscopic techniques. Spiropharanone exhibited significantly greater anti-inflammatory activity by attenuating pro-inflammatory 5-lipoxygenase (IC50 1.02 mM) than the non-steroidal drug ibuprofen (IC50 4.61 mM, p ≤ .05). Superior antioxidant properties of spiropharanone against free radicals (EC50 ~1.20 mM) and other oxidants (hydroxyl [EC50 0.97 mM] and superoxide [EC50 1.47 mM] scavenging) also reinforced its promising anti-inflammatory activity. The studied spiropharanone also exhibited significant attenuation toward insulin secretion regulating enzyme dipeptidyl peptidase-4 (IC50 0.92 mM) recognizing its anti-hyperglycemic potential. Significantly higher electronic properties (topological polar surface area ~100) combined with balanced hydrophilic-lipophilic properties (partition coefficient of logarithmic octanol-water ~3) and lesser docking parameters of spiropharanone demonstrated that the compound could be utilized as an important bioactive lead against oxidative stress, inflammation, and hyperglycemic-related ailments. PRACTICAL APPLICATIONS: Nutritionally rich edible marine pharaoh cuttlefish Sepia pharaonis occupies a prominent place among seafood fisheries owing to the presence of bioactive nutrients and functional food ingredients. These marine cuttlefish are widely distributed along the Asian and Mediterranean coasts, and consumed as culinary delicacy for decades. An undescribed trans-decalin spirolactone, spiropharanone was isolated from the organic extract of S. pharaonis based on bioactivity-assisted sequential chromatographic fractionation. Spiropharanone displayed promising antioxidant potential along with attenuation properties against inducible pro-inflammatory 5-lipoxygenase and insulin secretion regulating enzyme dipeptidyl peptidase-4. This study established the ameliorating potential of a naturally derived marine food constituent against inflammatory and diabetic ailments, and thus anticipated as functional food lead in pharmaceutical formulations towards inflammation and maintaining glucose homeostasis.


Assuntos
Sepia , Animais , Antioxidantes/farmacologia , Araquidonato 5-Lipoxigenase , Decapodiformes , Lactonas , Lipoxigenase , Naftalenos , Alimentos Marinhos , Serina Proteases
5.
Biomolecules ; 11(8)2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34439804

RESUMO

The bioprospecting of marine and brackish water systems has increased during the last decades. In this respect, microalgae, including cyanobacteria, and their metabolites are one of the most widely explored resources. Most of the bioactive compounds are isolated from ex situ cultures of microorganisms; however, analysis of field samples could also supply valuable information about the metabolic and biotechnological potential of microalgae communities. In this work, the activity of phytoplankton samples from the Curonian Lagoon was studied. The samples were active against antibiotic resistant clinical and environmental bacterial strains as well as against serine proteases and T47D human breast adenocarcinoma cells. No significant effect was found on Daphnia magna. In addition, using LC-MS/MS, we documented the diversity of metabolites present in field samples. A list of 117 detected cyanopeptides was presented. Cyanopeptolins constituted the largest class of cyanopeptides. As complex bloom samples were analyzed, no link between the observed activity and a specific sample component can be established. However, the results of the study showed a biotechnological potential of natural products from the Curonian Lagoon.


Assuntos
Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Produtos Biológicos/farmacologia , Cianobactérias/química , Microalgas/química , Fitoplâncton/química , Animais , Antibacterianos/química , Antineoplásicos/química , Oceano Atlântico , Países Bálticos , Baías/microbiologia , Produtos Biológicos/química , Linhagem Celular Tumoral , Misturas Complexas/química , Misturas Complexas/farmacologia , Cianobactérias/metabolismo , Daphnia/efeitos dos fármacos , Daphnia/fisiologia , Depsipeptídeos/química , Depsipeptídeos/farmacologia , Eutrofização , Água Doce/microbiologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/crescimento & desenvolvimento , Bactérias Gram-Positivas/efeitos dos fármacos , Bactérias Gram-Positivas/crescimento & desenvolvimento , Humanos , Microalgas/metabolismo , Testes de Sensibilidade Microbiana , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Fitoplâncton/metabolismo , Águas Salinas/química , Serina Proteases/metabolismo
6.
Molecules ; 26(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34361776

RESUMO

In this study, we examined aqueous extracts of the edible mushrooms Pleurotus ostreatus (oyster mushroom) and Lentinula edodes (shiitake mushroom). Proteome analysis was conducted using LC-Triple TOF-MS and showed the expression of 753 proteins by Pleurotus ostreatus, and 432 proteins by Lentinula edodes. Bioactive peptides: Rab GDP dissociation inhibitor, superoxide dismutase, thioredoxin reductase, serine proteinase and lectin, were identified in both mushrooms. The extracts also included promising bioactive compounds including phenolics, flavonoids, vitamins and amino acids. The extracts showed promising antiviral activities, with a selectivity index (SI) of 4.5 for Pleurotus ostreatus against adenovirus (Ad7), and a slight activity for Lentinula edodes against herpes simplex-II (HSV-2). The extracts were not cytotoxic to normal human peripheral blood mononuclear cells (PBMCs). On the contrary, they showed moderate cytotoxicity against various cancer cell lines. Additionally, antioxidant activity was assessed using DPPH radical scavenging, ABTS radical cation scavenging and ORAC assays. The two extracts showed potential antioxidant activities, with the maximum activity seen for Pleurotus ostreatus (IC50 µg/mL) = 39.46 ± 1.27 for DPPH; 11.22 ± 1.81 for ABTS; and 21.40 ± 2.20 for ORAC assays. This study encourages the use of these mushrooms in medicine in the light of their low cytotoxicity on normal PBMCs vis à vis their antiviral, antitumor and antioxidant capabilities.


Assuntos
Antineoplásicos/química , Antioxidantes/química , Antivirais/química , Proteínas Fúngicas/química , Pleurotus/química , Proteoma/química , Cogumelos Shiitake/química , Aminoácidos/química , Aminoácidos/isolamento & purificação , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Antivirais/isolamento & purificação , Antivirais/farmacologia , Benzotiazóis/antagonistas & inibidores , Compostos de Bifenilo/antagonistas & inibidores , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Misturas Complexas/química , Flavonoides/química , Flavonoides/isolamento & purificação , Proteínas Fúngicas/classificação , Proteínas Fúngicas/isolamento & purificação , Humanos , Lectinas/química , Lectinas/isolamento & purificação , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Especificidade de Órgãos , Fenóis/química , Fenóis/isolamento & purificação , Picratos/antagonistas & inibidores , Pleurotus/metabolismo , Cultura Primária de Células , Proteoma/classificação , Proteoma/isolamento & purificação , Serina Proteases/química , Serina Proteases/isolamento & purificação , Cogumelos Shiitake/metabolismo , Ácidos Sulfônicos/antagonistas & inibidores , Superóxido Dismutase/química , Superóxido Dismutase/isolamento & purificação , Tiorredoxina Dissulfeto Redutase/química , Tiorredoxina Dissulfeto Redutase/isolamento & purificação , Vitaminas/química , Vitaminas/isolamento & purificação , Água/química
7.
Acta Crystallogr D Struct Biol ; 77(Pt 8): 1040-1049, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-34342277

RESUMO

The ß-link is a composite protein motif consisting of a G1ß ß-bulge and a type II ß-turn, and is generally found at the end of two adjacent strands of antiparallel ß-sheet. The 1,2-positions of the ß-bulge are also the 3,4-positions of the ß-turn, with the result that the N-terminal portion of the polypeptide chain is orientated at right angles to the ß-sheet. Here, it is reported that the ß-link is frequently found in certain protein folds of the SCOPe structural classification at specific locations where it connects a ß-sheet to another area of a protein. It is found at locations where it connects one ß-sheet to another in the ß-sandwich and related structures, and in small (four-, five- or six-stranded) ß-barrels, where it connects two ß-strands through the polypeptide chain that crosses an open end of the barrel. It is not found in larger (eight-stranded or more) ß-barrels that are straightforward ß-meanders. In some cases it initiates a connection between a single ß-sheet and an α-helix. The ß-link also provides a framework for catalysis in serine proteases, where the catalytic serine is part of a conserved ß-link, and in cysteine proteases, including Mpro of human SARS-CoV-2, in which two residues of the active site are located in a conserved ß-link.


Assuntos
Estrutura Secundária de Proteína , Serina Proteases/química , Motivos de Aminoácidos , Animais , Domínio Catalítico , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/metabolismo , Cisteína Proteases/química , Cisteína Proteases/metabolismo , Bases de Dados de Proteínas , Humanos , Ligação de Hidrogênio , Modelos Moleculares , SARS-CoV-2/química , SARS-CoV-2/enzimologia , Serina Proteases/metabolismo , Homologia Estrutural de Proteína
8.
J Med Chem ; 64(16): 11972-11989, 2021 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-34405680

RESUMO

The three pan-genotypic HCV NS3/4A protease inhibitors (PIs) currently in clinical use-grazoprevir, glecaprevir, and voxilaprevir-are quinoxaline-based P2-P4 macrocycles and thus exhibit similar resistance profiles. Using our quinoxaline-based P1-P3 macrocyclic lead compounds as an alternative chemical scaffold, we explored structure-activity relationships (SARs) at the P2 and P4 positions to develop pan-genotypic PIs that avoid drug resistance. A structure-guided strategy was used to design and synthesize two series of compounds with different P2 quinoxalines in combination with diverse P4 groups of varying sizes and shapes, with and without fluorine substitutions. Our SAR data and cocrystal structures revealed the interplay between the P2 and P4 groups, which influenced inhibitor binding and the overall resistance profile. Optimizing inhibitor interactions in the S4 pocket led to PIs with excellent antiviral activity against clinically relevant PI-resistant HCV variants and genotype 3, providing potential pan-genotypic inhibitors with improved resistance profiles.


Assuntos
Antivirais/uso terapêutico , Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Compostos Macrocíclicos/uso terapêutico , Inibidores de Proteases/uso terapêutico , Quinoxalinas/uso terapêutico , Animais , Antivirais/síntese química , Antivirais/metabolismo , Antivirais/farmacocinética , Cristalografia por Raios X , Farmacorresistência Viral/efeitos dos fármacos , Compostos Macrocíclicos/síntese química , Compostos Macrocíclicos/metabolismo , Compostos Macrocíclicos/farmacocinética , Masculino , Estrutura Molecular , Inibidores de Proteases/síntese química , Inibidores de Proteases/metabolismo , Inibidores de Proteases/farmacocinética , Ligação Proteica , Quinoxalinas/síntese química , Quinoxalinas/metabolismo , Quinoxalinas/farmacocinética , Ratos Sprague-Dawley , Serina Proteases/metabolismo , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo
9.
Int J Mol Sci ; 22(12)2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34198546

RESUMO

Chronic obstructive pulmonary disease (COPD) is a debilitating heterogeneous disease characterised by unregulated proteolytic destruction of lung tissue mediated via a protease-antiprotease imbalance. In COPD, the relationship between the neutrophil serine protease, neutrophil elastase, and its endogenous inhibitor, alpha-1-antitrypsin (AAT) is the best characterised. AAT belongs to a superfamily of serine protease inhibitors known as serpins. Advances in screening technologies have, however, resulted in many members of the serpin superfamily being identified as having differential expression across a multitude of chronic lung diseases compared to healthy individuals. Serpins exhibit a unique suicide-substrate mechanism of inhibition during which they undergo a dramatic conformational change to a more stable form. A limitation is that this also renders them susceptible to disease-causing mutations. Identification of the extent of their physiological/pathological role in the airways would allow further expansion of knowledge regarding the complexity of protease regulation in the lung and may provide wider opportunity for their use as therapeutics to aid the management of COPD and other chronic airways diseases.


Assuntos
Doença Pulmonar Obstrutiva Crônica/metabolismo , Serina Proteases/metabolismo , Serpinas/metabolismo , Animais , Modelos Animais de Doenças , Humanos , Doença Pulmonar Obstrutiva Crônica/patologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Serpinas/química , Serpinas/uso terapêutico
10.
Int J Mol Sci ; 22(11)2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-34206113

RESUMO

Airway inflammation plays a central role in bronchiectasis. Protease-antiprotease balance is crucial in bronchiectasis pathophysiology and increased presence of unopposed proteases activity may contribute to bronchiectasis onset and progression. Proteases' over-reactivity and antiprotease deficiency may have a role in increasing inflammation in bronchiectasis airways and may lead to extracellular matrix degradation and tissue damage. Imbalances in serine proteases and matrix-metallo proteinases (MMPs) have been associated to bronchiectasis. Active neutrophil elastase has been associated with disease severity and poor long-term outcomes in this disease. Moreover, high levels of MMPs have been associated with radiological and disease severity. Finally, severe deficiency of α1-antitrypsin (AAT), as PiSZ and PiZZ (proteinase inhibitor SZ and ZZ) phenotype, have been associated with bronchiectasis development. Several treatments are under study to reduce protease activity in lungs. Molecules to inhibit neutrophil elastase activity have been developed in both oral or inhaled form, along with compounds inhibiting dipeptydil-peptidase 1, enzyme responsible for the activation of serine proteases. Finally, supplementation with AAT is in use for patients with severe deficiency. The identification of different targets of therapy within the protease-antiprotease balance contributes to a precision medicine approach in bronchiectasis and eventually interrupts and disrupts the vicious vortex which characterizes the disease.


Assuntos
Bronquiectasia/metabolismo , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/metabolismo , Deficiência de alfa 1-Antitripsina/metabolismo , Bronquiectasia/enzimologia , Bronquiectasia/genética , Bronquiectasia/patologia , Humanos , Elastase de Leucócito , Pulmão/metabolismo , Pulmão/patologia , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Neutrófilos/metabolismo , Neutrófilos/patologia , Serina Proteases/genética , Serina Proteases/metabolismo , Deficiência de alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/patologia
11.
Int J Mol Sci ; 22(11)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34200095

RESUMO

Inflammatory bowel diseases (IBD) are incurable disorders whose prevalence and global socioeconomic impact are increasing. While the role of host genetics and immunity is well documented, that of gut microbiota dysbiosis is increasingly being studied. However, the molecular basis of the dialogue between the gut microbiota and the host remains poorly understood. Increased activity of serine proteases is demonstrated in IBD patients and may contribute to the onset and the maintenance of the disease. The intestinal proteolytic balance is the result of an equilibrium between the proteases and their corresponding inhibitors. Interestingly, the serine protease inhibitors (serpins) encoded by the host are well reported; in contrast, those from the gut microbiota remain poorly studied. In this review, we provide a concise analysis of the roles of serine protease in IBD physiopathology and we focus on the serpins from the gut microbiota (gut serpinome) and their relevance as a promising therapeutic approach.


Assuntos
Microbioma Gastrointestinal , Doenças Inflamatórias Intestinais/fisiopatologia , Serina Proteases/química , Serpinas/metabolismo , Animais , Humanos
12.
Biomed Pharmacother ; 139: 111603, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34243596

RESUMO

The HtrA protein family is composed by evolutionally-conserved serine proteases, which are homologous to the HtrA protein of the model bacterium Escherichia coli. They are widely distributed in organisms including humans, prokaryotes and eukaryotes. Moreover, HtrA family proteins are important regulators of a variety of human physiological processes, which contains the maintenance of mitochondrial homeostasis, cellular signal transduction and apoptosis regulation. The HtrA family has been found to be associated with cancer and could be used as a target for future cancer treatments. The purpose of this article is to review the relationship between these HtrA and cancer and to summarize the latest researches on HtrA and cancer.


Assuntos
Neoplasias/metabolismo , Serina Proteases/metabolismo , Animais , Apoptose/fisiologia , Homeostase/fisiologia , Humanos , Mitocôndrias/metabolismo , Transdução de Sinais/fisiologia
13.
Angew Chem Int Ed Engl ; 60(40): 21789-21794, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34268844

RESUMO

A bicyclic peptide scaffold was chemically adapted to generate diarylethene-based photoswitchable inhibitors of serine protease Bos taurus trypsin 1 (T1). Starting from a prototype molecule-sunflower trypsin inhibitor-1 (SFTI-1)-we obtained light-controllable inhibitors of T1 with Ki in the low nanomolar range, whose activity could be modulated over 20-fold by irradiation. The inhibitory potency as well as resistance to proteolytic degradation were systematically studied on a series of 17 SFTI-1 analogues. The hydrogen bond network that stabilizes the structure of inhibitors and possibly the enzyme-inhibitor binding dynamics were affected by isomerization of the photoswitch. The feasibility of manipulating enzyme activity in time and space was demonstrated by controlled digestion of gelatin-based hydrogel and an antimicrobial peptide BP100-RW. Finally, our design principles of diarylethene photoswitches are shown to apply also for the development of other serine protease inhibitors.


Assuntos
Etilenos/farmacologia , Peptídeos Cíclicos/farmacologia , Serina Proteases/metabolismo , Inibidores de Serino Proteinase/farmacologia , Animais , Bovinos , Etilenos/química , Estrutura Molecular , Peptídeos Cíclicos/química , Inibidores de Serino Proteinase/química
14.
J Neurosci ; 41(30): 6430-6448, 2021 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-34210781

RESUMO

The adaptable transcriptional response to changes in food availability not only ensures animal survival but also lets embryonic development progress. Interestingly, the CNS is preferentially protected from periods of malnutrition, a phenomenon known as "brain sparing." However, the mechanisms that mediate this response remain poorly understood. To get a better understanding of this, we used Drosophila melanogaster as a model, analyzing the transcriptional response of neural stem cells (neuroblasts) and glia of the blood-brain barrier (BBB) from larvae of both sexes during nutrient restriction using targeted DamID. We found differentially expressed genes in both neuroblasts and glia of the BBB, although the effect of nutrient deficiency was primarily observed in the BBB. We characterized the function of a nutritional sensitive gene expressed in the BBB, the serine protease homolog, scarface (scaf). Scaf is expressed in subperineurial glia in the BBB in response to nutrition. Tissue-specific knockdown of scaf increases subperineurial glia endoreplication and proliferation of perineurial glia in the blood-brain barrier. Furthermore, neuroblast proliferation is diminished on scaf knockdown in subperineurial glia. Interestingly, reexpression of Scaf in subperineurial glia is able to enhance neuroblast proliferation and brain growth of animals in starvation. Finally, we show that loss of scaf in the blood-brain barrier increases sensitivity to drugs in adulthood, suggesting a physiological impairment. We propose that Scaf integrates the nutrient status to modulate the balance between neurogenesis and growth of the BBB, preserving the proper equilibrium between the size of the barrier and the brain.SIGNIFICANCE STATEMENT The Drosophila BBB separates the CNS from the open circulatory system. The BBB glia are not only acting as a physical segregation of tissues but participate in the regulation of the metabolism and neurogenesis during development. Here we analyze the transcriptional response of the BBB glia to nutrient deprivation during larval development, a condition in which protective mechanisms are switched on in the brain. Our findings show that the gene scarface reduces growth in the BBB while promoting the proliferation of neural stem, assuring the balanced growth of the larval brain. Thus, Scarface would link animal nutrition with brain development, coordinating neurogenesis with the growth of the BBB.


Assuntos
Barreira Hematoencefálica/metabolismo , Proteínas de Drosophila/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese/fisiologia , Neuroglia/metabolismo , Serina Proteases/metabolismo , Animais , Barreira Hematoencefálica/crescimento & desenvolvimento , Drosophila melanogaster , Feminino , Masculino , Desnutrição
15.
J Insect Physiol ; 133: 104285, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34284041

RESUMO

The digestive physiology of house dust mites (HDM) is of interest to understand their allergenicity towards humans since many of their allergens are digestive enzymes and/or are excreted into airborne fecal pellets. The aim of this study is to provide insight on the biochemical basis of proteolytic digestion in Dermatophagoides pteronyssinus, the most widespread HDM species. First, assays using non-specific protein substrates on purified fecal and body extracts determined that body-associated activity is almost exclusively dependent on cysteine proteases, and specifically on major allergen Der p 1. By contrast, cysteine and serine proteases contributed similarly to the activity estimated on fecal extracts. Second, the screening of group-specific peptide-based protease inhibitors followed by ingestion bioassays revealed that the human skin-derived cysteine protease inhibitor cystatin A produces a significant reduction in mite feeding (i.e. excreted guanine), and triggers the overproduction of Der p 1 (3-fold increase by ELISA). Noteworthy, the inhibition of cysteine proteases by cystatin A also resulted in a reduction in three non-target serine protease activities. Further incubation of these extracts with exogenous Der p 1, but not with other commercial cysteine proteases, restored trypsin (Der p 3) and chymotrypsin (Der p 6) activities, indicating that Der p 1 is responsible for their activation in vivo. Finally, the role of serine proteases on the mite's digestive physiology is discussed based on their remarkable activity in fecal extracts and the autocoprophagic behavior reported in mites in this study.


Assuntos
Proteínas de Artrópodes/metabolismo , Cisteína Proteases/metabolismo , Dermatophagoides pteronyssinus/metabolismo , Serina Proteases/metabolismo , Animais , Digestão , Hipersensibilidade , Proteólise
16.
Sci Rep ; 11(1): 14234, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244581

RESUMO

Glucantime (SbV) is the first-line treatment against American Tegumentary Leishmaniasis. Resistance cases to this drug have been reported and related to host characteristics and parasite phenotypes. In this study, 12 Leishmania (Viannia) braziliensis isolates from patients that presented clinical cure (Responders-R) and relapse or therapeutic failure (Non-responders-NR) after treatment with antimony, were analyzed. These parasites were assessed by in vitro susceptibility to SbIII and SbV, serine proteases activity measured with substrate (z-FR-AMC) and specific inhibitors (TLCK, AEBSF and PMSF). In vitro susceptibility of axenic amastigotes to SbIII showed a significant difference between R and NR groups. The protease assays showed that TLCK inhibited almost 100% of activity in both axenic amastigotes and promastigotes while AEBSF inhibited around 70%, and PMSF showed lower inhibition of some isolates. Principal component and clustering analysis performed with these data yielded one homogeneous cluster with only NR isolates and three heterogeneous clusters with R and NR isolates. Additionally, differential expression of subtilisins (LbrM.13.0860 and LbrM.28.2570) and TXNPx (LbrM.15.1080) was evaluated in promastigotes and axenic amastigotes from both groups. The results showed a higher expression of LbrM.13.0860 and LbrM.15.1080 genes in axenic amastigotes, while LbrM.28.2570 gene had the lowest expression in all isolates, regardless of the parasite form. The data presented here show a phenotypic heterogeneity among the parasites, suggesting that exploration of in vitro phenotypes based on SbIII and serine proteases profiles can aid in the characterization of L. (V.) braziliensis clinical isolates.


Assuntos
Antimônio/farmacologia , Leishmania braziliensis/efeitos dos fármacos , Leishmania braziliensis/enzimologia , Serina Proteases/metabolismo , Interações Hospedeiro-Parasita/efeitos dos fármacos , Parasitologia , Serina Proteases/genética
17.
Eur J Med Genet ; 64(8): 104259, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34126256

RESUMO

RNA sequencing (RNAseq) is emerging as a complementary tool to DNA sequencing, providing utility in diagnosis for disorders such as neuronal ceroid lipofuscinosis CLN2 disease. We describe an individual with a presentation suggestive of an attenuated CLN2 phenotype, including a history of regression, recent-onset microcephaly and spasticity from age five years. Exome sequencing revealed two variants inherited in trans in TPP1, NM_000391.4:c.225A>G; p.(Gln75 = ) and NM_000391.4:c.1012C>G; p.(Gln338Glu), both classified as variants of uncertain significance. TPP1 activity was found to be significantly reduced in fibroblasts of the affected individual. RNAseq was performed to assess the impact of compound heterozygous variants in TPP1 and enabled the identification of three aberrant splicing events. The c.225A>G variant introduces a 5 nucleotide truncation of exon 3 and a loss of reading frame. The majority of CLN2 transcripts exclude either exon 8 or exons 7-8, resulting in large in-frame deletions. Isoform specific RT-PCR confirmed the aberrant splicing events are mutually exclusive, suggesting that the paternal exon 8 c.1012C>G variant results in exon skipping. This case study demonstrates how RNAseq can be used as an orthogonal test to inform the interpretation of some variants of unknown significance and its particular importance in disorders where effective disease management requires early diagnosis.


Assuntos
Aminopeptidases/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Mutação , Lipofuscinoses Ceroides Neuronais/genética , Splicing de RNA , Serina Proteases/genética , Aminopeptidases/metabolismo , Células Cultivadas , Criança , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Feminino , Humanos , Lipofuscinoses Ceroides Neuronais/patologia , Serina Proteases/metabolismo
18.
J Vis Exp ; (171)2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-34096915

RESUMO

Proteases are regulators of countless physiological processes and the precise investigation of their activities remains an intriguing biomedical challenge. Among the ~600 proteases encoded by the human genome, neutrophil serine proteases (NSPs) are thoroughly investigated for their involvement in the onset and progression of inflammatory conditions including respiratory diseases. Uniquely, secreted NSPs not only diffuse within extracellular fluids but also localize to plasma membranes. During neutrophil extracellular trap (NETs) formation, NSPs become an integral part of the secreted chromatin. Such complex behavior renders the understanding of NSPs pathophysiology a challenging task. Here, detailed protocols are shown to visualize, quantify and discriminate free and membrane-bound neutrophil elastase (NE) and cathepsin G (CG) activities in sputum samples. NE and CG are NSPs whose activities have pleiotropic roles in the pathogenesis of cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD): they promote tissue remodeling, regulate downstream immune responses and correlate with lung disease severity. The protocols show how to separate fluid and cellular fraction, as well as the isolation of neutrophils from human sputum for enzymatic activity quantification via small-molecule Förster resonance energy transfer-based (FRET) reporters. To gather specific insights into the relative role of NE and CG activities, a FRET readout can be measured by different technologies: i) in vitro plate reader measurements allow for high-throughput and bulk detection of protease activity; ii) confocal microscopy spatiotemporally resolves membrane-bound activity at the cell surface; iii) small-molecule FRET flow cytometry enables for the rapid evaluation of anti-inflammatory treatments via single-cell protease activity quantification and phenotyping. The implementation of such methods opens the doors to explore NSPs pathobiology and their potential as biomarkers of disease severity for CF and COPD. Given their standardization potential, their robust readout and simplicity of transfer, the described techniques are immediately shareable for implementation across research and diagnostic laboratories.


Assuntos
Catepsina G , Fibrose Cística , Elastase de Leucócito , Doença Pulmonar Obstrutiva Crônica , Fibrose Cística/enzimologia , Humanos , Neutrófilos/enzimologia , Doença Pulmonar Obstrutiva Crônica/enzimologia , Serina Proteases , Escarro/enzimologia
19.
J Virol ; 95(19): e0086121, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34160253

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen causing the coronavirus disease 2019 (COVID-19) global pandemic. No effective treatment for COVID-19 has been established yet. The serine protease transmembrane protease serine 2 (TMPRSS2) is essential for viral spread and pathogenicity by facilitating the entry of SARS-CoV-2 into host cells. The protease inhibitor camostat, an anticoagulant used in the clinic, has potential anti-inflammatory and antiviral activities against COVID-19. However, the potential mechanisms of viral resistance and antiviral activity of camostat are unclear. Herein, we demonstrate high inhibitory potencies of camostat for a panel of serine proteases, indicating that camostat is a broad-spectrum inhibitor of serine proteases. In addition, we determined the crystal structure of camostat in complex with a serine protease (uPA [urokinase-type plasminogen activator]), which reveals that camostat is inserted in the S1 pocket of uPA but is hydrolyzed by uPA, and the cleaved camostat covalently binds to Ser195. We also generated a homology model of the structure of the TMPRSS2 serine protease domain. The model shows that camostat uses the same inhibitory mechanism to inhibit the activity of TMPRSS2, subsequently preventing SARS-CoV-2 spread. IMPORTANCE Serine proteases are a large family of enzymes critical for multiple physiological processes and proven diagnostic and therapeutic targets in several clinical indications. The serine protease transmembrane protease serine 2 (TMPRSS2) was recently found to mediate SARS-CoV-2 entry into the host. Camostat mesylate (FOY 305), a serine protease inhibitor active against TMPRSS2 and used for the treatment of oral squamous cell carcinoma and chronic pancreatitis, inhibits SARS-CoV-2 infection of human lung cells. However, the direct inhibition mechanism of camostat mesylate for TMPRSS2 is unclear. Herein, we demonstrate that camostat uses the same inhibitory mechanism to inhibit the activity of TMPRSS2 as uPA, subsequently preventing SARS-CoV-2 spread.


Assuntos
Antivirais/farmacologia , Ésteres/farmacologia , Guanidinas/farmacologia , SARS-CoV-2/efeitos dos fármacos , Serina Endopeptidases/química , Serina Endopeptidases/farmacologia , Serina Proteases/farmacologia , Antivirais/química , COVID-19/tratamento farmacológico , COVID-19/prevenção & controle , Carcinoma de Células Escamosas , Ésteres/química , Ésteres/metabolismo , Guanidinas/química , Guanidinas/metabolismo , Humanos , Simulação de Dinâmica Molecular , Neoplasias Bucais , Domínios Proteicos , Alinhamento de Sequência , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Serina Proteases/química , Serina Proteases/metabolismo , Inibidores de Serino Proteinase/química , Inibidores de Serino Proteinase/farmacologia , Internalização do Vírus/efeitos dos fármacos
20.
Exp Mol Pathol ; 121: 104658, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34102210

RESUMO

This paper was aimed at exploring the correlation of long non-coding RNA (lncRNA)-ABHD11 Antisense RNA1 (ABHD11-AS1) with the poor prognosis of patients with papillary thyroid carcinoma (PTC) and at investigating its effects on the survival of PTC cells. Serum was respectively collected from 64 PTC patients who were admitted to our hospital (PTC group) and from 50 healthy controls who underwent physical examinations (HC group) both from April 2011 to April 2015. The expression levels of ABHD11-AS1 in the serum were detected, and the values of it for diagnosis and prognosis (5-year follow-ups) were analyzed. The knockdown and overexpression models of ABHD11-AS1 in were constructed to explore the effects of the models on their proliferation, cycles and apoptosis. According to the data, the expression levels of serum ABHD11-AS1 in the PTC patients were remarkably higher than those in the healthy controls, and the area under the curve (AUC) for distinguishing the patients from the controls was 0.920. In the analysis of prognosis, the levels in patients with a poor prognosis were remarkably higher than those in patients with a good prognosis. According to the curves of overall survival rates (OSRs), the high levels of ABHD11-AS1 were remarkably correlated with the poor prognosis (a lower 5-year OSR). COX analysis showed that TNM staging, lymph node metastasis and ABHD11-AS1 were the independent prognostic factors of PTC patients. In the cell experiments, knocking down ABHD11-AS1 remarkably inhibited PTC cells from proliferation, arrested them in G0/G1 phase, and induced their apoptosis, negatively affecting their survival indices. Overexpressing this RNA had positive effects on the survival indices. Taken together, high levels of serum ABHD11-AS1 are related to the poor prognosis of PTC patients, and knocking down its expression can inhibit the survival of PTC cells.


Assuntos
Biomarcadores Tumorais/metabolismo , RNA Antissenso/genética , RNA Longo não Codificante/genética , Serina Proteases/genética , Câncer Papilífero da Tireoide/patologia , Neoplasias da Glândula Tireoide/patologia , Apoptose , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , Movimento Celular , Proliferação de Células , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Taxa de Sobrevida , Câncer Papilífero da Tireoide/genética , Câncer Papilífero da Tireoide/metabolismo , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Células Tumorais Cultivadas
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