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1.
AIDS Res Ther ; 18(1): 77, 2021 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-34702287

RESUMO

BACKGROUND: Targeting RNA is a promising yet underdeveloped modality for the selective killing of cells infected with HIV-1. The secretory ribonucleases (RNases) found in vertebrates have cytotoxic ribonucleolytic activity that is kept in check by a cytosolic ribonuclease inhibitor protein, RI. METHODS: We engineered amino acid substitutions that enable human RNase 1 to evade RI upon its cyclization into a zymogen that is activated by the HIV-1 protease. In effect, the zymogen has an HIV-1 protease cleavage site between the termini of the wild-type enzyme, thereby positioning a cleavable linker over the active site that blocks access to a substrate. RESULTS: The amino acid substitutions in RNase 1 diminish its affinity for RI by 106-fold and confer high toxicity for T-cell leukemia cells. Pretreating these cells with the zymogen leads to a substantial drop in their viability upon HIV-1 infection, indicating specific toxicity toward infected cells. CONCLUSIONS: These data demonstrate the utility of ribonuclease zymogens as biologic prodrugs.


Assuntos
Infecções por HIV , HIV-1 , Animais , Precursores Enzimáticos/genética , Infecções por HIV/tratamento farmacológico , HIV-1/genética , Humanos , Modelos Moleculares , Ribonucleases/genética
2.
Science ; 373(6561): eabj0486, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34529467
3.
J Biol Chem ; 297(4): 101227, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34562451

RESUMO

TMPRSS13, a member of the type II transmembrane serine protease (TTSP) family, harbors four N-linked glycosylation sites in its extracellular domain. Two of the glycosylated residues are located in the scavenger receptor cysteine-rich (SRCR) protein domain, while the remaining two sites are in the catalytic serine protease (SP) domain. In this study, we examined the role of N-linked glycosylation in the proteolytic activity, autoactivation, and cellular localization of TMPRSS13. Individual and combinatory site-directed mutagenesis of the glycosylated asparagine residues indicated that glycosylation of the SP domain is critical for TMPRSS13 autoactivation and catalytic activity toward one of its protein substrates, the prostasin zymogen. Additionally, SP domain glycosylation-deficient TMPRSS13 displayed impaired trafficking of TMPRSS13 to the cell surface, which correlated with increased retention in the endoplasmic reticulum. Importantly, we showed that N-linked glycosylation was a critical determinant for subsequent phosphorylation of endogenous TMPRSS13. Taken together, we conclude that glycosylation plays an important role in regulating TMPRSS13 activation and activity, phosphorylation, and cell surface localization.


Assuntos
Membrana Celular/enzimologia , Precursores Enzimáticos/metabolismo , Proteínas de Membrana/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise , Serina Endopeptidases/metabolismo , Animais , Células COS , Membrana Celular/genética , Chlorocebus aethiops , Precursores Enzimáticos/genética , Células HEK293 , Humanos , Proteínas de Membrana/genética , Domínios Proteicos , Transporte Proteico/genética , Serina Endopeptidases/genética
4.
Fish Shellfish Immunol ; 117: 124-135, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34343542

RESUMO

This study aims to assess and determine the oral-administration of probiotic, Lactobacillus pentosus BD6 on growth performance, immunity and disease resistance of white shrimp, Litopenaeus vannamei. Lac. pentosus BD6 effectively inhibited the growth of aquatic pathogens, which was used in the test. Shrimp were fed with the control diet (without probiotic supplement) for 60 days and the probiotic-containing diets at 107, 108, 109, and 1010 cfu kg-1, respectively. Shrimp fed with the diet containing probiotic at the doses of 109-10 cfu kg-1 showed significant increase in growth performance as well as feed efficiency than that of the control. After a challenge test with Vibrio alginolyticus, shrimp fed with a probiotic diet at a dose of 1010 cfu kg-1 showed a significantly lower mortality as compared to the control and that of shrimp fed the diet containing probiotic at the levels up to 107-8 cfu kg-1. In addition, a therapeutic potential of Lac. pentosus BD6 was discovered because the cumulative mortalities of shrimp fed with probiotic and pathogen V. parahaemolyticus simultaneously were significantly lower when compared to control shrimp. Probiotic in diet at a dose of 109-10 cfu kg-1 significantly increased PO activity of shrimp, while shrimp receiving probiotic at the doses of 108-10 cfu kg-1 showed significant increase in lysozyme activity and phagocytic activity. Shrimp fed with the diet containing probiotic at the level of 1010 cfu kg-1 also indicated higher gene expression of prophenoloxidase (proPO) I, but not proPO II, lipopolysaccharide and ß-1,3-glucan-binding protein and penaeidin 4. Analysis of the bacterial microbiota of the shrimp intestine revealed that oral administration of probiotic increased the relative abundance of beneficial bacteria and reduced the abundance of harmful pathogenic bacteria in the gut flora of shrimp. Despite no statistically significant difference, an analysis of microbial diversity recorded higher species richness, Shannon-Weaver diversity index and evenness in the probiotic group, compared to the control group. It was concluded that Lac. pentosus BD6 has great antibacterial ability against a wide range of pathogens and has therapeutic potential to reduce the mortality of shrimp infected with V. parahaemolyticus. Additionally, dietary Lac. pentosus BD6 at the level of 1010 cfu kg-1 was recommended to improve growth performance, immunity and disease resistance of shrimp against V. alginolyticus.


Assuntos
Lactobacillus pentosus , Penaeidae , Probióticos/administração & dosagem , Vibrioses/prevenção & controle , Vibrio alginolyticus , Administração Oral , Animais , Catecol Oxidase/imunologia , Resistência à Doença , Precursores Enzimáticos/imunologia , Microbioma Gastrointestinal , Expressão Gênica , Hemócitos/imunologia , Hemolinfa/imunologia , Muramidase/imunologia , Penaeidae/genética , Penaeidae/crescimento & desenvolvimento , Penaeidae/imunologia , Penaeidae/microbiologia , Fagocitose , Vibrioses/mortalidade , Vibrioses/veterinária
5.
Anticancer Res ; 41(8): 3891-3898, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34281851

RESUMO

BACKGROUND/AIM: Matrix metalloproteinases (MMPs) degrade extracellular matrix and process regulatory proteins. Recently, a membrane-bound 82kDa variant of proMMP-9 identified on myeloid blasts was shown to be associated with prognosis. PATIENTS AND METHODS: To investigate the role of 82kDa proMMP-9 with acute lymphoblastic leukemia (ALL) and chronic lymphoid leukemia (CLL), we performed flow-cytometry analysis of expression on ALL blasts (n=18) and CLL lymphocytes (n=21) from blood and correlated data with clinical parameters. RESULTS: In ALL, mature B-linear blasts expressed higher levels of 82kDa proMMP-9 compared to T-linear blasts. Elevated levels of 82kDa proMMP-9 were found in elderly patients and at patients with relapse. No correlation was observed on blood cells and extramedullary disease. In CLL, the 82kDa proMMP-9 expression did not correlate with any of the clinical parameters. CONCLUSION: Our findings suggest that higher levels of 82kDa proMMP-9 expression on blast cells may correlate with a more unfavorable ALL-subtype. Further studies are required to clarify the prognostic role of the 82kDa pro-MMP-9 expression.


Assuntos
Precursores Enzimáticos/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Linfócitos/imunologia , Metaloproteinase 9 da Matriz/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Adolescente , Adulto , Idoso , Células da Medula Óssea/citologia , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Adulto Jovem
6.
Sci Rep ; 11(1): 13376, 2021 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-34183752

RESUMO

MMP-9 plays a number of important physiological functions but is also responsible for many pathological processes, including cancer invasion, metastasis, and angiogenesis. It is, therefore, crucial to understand its enzymatic activity, including activation and inhibition mechanisms. This enzyme may also be partially involved in the "cytokine storm" that is characteristic of COVID-19 disease (SARS-CoV-2), as well as in the molecular mechanisms responsible for lung fibrosis. Due to the variety of processing pathways involving MMP-9 in biological systems and its uniqueness due to the O-glycosylated domain (OGD) and fibronectin-like (FBN) domain, specific interactions with its natural TIMP-1 inhibitor should be carefully studied, because they differ significantly from other homologous systems. In particular, earlier experimental studies have indicated that the newly characterised circular form of a proMMP-9 homotrimer exhibits stronger binding properties to TIMP-1 compared to its monomeric form. However, molecular structures of the complexes and the binding mechanisms remain unknown. The purpose of this study is to fill in the gaps in knowledge. Molecular modelling methods are applied to build the inhibitory and non-inhibitory MMP-9-TIMP-1 complexes, which allows for a detailed description of these structures and should allow for a better understanding of the regulatory processes in which MMP-9 is involved.


Assuntos
Metaloproteinase 9 da Matriz/metabolismo , Simulação de Dinâmica Molecular , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Precursores Enzimáticos/química , Precursores Enzimáticos/metabolismo , Humanos , Metaloproteinase 9 da Matriz/química , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Eletricidade Estática , Inibidor Tecidual de Metaloproteinase-1/antagonistas & inibidores
7.
Sci Rep ; 11(1): 12695, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-34135352

RESUMO

Caspase-6 (Casp6) is implicated in Alzheimer disease (AD) cognitive impairment and pathology. Hippocampal atrophy is associated with cognitive impairment in AD. Here, a rare functional exonic missense CASP6 single nucleotide polymorphism (SNP), causing the substitution of asparagine with threonine at amino acid 73 in Casp6 (Casp6N73T), was associated with hippocampal subfield CA1 volume preservation. Compared to wild type Casp6 (Casp6WT), recombinant Casp6N73T altered Casp6 proteolysis of natural substrates Lamin A/C and α-Tubulin, but did not alter cleavage of the Ac-VEID-AFC Casp6 peptide substrate. Casp6N73T-transfected HEK293T cells showed elevated Casp6 mRNA levels similar to Casp6WT-transfected cells, but, in contrast to Casp6WT, did not accumulate active Casp6 subunits nor show increased Casp6 enzymatic activity. Electrophysiological and morphological assessments showed that Casp6N73T recombinant protein caused less neurofunctional damage and neurodegeneration in hippocampal CA1 pyramidal neurons than Casp6WT. Lastly, CASP6 mRNA levels were increased in several AD brain regions confirming the implication of Casp6 in AD. These studies suggest that the rare Casp6N73T variant may protect against hippocampal atrophy due to its altered catalysis of natural protein substrates and intracellular instability thus leading to less Casp6-mediated damage to neuronal structure and function.


Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Região CA1 Hipocampal/patologia , Caspase 6/genética , Caspase 6/metabolismo , Polimorfismo de Nucleotídeo Único , Transmissão Sináptica , Doença de Alzheimer/enzimologia , Substituição de Aminoácidos , Encéfalo/enzimologia , Encéfalo/patologia , Caspase 1/genética , Caspase 1/metabolismo , Caspase 6/química , Precursores Enzimáticos/metabolismo , Células HEK293 , Hipocampo , Humanos , Lamina Tipo A/metabolismo , Mutação de Sentido Incorreto , Degeneração Neural , Células Piramidais/citologia , Células Piramidais/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Tubulina (Proteína)/metabolismo
8.
Ann Allergy Asthma Immunol ; 127(4): 420-426, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34175497

RESUMO

OBJECTIVE: To aid the clinician in correctly interpreting serum tryptase levels. DATA SOURCES: Primary peer-reviewed literature. STUDY SELECTIONS: Clinical and basic science peer-reviewed studies characterizing the genetic and physiological bases for tryptase generation, secretion, and elevation, including those describing serum tryptase levels in population-based cohort studies. RESULTS: Clinically measured basal serum tryptase (BST) consists of ostensibly inactive alpha- and beta-tryptase precursors. The autosomal dominant genetic trait hereditary alpha-tryptasemia is the most often cause for elevated BST levels, with other acquired causes, such as renal failure and clonal myeloid diseases being far less common. Acute increases in serum tryptase levels resulting from release of mature tryptase from secretory granules is specific to mast cell degranulation but is not detected in all cases of systemic anaphylaxis. CONCLUSION: Understanding the differences and distinguishing between acute increases in serum tryptase and chronic elevations in BST owing to inherited or acquired conditions is critical in the correct interpretation of this useful clinical biomarker.


Assuntos
Precursores Enzimáticos/sangue , Mastócitos/imunologia , Mastocitose/imunologia , Triptases/sangue , Anafilaxia/imunologia , Biomarcadores/sangue , Degranulação Celular/fisiologia , Humanos , Mastocitose/genética , Insuficiência Renal/sangue , Insuficiência Renal/patologia
10.
Adv Exp Med Biol ; 1269: 45-49, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33966193

RESUMO

This case report describes a major surgical procedure for a protein C-deficient, hypercoagulable patient who underwent two back-to-back invasive surgeries, hip replacement, and spinal stenosis correction. The patient, an 84-year-old male with a history of deep vein thromboses (DVT) and pulmonary emboli (PE), was treated pre-, peri-, and postoperatively with zymogen protein C (ZPC-Baxter, International) and recovered without clotting or increased bleeding. During the procedure, the patient was not administered any other anticoagulants. There have now been several case reports on different patients with unrelated teams in various locations worldwide using zymogen protein C during surgical procedures. Thus, this procedure is becoming a viable choice for patients with a high probability of clotting during and after invasive surgery. This case focuses on accomplishing safer surgery and reducing costs, by using less ZPC while accomplishing two surgeries in one procedure. As a result, this procedure might be useful for many medical situations where acquired protein C deficiency could be a problem (e.g., sepsis, pregnancy, etc.). This approach may have greater application to medical conditions other than protein C deficiency, where clotting and inflammation can become issues.


Assuntos
Deficiência de Proteína C , Proteína C , Idoso de 80 Anos ou mais , Anticoagulantes/uso terapêutico , Precursores Enzimáticos , Humanos , Masculino , Segurança do Paciente
11.
Acta Biochim Pol ; 68(2): 207-215, 2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-33945245

RESUMO

Pseudomonas aeruginosa, is an opportunistic bacterium with a high prevalence in diverse pulmonary infections. Although several genes are involved in the system of resistance and evasion of the immunological response of the host, little is known about the inflammatory, degradative, and cell-binding response induced by P. aeruginosa in human lung alveolar epithelial cells. The purpose of this study was to determine the cytokine expression (IL-1ß and TNFα), pro matrix metalloproteinases activation (proMMP-2 and proMMP-9), and the effects on the cell-binding adhesion protein (E-cadherin) in an in vitro model of human lung alveolar epithelial cells. A549 cells were stimulated with a different number of colony-forming units of P. aeruginosa for 3, 6, and 24 hours. Subsequently, the culture medium was collected, IL-1ß and TNFα levels were evaluated by ELISA; proMMP-2 and -9 levels were determined by substrate gel zymography, and the MMP-9 and E-cadherin assessed by immunostaining of A549 cells. Our results demonstrated that P. aeruginosa induces mainly the secretion of TNFα, increases actMMP-9 level, and significantly reduces the level of E-cadherin in the A549 cells. In summary, the inflammatory/degradative process induced by P. aeruginosa modulates the expression of the E-cadherin protein. The probable clinical implications of this study suggest the use of inhibitors that reduce the degradative activity of proMMP-9 which will be further explored in the next phase of this study.


Assuntos
Caderinas/metabolismo , Precursores Enzimáticos/metabolismo , Interleucina-1beta/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Pseudomonas aeruginosa/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Células A549 , Células Epiteliais Alveolares/metabolismo , Sobrevivência Celular , Citocinas/metabolismo , Gelatinases/metabolismo , Humanos , Pulmão/metabolismo , Pneumopatias/metabolismo , Pneumopatias/microbiologia , Infecções por Pseudomonas/metabolismo
12.
PLoS One ; 16(4): e0250454, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33914781

RESUMO

In the epididymis, lysosomal proteins of the epithelial cells are normally targeted from the Golgi apparatus to lysosomes for degradation, although their secretion into the epididymal lumen has been documented and associated with sperm maturation. In this study, cathepsin D (CatD) and prosaposin (PSAP) were examined in adult epididymis of control, and 2-day castrated rats without (Ct) and with testosterone replacement (Ct+T) to evaluate their expression and regulation within epididymal epithelial cells. By light microscope-immunocytochemistry, a quantitative increase in size of lysosomes in principal cells of Ct animals was noted from the distal initial segment to the proximal cauda. Androgen replacement did not restore the size of lysosomes to control levels. Western blot analysis revealed a significant increase in CatD expression in the epididymis of Ct animals, which suggested an upregulation of its expression in principal cells; androgens restored levels of CatD to that of controls. In contrast, PSAP expression in Ct animals was not altered from controls. Additionally, an increase in procathepsin D levels was noted from samples of the epididymal fluid of Ct compared to control animals, accompanied by an increased complex formation with PSAP. Moreover, an increased oligomerization of prosaposin was observed in the epididymal lumen of Ct rats, with changes reverted to controls in Ct+T animals. Taken together these data suggest castration causes an increased uptake of substrates that are acted upon by CatD in lysosomes of principal cells and in the lumen by procathepsin D. These substrates may be derived from apoptotic cells noted in the lumen of proximal regions and possibly by degenerating sperm in distal regions of the epididymis of Ct animals. Exploring the mechanisms by which lysosomal enzymes are synthesized and secreted by the epididymis may help resolve some of the issues originating from epididymal dysfunctions with relevance to sperm maturation.


Assuntos
Androgênios/genética , Catepsina D/genética , Precursores Enzimáticos/genética , Saposinas/genética , Androgênios/metabolismo , Animais , Castração/efeitos adversos , Epididimo/crescimento & desenvolvimento , Epididimo/metabolismo , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/genética , Lisossomos/genética , Lisossomos/fisiologia , Masculino , Ratos , Espermatozoides/metabolismo , Testosterona/genética , Testosterona/metabolismo
13.
Sci Rep ; 11(1): 7230, 2021 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-33790319

RESUMO

Generally, animals extract nutrients from food by degradation using digestive enzymes. Trypsin and chymotrypsin, one of the major digestive enzymes in vertebrates, are pancreatic proenzymes secreted into the intestines. In this investigation, we report the identification of a digestive teleost enzyme, a pancreatic astacin that we termed pactacin. Pactacin, which belongs to the astacin metalloprotease family, emerged during the evolution of teleosts through gene duplication of astacin family enzymes containing six cysteine residues (C6astacin, or C6AST). In this study, we first cloned C6AST genes from pot-bellied seahorse (Hippocampus abdominalis) and analyzed their phylogenetic relationships using over 100 C6AST genes. Nearly all these genes belong to one of three clades: pactacin, nephrosin, and patristacin. Genes of the pactacin clade were further divided into three subclades. To compare the localization and functions of the three pactacin subclades, we studied pactacin enzymes in pot-bellied seahorse and medaka (Oryzias latipes). In situ hybridization revealed that genes of all three subclades were commonly expressed in the pancreas. Western blot analysis indicated storage of pactacin pro-enzyme form in the pancreas, and conversion to the active forms in the intestine. Finally, we partially purified the pactacin from digestive fluid, and found that pactacin is novel digestive enzyme that is specific in teleosts.


Assuntos
Precursores Enzimáticos , Proteínas de Peixes , Regulação Enzimológica da Expressão Gênica , Metaloendopeptidases , Oryzias , Pâncreas/enzimologia , Smegmamorpha , Sequência de Aminoácidos , Animais , Clonagem Molecular , Precursores Enzimáticos/biossíntese , Precursores Enzimáticos/genética , Proteínas de Peixes/biossíntese , Proteínas de Peixes/genética , Metaloendopeptidases/biossíntese , Metaloendopeptidases/genética , Oryzias/genética , Oryzias/metabolismo , Homologia de Sequência de Aminoácidos , Smegmamorpha/genética , Smegmamorpha/metabolismo
14.
Mol Vis ; 27: 142-150, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33907369

RESUMO

Purpose: To evaluate the plasma levels of matrix metalloproteinase 9 (MMP9) and tissue inhibitors of metalloproteinase 3 (TIMP3) in neovascular age-related macular degeneration (nAMD) patients compared to controls, and to explore the potential effect of AMD-associated genetic variants on MMP9 and TIMP3 protein levels. Methods: nAMD and control patients were selected from the European Genetic Database (EUGENDA) based on different genotypes of rs142450006 near MMP9 and rs5754227 near TIMP3. Plasma total MMP9, active MMP9 and TIMP3 levels were measured using the enzyme linked immunosorbent assay (ELISA) and compared between nAMD patients and controls, as well as between different genotype groups. Results: nAMD patients had significantly higher total MMP9 levels compared to controls (median 46.58 versus 26.90 ng/ml; p = 0.0004). In addition, the median MMP9 level in the homozygous genotype group for the AMD-risk allele (44.23 ng/ml) was significantly higher than the median for the heterozygous genotype group (26.90 ng/ml; p = 0.0082) and the median for the homozygous group for the non-risk allele (28.55 ng/ml; p = 0.0355). No differences were detected for the active MMP9. TIMP3 levels did not significantly differ between the AMD and control groups, nor between the different genotype groups for rs5754227. Conclusions: The results of our MMP9 analyses indicate that nAMD patients have on average higher systemic MMP9 levels than control individuals, and that this is partly driven by the rs142450006 variant near MMP9. This finding might be an interesting starting point for further exploration of MMP9 as a therapeutic target in nAMD, particularly among individuals carrying the risk-conferring allele rs142450006.


Assuntos
Neovascularização de Coroide/enzimologia , Precursores Enzimáticos/sangue , Precursores Enzimáticos/genética , Metaloproteinase 9 da Matriz/sangue , Metaloproteinase 9 da Matriz/genética , Degeneração Macular Exsudativa/enzimologia , Idoso , Idoso de 80 Anos ou mais , Alelos , Neovascularização de Coroide/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Frequência do Gene , Técnicas de Genotipagem , Humanos , Masculino , Fenótipo , Polimorfismo de Nucleotídeo Único , Inibidor Tecidual de Metaloproteinase-3/sangue , Inibidor Tecidual de Metaloproteinase-3/genética , Degeneração Macular Exsudativa/genética
15.
Insect Mol Biol ; 30(4): 427-435, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33928689

RESUMO

The activation of immune pathways is triggered by the recognition of pathogens by pattern recognition receptors (PRRs). Gram-negative bacteria-binding proteins (GNBPs)/ß-1,3-glucan recognition proteins (ßGRPs) are a conserved family of PRRs in insects. Two GNBPs are predicted in the genome database of pea aphids; however, little is known about their functions in the aphid immune system. Here, we show that pea aphid GNBPs possess domain architectures and sequence features distinct from those of typical GNBPs/ßGRPs and that their expression is induced by bacterial infection. Knockdown of their expression by dsRNA resulted in lower phenoloxidase activity, higher bacterial loads and higher mortality in aphids after infection. Our data suggest that these two atypical GNBPs are involved in the antibacterial response in the pea aphid, likely acting as PRRs in the prophenoloxidase pathway.


Assuntos
Afídeos , Bactérias Gram-Negativas/imunologia , Imunidade , Receptores de Reconhecimento de Padrão , Animais , Afídeos/genética , Afídeos/imunologia , Catecol Oxidase/metabolismo , Precursores Enzimáticos/metabolismo , Genoma de Inseto , Glucanos/genética , Glucanos/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Interferência de RNA , Receptores de Reconhecimento de Padrão/genética , Receptores de Reconhecimento de Padrão/metabolismo
16.
J Biol Chem ; 296: 100565, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33745969

RESUMO

Rhodesain is the lysosomal cathepsin L-like cysteine protease of Trypanosoma brucei rhodesiense, the causative agent of Human African Trypanosomiasis. The enzyme is essential for the proliferation and pathogenicity of the parasite as well as its ability to overcome the blood-brain barrier of the host. Lysosomal cathepsins are expressed as zymogens with an inactivating prodomain that is cleaved under acidic conditions. A structure of the uncleaved maturation intermediate from a trypanosomal cathepsin L-like protease is currently not available. We thus established the heterologous expression of T. brucei rhodesiense pro-rhodesain in Escherichia coli and determined its crystal structure. The trypanosomal prodomain differs from nonparasitic pro-cathepsins by a unique, extended α-helix that blocks the active site and whose side-chain interactions resemble those of the antiprotozoal inhibitor K11777. Interdomain dynamics between pro- and core protease domain as observed by photoinduced electron transfer fluorescence correlation spectroscopy increase at low pH, where pro-rhodesain also undergoes autocleavage. Using the crystal structure, molecular dynamics simulations, and mutagenesis, we identify a conserved interdomain salt bridge that prevents premature intramolecular cleavage at higher pH values and may thus present a control switch for the observed pH sensitivity of proenzyme cleavage in (trypanosomal) CathL-like proteases.


Assuntos
Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Precursores Enzimáticos/química , Precursores Enzimáticos/metabolismo , Trypanosoma brucei rhodesiense/enzimologia , Ativação Enzimática , Concentração de Íons de Hidrogênio , Modelos Moleculares , Domínios Proteicos
17.
Int J Mol Sci ; 22(4)2021 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-33672594

RESUMO

High alcohol intake results in the accumulation of non-oxidative ethanol metabolites such as fatty acid ethyl esters (FAEEs) in the pancreas. High FAEE concentrations mediate pancreatic acinar cell injury and are associated with alcoholic pancreatitis. Treatment with ethanol and the fatty acid palmitoleic acid (EtOH/POA) increased the levels of palmitoleic acid ethyl ester and induced zymogen activation and cytokine expression in pancreatic acinar cells. EtOH/POA induces nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated reactive oxygen species (ROS) production and pancreatic acinar cell injury. Lycopene, a bright-red carotenoid, is a potent antioxidant due to its high number of conjugated double bands. This study aimed to investigate whether lycopene inhibits the EtOH/POA-induced increase in ROS production, zymogen activation, and expression of the inflammatory cytokine IL-6 in EtOH/POA-stimulated pancreatic acinar AR42J cells. EtOH/POA increased the ROS levels, NADPH oxidase and NF-κB activities, zymogen activation, IL-6 expression, and mitochondrial dysfunction, which were inhibited by lycopene. The antioxidant N-acetylcysteine and NADPH oxidase 1 inhibitor ML171 suppressed the EtOH/POA-induced increases in ROS production, NF-κB activation, zymogen activation, and IL-6 expression. Therefore, lycopene inhibits EtOH/POA-induced mitochondrial dysfunction, zymogen activation, and IL-6 expression by suppressing NADPH oxidase-mediated ROS production in pancreatic acinar cells.


Assuntos
Células Acinares/patologia , Inflamação/patologia , Licopeno/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Pâncreas Exócrino/patologia , Acetilcisteína/farmacologia , Células Acinares/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Precursores Enzimáticos/metabolismo , Etanol , Ácidos Graxos Monoinsaturados , Interleucina-6/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , NADPH Oxidases/metabolismo , NF-kappa B/metabolismo , Ligação Proteica/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio/metabolismo
19.
Acta Physiol (Oxf) ; 232(1): e13640, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33650216

RESUMO

AIM: The serine protease prostasin (Prss8) is expressed in the distal tubule and stimulates proteolytic activation of the epithelial sodium channel (ENaC) in co-expression experiments in vitro. The aim of this study was to explore the role of prostasin in proteolytic ENaC activation in the kidney in vivo. METHODS: We used genetically modified knockin mice carrying a Prss8 mutation abolishing proteolytic activity (Prss8-S238A) or a mutation leading to a zymogen-locked state (Prss8-R44Q). Mice were challenged with low sodium diet and diuretics. Regulation of ENaC activity by Prss8-S238A and Prss8-R44Q was studied in vitro using the Xenopus laevis oocyte expression system. RESULTS: Co-expression of murine ENaC with Prss8-wt or Prss8-S238A in oocytes caused maximal proteolytic ENaC activation, whereas ENaC was activated only partially in oocytes co-expressing Prss8-R44Q. This was paralleled by a reduced proteolytic activity at the cell surface of Prss8-R44Q expressing oocytes. Sodium conservation under low sodium diet was preserved in Prss8-S238A and Prss8-R44Q mice but with higher plasma aldosterone concentrations in Prss8-R44Q mice. Treatment with the ENaC inhibitor triamterene over four days was tolerated in Prss8-wt and Prss8-S238A mice, whereas Prss8-R44Q mice developed salt wasting and severe weight loss associated with hyperkalemia and acidosis consistent with impaired ENaC function and renal failure. CONCLUSION: Unlike proteolytically inactive Prss8-S238A, zymogen-locked Prss8-R44Q produces incomplete proteolytic ENaC activation in vitro and causes a severe renal phenotype in mice treated with the ENaC inhibitor triamterene. This indicates that Prss8 plays a role in proteolytic ENaC activation and renal function independent of its proteolytic activity.


Assuntos
Precursores Enzimáticos , Canais Epiteliais de Sódio , Animais , Camundongos , Oócitos/metabolismo , Serina Endopeptidases/metabolismo , Triantereno , Xenopus laevis/metabolismo
20.
J Parasitol ; 107(1): 115-124, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33592628

RESUMO

The immune response of animals, including insects, is overcome by some parasites. For example, dauer larvae (DL) of the obligate entomopathogenic nematodes (EPNs) Heterorhabditis and Steinernema can invade insects, evade their defenses, and cause death. Although DL were long assumed to be the only infective stage of nematodes, recent reports suggest that L2-L3 larvae of facultative EPNs are also capable of killing insects. There are no studies, to our knowledge, about the role of nonimmunological barriers (the exoskeleton and its openings) in avoiding infection by DL and L2-L3 larvae, or whether these larval stages evade the host immune system in the same way. The objective of this study was to examine these questions by infecting Galleria mellonella with the facultative parasitic nematode Rhabditis regina. DL or L2-L3 larvae were either deposited on or near the moths or injected into their hemocoel. Once nematodes reached the hemocoel, the following host immune response parameters were quantified: prophenoloxidase, phenoloxidase, lytic activity, and the number of granular hemocytes. DL showed a greater ability to penetrate the exoskeleton than L2-L3 larvae. Once inside, however, both went unnoticed by the immune system and killed the insect. A higher number of granular hemocytes was activated by L2-L3 larvae than DL. We show for the first time that L2-L3 larvae can penetrate and evade the insect immune system. Further research is needed to compare facultative and specialized EPNs to determine which is more likely, with both DL and L2-L3 larvae, to evade insect defense barriers and produce death. The results will contribute to understanding the evolution of virulence in entomopathogenic nematodes.


Assuntos
Evasão da Resposta Imune/fisiologia , Lepidópteros/parasitologia , Strongyloidea/imunologia , Análise de Variância , Animais , Catecol Oxidase/metabolismo , Proteínas Cardiotóxicas de Elapídeos/metabolismo , Precursores Enzimáticos/metabolismo , Larva/imunologia , Lepidópteros/enzimologia , Lepidópteros/imunologia , Monofenol Mono-Oxigenase/metabolismo , Strongyloidea/patogenicidade , Strongyloidea/ultraestrutura , Fatores de Tempo , Virulência
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