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1.
Mol Immunol ; 109: 1-11, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30836204

RESUMO

Protease activity of allergens has been suggested to be involved in the pathogenesis of allergic diseases. The major allergen Der f 3 from Dermatophagoides farinae harbors serine protease activity, but its immunopathogenesis remains unclear. This study aims to explore the effect of Der f 3 on the airway epithelial barrier and on the molecular pathways by which Der f 3 induces inflammation. RNA-seq was performed to identify differentially expressed genes in bronchial airway epithelial cells (AEC) between native Der f 3 and heat-inactivated (H) Der f 3, coupled with real-time PCR (RT-PCR) and ELISA for validation. Unlike other protease allergens such as that induce Th2-promoting alarmins (IL-25, IL-33, TSLP) in AECs, Der f 3 induced pro-inflammatory cytokines and chemokines including IL-6, IL-8 and GM-CSF, which are known to promote Th17 response. These pro-inflammatory mediators were induced by Der f 3 via the MAPK and NF-κB pathways as well as the store-operated calcium signaling. Gene silencing with small interfering RNA in A549 and BEAS-2B cells indicated that activation of AECs by Der f 3 was mainly dependent on protease-activated receptor 2 (PAR-2), while PAR-1 was also required for the full activation of AECs. Double knock-down of PAR-1 and PAR-2 largely impaired Der f 3-inducecd IL-8 production and subsequent signaling pathways. Our data suggest that Der f 3 induces pro-inflammatory mediators in human epithelial cell lines via the PARs-MAPK-NF-κB axis. Our results provide a molecular mechanism by which Der f 3 may trigger the Th17-skewed allergic response toward house dust mites.


Assuntos
Alérgenos/imunologia , Proteínas de Artrópodes/imunologia , Células Epiteliais/imunologia , Pyroglyphidae/imunologia , Receptor PAR-1/imunologia , Receptor PAR-2/imunologia , Mucosa Respiratória/imunologia , Serina Endopeptidases/imunologia , Células A549 , Alérgenos/farmacologia , Animais , Proteínas de Artrópodes/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/genética , Sinalização do Cálcio/imunologia , Citocinas/genética , Citocinas/imunologia , Células Epiteliais/patologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Técnicas de Silenciamento de Genes , Humanos , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Mediadores da Inflamação/imunologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/imunologia , NF-kappa B/genética , NF-kappa B/imunologia , Receptor PAR-1/genética , Receptor PAR-2/genética , Serina Endopeptidases/farmacologia , Células Th17/imunologia , Células Th17/patologia , Células Th2/imunologia , Células Th2/patologia
2.
PLoS Negl Trop Dis ; 12(4): e0006446, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29677188

RESUMO

BACKGROUND: Serine proteases are important virulence factors for many pathogens. Recently, we discovered a group of trypsin-like serine proteases with domain organization unique to flatworm parasites and containing a thrombospondin type 1 repeat (TSR-1). These proteases are recognized as antigens during host infection and may prove useful as anthelminthic vaccines, however their molecular characteristics are under-studied. Here, we characterize the structural and proteolytic attributes of serine protease 2 (SmSP2) from Schistosoma mansoni, one of the major species responsible for the tropical infectious disease, schistosomiasis. METHODOLOGY/PRINCIPAL FINDINGS: SmSP2 comprises three domains: a histidine stretch, TSR-1 and a serine protease domain. The cleavage specificity of recombinant SmSP2 was determined using positional scanning and multiplex combinatorial libraries and the determinants of specificity were identified with 3D homology models, demonstrating a trypsin-like endopeptidase mode of action. SmSP2 displayed restricted proteolysis on protein substrates. It activated tissue plasminogen activator and plasminogen as key components of the fibrinolytic system, and released the vasoregulatory peptide, kinin, from kininogen. SmSP2 was detected in the surface tegument, esophageal glands and reproductive organs of the adult parasite by immunofluorescence microscopy, and in the excretory/secretory products by immunoblotting. CONCLUSIONS/SIGNIFICANCE: The data suggest that SmSP2 is secreted, functions at the host-parasite interface and contributes to the survival of the parasite by manipulating host vasodilatation and fibrinolysis. SmSP2 may be, therefore, a potential target for anti-schistosomal therapy.


Assuntos
Hemostáticos/antagonistas & inibidores , Schistosoma mansoni/enzimologia , Esquistossomose mansoni/parasitologia , Serina Endopeptidases/farmacologia , Sequência de Aminoácidos , Animais , Coagulação Sanguínea/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Feminino , Fibrinólise/efeitos dos fármacos , Proteínas de Helminto/química , Proteínas de Helminto/genética , Proteínas de Helminto/farmacologia , Masculino , Modelos Moleculares , Plasminogênio/efeitos dos fármacos , Domínios Proteicos , Proteólise/efeitos dos fármacos , Proteínas Recombinantes , Serina Endopeptidases/química , Serina Endopeptidases/genética , Ativador de Plasminogênio Tecidual/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos
3.
Magy Seb ; 70(3): 221-231, 2017 09.
Artigo em Húngaro | MEDLINE | ID: mdl-28876118

RESUMO

INTRODUCTION: Non-occlusive mesenteric ischemia (NOMI) develops without anatomical causes. Early diagnosis is challenging and treatments are of questionable effectiveness. We investigated the role of complement activation in the pathophysiology of NOMI in animal models through the inhibition of complement C5a. MATERIALS AND METHODS: 60-min partial aortic occlusion (PAO; abdominal aorta, proximal to celiac trunk; mean arterial pressure: 30-40 mmHg) was established in Sprague-Dawley rats (n = 28) and 60-min cardiac tamponade in minipigs (n = 19; mean arterial pressure: 40-50 mmHg) to observe short- and long-term circulatory and inflammatory consequences of NOMI. Macro- and microhemodynamics, leukocyte infiltration, plasma levels of inflammatory mediators (endothelin, HMGB-1) were measured. C5a inhibitor (Acetyl-Peptid-A; 4 mg/kg iv) was administered at the 45th min of PAO or tamponade, respectively. RESULTS: Twenty-four hours after PAO systemic inflammatory response increased cardiac output and superior mesenteric artery flow (SMAF). C5a inhibition reduced the elevated cardiac output (203.1 ± 5 vs 269.6 ± 8.1 ml/min/kg) and SMAF and increased ileal microcirculation (833.5 ± 33.8 vs 441.9 ± 22.4 µm/s). In pigs, after the tamponade, C5a inhibition reduced the immediate hemodynamic disturbances, temporarily increased SMAF and permanently the ileal microcirculation. The Acetyl-Peptid-A treatment reduced leukocyte infiltration and plasma levels of inflammatory mediators in both NOMI models. CONCLUSIONS: Complement activation plays central role in the macro- and microcirculatory disturbance during NOMI. C5a inhibition reduces the inflammatory activation and influences the hemodynamic consequences of experimental NOMI.


Assuntos
Hemodinâmica/efeitos dos fármacos , Isquemia Mesentérica/tratamento farmacológico , Serina Endopeptidases/farmacologia , Animais , Modelos Animais de Doenças , Hemodinâmica/fisiologia , Isquemia Mesentérica/fisiopatologia , Microcirculação/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Suínos
4.
Biochem Biophys Res Commun ; 491(2): 455-462, 2017 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-28709869

RESUMO

Bacillopeptidase is a serine peptidase, known for its fibrinolytic activity. However, a very little information is known about its in vivo inflammatory and/or anti-inflammatory properties. Thus, to understand whether bacillopeptidase incorporation can regulate pancreatitis or not, the cerulein-induced pancreatitis model was used, and the role of bacillopeptidase on pancreatitis was studied. In this study, 46 kDa protein was purified from Bacillus subtilis and identified as bacillopeptidase CFR5 (BPC) through MS/MS analysis. The nutritional prophylactic group was orally fed with two doses of BPC (100 µg/Kg/BW of rat) 6 h before cerulein administration and analyzed for its effect on intestine and pancreas inflammation, cytokines, and pancreatitis marker gene expression. BPC administration significantly reduced the severity of pancreatitis by decreasing serum amylase, lipase, pancreatic edema and myeloperoxidase activity. The pretreatment with BPC suppressed the pancreatic pro-inflammatory and inflammatory cytokines production including IL-6, IL-1ß, TNF-α, IL-2, IL-4, IL-5, IL-10, and IL-13 in both pancreas and serum samples. Moreover, BPC supplementation restored pancreatitis mediated disruption of intestinal barrier integrity by upregulating tight junction proteins (ZO-1, occludin), antimicrobial peptides (DEFB1, CRAMP), MUC-2, TFF3 expression and by enhancing SCFA's production. Pretreatment with BPC suppressed the intestinal inflammation with reduced cytokines production in the colon and ileal region of cerulein-induced pancreatitis. Thus, BPC based pretreatment protocol is a novel intervention to prevent acute pancreatitis.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Bacillus subtilis/química , Proteínas de Bactérias/farmacologia , Edema/tratamento farmacológico , Pancreatite/tratamento farmacológico , Serina Endopeptidases/farmacologia , Administração Oral , Animais , Anti-Inflamatórios não Esteroides/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos , Proteínas de Bactérias/isolamento & purificação , Catelicidinas/genética , Catelicidinas/metabolismo , Ceruletídeo , Citocinas/biossíntese , Defensinas/genética , Defensinas/metabolismo , Edema/induzido quimicamente , Edema/genética , Edema/patologia , Regulação da Expressão Gênica , Masculino , Mucina-2/genética , Mucina-2/metabolismo , Ocludina/genética , Ocludina/metabolismo , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Pâncreas/patologia , Pancreatite/induzido quimicamente , Pancreatite/genética , Pancreatite/patologia , Ratos , Ratos Wistar , Serina Endopeptidases/isolamento & purificação , Fator Trefoil-3/genética , Fator Trefoil-3/metabolismo , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismo
5.
Arthritis Rheumatol ; 69(8): 1601-1611, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28464560

RESUMO

OBJECTIVE: To assess the ability of matriptase, a type II transmembrane serine proteinase, to promote aggrecan loss from the cartilage of patients with osteoarthritis (OA) and to determine whether its inhibition can prevent aggrecan loss and cartilage damage in experimental OA. METHODS: Aggrecan release from human OA cartilage explants and human stem cell-derived cartilage discs was evaluated, and cartilage-conditioned media were used for Western blotting. Gene expression was analyzed by real-time polymerase chain reaction. Murine OA was induced by surgical destabilization of the medial meniscus, and matriptase inhibitors were administered via osmotic minipump or intraarticular injection. Cartilage damage was scored histologically and aggrecan cleavage was visualized immunohistochemically using specific neoepitope antibodies. RESULTS: The addition of soluble recombinant matriptase promoted a time-dependent release of aggrecan (and collagen) from OA cartilage, which was sensitive to metalloproteinase inhibition and protease-activated receptor 2 antagonism. Although engineered human (normal) cartilage discs failed to release aggrecan following matriptase addition, both matrix metalloproteinase- and aggrecanase-mediated cleavages of aggrecan were detected in human OA cartilage. Additionally, while matriptase did not directly degrade aggrecan, it promoted the accumulation of low-density lipoprotein receptor-related protein 1 (LRP-1) in conditioned media of the OA cartilage explants. Matriptase inhibition via neutralizing antibody or small molecule inhibitor significantly reduced cartilage damage scores in murine OA, which was associated with reduced generation of metalloproteinase-mediated aggrecan cleavage. CONCLUSION: Matriptase potently induces the release of metalloproteinase-generated aggrecan fragments as well as soluble LRP-1 from OA cartilage. Therapeutic targeting of matriptase proteolytic activity reduces metalloproteinase activity, further suggesting that this serine proteinase may have potential as a disease-modifying therapy in OA.


Assuntos
Agrecanas/efeitos dos fármacos , Cartilagem Articular/efeitos dos fármacos , Osteoartrite do Joelho/metabolismo , Serina Endopeptidases/farmacologia , Proteína ADAMTS4/efeitos dos fármacos , Proteína ADAMTS4/metabolismo , Proteína ADAMTS5/efeitos dos fármacos , Proteína ADAMTS5/metabolismo , Idoso , Idoso de 80 Anos ou mais , Agrecanas/metabolismo , Animais , Anticorpos Neutralizantes/farmacologia , Western Blotting , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Endopeptidases/efeitos dos fármacos , Endopeptidases/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/efeitos dos fármacos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Masculino , Metaloproteinases da Matriz/efeitos dos fármacos , Metaloproteinases da Matriz/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Meniscos Tibiais/cirurgia , Camundongos , Pessoa de Meia-Idade , Osteoartrite do Joelho/patologia , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/farmacologia , Serina Endopeptidases/metabolismo
6.
Cell Rep ; 19(3): 479-486, 2017 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-28423312

RESUMO

The activation of quiescent stem cells into the cell cycle is a key step in initiating the process of tissue repair. We recently reported that quiescent stem cells can transition into GAlert, a cellular state in which they have an increased functional ability to activate and participate in tissue repair. However, the precise molecular signals that induce GAlert in stem cells have remained elusive. Here, we show that the injury-induced regulation of hepatocyte growth factor (HGF) proteolytic processing via the systemic protease, hepatocyte growth factor activator (HGFA), stimulates GAlert in skeletal muscle stem cells (MuSCs) and fibro-adipogenic progenitors (FAPs). We demonstrate that administering active HGFA to animals is sufficient to induce GAlert in stem cells throughout the body and to significantly accelerate the processes of stem cell activation and tissue repair. Our data suggest that factors that induce GAlert will have broad therapeutic applications for regenerative medicine and wound healing.


Assuntos
Ciclo Celular/efeitos dos fármacos , Serina Endopeptidases/farmacologia , Células-Tronco/citologia , Cicatrização/efeitos dos fármacos , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Animais , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Cinética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/citologia , Serina Endopeptidases/administração & dosagem , Soro/metabolismo , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo
7.
Immunopharmacol Immunotoxicol ; 39(1): 37-44, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28004985

RESUMO

Cyclophosphamide (CY) is a DNA alkylating agent, which is widely used with other chemotherapy drugs in the treatment of various types of cancer. It can be used not only as a chemotherapeutic but also as an immunomodulatory agent to inhibit IL-10 expression and T regulatory cells (Tregs). Fibroblast activation protein α (FAPα) is expressed in cancer-associated fibroblasts in the tumor microenvironment. Immunotherapy based on FAPα, as a tumor stromal antigen, typically induces specific immune response targeting the tumor microenvironment. This study evaluated the efficacy of a previously unreported CY combination strategy to enhance the limited anti-tumor effect of a DNA vaccine targeting FAPα. The results suggested CY administration could promote the percentage of splenic CD8+ T cells and decrease the proportion of CD4 + CD25 + Foxp3+ Tregs in spleen. In tumor tissues, levels of immunosuppressive cytokines including IL-10 and CXCL-12 were also reduced. Meanwhile, the CY combination did not impair the FAPα-specific immunity induced by the DNA vaccine and further reduced tumor stromal factors. Most importantly, FAP-vaccinated mice also treated with CY chemotherapy showed a marked suppression of tumor growth (inhibition ratio =80%) and a prolongation of survival time. Thus, the combination of FAPα immunotherapy and chemotherapy with CY offers new insights into improving cancer therapies.


Assuntos
Vacinas Anticâncer/farmacologia , Ciclofosfamida/farmacocinética , Gelatinases/farmacologia , Imunidade Celular/efeitos dos fármacos , Neoplasias Mamárias Experimentais/terapia , Proteínas de Membrana/farmacologia , Serina Endopeptidases/farmacologia , Vacinas de DNA/farmacocinética , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Vacinas Anticâncer/imunologia , Feminino , Gelatinases/imunologia , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/patologia , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Serina Endopeptidases/imunologia , Vacinas de DNA/imunologia
8.
Am J Physiol Gastrointest Liver Physiol ; 311(3): G466-79, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27492333

RESUMO

Barrier dysfunction is a characteristic of the inflammatory bowel diseases (IBD), Crohn's disease and ulcerative colitis. Understanding how the tight junction is modified to maintain barrier function may provide avenues for treatment of IBD. We have previously shown that the apical addition of serine proteases to intestinal epithelial cell lines causes a rapid and sustained increase in transepithelial electrical resistance (TER), but the mechanisms are unknown. We hypothesized that serine proteases increase barrier function through trafficking and insertion of tight junction proteins into the membrane, and this could enhance recovery of a disrupted monolayer after calcium switch or cytokine treatment. In the canine epithelial cell line, SCBN, we showed that matriptase, an endogenous serine protease, could potently increase TER. Using detergent solubility-based cell fractionation, we found that neither trypsin nor matriptase treatment changed levels of tight junction proteins at the membrane. In a fast calcium switch assay, serine proteases did not enhance the rate of recovery of the junction. In addition, serine proteases could not reverse barrier disruption induced by IFNγ and TNFα. We knocked down occludin in our cells using siRNA and found this prevented the serine protease-induced increase in TER. Using fluorescence recovery after photobleaching (FRAP), we found serine proteases induce a greater mobile fraction of occludin in the membrane. These data suggest that a functional tight junction is needed for serine proteases to have an effect on TER, and that occludin is a crucial tight junction protein in this mechanism.


Assuntos
Células Epiteliais/enzimologia , Mucosa Intestinal/citologia , Ocludina/metabolismo , Junções Íntimas/fisiologia , Animais , Linhagem Celular , Cães , Impedância Elétrica , Fenômenos Eletrofisiológicos , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Ocludina/genética , Transporte Proteico , Serina Endopeptidases/farmacologia , Serina Proteases , Proteínas de Junções Íntimas/metabolismo , Tripsina/farmacologia
9.
J Neurosci ; 36(24): 6538-52, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27307241

RESUMO

UNLABELLED: How the integrity of laminar structures in the postnatal brain is maintained impacts neuronal functions. Ndel1, the mammalian homolog of NuDE from the filamentous fungus Aspergillus nidulans, is an atypical microtubule (MT)-associated protein that was initially investigated in the contexts of neurogenesis and neuronal migration. Constitutive knock-out mice for Ndel1 are embryonic lethal, thereby necessitating the creation a conditional knock-out to probe the roles of Ndel1 in postnatal brains. Here we report that CA1 pyramidal neurons from mice postnatally lacking Ndel1 (Ndel1 conditional knock-out) exhibit fragmented MTs, dendritic/synaptic pathologies, are intrinsically hyperexcitable and undergo dispersion independently of neuronal migration defect. Secondary to the pyramidal cell changes is the decreased inhibitory drive onto pyramidal cells from interneurons. Levels of the glycoprotein Reelin that regulates MTs, neuronal plasticity, and cell compaction are significantly reduced in hippocampus of mutant mice. Strikingly, a single injection of Reelin into the hippocampus of Ndel1 conditional knock-out mice ameliorates ultrastructural, cellular, morphological, and anatomical CA1 defects. Thus, Ndel1 and Reelin contribute to maintain postnatal CA1 integrity. SIGNIFICANCE STATEMENT: The significance of this study rests in the elucidation of a role for Nde1l and Reelin in postnatal CA1 integrity using a new conditional knock-out mouse model for the cytoskeletal protein Ndel1, one that circumvents the defects associated with neuronal migration and embryonic lethality. Our study serves as a basis for understanding the mechanisms underlying postnatal hippocampal maintenance and function, and the significance of decreased levels of Ndel1 and Reelin observed in patients with neurological disorders.


Assuntos
Região CA1 Hipocampal/crescimento & desenvolvimento , Região CA1 Hipocampal/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Serina Endopeptidases/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Região CA1 Hipocampal/ultraestrutura , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/farmacologia , Proteínas de Ciclo Celular/genética , Dendritos/metabolismo , Dendritos/ultraestrutura , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Glutamato Descarboxilase/metabolismo , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurotransmissores/farmacologia , Serina Endopeptidases/genética , Serina Endopeptidases/farmacologia , Coloração pela Prata , Sinapses/metabolismo , Sinapses/ultraestrutura
10.
J Am Soc Nephrol ; 27(9): 2622-9, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-26850635

RESUMO

IgA nephropathy (IgAN), characterized by mesangial IgA1 deposits, is a leading cause of renal failure worldwide. IgAN pathogenesis involves circulating hypogalactosylated IgA1 complexed with soluble IgA Fc receptor I (sCD89) and/or anti-hypogalactosylated-IgA1 autoantibodies, but no specific treatment is available for IgAN. The absence of IgA1 and CD89 homologs in the mouse has precluded in vivo proof-of-concept studies of specific therapies targeting IgA1. However, the α1KI­CD89Tg mouse model of IgAN, which expresses human IgA1 and human CD89, allows in vivo testing of recombinant IgA1 protease (IgA1­P), a bacterial protein that selectively cleaves human IgA1. Mice injected with IgA1­P (1-10 mg/kg) had Fc fragments of IgA1 in both serum and urine, associated with a decrease in IgA1-sCD89 complexes. Levels of mesangial IgA1 deposits and the binding partners of these deposits (sCD89, transferrin receptor, and transglutaminase 2) decreased markedly 1 week after treatment, as did the levels of C3 deposition, CD11b(+) infiltrating cells, and fibronectin. Antiprotease antibodies did not significantly alter IgA1­P activity. Moreover, hematuria consistently decreased after treatment. In conclusion, IgA1­P strongly diminishes human IgA1 mesangial deposits and reduces inflammation, fibrosis, and hematuria in a mouse IgAN model, and therefore may be a plausible treatment for patients with IgAN.


Assuntos
Mesângio Glomerular/metabolismo , Glomerulonefrite por IGA/tratamento farmacológico , Hematúria/tratamento farmacológico , Imunoglobulina A/efeitos dos fármacos , Imunoglobulina A/metabolismo , Serina Endopeptidases/farmacologia , Animais , Modelos Animais de Doenças , Camundongos , Serina Endopeptidases/uso terapêutico
11.
J Biosci Bioeng ; 121(6): 614-618, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26896861

RESUMO

A rhizosphere strain of the bacterium Stenotrophomonas maltophilia N4 secretes the serine protease PN4, whose molecular mass is approximately 42 kDa. The optimal temperature for the enzyme activity of the 11-fold purified protein was 50°C and the optimal pH was 10.5. The activity of the enzyme was strongly inhibited by specific serine protease inhibitors, which allowed for its classification as an alkaline serine protease family. Ca(2+) ions stimulated the activity of the protease PN4, while Mg(2+) ions stabilized its activity, and Zn(2+) and Cd(2+) ions strongly inhibited its activity. The enzyme has broad substrate specificity. For example, it is able to hydrolyse casein, keratin, albumin, haemoglobin, and gelatin, as well as the insoluble modified substrates azure keratin and azocoll. The gene that encodes the 1740 bp precursor form of the enzyme (accession number: LC031815) was cloned. We then deduced that its amino acid sequence includes the region of the conserved domain of the S8 family of peptidases as well as the catalytic triad Asp/His/Ser. The bacterial culture fluid as well as the purified protease PN4 demonstrated biocidal activity with regard to the nematodes Caenorhabditis elegans and Panagrellus spp.


Assuntos
Serina Endopeptidases/metabolismo , Stenotrophomonas maltophilia/enzimologia , Stenotrophomonas maltophilia/genética , Sequência de Aminoácidos , Animais , Antinematódeos/química , Antinematódeos/metabolismo , Antinematódeos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Clonagem Molecular , Endopeptidases/química , Endopeptidases/genética , Endopeptidases/metabolismo , Endopeptidases/farmacologia , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Peso Molecular , Domínios Proteicos , Rabditídios/efeitos dos fármacos , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/farmacologia , Especificidade por Substrato , Temperatura Ambiente
12.
Microbiol Immunol ; 60(1): 35-46, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26576826

RESUMO

Acute mesenteric ischemia (AMI) is caused by considerable intestinal injury, which is associated with intestinal ischemia followed by reperfusion. To elucidate the mechanisms of ischemia/reperfusion injuries, a C5a inhibitory peptide termed AcPepA was used to examine the role of C5a anaphylatoxin, induction of inflammatory cells, and cell proliferation of the intestinal epithelial cells in an experimental AMI model. In this rat model, the superior mesenteric artery was occluded and subsequently reperfused (Induce-I/R). Other groups were treated with AcPepA before ischemia or reperfusion. Induce-I/R induced injuries in the intestine and AcPepA significantly decreased the proportion of severely injured villi. Induce-I/R induced secondary receptor for C5a-positive polymorphonuclear leukocytes in the vessels and CD204-positive macrophages near the injured site; this was correlated with hypoxia-induced factor 1-alpha-positive cells. Induction of these inflammatory cells was attenuated by AcPepA. In addition, AcPepA increased proliferation of epithelial cells in the villi, possibly preventing further damage. Therefore, Induce-I/R activates C5a followed by the accumulation of polymorphonuclear leukocyte and hypoxia-induced factor 1-alpha-producing macrophages, leading to villus injury. AcPepA, a C5a inhibitory peptide, blocks the deleterious effects of C5a, indicating it has a therapeutic effect on the inflammatory consequences of experimental AMI.


Assuntos
Enteropatias/prevenção & controle , Intestino Delgado/irrigação sanguínea , Receptor da Anafilatoxina C5a/antagonistas & inibidores , Traumatismo por Reperfusão/prevenção & controle , Serina Endopeptidases/farmacologia , Animais , Antígenos CD/imunologia , Antígenos de Diferenciação Mielomonocítica/imunologia , Proliferação de Células , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Imuno-Histoquímica , Mucosa Intestinal/patologia , Intestino Delgado/patologia , Masculino , Neutrófilos , Ratos , Ratos Sprague-Dawley , Receptor da Anafilatoxina C5a/imunologia , Fator de Necrose Tumoral alfa/imunologia
13.
Surgery ; 159(3): 960-71, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26632492

RESUMO

BACKGROUND: Nonocclusive mesenteric ischemia (NOMI) can evolve in a variety of low-flow states. Although the mechanisms leading to NOMI-related intestinal necrosis are largely unknown, circumstantial evidence suggests that excessive vasoconstriction and complement activation both play important roles in this process. Because targeting of the circulatory malfunction of the splanchnic area could be of therapeutic relevance, we set out to investigate the long-term effects of treatment with a complement C5a antagonist in a rat model of partial aortic occlusion (PAO)-induced transient mesenteric hypoperfusion. METHODS: The mean arterial pressure of the splanchnic area was kept between 30 and 40 mm Hg by 60 minutes of PAO in anesthetized male Sprague-Dawley rats. C5a inhibitor acetyl-peptide-A (AcPepA; 4 mg kg(-1) intravenously) or vehicle administration was initiated at the 45th minute of PAO. After 24 hours, the animals were reanesthetized to record the macrohemodynamics and ileal microcirculation, and plasma and tissue samples were taken for determination of high-mobility group box protein-1 (HMGB-1), endothelin-1, tumor necrosis factor (TNF)-α levels, and small intestinal leukocyte infiltration. Epithelial structural changes were visualized by in vivo confocal laser scanning endomicroscopy. RESULTS: At 24 hours after PAO, mean arterial pressure, heart rate, and cardiac output were significantly greater, the intestinal intramural microcirculation was significantly impaired, and plasma HMGB-1, endothelin-1, TNF-α levels, the degree of epithelial damage and leukocyte infiltration was increased. The AcPepA treatment moderated the hemodynamic and microcirculatory changes, and decreased inflammatory activation and histologic signs of mucosal damage. CONCLUSION: C5a inhibition ameliorated the potentially harmful local mesenteric hypoperfusion and global long-term inflammatory consequences of PAO. This approach is of promise for use in NOMI-associated situations.


Assuntos
Hemodinâmica/efeitos dos fármacos , Íleo/irrigação sanguínea , Inflamação/sangue , Isquemia Mesentérica/tratamento farmacológico , Serina Endopeptidases/farmacologia , Análise de Variância , Animais , Biomarcadores/sangue , Modelos Animais de Doenças , Endotelina-1/sangue , Proteína HMGB1/sangue , Hemodinâmica/fisiologia , Inflamação/fisiopatologia , Masculino , Isquemia Mesentérica/patologia , Microcirculação/efeitos dos fármacos , Microscopia de Vídeo , Análise Multivariada , Projetos Piloto , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Estatísticas não Paramétricas , Fator de Necrose Tumoral alfa/sangue , Grau de Desobstrução Vascular/efeitos dos fármacos
14.
Immunopharmacol Immunotoxicol ; 37(5): 413-20, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26369367

RESUMO

CONTEXT: Endotoxins including lipopolysaccharide (LPS) could cause endotoxemia which often results in excessive inflammation, organ dysfunction, sepsis, disseminated intravascular coagulation (DIC) or even death. Previously, a novel fibrinogenase (FII) showed protective effects on LPS-induced DIC via activating protein C and suppressing inflammatory cytokines. OBJECTIVE: To evaluate whether FII has protective effect on LPS-induced endotoxemia in mice and learn about the role of NF-κB pathway in TNF-α producing process. METHODS: BALB/C mice were intraperitoneally injected (i.p.) with (a) 30 mg/kg LPS, (b) LPS + 0.3 mg/kg FII, (c) LPS + 1.0 mg/kg FII, (d) LPS + 3.0 mg/kg FII or (e) saline. Both survival rate and organ function were tested, including alanine aminotransferase (ALT), blood urine nitrogen (BUN) and tissue section, and TNF-α was examined by ELISA. RAW 264.7 macrophage was administered with (a) LPS, (b) LPS + FII, (c) FII alone or (d) saline, and TNF-α and phosphorylation (P)-NF-κB (P65) were determined by Western blot. RESULTS: The administration of LPS led to 65% mortality rate, a rise of serum TNF-α, BUN and ALT levels, and both liver and renal tissue damage were observed. While FII treatment significantly increased the survival rate of LPS-induced endotoxemia mice model, histopathology and protein analysis results also revealed that FII remarkably protected liver and renal from LPS damage as well as decreasing TNF-α level. In vitro, FII significantly decreased LPS-induced TNF-α production and the expression of P-NF-κB (P65). CONCLUSIONS: Our findings suggested that FII had protective effect on LPS-induced endotoxemia and organ injuries by suppressing the activation of NF-κB which decreased TNF-α level.


Assuntos
Venenos de Crotalídeos/química , Endotoxemia/induzido quimicamente , Endotoxemia/prevenção & controle , Lipopolissacarídeos/toxicidade , Serina Endopeptidases/farmacologia , Viperidae , Animais , Endotoxemia/imunologia , Camundongos , Camundongos Endogâmicos BALB C
15.
Oncotarget ; 6(32): 33534-53, 2015 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-26392335

RESUMO

The membrane-anchored serine proteases are a unique group of trypsin-like serine proteases that are tethered to the cell surface via transmembrane domains or glycosyl-phosphatidylinositol-anchors. Overexpressed in tumors, with pro-tumorigenic properties, they are attractive targets for protease-activated prodrug-like anti-tumor therapies. Here, we sought to engineer anthrax toxin protective antigen (PrAg), which is proteolytically activated on the cell surface by the proprotein convertase furin to instead be activated by tumor cell-expressed membrane-anchored serine proteases to function as a tumoricidal agent. PrAg's native activation sequence was mutated to a sequence derived from protein C inhibitor (PCI) that can be cleaved by membrane-anchored serine proteases, to generate the mutant protein PrAg-PCIS. PrAg-PCIS was resistant to furin cleavage in vitro, yet cytotoxic to multiple human tumor cell lines when combined with FP59, a chimeric anthrax toxin lethal factor-Pseudomonas exotoxin fusion protein. Molecular analyses showed that PrAg-PCIS can be cleaved in vitro by several serine proteases including the membrane-anchored serine protease testisin, and mediates increased killing of testisin-expressing tumor cells. Treatment with PrAg-PCIS also potently attenuated the growth of testisin-expressing xenograft tumors in mice. The data indicates PrAg can be engineered to target tumor cell-expressed membrane-anchored serine proteases to function as a potent tumoricidal agent.


Assuntos
Antígenos de Bactérias/farmacologia , Toxinas Bacterianas/farmacologia , Pró-Fármacos/farmacologia , Serina Endopeptidases/farmacologia , Sequência de Aminoácidos , Animais , Antígenos de Bactérias/genética , Antineoplásicos/farmacologia , Toxinas Bacterianas/genética , Linhagem Celular Tumoral , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/farmacologia , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Nus , Engenharia de Proteínas , Serina Endopeptidases/genética , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Kidney Int ; 88(4): 764-75, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26061547

RESUMO

Neutrophil serine proteases (NSPs) are released from activated neutrophils during inflammation. Here we studied the transfer of the three major NSPs, namely proteinase 3, human neutrophil elastase, and cathepsin G, from neutrophils to endothelial cells and used an unbiased approach to identify novel endothelial NSP substrates. Enzymatically active NSPs were released from stimulated neutrophils and internalized by endothelial cells in a dose- and time-dependent manner as shown by immunoblotting, flow cytometry, and the Boc-Ala substrate assay. Using terminal-amine isotopic labeling of substrates in endothelial cells, we identified 121 peptides from 82 different proteins consisting of 36 substrates for proteinase 3, 30 for neutrophil elastase, and 28 for cathepsin G, respectively. We characterized the extended cleavage pattern and provide corresponding IceLogos. Gene ontology analysis showed significant cytoskeletal substrate enrichment and confirmed several cytoskeletal protein substrates by immunoblotting. Finally, ANCA-stimulated neutrophils released all three active NSPs into the supernatant. Supernatants increased endothelial albumin flux and disturbed the endothelial cell cytoskeletal architecture. Serine protease inhibition abrogated this effect. Longer exposure to NSPs reduced endothelial cell viability and increased apoptosis. Thus, we identified novel NSP substrates and suggest NSP inhibition as a therapeutic measure to inhibit neutrophil-mediated inflammatory vascular diseases.


Assuntos
Células Endoteliais/enzimologia , Neutrófilos/enzimologia , Comunicação Parácrina , Serina Endopeptidases/metabolismo , Citoesqueleto de Actina/enzimologia , Albuminas/metabolismo , Apoptose , Catepsina G/metabolismo , Linhagem Celular , Permeabilidade da Membrana Celular , Sobrevivência Celular , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Humanos , Elastase de Leucócito/metabolismo , Mieloblastina/metabolismo , Ativação de Neutrófilo , Proteólise , Serina Endopeptidases/genética , Serina Endopeptidases/farmacologia , Transdução de Sinais , Especificidade por Substrato , Fatores de Tempo
17.
Neurosci Lett ; 599: 97-101, 2015 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-26003447

RESUMO

Reelin, an extracellular matrix protein, plays an important role in brain development as well as synaptic plasticity. Interestingly, several recent studies have found that Reelin is important for dendritic spine formation in vitro and in vivo. However, the molecular mechanism by which Reelin regulates the dendritic spine density has not been studied well yet. In this study, we found that exogenous Reelin treatment was significantly increased the dendritic spine density in the primary hippocampal neurons. In addition, Reelin was increased the puncta numbers of synaptophysin and PSD-95. Moreover, we found that Reelin modulated the levels of CaMKIIß, and CaMKIIß siRNA prevented Reelin's effect on the dendritic spine density. Overall, our results are the first to demonstrate that CaMKIIß might be required to enable Reelin to alter the dendritic spine density.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Espinhas Dendríticas/ultraestrutura , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Serina Endopeptidases/metabolismo , Animais , Moléculas de Adesão Celular Neuronais/farmacologia , Células Cultivadas , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/metabolismo , Proteínas da Matriz Extracelular/farmacologia , Hipocampo/citologia , Humanos , Proteínas do Tecido Nervoso/farmacologia , Ratos Sprague-Dawley , Serina Endopeptidases/farmacologia
18.
Biochim Biophys Acta ; 1853(5): 904-17, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25644714

RESUMO

Very Low Density Lipoprotein Receptor (VLDLR) is an apolipoprotein E receptor involved in synaptic plasticity, learning, and memory. However, it is unknown how VLDLR can regulate synaptic and cognitive function. In the present study, we found that VLDLR is present at the synapse both pre- and post-synaptically. Overexpression of VLDLR significantly increases, while knockdown of VLDLR decreases, dendritic spine number in primary hippocampal cultures. Additionally, knockdown of VLDLR significantly decreases synaptophysin puncta number while differentially regulating cell surface and total levels of glutamate receptor subunits. To identify the mechanism by which VLDLR induces these synaptic effects, we investigated whether VLDLR affects dendritic spine formation through the Ras signaling pathway, which is involved in spinogenesis and neurodegeneration. Interestingly, we found that VLDLR interacts with RasGRF1, a Ras effector, and knockdown of RasGRF1 blocks the effect of VLDLR on spinogenesis. Moreover, we found that VLDLR did not rescue the deficits induced by the absence of Ras signaling proteins CaMKIIα or CaMKIIß. Taken together, our results suggest that VLDLR requires RasGRF1/CaMKII to alter dendritic spine formation.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Espinhas Dendríticas/metabolismo , Receptores de LDL/metabolismo , ras-GRF1/metabolismo , Animais , Células COS , Moléculas de Adesão Celular Neuronais/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Cercopithecus aethiops , Espinhas Dendríticas/efeitos dos fármacos , Proteínas da Matriz Extracelular/farmacologia , Técnicas de Silenciamento de Genes , Hipocampo/citologia , Camundongos Knockout , Modelos Biológicos , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ligação Proteica/efeitos dos fármacos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismo , Serina Endopeptidases/farmacologia , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Sinaptofisina/metabolismo
19.
Cell Biochem Biophys ; 72(1): 93-8, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25424359

RESUMO

Hypodermins A (HA), B (HB) and C (HC) are the major proteases secreted by first-instar larvae of Hypoderma lineatum (Diptera: Oestridae). These proteases are involved in the larval migration in the tissue, and prevent the activation of the host immune response. We previously showed that the recombinant HA functions as an immunosuppressive agent which could inhibit the rejection of xenotransplants. In the current study, we cloned the cDNA sequence of HC, which was transfected in Cos7 cells using the pEF1α-IRES-AcGFP expression vector. The Cos7 cells stably expressed HC, and were more resistant to lysis by guinea pig C3 than the control cells. The HC protease degraded the guinea pig C3, and inhibited the complement pathway in vitro. The DNA binding sites of HC were identified using an electrophoretic mobility shift assay. Our findings suggest that the recombinant HC might be useful as an immunosuppressive agent for the inhibition of the xenotransplant rejection.


Assuntos
Complemento C3/química , Imunossupressores/farmacologia , Serina Endopeptidases/farmacologia , Animais , Sítios de Ligação , Células COS , Cercopithecus aethiops , Proteínas do Sistema Complemento , Dípteros/enzimologia , Ensaio de Imunoadsorção Enzimática , Rejeição de Enxerto , Cobaias , Sistema Imunitário , Larva/enzimologia , Reação em Cadeia da Polimerase , Proteínas Recombinantes/farmacologia , Transfecção , Transplante Heterólogo
20.
PLoS One ; 9(10): e111363, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25347319

RESUMO

EspPα and EspI are serine protease autotransporters found in enterohemorrhagic Escherichia coli. They both belong to the SPATE autotransporter family and are believed to contribute to pathogenicity via proteolytic cleavage and inactivation of different key host proteins during infection. Here, we describe the specific cleavage and functional inactivation of serine protease inhibitors (serpins) by EspPα and compare this activity with the related SPATE EspI. Serpins are structurally related proteins that regulate vital protease cascades, such as blood coagulation and inflammatory host response. For the rapid determination of serpin cleavage sites, we applied direct MALDI-TOF-MS or ESI-FTMS analysis of coincubations of serpins and SPATE proteases and confirmed observed cleavage positions using in-gel-digest of SDS-PAGE-separated degradation products. Activities of both serpin and SPATE protease were assessed in a newly developed photometrical assay using chromogenic peptide substrates. EspPα cleaved the serpins α1-protease inhibitor (α1-PI), α1-antichymotrypsin, angiotensinogen, and α2-antiplasmin. Serpin cleavage led to loss of inhibitory function as demonstrated for α1-PI while EspPα activity was not affected. Notably, EspPα showed pronounced specificity and cleaved procoagulatory serpins such as α2-antiplasmin while the anticoagulatory antithrombin III was not affected. Together with recently published research, this underlines the interference of EspPα with hemostasis or inflammatory responses during infection, while the observed interaction of EspI with serpins is likely to be not physiologically relevant. EspPα-mediated serpin cleavage occurred always in flexible loops, indicating that this structural motif might be required for substrate recognition.


Assuntos
Proteínas de Escherichia coli/farmacologia , Proteólise , Serina Endopeptidases/farmacologia , Serpinas/química , Sequência de Aminoácidos , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Humanos , Dados de Sequência Molecular , Serina Endopeptidases/química , Serpinas/metabolismo
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