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1.
Mol Biol (Mosk) ; 54(1): 103-113, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32163394

RESUMO

The degradation of the extracellular matrix plays an important role in the processes of morphogenesis, angio- and neurogenesis, wound healing, inflammation, carcinogenesis and others. The urokinase receptor uPAR is an important participant in processes that regulate extracellular proteolysis, cell adhesion to the extracellular matrix, cell migration along the chemokine gradient, proliferation and survival involving growth factor receptors. The presence of the GPI anchor and the absence of transmembrane and cytoplasmic domains in uPAR promote involvement of membrane partners for the realization of uPAR signal effects. In some studies, involvement of the fMLP chemokine receptor FPRL in the regulation of uPAR-dependent directed migration has been shown. Moreover, the migration of neural progenitors and their maturation into neurons during the formation of brain structures are regulated by chemokine receptors. Despite the data on the role of uPARin the processes of morphogenesis, little is known about the interactions between uPAR and chemokine receptors in guidance processes during nerve growth and regeneration. In the present work, it was shown for the first time that the soluble form of uPAR (suPAR) regulates the trajectory of axon outgrowth, and this effect does not depend on the presence of urokinase. It was also shown that regulation of the directed axon growth is based on the interaction of suPAR with the chemokine receptor FPRL1. These data show new mechanisms for the participation of the urokinase system in the regulation of axon guidance.


Assuntos
Crescimento Neuronal/fisiologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Orientação de Axônios/fisiologia , Movimento Celular , Matriz Extracelular/metabolismo , Humanos , Receptores de Formil Peptídeo/metabolismo , Receptores de Lipoxinas/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
2.
Cell Prolif ; 53(2): e12745, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31889361

RESUMO

OBJECTIVES: The transformation of cytotrophoblasts into mesenchymal-like extravillous trophoblasts is necessary for successful embryo implantation, and the inadequate transformation may cause abortion. Epiregulin, which is a new growth factor, plays important roles in the reproductive processes. The glycosylation of many proteins in reproduction processes is critical. Protein O-fucosyltransferase 1 (poFUT1) is the key enzyme for the biosynthesis of O-fucosylation on the specific glycoproteins. Urokinase-type plasminogen activator (uPA) contains O-fucosylated domain on Thr18 . However, the functions of epiregulin and poFUT1 in the trophoblast epithelial-mesenchymal transition (EMT) process, the regulatory mechanism of epiregulin on poFUT1 and the resulting O-fucosylated uPA remain unclear. MATERIALS AND METHODS: We employed ELISA and Western blot to detect serum levels of epiregulin and poFUT1 from non-pregnancy women, pregnancy women and abortion patients. Using two trophoblast cell lines and a mouse pregnancy model, we investigated the underlying mechanisms of epiregulin and poFUT1 in trophoblast EMT process. RESULTS: Serum levels of epiregulin and poFUT1 were higher in pregnant women compared with non-pregnant women, and their levels were significantly decreased in abortion patients compared with pregnant women. The results showed that epiregulin upregulated poFUT1 expression and increased O-fucosylation on uPA, which further activated the PI3K/Akt signalling pathway, facilitating EMT behaviour of trophoblast cells and embryo implantation in the mouse pregnant model. CONCLUSIONS: Level of epiregulin and poFUT1 is lower in abortion patients than early pregnancy women. Epiregulin promotes trophoblast EMT through O-fucosylation on uPA catalysed by poFUT1. Epiregulin and poFUT1 may be suggested as the potential diagnostic biomarkers and useful treatment targets for abortion.


Assuntos
Epirregulina/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Fucosiltransferases/metabolismo , Trofoblastos/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Adulto , Animais , Linhagem Celular , Feminino , Glicosilação , Humanos , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Gravidez , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Regulação para Cima/fisiologia
3.
J Photochem Photobiol B ; 203: 111773, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31931385

RESUMO

Glioma is the prime cause of cancer allied mortality in adolescent people and it accounts about 80% of all malignant tumours. Eugenol is a major bioactive constituent present in the essential oils with numerous pharmacological benefits including nueroprotective activity. The major drawback of eugenol is its extreme volatile property and oxygen sensitivity therefore we increased the efficacy of drug; eugenol by encapsulating with chitosan polymer. Eugenol loaded chitosan polymer (EuCs) was characterized using FTIR, XRD, SEM, HR-TEM analysis and the encapsulation, drug release efficacy was assessed at in vitro condition. The induction of autophagy and anticancer efficacy of EuCs on glioma cells was evaluated with rat C6 glioma cells using MTT assay, acridine orange staining, immunocytochemical analysis of NFκß protein expression and FLOW cytometric analysis. The anti-metastatic property of Eu-CS was assessed by immunoblotting and RT-PCR analysis of epithelial mesenchymal transition protein expression in EuCs treated rat C6 glioma cells. Our characterization analysis proves that EuCs possess essential physical and functional properties of copolymer to be utilized as a drug. Further the MTT analysis and AO staining confirms even in the presence of oncogenic inducer and autophagic inhibitors, EuCs exhibits apoptotic potency on rat C6 glioma cells. The result of immunocytochemical studies depicts the inhibition of NFκß protein expression and flow cytometry studies confirm apoptosis induction by EuCs. The inhibition of metastasis by EuCs was proven by the decrease in epithelial mesenchymal transition protein expression in Eu-Cs treated rat C6 glioma cells. Over all our results authentically confirms eugenol loaded chitosan nanopolymer persuasively induces apoptosis and inhibits metastasis in rat C6 glioma cells.


Assuntos
Antineoplásicos/química , Apoptose/efeitos dos fármacos , Quitosana/química , Eugenol/química , Metaloproteinase 9 da Matriz/metabolismo , Nanoestruturas/química , Transdução de Sinais/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Portadores de Fármacos/química , Eugenol/farmacologia , Glioma/metabolismo , Glioma/patologia , NF-kappa B/metabolismo , Ratos , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
4.
Res Vet Sci ; 127: 18-26, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31670051

RESUMO

The present study was aimed to understand the development of bovine urolithiasis through measuring oxidative/nitrosative, cortisol and urokinase status and their relationship with disease severity. The cases of buffalo calves with obstructive urolithiasis were selected based on clinical signs, ultrasonography and laboratory examination of blood and urine (creatinine, urea nitrogen). Total 35 urolithiatic buffalo calves (urolithiatic controls) and 6 healthy calves (healthy controls) were used for study. Further, calves of urolithiatic control were sub-divided into two groups based on disease severity: mild (n = 10) and severe (n = 25) form. Oxidative/nitrosative stress were evaluated based on serum malondialdehyde (MDA), glutathione-S-transferase (GST), nitric oxide (NO) parameters. Serum cortisol was evaluated to measure stress hormone status. Serum and urine urokinase were measured and its relationship with disease severity and oxidative/nitrosative stress were established. Obstructive urolithiasis resulted in significant (p < .05) increase in biochemical parameters (creatinine, urea nitrogen), oxidant/antioxidant imbalance (increased MDA, and increased GST), nitrosative stress (increased nitric oxide), upregulated stress hormone (cortisol) in serum and elevated urokinase in serum and urine (p < .05) as compared to healthy controls. In non-parametric Kendall rank correlation (p < .01), a positive correlation was established between urokinase level and disease severity (urolithiasis). It is concluded that in bovine obstructive urolithiasis, increased oxidative/nitrosative stress, cortisol and urokinase play a significant role. The urokinase can help to understand pathophysiology of bovine urolithiasis because of having positive correlation with disease severity (urolithiasis) and stress markers.


Assuntos
Búfalos , Hidrocortisona/sangue , Estresse Nitrosativo/fisiologia , Estresse Oxidativo/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Urolitíase/sangue , Animais , Masculino , Soro/química , Urolitíase/enzimologia
5.
Invest Ophthalmol Vis Sci ; 60(13): 4205-4214, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31618424

RESUMO

Purpose: Plasminogen has been detected in the corneal stroma after tissue injury and interacts with corneal fibroblasts during wound healing. We examined the effect of plasminogen on phagocytic activity of corneal fibroblasts. Methods: Cultured human corneal fibroblasts were exposed to plasminogen and then incubated with fluorescent microparticles before measurement of phagocytic activity by confocal microscopy or flow cytometry. The binding of corneal fibroblasts to immobilized plasminogen was quantitated with a real-time biomolecular interaction assay. The production of urokinase-type plasminogen activator (uPA), matrix metalloproteinases (MMPs), and IL-1ß by corneal fibroblasts was measured by fibrin zymography, by immunoblot analysis or gelatin zymography, or with an enzyme-linked immunosorbent assay, respectively. Results: Plasminogen increased phagocytic activity of corneal fibroblasts in a concentration- and time-dependent manner, with the maximal effect apparent at 30 µg/mL and 24 hours. Corneal fibroblasts bound to immobilized plasminogen in a manner dependent on time and cell number, and the stimulatory effect of plasminogen on phagocytic activity was blocked in the presence of epsilon-aminocaproic acid, an inhibitor of plasminogen binding to cell surface receptors. Plasminogen-induced phagocytic activity was not associated with changes in the production of uPA, MMPs, or IL-1ß by corneal fibroblasts. Conclusions: Plasminogen induced phagocytic activity in corneal fibroblasts in a manner dependent on its binding to the cell surface. This effect was not associated with increased production of proteases or IL-1ß. Thus, plasminogen may promote the clearance of foreign particles or damaged tissue components by corneal fibroblasts early after tissue injury.


Assuntos
Córnea/citologia , Fibroblastos/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Plasminogênio/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Fibroblastos/metabolismo , Humanos , Interleucina-1beta/metabolismo , Metaloproteinases da Matriz/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
6.
BMC Cancer ; 19(1): 692, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31307406

RESUMO

BACKGROUND: The protease uPA and its inhibitor PAI-1 play major roles in hemostasis and are also involved in cancer progression. This is mainly caused by their ability to degrade extracellular matrix-facilitating tumor cell migration. This study aimed to investigate the impact of uPA/PAI-1 and disseminated cytokeratin-positive cells (dCK+) on the outcome and the existence of synergistic effects. METHODS: We retrospectively analyzed a cohort of 480 breast cancer cases with known uPA/PAI-1 and dCK+ status. uPA/PAI-1 was tested on fresh tumor samples using a commercial ELISA test. Bone marrow aspirates were investigated immunocytochemically for CK18. RESULTS: DCK+ cells were identified in 23% of cases. uPA positivity was significantly associated with the occurrence of dCK+ cells (P = 0.028). uPA and PAI-1 were significantly associated with outcome in the subgroup of early-stage cases without chemotherapy. DCK+ cells alone were not prognostic. However, we found synergistic effects. In the subgroup of node-negative cases with and without chemotherapy, the prognostic impact of uPA and PAI-1 was enhanced in cases with additional dCK-positivity (triple +). In cases without chemotherapy, triple-positive status was independently prognostic (HR: 9.3 CI: 1.1-75) next to T stage. CONCLUSIONS: uPA and PAI-1 seem to influence the metastatic potential of dCK+ cells, which underlines its important role in tumor progression.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Queratinas/metabolismo , Células Neoplásicas Circulantes/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Idoso , Idoso de 80 Anos ou mais , Medula Óssea/patologia , Progressão da Doença , Feminino , Seguimentos , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Estudos Retrospectivos
7.
J Nutr Sci Vitaminol (Tokyo) ; 65(2): 171-176, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31061286

RESUMO

Dysfunction of vascular endothelial cells causes the risk of thrombosis. Aim of this study is to evaluate the antithrombotic effect of Okinawa mozuku (brown seaweed, Cladosiphon okamuranus) extract by using cultured vascular endothelial cells and rat carotid arterial thrombosis model induced by ferric chloride (FeCl3). The cell line (TKM-33) established from human umbilical vein endothelial cells were cultured with or without Okinawa mozuku extract. After incubation for 24 h, the conditioned medium was collected to evaluate urokinase-type plasminogen activator (u-PA) activity. Next, rats were fed with water or water containing 5% of Okinawa mozuku extract for 8 wk. After 8 wk of treatments, the rats were provided for the carotid arterial thrombosis model, and fibrinolytic factor and coagulation factor in blood were measured. Okinawa mozuku extract significantly augmented u-PA activity in the conditioned medium. The decrease of carotid artery blood flow induced by 40% FeCl3 injury in rats fed with Okinawa mozuku extract was less than that in control rats. Thus, oral administration of Okinawa mozuku extract prevented thrombus formation in this model. Oral administration of Okinawa mozuku extract significantly increased u-PA activity in euglobulin fraction, compared with control group. On the other hand, platelet aggregation activity, activated partial thromboplastin time, and active PAI-1 level in plasma exhibited no significant differences between control and Okinawa mozuku groups. These results indicate that oral administration of Okinawa mozuku enhances fibrinolytic activity in plasma and prevents thrombus formation which is induced by injury of vascular endothelial cells.


Assuntos
Trombose das Artérias Carótidas/metabolismo , Fibrinolíticos/farmacologia , Feófitas , Extratos Vegetais/farmacologia , Administração Oral , Animais , Modelos Animais de Doenças , Fibrinolíticos/administração & dosagem , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Extratos Vegetais/administração & dosagem , Ratos , Ratos Sprague-Dawley , Ativador de Plasminogênio Tipo Uroquinase/efeitos dos fármacos , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
8.
Breast ; 46: 101-107, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31132475

RESUMO

BACKGROUND: To evaluate whether uPA/PAI-1 protein in hormone receptor-positive (HR+) breast tumor can predict prognosis in early breast cancer (BC). METHODS: 606 women with HR + BC who had ≥5 years of endocrine therapy and in whom tumor tissue was available were included in this analysis. Stromal uPA/PAI-1 protein expression was evaluated by immunohistochemistry and correlated with distant recurrence-free survival (DRFS) and overall survival (OS). RESULTS: Stromal uPA was detected in 292/538 tumors (54.3%) while 269/505 samples (53.3%) exhibited stromal PAI-1. Co-expression of both proteins was found in 163/437 (37.3%) samples. Stromal uPA/PAI-1 co-expression was not associated with tumor size, age, nodal status, grading, or receptor status. Tumor stroma with both uPA and PAI-1 protein expression were more likely to have a shorter DRFS (HR: 1.87; 95%CI 1.18-2.96; p = 0.007) and OS (HR: 1.29; 95%CI 0.93-1.80; p = 0.129) than women without uPA/PAI-1 co-expression. After a median follow-up of 10 years, women with uPA/PAI-1-positive tumors experienced a significantly shorter DRFS (86.5% vs 72.4%; p < 0.001) and OS (70.4% vs 58.9%; p = 0.020) compared to women with uPA/PAI-1 negative tumors. CONCLUSION: Stromal co-expression of uPA and PAI-1 in breast cancer predicts poor DRFS and OS in postmenopausal women with HR + early-stage BC who receive endocrine therapy.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Mama/citologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Idoso , Antineoplásicos Hormonais/uso terapêutico , Biomarcadores Tumorais/genética , Neoplasias da Mama/tratamento farmacológico , Intervalo Livre de Doença , Receptores ErbB/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Pós-Menopausa , Prognóstico , Fatores de Risco , Células Estromais/metabolismo , Resultado do Tratamento
9.
Chem Biol Interact ; 306: 62-69, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30980805

RESUMO

Myocardial fibrosis is a critical event during septic shock. Upregulation in the fibrosis signaling cascade proteins such as fibroblast growth factor (FGF), urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA) and activation of matrix metalloproteinases (MMPs) are widely associated with the development of myocardial infarction, dilated cardiomyopathy, cardiac fibrosis and heart failure. However, evidences suggest that the common upstream mediators of fibrosis cascade play little role in cardiac fibrosis induced by LPS; further, it is unknown if LPS directly triggers the expressions and/or activity of FGF-2, uPA, tPA, MMP-2 and MMP-9 in cardiac fibroblasts. In the present study, we treated primary cultures of cardiac fibroblasts with LPS to explore whether LPS upregulates FGF-2, uPA, tPA, MMP-2, MMP-9 and enhance cellular migration. Further the precise molecular and cellular mechanisms behind these LPS induced responses were identified. Inhibition assays on MAPKs using U0126 (ERK1/2 inhibitor), SB203580 (p38 MAPK inhibitor), SP600125 (JNK1/2 inhibitor), CsA (calcineurin inhibitor) and QNZ (NFκB inhibitor) show that LPS-induced upregulation of FGF-2, uPA, MMP-2 and MMP-9 in cardiac fibroblasts was mediated through ERK1/2 signaling. Collectively, our results provide a link between LPS-induced cardiac dysfunction and ERK1/2 signaling pathway and thereby implies ERK1/2 as a possible target to regulate LPS induced upregulation of FGF-2, uPA, MMP-2, MMP-9 and cellular migration in cardiac fibroblasts.


Assuntos
Movimento Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Miocárdio/citologia , Regulação para Cima/efeitos dos fármacos , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fibroblastos/citologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
10.
Anal Chim Acta ; 1064: 1-10, 2019 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-30982506

RESUMO

The unraveling of enzymatic reactions, especially identification of enzymatic substrates or products, is important to elucidate biological processes. Here a selenium-isotopic signature for mass spectrometric identification of enzymatic-related species is demonstrated by using selenium-containing peptides (SePeps) as substrates. Thus a strategy is proposed for rapid and precise assay of multiple enzyme activity. These SePeps can be synthesized by introduction of one selenomethionine residue in the sequence and simply identified in the full-scan mode with the feature of distinctive selenium-isotopic distribution without MS/MS verifications, which proposes a novel solution to the specific identification of enzyme-related species, allows to exclude the interferences of species with tiny mass differences in bio-samples, and meanwhile can offer a judgement on data accuracy for the analysis of enzyme activities. As a proof-of-concept, a method for multiple analysis of two representative enzymes in MCF-7 cell lysate has been developed with the isotopic peak areas of either SePep substrates or enzymatic products with the top intensities. These results could be the foundation to extend the method for more complicated enzyme systems. The selenium-isotopic signature provides a powerful protocol for high-throughput assays of peptide-metabolizing enzymes with enhanced confidence and can be extended to screen enzymatic reaction-related substrates.


Assuntos
Proteína Quinase 3 Ativada por Mitógeno/análise , Peptídeos/química , Selênio/química , Ativador de Plasminogênio Tipo Uroquinase/análise , Ensaios de Triagem em Larga Escala , Humanos , Marcação por Isótopo , Células MCF-7 , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Radioisótopos de Selênio , Espectrometria de Massas em Tandem , Células Tumorais Cultivadas , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
11.
In Vivo ; 33(3): 801-810, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31028200

RESUMO

BACKGROUND/AIM: Evidence has indicated that fisetin induces cytotoxic effects in human cancer cell lines, including the inhibition of cell migration and invasion, however, the exact molecular mechanism of action of fisetin in human osteosarcoma cells remains unclear. MATERIALS AND METHODS: The anti-metastatic mechanisms of fisetin in human osteosarcoma U-2 OS cells were investigated in vitro. RESULTS: Fisetin reduced the viability of cells at different concentrations (2.5, 5 and 10 µM) as measured by flow cytometric assay. Fisetin suppressed cell mobility, migration and invasion of U-2 OS cells, as shown by wound healing assay and transwell filter chambers, respectively. The gelatin zymography assay showed that fisetin inhibited MMP-2 activity in U-2 OS cells. Results from western blotting indicated that fisetin reduced the levels of pEGFR, SOS-1, GRB2, Ras, PKC, p-ERK1/2, p-JNK, p-p-38, VEGF, FAK, RhoA, PI3K, p-AKT, NF-ĸB, uPA, MMP-7, MMP-9, and MMP-13, but increased GSK3ß and E-cadherin in U-2 OS cells after 48 h of treatment. CONCLUSION: Fisetin can be used in the future, as a target for the treatment of metastasis of human osteosarcoma cells.


Assuntos
Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Flavonoides/farmacologia , Quinase 1 de Adesão Focal/metabolismo , NF-kappa B/metabolismo , Osteossarcoma/genética , Osteossarcoma/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Modelos Biológicos , Transdução de Sinais
12.
J Biol Chem ; 294(18): 7403-7418, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-30894413

RESUMO

The urokinase receptor (uPAR) is a founding member of a small protein family with multiple Ly6/uPAR (LU) domains. The motif defining these LU domains contains five plesiotypic disulfide bonds stabilizing its prototypical three-fingered fold having three protruding loops. Notwithstanding the detailed knowledge on structure-function relationships in uPAR, one puzzling enigma remains unexplored. Why does the first LU domain in uPAR (DI) lack one of its consensus disulfide bonds, when the absence of this particular disulfide bond impairs the correct folding of other single LU domain-containing proteins? Here, using a variety of contemporary biophysical methods, we found that reintroducing the two missing half-cystines in uPAR DI caused the spontaneous formation of the corresponding consensus 7-8 LU domain disulfide bond. Importantly, constraints due to this cross-link impaired (i) the binding of uPAR to its primary ligand urokinase and (ii) the flexible interdomain assembly of the three LU domains in uPAR. We conclude that the evolutionary deletion of this particular disulfide bond in uPAR DI may have enabled the assembly of a high-affinity urokinase-binding cavity involving all three LU domains in uPAR. Of note, an analogous neofunctionalization occurred in snake venom α-neurotoxins upon loss of another pair of the plesiotypic LU domain half-cystines. In summary, elimination of the 7-8 consensus disulfide bond in the first LU domain of uPAR did have significant functional and structural consequences.


Assuntos
Evolução Biológica , Deleção de Sequência , Sulfetos/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Fenômenos Biofísicos , Quimotripsina/metabolismo , Glicosilação , Cinética , Ligantes , Dobramento de Proteína , Proteólise , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Ativador de Plasminogênio Tipo Uroquinase/química
13.
BMC Vet Res ; 15(1): 82, 2019 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-30849965

RESUMO

BACKGROUND: The Shimen strain of classical swine fever (CSF) virus (CSFV) causes CSF, which is mainly characterised by disseminated intravascular haemorrhage. Macrophages are an essential component of innate immunity against pathogenic microorganisms; however, the role of macrophages in CSF pathogenesis remains unclear. To illuminate the infective mechanism of CSFV, we used gene co-expression networks derived from macrophages infected with CSFV Shimen and CSFV C as well as uninfected macrophages to screen key regulatory genes, and their contributions to the pathogenesis of CSF were discussed. RESULTS: Vascular endothelial growth factor A (VEGFA) and plasminogen activator, urokinase (PLAU, which encodes urokinase-type plasminogen activator [uPA]) were identified as coordinated genes expressed in macrophages by gene co-expression networks. Quantitative polymerase chain reaction and western blot analysis confirmed that VEGFA and PLAU were significantly up-regulated at both the transcription and translation levels after infection. Further, confocal microscopy analysis proposed that the VEGFA and uPA proteins were temporally co-localised with the CSFV protein E2. CONCLUSIONS: Our findings suggest that co-expression of VEGFA and PLAU in macrophages contributes to CSFV Shimen infection and serves as a significant avenue for the strain to form an inflammatory microenvironment, providing new insight into the mechanisms of CSF caused by a virulent strain.


Assuntos
Peste Suína Clássica/virologia , Macrófagos/virologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Vírus da Febre Suína Clássica/fisiologia , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Macrófagos/metabolismo , Sus scrofa , Suínos , Ativador de Plasminogênio Tipo Uroquinase/genética , Fator A de Crescimento do Endotélio Vascular/genética , Virulência
14.
Biochem Biophys Res Commun ; 512(2): 256-262, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-30879770

RESUMO

Secreted frizzled-related protein 5 (Sfrp5), an anti-inflammatory adipocytokine secreted by adipocytes, plays an important role in energy metabolism. Studies have shown that Sfrp5 plays a salutary role in the regulation of lipid metabolism, but its specific mechanism needs further study. In this study, we showed a lower level of Sfrp5 in subjects with diet-induced obesity than in normal-diet C57BL/6J mice. To further investigate Sfrp5-associated proteins in HepG2 cells, the immunoprecipitation assay and silver staining assay were performed. By mass spectrometry analysis, secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide (Slurp-1) was found to interact with Sfrp5. Further verification was obtained through the positive and reverse immunoprecipitation assay. In this study, we found that the sole over-expression of Slurp1 promoted the expression of Sfrp5 in palmitate-induced HepG2 cells. In addition, our experimental evidence shows that the role of Slurp1 in decreasing TG level was greatly reduced in the case of suppression of expression of Sfrp5 in palmitate-induced model cells. Our study further found that Slurp1 regulates the synthesis pathway of triglyceride by interacting with Sfrp5 to alleviate triglyceride accumulation in palmitate-induced model cells. In summary, we are the first to discover the interaction between Sfrp5 and Slurp1, and we found that Slurp1 may regulate the accumulation of TG through Sfrp5.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antígenos Ly/metabolismo , Fígado Gorduroso/metabolismo , Hepatócitos/metabolismo , Triglicerídeos/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Fígado Gorduroso/patologia , Células Hep G2 , Hepatócitos/patologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , Mapas de Interação de Proteínas
15.
Int J Mol Sci ; 20(3)2019 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-30709025

RESUMO

Systemic sclerosis (SSc) is a connective tissue disease of autoimmune origin characterized by vascular dysfunction and extensive fibrosis of the skin and visceral organs. Vascular dysfunction is caused by endothelial cell (EC) apoptosis, defective angiogenesis, defective vasculogenesis, endothelial-to-mesenchymal transition (EndoMT), and coagulation abnormalities, and exacerbates the disease. Fibrinolytic regulators, such as plasminogen (Plg), plasmin, α2-antiplasmin (α2AP), tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA) and its receptor (uPAR), plasminogen activator inhibitor 1 (PAI-1), and angiostatin, are considered to play an important role in the maintenance of endothelial homeostasis, and are associated with the endothelial dysfunction of SSc. This review considers the roles of fibrinolytic factors in vascular dysfunction of SSc.


Assuntos
Endotélio/citologia , Fibrinolíticos/metabolismo , Escleroderma Sistêmico/patologia , Angiostatinas/metabolismo , Apoptose , Endotélio/metabolismo , Endotélio/patologia , Fibrinolisina/metabolismo , Humanos , Plasminogênio/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Escleroderma Sistêmico/metabolismo , Transdução de Sinais , Ativador de Plasminogênio Tecidual/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , alfa 2-Antiplasmina/metabolismo
16.
Cell Biol Int ; 43(3): 313-322, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30632648

RESUMO

The functional and physical interaction between mitochondria and the endoplasmic reticulum (ER) has been the subject of intense study. To test the effect of this interaction on macrophage inflammatory activation, the human macrophage-like monocytic leukemia cell line THP-1 was treated with oligomycin, rotenone, or sodium azide, which induce mitochondrial dysfunction (MD) by blocking the electron transport chain (ETC). MD induced by these agents triggered activation of various sensors and markers of ER stress. This linkage affected macrophage function since LPS-induced expression of IL-23 was enhanced by the MD inducers, and this enhancing effect was abolished by inhibition of pancreatic endoplasmic reticulum kinase (PERK) activity. This MD-mediated ER stress may be universal since it was observed in human embryonic kidney HEK293 cells and colon cancer SW480 cells. On the other hand, MD regulated LPS-induced activation of the AKT/GSK3ß/ß-catenin pathway in a manner not affected by inhibition of PERK or inositol-requiring enzyme 1α (IRE1α) activities. These results indicate that the occurrence of MD can lead to ER stress and these two events, separately or in combination, can affect various cellular processes.


Assuntos
Estresse do Retículo Endoplasmático , Mediadores da Inflamação/metabolismo , Mitocôndrias/patologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/metabolismo , Células HEK293 , Humanos , Interleucina-23/metabolismo , Lipopolissacarídeos/farmacologia , Mitocôndrias/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células THP-1 , Fator de Transcrição CHOP/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , beta Catenina/metabolismo
17.
Chem Commun (Camb) ; 55(11): 1651-1654, 2019 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-30657484

RESUMO

We develop a novel peptide-based four-color fluorescent polydopamine nanoprobe for multiplexed sensing and imaging of tumor-related proteases in living cells. This nanoprobe responds rapidly and selectively, enabling simultaneous and high-contrast imaging of multiple proteases in living cells. Furthermore, it could detect the changes of protease expression in living cells.


Assuntos
Corantes Fluorescentes/química , Indóis/química , Nanopartículas/química , Peptídeo Hidrolases/metabolismo , Peptídeos/química , Polímeros/química , Carbocianinas/química , Catepsina B/metabolismo , Linhagem Celular Tumoral , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Microscopia de Fluorescência , Sondas Moleculares/química , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
18.
J Biol Chem ; 294(10): 3794-3805, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30651349

RESUMO

Protein sequences of members of the plasminogen activation system are present throughout the entire vertebrate phylum. This important and well-described proteolytic cascade is governed by numerous protease-substrate and protease-inhibitor interactions whose conservation is crucial to maintaining unchanged protein function throughout evolution. The pressure to preserve protein-protein interactions may lead to either co-conservation or covariation of binding interfaces. Here, we combined covariation analysis and structure-based prediction to analyze the binding interfaces of urokinase (uPA):plasminogen activator inhibitor-1 (PAI-1) and uPA:plasminogen complexes. We detected correlated variation between the S3-pocket-lining residues of uPA and the P3 residue of both PAI-1 and plasminogen. These residues are known to form numerous polar interactions in the human uPA:PAI-1 Michaelis complex. To test the effect of mutations that correlate with each other and have occurred during mammalian diversification on protein-protein interactions, we produced uPA, PAI-1, and plasminogen from human and zebrafish to represent mammalian and nonmammalian orthologs. Using single amino acid point substitutions in these proteins, we found that the binding interfaces of uPA:plasminogen and uPA:PAI-1 may have coevolved to maintain tight interactions. Moreover, we conclude that although the interaction areas between protease-substrate and protease-inhibitor are shared, the two interactions are mechanistically different. Compared with a protease cleaving its natural substrate, the interaction between a protease and its inhibitor is more complex and involves a more fine-tuned mechanism. Understanding the effects of evolution on specific protein interactions may help further pharmacological interventions of the plasminogen activation system and other proteolytic systems.


Assuntos
Evolução Molecular , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ativadores de Plasminogênio/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Modelos Moleculares , Ativadores de Plasminogênio/antagonistas & inibidores , Ativadores de Plasminogênio/química , Ligação Proteica , Conformação Proteica , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
19.
Hepatol Int ; 13(2): 180-189, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30600477

RESUMO

BACKGROUND: Plasminogen activator inhibitor 2 (PAI2) has been shown to be associated with invasive phenotypes and prognosis in hepatocellular carcinoma (HCC). However, its biological roles and underlying mechanisms in invasion of HCC have not been explored. The present study aimed to address the issues. METHODS: First, sub-lines in that PAI2 was stably overexpressed and silenced were established based on MHCC97H and BEL7402 cell lines, respectively. Wound-healing and transwell assays were applied to evaluate cell migration and invasion. Urokinase-type plasminogen activator (uPA) activity was measured using an ELISA kit. Real-time RT-PCR and western blotting were used to show gene expression at mRNA and protein levels. E2F1 expression in human specimens was determined by tissue microarray-based immunohistochemical staining. RESULTS: The sub-lines, MHCC97H-PAI2 and BEL7402-siPAI2, were successfully established. The two sub-lines carried much lower and higher migration and invasion powers, respectively, in contrast to the controls. In MHCC97H-PAI2 sub-line, intra-medium uPA activity was significantly decreased, while RB expression was obviously elevated, compared with the controls. The BEL7402-siPAI2 sub-line presented the opposite trend. To identify the role of RB/E2F1 pathway, we transiently overexpressed E2F1 in MHCC97H-PAI2 sub-line, and largely reversed the inhibitory effects of PAI2 on cell migration and invasion, through regulating multiple matrix metalloproteinases and epithelial-mesenchymal transition. In HCC specimens, E2F1 expression was much higher in tumor than in non-tumor tissues, and was significantly related to Edmondson-Steiner grade, overall as well as tumor-free survival. CONCLUSIONS: Our data suggest that PAI2 inhibits invasive potential of HCC cells via uPA- and RB/E2F1-related mechanisms.


Assuntos
Carcinoma Hepatocelular/patologia , Movimento Celular/genética , Fator de Transcrição E2F1/genética , Neoplasias Hepáticas/patologia , Inibidor 2 de Ativador de Plasminogênio/genética , Adulto , Idoso , Linhagem Celular Tumoral , Citoproteção/genética , Fator de Transcrição E2F1/metabolismo , Feminino , Expressão Gênica , Inativação Gênica , Humanos , Masculino , Metaloproteinase 14 da Matriz/genética , Metaloproteinase 14 da Matriz/metabolismo , Metaloproteinase 15 da Matriz/genética , Metaloproteinase 15 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Pessoa de Meia-Idade , Invasividade Neoplásica , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
20.
ChemMedChem ; 14(1): 119-131, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30548204

RESUMO

There is growing interest in the use of structure-based virtual screening to identify small molecules that inhibit challenging protein-protein interactions (PPIs). In this study, we investigated how effectively chemical library members docked at the PPI interface mimic the position of critical side-chain residues known as "hot spots". Three compound collections were considered, a commercially available screening collection (ChemDiv), a collection of diversity-oriented synthesis (DOS) compounds that contains natural-product-like small molecules, and a library constructed using established reactions (the "screenable chemical universe based on intuitive data organization", SCUBIDOO). Three different tight PPIs for which hot-spot residues have been identified were selected for analysis: uPAR⋅uPA, TEAD4⋅Yap1, and CaV α⋅CaV ß. Analysis of library physicochemical properties was followed by docking to the PPI receptors. A pharmacophore method was used to measure overlap between small-molecule substituents and hot-spot side chains. Fragment-like conformationally restricted small molecules showed better hot-spot overlap for interfaces with well-defined pockets such as uPAR⋅uPA, whereas better overlap was observed for more complex DOS compounds in interfaces lacking a well-defined binding site such as TEAD4⋅Yap1. Virtual screening of conformationally restricted compounds targeting uPAR⋅uPA and TEAD4⋅Yap1 followed by experimental validation reinforce these findings, as the best hits were fragment-like and had few rotatable bonds for the former, while no hits were identified for the latter. Overall, such studies provide a framework for understanding PPIs in the context of additional chemical matter and new PPI definitions.


Assuntos
Produtos Biológicos/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Produtos Biológicos/síntese química , Produtos Biológicos/química , Canais de Cálcio/química , Canais de Cálcio/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Proteínas Musculares/antagonistas & inibidores , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , Receptores de Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
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