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1.
PLoS Pathog ; 17(1): e1009233, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33476327

RESUMO

The spike (S) protein of Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) binds to a host cell receptor which facilitates viral entry. A polybasic motif detected at the cleavage site of the S protein has been shown to broaden the cell tropism and transmissibility of the virus. Here we examine the properties of SARS-CoV-2 variants with mutations at the S protein cleavage site that undergo inefficient proteolytic cleavage. Virus variants with S gene mutations generated smaller plaques and exhibited a more limited range of cell tropism compared to the wild-type strain. These alterations were shown to result from their inability to utilize the entry pathway involving direct fusion mediated by the host type II transmembrane serine protease, TMPRSS2. Notably, viruses with S gene mutations emerged rapidly and became the dominant SARS-CoV-2 variants in TMPRSS2-deficient cells including Vero cells. Our study demonstrated that the S protein polybasic cleavage motif is a critical factor underlying SARS-CoV-2 entry and cell tropism. As such, researchers should be alert to the possibility of de novo S gene mutations emerging in tissue-culture propagated virus strains.


Assuntos
/genética , Serina Endopeptidases/deficiência , Glicoproteína da Espícula de Coronavírus/genética , Sequência de Aminoácidos , Animais , Células CACO-2 , Linhagem Celular , Chlorocebus aethiops , Células HEK293 , Humanos , Mutação , /crescimento & desenvolvimento , Alinhamento de Sequência , Inoculações Seriadas , Células Vero , Tropismo Viral
2.
Arterioscler Thromb Vasc Biol ; 40(10): 2391-2403, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32787521

RESUMO

OBJECTIVE: Reelin, a secreted glycoprotein, was originally identified in the central nervous system, where it plays an important role in brain development and maintenance. In the cardiovascular system, reelin plays a role in atherosclerosis by enhancing vascular inflammation and in arterial thrombosis by promoting platelet adhesion, activation, and thrombus formation via APP (amyloid precursor protein) and GP (glycoprotein) Ib. However, the role of reelin in hemostasis and arterial thrombosis is not fully understood to date. Approach and Results: In the present study, we analyzed the importance of reelin for cytoskeletal reorganization of platelets and thrombus formation in more detail. Platelets release reelin to amplify alphaIIb beta3 integrin outside-in signaling by promoting platelet adhesion, cytoskeletal reorganization, and clot retraction via activation of Rho GTPases RAC1 (Ras-related C3 botulinum toxin substrate) and RhoA (Ras homolog family member A). Reelin interacts with the collagen receptor GP (glycoprotein) VI with subnanomolar affinity, induces tyrosine phosphorylation in a GPVI-dependent manner, and supports platelet binding to collagen and GPVI-dependent RAC1 activation, PLC gamma 2 (1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase gamma-2) phosphorylation, platelet activation, and aggregation. When GPVI was deleted from the platelet surface by antibody treatment in reelin-deficient mice, thrombus formation was completely abolished after injury of the carotid artery while being only reduced in either GPVI-depleted or reelin-deficient mice. CONCLUSIONS: Our study identified a novel signaling pathway that involves reelin-induced GPVI activation and alphaIIb beta3 integrin outside-in signaling in platelets. Loss of both, GPVI and reelin, completely prevents stable arterial thrombus formation in vivo suggesting that inhibiting reelin-platelet-interaction might represent a novel strategy to avoid arterial thrombosis in cardiovascular disease.


Assuntos
Plaquetas/enzimologia , Lesões das Artérias Carótidas/enzimologia , Moléculas de Adesão Celular Neuronais/sangue , Proteínas da Matriz Extracelular/sangue , Proteínas do Tecido Nervoso/sangue , Neuropeptídeos/sangue , Fosfolipase C gama/sangue , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Serina Endopeptidases/sangue , Trombose/enzimologia , Proteínas rac1 de Ligação ao GTP/sangue , Proteína rhoA de Ligação ao GTP/sangue , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Coagulação Sanguínea , Lesões das Artérias Carótidas/sangue , Lesões das Artérias Carótidas/etiologia , Moléculas de Adesão Celular Neuronais/deficiência , Moléculas de Adesão Celular Neuronais/genética , Retração do Coágulo , Citoesqueleto/enzimologia , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/deficiência , Proteínas da Matriz Extracelular/genética , Camundongos da Linhagem 129 , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Ativação Plaquetária , Serina Endopeptidases/deficiência , Serina Endopeptidases/genética , Transdução de Sinais , Trombose/sangue , Trombose/etiologia
3.
PLoS One ; 15(6): e0234407, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32511276

RESUMO

Testisin (encoded by PRSS21) is a membrane anchored serine protease, which is tethered to the cell surface via a glycosylphosphatidylinositol (GPI)-anchor. While testisin is found in abundance in spermatozoa, it is also expressed in microvascular endothelial cells where its function is unknown. Here we identify testisin as a novel regulator of physiological hormone-induced angiogenesis and microvascular endothelial permeability. Using a murine model of rapid physiological angiogenesis during corpus luteal development in the ovary, we found that mice genetically deficient in testisin (Prss21-/-) show a substantially increased incidence of hemorrhages which are significantly more severe than in littermate control Prss21+/+ mice. This phenotype was associated with increased vascular leakiness, demonstrated by a greater accumulation of extravasated Evans blue dye in Prss21-/- ovaries. Live cell imaging of in vitro cultured microvascular endothelial cells depleted of testisin by siRNA knockdown revealed that loss of testisin markedly impaired reorganization and tubule-like formation on Matrigel basement membranes. Moreover testisin siRNA knockdown increased the paracellular permeability to FITC-albumin across endothelial cell monolayers, which was associated with decreased expression of the adherens junction protein VE-cadherin and increased levels of phospho(Tyr658)-VE-cadherin, without affecting the levels of the tight junction proteins occludin and claudin-5, or ZO-1. Decreased expression of VE-cadherin in the neovasculature of Prss21-/- ovaries was also observed without marked differences in endothelial cell content, vascular claudin-5 expression or pericyte recruitment. Together, these data identify testisin as a novel regulator of VE-cadherin adhesions during angiogenesis and indicate a potential new target for regulating neovascular integrity and associated pathologies.


Assuntos
Permeabilidade Capilar/fisiologia , Corpo Lúteo/irrigação sanguínea , Neovascularização Fisiológica , Serina Endopeptidases/deficiência , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Permeabilidade Capilar/genética , Células Cultivadas , Corpo Lúteo/patologia , Corpo Lúteo/fisiopatologia , Feminino , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/deficiência , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/fisiologia , Técnicas de Silenciamento de Genes , Hemorragia/etiologia , Hemorragia/genética , Hemorragia/fisiopatologia , Humanos , Luteinização/genética , Luteinização/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Fisiológica/genética , Fenótipo , Serina Endopeptidases/genética , Serina Endopeptidases/fisiologia
4.
J Virol ; 94(1)2019 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-31597759

RESUMO

Influenza A virus (IAV) and influenza B virus (IBV) cause yearly epidemics with significant morbidity and mortality. When zoonotic IAVs enter the human population, the viral hemagglutinin (HA) requires adaptation to achieve sustained virus transmission. In contrast, IBV has been circulating in humans, its only host, for a long period of time. Whether this entailed adaptation of IBV HA to the human airways is unknown. To address this question, we compared two seasonal IAVs (A/H1N1 and A/H3N2) and two IBVs (B/Victoria and B/Yamagata lineages) with regard to host-dependent activity of HA as the mediator of membrane fusion during viral entry. We first investigated proteolytic activation of HA by covering all type II transmembrane serine protease (TTSP) and kallikrein enzymes, many of which proved to be present in human respiratory epithelium. The IBV HA0 precursor is cleaved by a broader panel of TTSPs and activated with much higher efficiency than IAV HA0. Accordingly, knockdown of a single protease, TMPRSS2, abrogated spread of IAV but not IBV in human respiratory epithelial cells. Second, the HA fusion pH values proved similar for IBV and human-adapted IAVs (with one exception being the HA of 1918 IAV). Third, IBV HA exhibited higher expression at 33°C, a temperature required for membrane fusion by B/Victoria HA. This indicates pronounced adaptation of IBV HA to the mildly acidic pH and cooler temperature of human upper airways. These distinct and intrinsic features of IBV HA are compatible with extensive host adaptation during prolonged circulation of this respiratory virus in the human population.IMPORTANCE Influenza epidemics are caused by influenza A and influenza B viruses (IAV and IBV, respectively). IBV causes substantial disease; however, it is far less studied than IAV. While IAV originates from animal reservoirs, IBV circulates in humans only. Virus spread requires that the viral hemagglutinin (HA) is active and sufficiently stable in human airways. We resolve here how these mechanisms differ between IBV and IAV. Whereas human IAVs rely on one particular protease for HA activation, this is not the case for IBV. Superior activation of IBV by several proteases should enhance shedding of infectious particles. IBV HA exhibits acid stability and a preference for 33°C, indicating pronounced adaptation to the human upper airways, where the pH is mildly acidic and a cooler temperature exists. These adaptive features are rationalized by the long existence of IBV in humans and may have broader relevance for understanding the biology and evolution of respiratory viruses.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza B/genética , Influenza Humana/virologia , Pulmão/virologia , Replicação Viral/genética , Linhagem Celular , Células Epiteliais/patologia , Células Epiteliais/virologia , Regulação da Expressão Gênica , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Interações Hospedeiro-Patógeno/genética , Humanos , Concentração de Íons de Hidrogênio , Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vírus da Influenza A Subtipo H3N2/metabolismo , Vírus da Influenza A Subtipo H3N2/patogenicidade , Vírus da Influenza B/metabolismo , Vírus da Influenza B/patogenicidade , Influenza Humana/patologia , Calicreínas/classificação , Calicreínas/genética , Calicreínas/metabolismo , Pulmão/patologia , Fusão de Membrana , Proteínas de Membrana/classificação , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteólise , Mucosa Respiratória/patologia , Mucosa Respiratória/virologia , Serina Endopeptidases/deficiência , Serina Endopeptidases/genética , Serina Proteases/classificação , Serina Proteases/genética , Serina Proteases/metabolismo , Especificidade da Espécie , Temperatura , Internalização do Vírus
5.
J Pharmacol Exp Ther ; 370(3): 437-446, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31248979

RESUMO

Multiple sclerosis is a neurodegenerative disease affecting predominantly female patients between 20 and 45 years of age. We previously reported the significant contribution of mouse mast cell protease 4 (mMCP-4) in the synthesis of endothelin-1 (ET-1) in healthy mice and in a murine model of experimental autoimmune encephalomyelitis (EAE). In the current study, the cardiovascular effects of ET-1 and big endothelin-1 (big-ET-1) administered systemically or intrathecally were assessed in the early preclinical phase of EAE in telemetry instrumented/conscious mice. Chymase-specific enzymatic activity was also measured in the lung, brain, and mast cell extracts in vitro. Finally, the impact of EAE immunization was studied on the pulmonary and brain mRNA expression of different genes of the endothelin pathway, interleukin-33 (IL-33), and monitoring of immunoreactive tumor necrosis factor-α (TNF-α). Systemically or intrathecally administered big-ET-1 triggered increases in blood pressure in conscious mice. One week post-EAE, the pressor responses to big-ET-1 were potentiated in wild-type (WT) mice but not in mMCP-4 knockout (KO) mice. EAE triggered mMCP-4-specific activity in cerebral homogenates and peritoneal mast cells. Enhanced pulmonary, but not cerebral preproendothelin-1 and IL-33 mRNA were found in KO mice and further increased 1 week post-EAE immunization, but not in WT animals. Finally, TNF-α levels were also increased in serum from mMCP-4 KO mice, but not WT, 1 week post-EAE. Our study suggests that mMCP-4 activity is enhanced both centrally and systemically in a mouse model of EAE.


Assuntos
Encefalomielite Autoimune Experimental/metabolismo , Endotelina-1/administração & dosagem , Endotelina-1/farmacologia , Serina Endopeptidases/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , Encefalomielite Autoimune Experimental/fisiopatologia , Técnicas de Inativação de Genes , Hemodinâmica/efeitos dos fármacos , Injeções Espinhais , Interleucina-33/deficiência , Interleucina-33/genética , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Serina Endopeptidases/deficiência , Serina Endopeptidases/genética , Regulação para Cima/efeitos dos fármacos
6.
J Gen Virol ; 100(7): 1073-1078, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31099738

RESUMO

The surface protein haemagglutinin (HA) of influenza A viruses (IAV) needs to be cleaved by a host protease to become functional. Here, we investigated if IAV of the H10 subtype also requires TMPRSS2 for replication and pathogenesis in mice. We first showed in cell culture that TMPRSS2 is able to cleave H10-HA. When Tmprss2-/- deficient mice were infected with a re-assorted virus H10-HA, they did not lose body weight and no viral replication was observed in contrast to wild-type mice. Histopathological analysis showed that inflammatory lesions in the lung of Tmprss2-/- mice were reduced compared to wild-type mice. In addition, no viral antigen was detected in the lungs of Tmprss2-/- mice and no evidence for HA cleavage was observed. We conclude from these studies that TMPRSS2 activity is also essential for in vivo replication and pathogenesis of H10 IAV.


Assuntos
Vírus da Influenza A/fisiologia , Influenza Humana/enzimologia , Serina Endopeptidases/genética , Animais , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/patogenicidade , Influenza Humana/genética , Influenza Humana/imunologia , Influenza Humana/virologia , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Serina Endopeptidases/deficiência , Virulência , Replicação Viral
7.
Metallomics ; 11(5): 959-967, 2019 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-30888356

RESUMO

Manganese is an essential metal that is required for a wide range of biological functions. Ferroportin (FPN), the only known cellular exporter of iron, has also been proposed to play a role in manganese export, but this relationship is incompletely understood. To investigate this in more detail in vivo, we examined the relative distributions of manganese and iron in TMPRSS6 deficient mice, which are characterized by constitutively high expression of the iron regulatory hormone hepcidin and, consequently, very low FPN levels in their tissues. Tmprss6-/- mice showed frank iron deficiency and reduced iron levels in most tissues, consistent with FPN playing an important role in the distribution of this metal, but manganese levels were largely unaffected. Associated studies using intestine-specific FPN knockout mice showed that loss of FPN significantly reduced the dietary absorption of iron, but had no effect on manganese intake. Taken together, our data suggest that FPN does not play a major role in Mn transport in vivo. They do not exclude a minor role for FPN in manganese homeostasis, nor the possibility that the transporter may be relevant at high Mn levels, but at physiological levels of this metal, other transport proteins appear to be more important.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Hepcidinas/metabolismo , Homeostase , Manganês/metabolismo , Animais , Regulação da Expressão Gênica , Hepcidinas/genética , Ferro/metabolismo , Manganês/sangue , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade de Órgãos , Serina Endopeptidases/deficiência , Serina Endopeptidases/metabolismo
8.
J Bacteriol ; 201(11)2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30858298

RESUMO

In Escherichia coli, the periplasmic protease DegP plays a critical role in degrading misfolded outer membrane proteins (OMPs). Consequently, mutants lacking DegP display a temperature-sensitive growth defect, presumably due to the toxic accumulation of misfolded OMPs. The Tol-Pal complex plays a poorly defined but an important role in envelope biogenesis, since mutants defective in this complex display a classical periplasmic leakage phenotype. Double mutants lacking DegP and an intact Tol-Pal complex display exaggerated temperature-sensitive growth defects and the leaky phenotype. Two revertants that overcome the temperature-sensitive growth phenotype carry missense mutations in the degS gene, resulting in D102V and D320A substitutions. D320 and E317 of the PDZ domain of DegS make salt bridges with R178 of DegS's protease domain to keep the protease in the inactive state. However, weakening of the tripartite interactions by D320A increases DegS's basal protease activity. Although the D102V substitution is as effective as D320A in suppressing the temperature-sensitive growth phenotype, the molecular mechanism behind its effect on DegS's protease activity is unclear. Our data suggest that the two DegS variants modestly activate RseA-controlled, σE-mediated envelope stress response pathway and elevate periplasmic protease activity to restore envelope homeostasis. Based on the release of a cytoplasmic enzyme in the culture supernatant, we conclude that the conditional lethal phenotype of ΔtolB ΔdegP mutants stems from a grossly destabilized envelope structure that causes excessive cell lysis. Together, the data point to a critical role for periplasmic proteases when the Tol-Pal complex-mediated envelope structure and/or functions are compromised.IMPORTANCE The Tol-Pal complex plays a poorly defined role in envelope biogenesis. The data presented here show that DegP's periplasmic protease activity becomes crucial in mutants lacking the intact Tol-Pal complex, but this requirement can be circumvented by suppressor mutations that activate the basal protease activity of a regulatory protease, DegS. These observations point to a critical role for periplasmic proteases when Tol-Pal-mediated envelope structure and/or functions are perturbed.


Assuntos
Adaptação Fisiológica/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico/genética , Proteínas Periplásmicas/genética , Serina Endopeptidases/genética , Substituição de Aminoácidos , Parede Celular/genética , Parede Celular/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Mutação , Proteínas Periplásmicas/metabolismo , Fenótipo , Ligação Proteica , Estrutura Secundária de Proteína , Serina Endopeptidases/deficiência , Fator sigma/genética , Fator sigma/metabolismo , Estresse Fisiológico , Temperatura , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
10.
J Bacteriol ; 201(11)2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30858299

RESUMO

The Gram-negative outer membrane (OM) is a selectively permeable asymmetric bilayer that allows vital nutrients to diffuse into the cell but prevents toxins and hydrophobic molecules from entering. Functionally and structurally diverse ß-barrel outer membrane proteins (OMPs) build and maintain the permeability barrier, making the assembly of OMPs crucial for cell viability. In this work, we characterize an assembly-defective mutant of the maltoporin LamB, LamBG439D We show that the folding defect of LamBG439D results in an accumulation of unfolded substrate that is toxic to the cell when the periplasmic protease DegP is removed. Selection for suppressors of this toxicity identified the novel mutant degSA323E allele. The mutant DegSA323E protein contains an amino acid substitution at the PDZ/protease domain interface that results in a partially activated conformation of this protein. This activation increases basal levels of downstream σE stress response signaling. Furthermore, the enhanced σE activity of DegSA323E suppresses a number of other assembly-defective conditions without exhibiting the toxicity associated with high levels of σE activity. We propose that the increased basal levels of σE signaling primes the cell to respond to envelope stress before OMP assembly defects threaten cell viability. This finding addresses the importance of envelope stress responses in monitoring the OMP assembly process and underpins the critical balance between envelope defects and stress response activation.IMPORTANCE Gram-negative bacteria, such as Escherichia coli, inhabit a natural environment that is prone to flux. In order to cope with shifting growth conditions and the changing availability of nutrients, cells must be capable of quickly responding to stress. Stress response pathways allow cells to rapidly shift gene expression profiles to ensure survival in this unpredictable environment. Here we describe a mutant that partially activates the σE stress response pathway. The elevated basal level of this stress response allows the cell to quickly respond to overwhelming stress to ensure cell survival.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico/genética , Proteínas Periplásmicas/genética , Porinas/genética , Receptores Virais/genética , Serina Endopeptidases/genética , Fator sigma/genética , Adaptação Fisiológica/genética , Substituição de Aminoácidos , Proteínas da Membrana Bacteriana Externa/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico/deficiência , Viabilidade Microbiana , Modelos Moleculares , Mutação , Periplasma/genética , Periplasma/metabolismo , Porinas/química , Porinas/deficiência , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Receptores Virais/química , Receptores Virais/deficiência , Serina Endopeptidases/deficiência , Fator sigma/metabolismo , Transdução de Sinais , Estresse Fisiológico
11.
Biochim Biophys Acta Mol Basis Dis ; 1865(6): 1170-1181, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30639224

RESUMO

Mouse mast cell protease-4 (mMCP4) is a chymase that has been implicated in cardiovascular diseases, including myocardial infarction (MI). This study tested a direct role of mMCP4 in mouse post-MI cardiac dysfunction and myocardial remodeling. Immunoblot and immunofluorescent double staining demonstrated mMCP4 expression in cardiomyocytes from the infarct zone from mouse heart at 28 day post-MI. At this time point, mMCP4-deficient Mcpt4-/- mice showed no difference in survival from wild-type (WT) control mice, yet demonstrated smaller infarct size, improved cardiac functions, reduced macrophage content but increased T-cell accumulation in the infarct region compared with those of WT littermates. mMCP4-deficiency also reduced cardiomyocyte apoptosis and expression of TGF-ß1, p-Smad2, and p-Smad3 in the infarct region, but did not affect collagen deposition or α-smooth muscle actin expression in the same area. Gelatin gel zymography and immunoblot analysis revealed reduced activities of matrix metalloproteinases and expression of cysteinyl cathepsins in the myocardium, macrophages, and T cells from Mcpt4-/- mice. Immunoblot analysis also found reduced p-Smad2 and p-Smad3 in the myocardium from Mcpt4-/- mice, yet fibroblasts from Mcpt4-/- mice showed comparable levels of p-Smad2 and p-Smad3 to those of WT fibroblasts. Flow cytometry, immunoblot analysis, and immunofluorescent staining demonstrated that mMCP4-deficiency reduced the expression of proapoptotic cathepsins in cardiomyocytes and protected cardiomyocytes from H2O2-induced apoptosis. This study established a role of mMCP4 in mouse post-MI dysfunction by regulating myocardial protease expression and cardiomyocyte death without significant impact on myocardial fibrosis or survival post-MI in mice.


Assuntos
Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Serina Endopeptidases/deficiência , Remodelação Ventricular , Animais , Apoptose/genética , Células Cultivadas , Fibroblastos/metabolismo , Fibrose , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Serina Endopeptidases/genética , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad3/genética , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo
12.
Hippocampus ; 29(6): 550-565, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30394609

RESUMO

Cajal-Retzius (CR) cells are early-born glutamatergic neurons that are primarily known as the early main source of the signal protein Reelin. In the reeler mutant, the absence of Reelin causes severe defects in the radial migration of neurons, resulting in abnormal cortical layering. To date, the exact morphological properties of CR-cells independent of Reelin are unknown. With this in view, we studied the ontogenesis, density, and distribution of CR-cells in reeler mice that were cross-bred with a CXCR4-EGFP reporter mouse line, thus enabling us to clearly identify CR-cells positions in the disorganized hippocampus of the reeler mouse. As evidenced by morphological analysis, differences were found regarding CR-cell distribution and density: generally, we found fewer CR-cells in the developing and adult reeler hippocampus as compared to the hippocampus of wild-type animals (WT); however, in reeler mice, CR-cells were much more closely associated to the hippocampal fissure (HF), resulting in relatively higher local CR-cell densities. This higher local cell density was accompanied by stronger immunoreactivity of the CXCR4 ligand, stroma-derived factor-1 (SDF-1) that is known to regulate CR-cell positioning. Importantly, confocal microscopy indicates an integration of CR-cells into the developing and adult hippocampal network in reeler mice, raising evidence that network integration of CR-cells might be independent of Reelin.


Assuntos
Moléculas de Adesão Celular Neuronais/deficiência , Moléculas de Adesão Celular Neuronais/genética , Proteínas da Matriz Extracelular/deficiência , Proteínas da Matriz Extracelular/genética , Hipocampo/patologia , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Neurônios/patologia , Serina Endopeptidases/deficiência , Serina Endopeptidases/genética , Animais , Contagem de Células , Movimento Celular , Quimiocina CXCL12/metabolismo , Giro Denteado/metabolismo , Giro Denteado/patologia , Ácido Glutâmico/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/crescimento & desenvolvimento , Hipocampo/metabolismo , Camundongos , Camundongos Mutantes Neurológicos , Camundongos Transgênicos , Microscopia Confocal , Rede Nervosa/metabolismo , Rede Nervosa/patologia , Neurogênese , Neurônios/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Transdução de Sinais
13.
Oncogene ; 38(4): 497-517, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30115975

RESUMO

The serine protease PRSS8 has shown important physiological and pathological functions, but its roles in cancer initiation and progression are unclear. We developed and dynamically characterized a conditional knockout Prss8fl/fl, p-Villin-Cre+ mouse model. We found that genetic deficiency of the Prss8 gene caused spontaneous colitis and an inflamed rectum at an early age and caused intestinal tumors at a late age, which were linked to increased intestinal cell proliferation and migration but decreased cell differentiation. Increased PRSS8 expression inhibited cancer cell growth and metastasis in nude mice and inhibited cancer cell migration, invasion, colony formation and tumor sphere formation in vitro, but decreased PRSS8 expression facilitated malignancies in vivo and in vitro. Gene profiling on manipulated cancer cells and intestinal epithelial cells of Prss8 mouse models, gene set enrichment analysis and mechanistic studies revealed that PRSS8 targeted the Wnt/ß-catenin, epithelial-mesenchymal transition, and stem cell signaling pathways, which were further supported by the results from the TCGA data mining and validated by immunohistochemical staining on colorectal cancer tissue microarrays. In conclusion, PRSS8 is a novel tumor suppressor that plays critical roles in the suppression of colorectal carcinogenesis and metastasis.


Assuntos
Colite/genética , Neoplasias Colorretais/genética , Proteínas de Neoplasias/fisiologia , Serina Endopeptidases/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenoma/genética , Animais , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Cruzamentos Genéticos , Neoplasias Duodenais/genética , Transição Epitelial-Mesenquimal/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos Transgênicos , Metástase Neoplásica , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/fisiologia , Interferência de RNA , RNA Interferente Pequeno/genética , Serina Endopeptidases/deficiência , Serina Endopeptidases/genética , Organismos Livres de Patógenos Específicos , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Via de Sinalização Wnt
14.
Stem Cell Reports ; 11(5): 1092-1105, 2018 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-30392974

RESUMO

Hematopoietic stem and progenitor cells (HSPCs) undergo self-renewal and differentiation to guarantee a constant supply of short-lived blood cells. Both intrinsic and extrinsic factors determine HSPC fate, but the underlying mechanisms remain elusive. Here, we report that Proteinase 3 (PR3), a serine protease mainly confined to granulocytes, is also expressed in HSPCs. PR3 deficiency intrinsically suppressed cleavage and activation of caspase-3, leading to expansion of the bone marrow (BM) HSPC population due to decreased apoptosis. PR3-deficient HSPCs outcompete the long-term reconstitution potential of wild-type counterparts. Collectively, our results establish PR3 as a physiological regulator of HSPC numbers. PR3 inhibition is a potential therapeutic target to accelerate and increase the efficiency of BM reconstitution during transplantation.


Assuntos
Medula Óssea/enzimologia , Células-Tronco Hematopoéticas/enzimologia , Serina Endopeptidases/metabolismo , Animais , Apoptose , Medula Óssea/efeitos da radiação , Contagem de Células , Proliferação de Células , Sobrevivência Celular , Hematopoese , Células-Tronco Hematopoéticas/citologia , Camundongos Endogâmicos C57BL , Serina Endopeptidases/deficiência
15.
Cell Commun Signal ; 16(1): 65, 2018 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-30286765

RESUMO

BACKGROUND: Our previous work revealed that rhomboid domain-containing protein 1 (RHBDD1) participates in the modulation of cell growth and apoptosis in colorectal cancer cells. This study aimed to investigate the function of RHBDD1 in regulating breast cancer progression and its underlying molecular basis. METHODS: Immunohistochemistry was performed to evaluate RHBDD1 expression in 116 breast cancer tissue and 39 adjacent normal tissue and expression of RHBDD1, phospho-Akt (p-Akt) and cyclin-dependent kinase 2 (CDK2) in the same 84 breast cancer specimens. RHBDD1-knock-out cells were established using breast cancer cell lines. In vitro studies were carried out to estimate the function of RHBDD1 in cell proliferation, migration and invasion. Fluorescence microscopy assay and flow cytometric analysis were used to measure apoptosis and cell cycle regulation. RNA sequencing and western blot analysis were used to investigate the molecular mechanisms of RHBDD1. RESULTS: RHBDD1 was highly up-regulated in breast cancer tissue compared with that in normal tissue and associated with pathological tumor (pT) stage, pathological tumor-node-metastasis (pTNM) stage and estrogen receptor (ER) expression. RHBDD1 up-regulation was associated with poor prognosis in several subtypes of breast cancer. Deletion of RHBDD1 promoted apoptosis and suppressed proliferation, migration and invasion in breast cancer cells. RHBDD1 deletion suppressed Akt activation and decreased CDK2 protein level via proteasome pathway, thus inhibited cell cycle progression and G1/S phase transition. Moreover, the protein level of RHBDD1, p-Akt and CDK2 was significantly positively correlated in breast cancer tissue. CONCLUSIONS: Our study reveals that RHBDD1 promotes breast cancer progression by regulating p-Akt and CDK2 protein levels, and might be a potential biomarker and prognostic indicator for breast cancer patients.


Assuntos
Neoplasias da Mama/patologia , Quinase 2 Dependente de Ciclina/metabolismo , Progressão da Doença , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina Endopeptidases/metabolismo , Apoptose , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Regulação para Baixo , Pontos de Checagem da Fase G2 do Ciclo Celular , Deleção de Genes , Humanos , Pontos de Checagem da Fase M do Ciclo Celular , Células MCF-7 , Invasividade Neoplásica , Serina Endopeptidases/deficiência , Serina Endopeptidases/genética
16.
Biochem Biophys Res Commun ; 505(1): 81-86, 2018 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-30241938

RESUMO

Reelin is a secreted protein essential for the development and function of the mammalian brain. The receptors for Reelin, apolipoprotein E receptor 2 and very low-density lipoprotein receptor, belong to the low-density lipoprotein receptor family, but it is not known whether Reelin is involved in the brain lipid metabolism. In the present study, we performed lipidomic analysis of the cerebral cortex of wild-type and Reelin-deficient (reeler) mice, and found that reeler mice exhibited several compositional changes in phospholipids. First, the ratio of phospholipids containing one saturated fatty acid (FA) and one docosahexaenoic acid (DHA) or arachidonic acid (ARA) decreased. Secondly, the ratio of phospholipids containing one monounsaturated FA (MUFA) and one DHA or ARA increased. Thirdly, the ratio of phospholipids containing 5,8,11-eicosatrienoic acid, or Mead acid (MA), increased. Finally, the expression of stearoyl-CoA desaturase-1 (SCD-1) increased. As the increase of MA is seen as an index of polyunsaturated FA (PUFA) deficiency, and the expression of SCD-1 is suppressed by PUFA, these results strongly suggest that the loss of Reelin leads to PUFA deficiency. Hence, MUFA and MA are synthesized in response to this deficiency, in part by inducing SCD-1 expression. This is the first report of changes of FA composition in the reeler mouse brain and provides a basis for further investigating the new role of Reelin in the development and function of the brain.


Assuntos
Encéfalo/metabolismo , Moléculas de Adesão Celular Neuronais/deficiência , Proteínas da Matriz Extracelular/deficiência , Lipídeos/química , Proteínas do Tecido Nervoso/deficiência , Serina Endopeptidases/deficiência , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/metabolismo , Animais , Ácido Araquidônico/metabolismo , Encéfalo/embriologia , Moléculas de Adesão Celular Neuronais/genética , Ácidos Docosa-Hexaenoicos/metabolismo , Proteínas da Matriz Extracelular/genética , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Metabolismo dos Lipídeos , Camundongos Endogâmicos ICR , Camundongos Mutantes Neurológicos , Proteínas do Tecido Nervoso/genética , Fosfolipídeos/metabolismo , Serina Endopeptidases/genética , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismo
17.
Sci Rep ; 8(1): 13046, 2018 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-30158644

RESUMO

Reelin protein (RELN), an extracellular matrix protein, plays multiple roles that range from embryonic neuronal migration to spine formation in the adult brain. Results from genetic studies have suggested that RELN is associated with the risk of psychiatric disorders, including schizophrenia (SCZ). We previously identified a novel exonic deletion of RELN in a patient with SCZ. High-resolution copy number variation analysis revealed that this deletion included exons 52 to 58, which truncated the RELN in a similar manner to the Reln Orleans mutation (Relnrl-Orl). We examined the clinical features of this patient and confirmed a decreased serum level of RELN. To elucidate the pathophysiological role of the exonic deletion of RELN in SCZ, we conducted behavioral and neurochemical analyses using heterozygous Relnrl-Orl/+ mice. These mice exhibited abnormalities in anxiety, social behavior, and motor learning; the deficits in motor learning were ameliorated by antipsychotics. Methamphetamine-induced hyperactivity and dopamine release were significantly reduced in the Relnrl-Orl/+ mice. In addition, the levels of GABAergic markers were decreased in the brain of these mice. Taken together, our results suggest that the exonic deletion of RELN plays a pathological role, implicating functional changes in the dopaminergic and GABAergic systems, in the pathophysiology of SCZ.


Assuntos
Moléculas de Adesão Celular Neuronais/deficiência , Proteínas da Matriz Extracelular/deficiência , Proteínas do Tecido Nervoso/deficiência , Esquizofrenia/genética , Esquizofrenia/fisiopatologia , Serina Endopeptidases/deficiência , Animais , Análise do Comportamento Aplicada , Moléculas de Adesão Celular Neuronais/sangue , Códon sem Sentido , Proteínas da Matriz Extracelular/sangue , Humanos , Camundongos , Modelos Animais , Proteínas do Tecido Nervoso/sangue , Testes Neuropsicológicos , Esquizofrenia/patologia , Deleção de Sequência , Serina Endopeptidases/sangue
19.
Nat Commun ; 9(1): 1350, 2018 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-29636509

RESUMO

Alterations in iron status have frequently been associated with obesity and other metabolic disorders. The hormone hepcidin stands out as a key regulator in the maintenance of iron homeostasis by controlling the main iron exporter, ferroportin. Here we demonstrate that the deficiency in the hepcidin repressor matriptase-2 (Tmprss6) protects from high-fat diet-induced obesity. Tmprss6 -/- mice show a significant decrease in body fat, improved glucose tolerance and insulin sensitivity, and are protected against hepatic steatosis. Moreover, these mice exhibit a significant increase in fat lipolysis, consistent with their dramatic reduction in adiposity. Rescue experiments that block hepcidin up-regulation and restore iron levels in Tmprss6-/- mice via anti-hemojuvelin (HJV) therapy, revert the obesity-resistant phenotype of Tmprss6-/- mice. Overall, this study describes a role for matritpase-2 and hepcidin in obesity and highlights the relevance of iron regulation in the control of adipose tissue function.


Assuntos
Tecido Adiposo/metabolismo , Hepcidinas/genética , Ferro/metabolismo , Proteínas de Membrana/genética , Obesidade/genética , Serina Endopeptidases/genética , Tecido Adiposo/patologia , Animais , Anticorpos Monoclonais/farmacologia , Dieta Hiperlipídica/efeitos adversos , Proteínas Ligadas por GPI , Regulação da Expressão Gênica , Proteína da Hemocromatose , Hepcidinas/metabolismo , Homeostase , Metabolismo dos Lipídeos , Masculino , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Obesidade/etiologia , Obesidade/patologia , Obesidade/prevenção & controle , Serina Endopeptidases/deficiência , Transdução de Sinais
20.
Mol Neurobiol ; 55(8): 7062-7071, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29383685

RESUMO

Several pieces of evidence indicate that elastase-2 (ELA2; chymotrypsin-like ELA2) is an alternative pathway to the generation of angiotensin II (ANGII). Elastase-2 knockout mice (ELA2KO) exhibit alterations in the arterial blood pressure and heart rate. However, there is no data on the behavioral consequences of ELA2 deletion. In this study, we addressed this question, submitting ELA2KO and wild-type (WT) mice to several models sensitive to anxiety- and depression-like, memory, and repetitive behaviors. Our data indicates a higher incidence of barbering behavior in ELA2KO compared to WT, as well as an anxiogenic phenotype, evaluated in the elevated plus maze (EPM). While a decrease in locomotor activity was observed in ELA2KO in EPM, this feature was not the main source of variation in the other parameters analyzed. The marble-burying test (MBT) indicated increase in repetitive behavior, observed by a higher number of buried marbles. The actimeter test indicated a decrease in total activity and confirmed the increase in repetitive behavior. The spatial memory was tested by repeated exposure to the actimeter in a 24-h interval. Both ELA2KO and WT exhibited decreased activity compared to the first exposure, without any distinction between the genotypes. However, when submitted to the cued fear conditioning, ELA2KO displayed lower levels of freezing behavior in the extinction session when compared to WT, but no difference was observed during the conditioning phase. Increased levels of BDNF were found in the prefrontal cortex but not in the hippocampus of ELA2KO mice compared to WT. Finally, in silico analysis indicates that ELA2 is putatively able to cleave BDNF, and incubation of the purified enzyme with BDNF led to the degradation of the latter. Our data suggested an anxiogenic- and antidepressant-like phenotype of ELA2KO, possibly associated with increased levels of BDNF in the prefrontal cortex.


Assuntos
Antidepressivos/metabolismo , Ansiedade/enzimologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Córtex Pré-Frontal/metabolismo , Serina Endopeptidases/deficiência , Animais , Comportamento Animal , Simulação por Computador , Condicionamento Psicológico , Medo , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/farmacologia , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo
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