RESUMO
Furin is an important mammalian proprotein convertase that catalyzes the proteolytic maturation of a variety of prohormones and proproteins in the secretory pathway. In the brain, the substrates of furin include the proproteins of growth factors, receptors and enzymes. Emerging evidence, such as reduced FURIN mRNA expression in the brains of Alzheimer's disease patients or schizophrenia patients, has implicated a crucial role of furin in the pathophysiology of neurodegenerative and neuropsychiatric diseases. Currently, compared to cancer and infectious diseases, the aberrant expression of furin and its pharmaceutical potentials in neurological diseases remain poorly understood. In this article, we provide an overview on the physiological roles of furin and its substrates in the brain, summarize the deregulation of furin expression and its effects in neurodegenerative and neuropsychiatric disorders, and discuss the implications and current approaches that target furin for therapeutic interventions. This review may expedite future studies to clarify the molecular mechanisms of furin deregulation and involvement in the pathogenesis of neurodegenerative and neuropsychiatric diseases, and to develop new diagnosis and treatment strategies for these diseases.
Assuntos
Furina , Doenças Neurodegenerativas , Animais , Furina/genética , Furina/fisiologia , Humanos , Pró-Proteína Convertases/genéticaRESUMO
Recently, a mini-review was published in the Medical Hypotheses journal by Usul Afsar entitled 2019-nCoV-SARS-CoV-2 (COVID-19) infection: Cruciality of Furin and relevance with cancer. Previous studies have pointed out that disruption of the proteolytic cleavage of proteins can promote infectious and non-infectious diseases. The last few weeks have been marked by an important revelation concerning the pathophysiology of SARS-CoV-2. This new coronavirus disease (COVID-19) is a highly contagious and transmissible acute respiratory infectious disorder. SARS-CoV-2 is composed of RNA-dependent RNA polymerase and structural proteins including Spike protein (S protein). Interestingly, the FURIN, one of the proproteins of the convertase family, plays a crucial role in the maturation of viral glycoproteins. In addition, many viruses including coronaviruses, exploit FURIN for the activation of their glycoproteins. Recent data indicate that SARS-CoV-2 enters human cells by binding to angiotensin-converting enzyme 2. Subsequently, the S protein is cleaved by transmembrane protease serine 2 with the help of FURIN which facilitates the entry of the virus into the cell after binding. Furthermore, it seems that FURIN is implicated in the pathogenesis of SARS-CoV-2 and potentially in the increased rates of human-to-human transmission.
Assuntos
Betacoronavirus , Infecções por Coronavirus/enzimologia , Infecções por Coronavirus/etiologia , Furina/fisiologia , Peptidil Dipeptidase A/fisiologia , Pneumonia Viral/enzimologia , Pneumonia Viral/etiologia , Glicoproteína da Espícula de Coronavírus/fisiologia , Enzima de Conversão de Angiotensina 2 , Betacoronavirus/patogenicidade , Betacoronavirus/fisiologia , COVID-19 , Infecções por Coronavirus/imunologia , Furina/genética , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Tolerância Imunológica , Imunidade Celular , Pandemias , Pneumonia Viral/imunologia , Receptores Virais/fisiologia , SARS-CoV-2 , Linfócitos T/imunologia , Linfócitos T/fisiologia , Internalização do VírusRESUMO
Objective- Atherosclerotic coronary artery disease is the leading cause of death worldwide, and current treatment options are insufficient. Using systems-level network cluster analyses on a large coronary artery disease case-control cohort, we previously identified PCSK3 (proprotein convertase subtilisin/kexin family member 3; FURIN) as a member of several coronary artery disease-associated pathways. Thus, our objective is to determine the role of FURIN in atherosclerosis. Approach and Results- In vitro, FURIN inhibitor treatment resulted in reduced monocyte migration and reduced macrophage and vascular endothelial cell inflammatory and cytokine gene expression. In vivo, administration of an irreversible inhibitor of FURIN, α-1-PDX (α1-antitrypsin Portland), to hyperlipidemic Ldlr-/- mice resulted in lower atherosclerotic lesion area and a specific reduction in severe lesions. Significantly lower lesional macrophage and collagen area, as well as systemic inflammatory markers, were observed. MMP2 (matrix metallopeptidase 2), an effector of endothelial function and atherosclerotic lesion progression, and a FURIN substrate was significantly reduced in the aorta of inhibitor-treated mice. To determine FURIN's role in vascular endothelial function, we administered α-1-PDX to Apoe-/- mice harboring a wire injury in the common carotid artery. We observed significantly decreased carotid intimal thickness and lower plaque cellularity, smooth muscle cell, macrophage, and inflammatory marker content, suggesting protection against vascular remodeling. Overexpression of FURIN in this model resulted in a significant 67% increase in intimal plaque thickness, confirming that FURIN levels directly correlate with atherosclerosis. Conclusions- We show that systemic inhibition of FURIN in mice decreases vascular remodeling and atherosclerosis. FURIN-mediated modulation of MMP2 activity may contribute to the atheroprotection observed in these mice.
Assuntos
Aterosclerose/prevenção & controle , Furina/antagonistas & inibidores , Placa Aterosclerótica/tratamento farmacológico , alfa 1-Antitripsina/uso terapêutico , Animais , Aorta/enzimologia , Aterosclerose/genética , Aterosclerose/patologia , Artéria Carótida Primitiva , Progressão da Doença , Avaliação Pré-Clínica de Medicamentos , Indução Enzimática/efeitos dos fármacos , Furina/genética , Furina/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Macrófagos/fisiologia , Masculino , Metaloproteinase 2 da Matriz/análise , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/fisiologia , Placa Aterosclerótica/patologia , Receptores de LDL/deficiência , Túnica Íntima/efeitos dos fármacos , Túnica Íntima/patologia , Remodelação Vascular , alfa 1-Antitripsina/farmacologiaRESUMO
Ischemic retinopathy is a vision-threatening disease associated with chronic retinal inflammation and hypoxia leading to abnormal angiogenesis. Furin, a member of the proprotein convertase family of proteins, has been implicated in the regulation of angiogenesis due to its essential role in the activation of several angiogenic growth factors, including vascular endothelial growth factor-C (VEGF-C), VEGF-D and transforming growth factor - ß (TGF- ß). In the present study, we evaluated expression of furin in the retina and its role in retinal angiogenesis. As both inflammation and hypoxia contribute to angiogenesis, the role of furin was evaluated using myeloid-cell specific furin knockout (KO) mice (designated LysMCre-fur(fl/fl)) both in developmental retinal angiogenesis as well as in hypoxia-driven angiogenesis using the oxygen-induced retinopathy (OIR) model. In the retina, furin expression was detected in endothelial cells, macrophages and, to some extent, in neurons. The rate of angiogenesis was not different in LysMCre-fur(fl/fl) mice when compared to their wild-type littermates during development. In the OIR model, the revascularization of retina was significantly delayed in LysMCre-fur(fl/fl) mice compared to their wild-type littermates, while there was no compensatory increase in the preretinal neovascularization in LysMCre-fur(fl/fl) mice. These results demonstrate that furin expression in myeloid cells plays a significant role in hypoxia-induced angiogenesis in retina.
Assuntos
Furina/fisiologia , Células Mieloides/metabolismo , Retina/metabolismo , Neovascularização Retiniana/metabolismo , Animais , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Furina/deficiência , Furina/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Microglia/metabolismo , Neurônios Retinianos/metabolismoRESUMO
Osteocalcin (OCN) is an osteoblast-derived hormone that increases energy expenditure, insulin sensitivity, insulin secretion, and glucose tolerance. The cDNA sequence of OCN predicts that, like many other peptide hormones, OCN is first synthesized as a prohormone (pro-OCN). The importance of pro-OCN maturation in regulating OCN and the identity of the endopeptidase responsible for pro-OCN cleavage in osteoblasts are still unknown. Here, we show that the proprotein convertase furin is responsible for pro-OCN maturation in vitro and in vivo. Using pharmacological and genetic experiments, we also determined that furin-mediated pro-OCN cleavage occurred independently of its γ-carboxylation, a posttranslational modification that is known to hamper OCN endocrine action. However, because pro-OCN is not efficiently decarboxylated and activated during bone resorption, inactivation of furin in osteoblasts in mice resulted in decreased circulating levels of undercarboxylated OCN, impaired glucose tolerance, and reduced energy expenditure. Furthermore, we show that Furin deletion in osteoblasts reduced appetite, a function not modulated by OCN, thus suggesting that osteoblasts may secrete additional hormones that regulate different aspects of energy metabolism. Accordingly, the metabolic defects of the mice lacking furin in osteoblasts became more apparent under pair-feeding conditions. These findings identify furin as an important regulator of bone endocrine function.
Assuntos
Osso e Ossos/enzimologia , Furina/fisiologia , Osteocalcina/metabolismo , Sequência de Aminoácidos , Animais , Osso e Ossos/citologia , Células Cultivadas , Sistema Endócrino , Metabolismo Energético , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Osteoblastos/enzimologia , Pró-Proteína Convertase 5/metabolismo , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteólise , Células RAW 264.7RESUMO
The proprotein convertase enzyme FURIN processes immature pro-proteins into functional end- products. FURIN is upregulated in activated immune cells and it regulates T-cell dependent peripheral tolerance and the Th1/Th2 balance. FURIN also promotes the infectivity of pathogens by activating bacterial toxins and by processing viral proteins. Here, we evaluated the role of FURIN in LysM+ myeloid cells in vivo. Mice with a conditional deletion of FURIN in their myeloid cells (LysMCre-fur(fl/fl)) were healthy and showed unchanged proportions of neutrophils and macrophages. Instead, LysMCre-fur(fl/fl) mice had elevated serum IL-1ß levels and reduced numbers of splenocytes. An LPS injection resulted in accelerated mortality, elevated serum pro-inflammatory cytokines and upregulated numbers of pro-inflammatory macrophages. A genome-wide gene expression analysis revealed the overexpression of several pro-inflammatory genes in resting FURIN-deficient macrophages. Moreover, FURIN inhibited Nos2 and promoted the expression of Arg1, which implies that FURIN regulates the M1/M2-type macrophage balance. FURIN was required for the normal production of the bioactive TGF-ß1 cytokine, but it inhibited the maturation of the inflammation-provoking TACE and Caspase-1 enzymes. In conclusion, FURIN has an anti-inflammatory function in LysM+ myeloid cells in vivo.
Assuntos
Furina/fisiologia , Inflamação/prevenção & controle , Células Mieloides/enzimologia , Proteína ADAM17/metabolismo , Animais , Caspase 1/metabolismo , Regulação da Expressão Gênica , Interleucina-1beta/sangue , Lipopolissacarídeos/toxicidade , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Antigen emergence rapidly stimulates T cells, which leads to changes in cytokine production, cell proliferation, and differentiation. Some of the key molecules involved in these events, such as TGF-ß1 and NOTCH1, are synthesized initially as inactive precursors and are proteolytically activated during T cell activation. PCSKs regulate proprotein maturation by catalyzing the proteolytic cleavage of their substrates. The prototype PCSK FURIN is induced upon TCR activation, and its expression in T cells is critical for the maintenance of peripheral immune tolerance. In this study, we tested the hypothesis that FURIN regulates T cell activation. Our data demonstrate that IL-2 is increased initially in FURIN-deficient mouse CD4(+) T cells, but the TCR-induced IL-2 mRNA expression is not sustained in the absence of FURIN. Accordingly, the inhibition of FURIN in human Jurkat T cell lines also results in a decrease in IL-2 production, whereas the overexpression of WT FURIN is associated with elevated IL-2 levels. In Jurkat cells, FURIN is dispensable for immediate TCR signaling steps, such as ERK, ZAP70, or LAT phosphorylation. However, with the use of gene reporter assays, we demonstrate that FURIN regulates the AP-1, NFAT, and NF-κB transcription factors. Finally, by performing a transcription factor-binding site enrichment analysis on FURIN-dependent transcriptomes, we identify the FURIN-regulated transcription factors in mouse CD4(+) T cell subsets. Collectively, our work confirms the hypothesis that the TCR-regulated protease FURIN plays an important role in T cell activation and that it can specifically modulate TCR-activated transactivation.
Assuntos
Furina/fisiologia , Receptores de Antígenos de Linfócitos T/genética , Ativação Transcricional/fisiologia , Sequência de Bases , Linfócitos T CD4-Positivos/imunologia , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Humanos , Interleucina-2/biossíntese , Células Jurkat , Reação em Cadeia da Polimerase em Tempo RealRESUMO
OBJECTIVE: Atherosclerosis is a complex disease, involving elevated LDL-c, lipid accumulation in the blood vessel wall, foam cell formation and vascular dysfunction. Lowering plasma LDL-c is the cornerstone of current management of cardiovascular disease. However, new approaches which reduce plasma LDL-c and lessen the pathological vascular remodeling occurring in the disease should also have therapeutic value. Previously, we found that overexpression of profurin, the 83-amino acid prodomain of the proprotein convertase furin, lowered plasma HDL levels in wild-type mice. The question that remained was whether it had effects on apolipoprotein B (ApoB)-containing lipoproteins. METHODS: Adenovirus mediated overexpression of hepatic profurin in Ldlr(-/-)mice and wild-type mice were used to evaluate effects of profurin on ApoB-containing lipoproteins, atherosclerosis and vascular remodeling. RESULTS: Hepatic profurin overexpression resulted in a significant reduction in atherosclerotic lesion development in Ldlr(-/-)mice and a robust reduction in plasma LDL-c. Metabolic studies revealed lower secretion of ApoB and triglycerides in VLDL particles. Mechanistic studies showed that in the presence of profurin, hepatic ApoB, mainly ApoB100, was degraded by proteasomes. There was no effect on ApoB mRNA expression. Importantly, short-term hepatic profurin overexpression did not result in hepatic lipid accumulation. Blood vessel wall thickening caused by either wire-induced femoral artery injury or common carotid artery ligation was reduced. Profurin expression inhibited proliferation and migration in vascular smooth muscle cells in vitro. CONCLUSION: These results indicate that a profurin-based therapy has the potential to treat atherosclerosis by improving metabolic lipid profiles and reducing both atherosclerotic lesion development and pathological vascular remodeling.
Assuntos
Aterosclerose/prevenção & controle , Furina/fisiologia , Terapia Genética , Fígado/metabolismo , Precursores de Proteínas/fisiologia , Adenoviridae/genética , Animais , Apolipoproteínas B/metabolismo , Aterosclerose/sangue , Aterosclerose/genética , Lesões das Artérias Carótidas/patologia , Artéria Carótida Primitiva/patologia , LDL-Colesterol/sangue , Dieta Ocidental/efeitos adversos , Endotélio Vascular/lesões , Endotélio Vascular/patologia , Artéria Femoral/lesões , Artéria Femoral/patologia , Furina/biossíntese , Furina/genética , Vetores Genéticos/uso terapêutico , Hemorreologia , Hepatócitos/metabolismo , Humanos , Ligadura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexo de Endopeptidases do Proteassoma/metabolismo , Precursores de Proteínas/biossíntese , Precursores de Proteínas/genética , Estrutura Terciária de Proteína , Receptores de LDL/deficiência , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Exosomes are extracellular vesicles that mediate intercellular communication and are involved in several biological processes. The objective of our study was to determine whether endogenous retrovirus group WE, member l (ERVWE1)/syncytin-1 and endogenous retrovirus group FRD, member 1 (ERVFRDE1)/syncytin-2, encoded by human endogenous retrovirus (HERV) envelope (env) genes, are present at the surface of exosomes produced by placenta-derived villous cytotrophoblasts and whether they play a role in cellular uptake of exosomes. In addition, we sought to determine whether these proteins are present in various abundances in serum-derived exosomes from normal pregnant women vs. women with preeclampsia (PE). Isolated exosomes were analyzed for their content by Western blot, a bead-associated flow cytometry approach, and a syncytin-2 ELISA. Binding and uptake were tested through confocal and electron microscopy using the BeWo choriocarcinoma cell line. Quality control of exosome preparations consisted of detection of exosomal and nonexosomal markers. Exosome-cell interactions were compared between cells incubated in the presence of control exosomes, syncytin-1 or syncytin-2-deprived exosomes, or exosomes solely bearing the uncleaved forms of these HERV env proteins. From our data, we conclude that villous cytotrophoblast exosomes are positive for both env proteins and are rapidly taken up by BeWo cells in a syncytin-1- and syncytin-2-dependent manner and that syncytin-2 is reduced in serum-derived exosomes from women with PE when compared to exosomes from normal pregnant women.
Assuntos
Exossomos/metabolismo , Produtos do Gene env/fisiologia , Pré-Eclâmpsia/sangue , Proteínas da Gravidez/fisiologia , Trofoblastos/metabolismo , Adulto , Sistema ASC de Transporte de Aminoácidos/antagonistas & inibidores , Sistema ASC de Transporte de Aminoácidos/genética , Sistema ASC de Transporte de Aminoácidos/fisiologia , Comunicação Celular , Fusão Celular , Linhagem Celular Tumoral , Coriocarcinoma/patologia , Endocitose , Retrovirus Endógenos/genética , Retrovirus Endógenos/fisiologia , Endossomos/metabolismo , Feminino , Furina/antagonistas & inibidores , Furina/fisiologia , Produtos do Gene env/sangue , Humanos , Microscopia Confocal , Antígenos de Histocompatibilidade Menor , Gravidez , Proteínas da Gravidez/sangue , Proteínas da Gravidez/deficiência , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Simportadores , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/fisiologia , Neoplasias Uterinas/patologiaRESUMO
Proprotein convertases (PCs) are crucial in the processing and entry of viral or bacterial protein precursors and confer increased infectivity of pathogens bearing a PC activation site, which results in increased symptom severity and lethality. Previously, we developed a nanomolar peptide inhibitor of PCs to prevent PC activation of infectious agents. Herein, we describe a peptidomimetic approach that increases the stability of this inhibitor for use in vivo to prevent systemic infections and cellular damage, such as that caused by influenza H5N1 and Shiga toxin. The addition of azaß(3)-amino acids to both termini of the peptide successfully prevented influenza hemagglutinin 5 fusogenicity and Shiga toxin Vero toxicity in cell-based assays. The results from a cell-based model using stable shRNA-induced proprotein convertase knockdown indicate that only furin is the major proprotein convertase required for HA5 cleavage.
Assuntos
Furina/antagonistas & inibidores , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Peptidomiméticos , Toxina Shiga/metabolismo , Furina/fisiologia , Relação Estrutura-AtividadeRESUMO
ADAMTS5 (aggrecanase-2) is an extracellular matrix-degrading protease implicated in cartilage destruction in arthritis. Our goals were to determine expression sites of Adamts5 in the murine musculoskeletal system and in an ex vivo joint inflammation model. In mice with an intragenic LacZ reporter controlled by the Adamts5 promoter, ß-galactosidase staining was used to identify Adamts5 expressing cells. Mice expressing one wild-type Adamts5 allele were used to determine distribution of Adamts5 mRNA, cleaved aggrecan and versican, and the ADAMTS5 activating enzymes furin and PACE4. Quantitative RT-PCR and immunoblotting were used to validate the immunohistochemistry results. Adamts5 was expressed in mouse synovium, tenosynovium, bone marrow sinusoids, tendons, ligaments, ligament insertions, periosteal cells, and bone vasculature. In knee joint explants treated with IL-1α and TNFα, Adamts5 expression was induced in tenocytes, synovium, and in patellar, but not femoral or tibial articular cartilage. In contrast, increased proteoglycan breakdown in tibial and femoral articular cartilage was associated with increased immunohistochemical staining of PACE4 and furin. These studies identify diverse cell types in the musculoskeletal system that express Adamts5. They also suggest that Adamts5 induction in joint components other than cartilage, and its post-translational activation by PACE4 and/or furin may be important in the pathophysiology of arthritis.
Assuntos
Proteínas ADAM/fisiologia , Artrite/etiologia , Osso e Ossos/química , Interleucina-1alfa/farmacologia , Articulação do Joelho/metabolismo , Articulação do Joelho/cirurgia , Fator de Necrose Tumoral alfa/farmacologia , Proteínas ADAM/análise , Proteínas ADAM/genética , Proteína ADAMTS5 , Animais , Furina/análise , Furina/fisiologia , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pró-Proteína Convertases/análise , Pró-Proteína Convertases/fisiologia , Proteoglicanas/metabolismo , RNA Mensageiro/análiseRESUMO
BMP4 is synthesized as an inactive precursor that is cleaved at two sites during maturation: initially at a site (S1) adjacent to the ligand domain, and then at an upstream site (S2) within the prodomain. Cleavage at the second site regulates the stability of mature BMP4 and this in turn influences its signaling intensity and range of action. The Drosophila ortholog of BMP4, Dpp, functions as a long- or short-range signaling molecule in the wing disc or embryonic midgut, respectively but mechanisms that differentially regulate its bioactivity in these tissues have not been explored. In the current studies we demonstrate, by dpp mutant rescue, that cleavage at the S2 site of proDpp is required for development of the wing and leg imaginal discs, whereas cleavage at the S1 site is sufficient to rescue Dpp function in the midgut. Both the S1 and S2 sites of proDpp are cleaved in the wing disc, and S2-cleavage is essential to generate sufficient ligand to exceed the threshold for pMAD activation at both short- and long-range in most cells. By contrast, proDpp is cleaved at the S1 site alone in the embryonic mesoderm and this generates sufficient ligand to activate physiological target genes in neighboring cells. These studies provide the first biochemical and genetic evidence that selective cleavage of the S2 site of proDPP provides a tissue-specific mechanism for regulating Dpp activity, and that differential cleavage can contribute to, but is not an absolute determinant of signaling range.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Sequência de Aminoácidos , Animais , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 4/metabolismo , Proteínas de Ligação a DNA/análise , Proteínas de Drosophila/análise , Proteínas de Drosophila/química , Furina/fisiologia , Humanos , Mesoderma/metabolismo , Dados de Sequência Molecular , Especificidade de Órgãos , Precursores de Proteínas/metabolismo , Estrutura Terciária de Proteína , Transdução de Sinais , Fatores de Transcrição/análise , Asas de Animais/embriologia , XenopusRESUMO
Integrins link the cytoskeleton to the extracellular matrix, providing outside-in/inside-out signalling essential for vascular smooth muscle cell (VSMC) migration in atherosclerosis. The integrin av subunit is synthesised from its precursor via furin-dependent endoproteolytic cleavage. Furin is a proprotein convertase (PC) highly expressed in VSMCs and in human atherosclerotic lesions. Inhibition of av processing inhibits binding to vitronectin and migration. However, the precise role of furin-dependent av cleavage on integrin bidirectional signalling and subsequent VSMC functions is unknown. Our present study demonstrates that the furin-like PC inhibitor decanoyl-RVKR-chloromethylketone (dec-CMK) inhibited av cleavage. This reduced vitronectin-induced (outside-in) focal adhesion kinase (FAK)- and paxillin-phosphorylation, and VSMC motility. Inside-out-stimulated, integrin- mediated VSMC adhesion/migration relied on integrin-adaptor protein activation following protein kinase C (PKC) and ERK1/2 phosphorylation. In contrast to outside-in signalling, PKC-dependent phosphorylation of FAK and paxillin was unaffected by the status of integrin cleavage. Still, cytoskeleton and focal adhesion site rearrangements were modulated by the inhibition of furin-dependent integrin cleavage, thereby lessening inside-out dependent migration. Hence, we find that integrin bidirectional signalling is critically controlled by furin. Furin- dependent integrin processing modulates rapid adaptive integrin/cytoskeleton changes, essential to VSMC motility, which represents a crucial component in atherosclerosis and restenosis.
Assuntos
Furina/fisiologia , Integrinas/metabolismo , Miócitos de Músculo Liso/metabolismo , Transdução de Sinais , Animais , Aterosclerose/patologia , Adesão Celular , Movimento Celular , Células Cultivadas , Citoesqueleto , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Furina/antagonistas & inibidores , Furina/metabolismo , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , RatosRESUMO
Furin, a proprotein convertase (PC), is ubiquitously expressed and implicated in many physiological and pathological processes. This study is aimed to identify the role of furin in human trophoblast invasion and migration. Furin was found to be highly expressed in placental villi of both rhesus monkeys and human beings during early pregnancy. Specifically, furin was found in trophoblast column and trophoblast shell, regions where highly invasive cytotrophoblast cells invade the maternal decidua during human placentation. To determine whether furin plays any role in trophoblast invasion and migration, we employed human extravillous HTR8/SVneo cells in Matrigel invasion and transwell migration assays. Knocking-down furin expression by siRNA significantly inhibited invasion and migration of HTR8/SVneo cells (P<0.01), with corresponding decrease of matrix metalloproteinase-9 (MMP-9) activities. In contrast, over-expression of furin markedly increased cell invasion and migration (P<0.01), accompanied by significant increase of MMP-9 activities. Furthermore, furin siRNA significantly increased the levels of both tissue inhibitors of MMPs (TIMP)-1 and -2. Our results suggest that furin may play an important role in the invasion and migration of human trophoblast cells during early pregnancy.
Assuntos
Furina/fisiologia , Placentação/fisiologia , Trofoblastos/enzimologia , Animais , Linhagem Celular , Movimento Celular/fisiologia , Feminino , Furina/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Macaca mulatta , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Gravidez , Trofoblastos/fisiologiaRESUMO
Most pathogen-derived peptides recognized by CD8+ CTL are produced by proteasomes and delivered to the endoplasmic reticulum by the TAP transporters associated with Ag processing. Alternative proteases also produce antigenic peptides, but their actual relevance is unclear. There is a need to quantify the contribution of these supplementary pathways in vitro and in vivo. A well-defined TAP-independent secretory route of Ag processing involves the trans-Golgi network protease furin. Quantitation of this route by using OVA constructs encoded by vaccinia viruses indicates that it provides approximately one-third of all surface complexes of peptide and MHC class I molecules. Generation of the epitope carboxyl terminus is a dramatic rate-limiting step, since bypassing it increased efficiency by at least 1000-fold. Notably, the secretory construct activated a similar percentage of Ag-specific CD8+ T cells in wild type as in TAP1-deficient mice, which allow only secretory routes but which have a 10- to 20-fold smaller CD8 compartment. Moreover, these TAP1(-/-) OVA-specific CD8+ T lymphocytes accomplished elimination of epitope-bearing cells in vivo. The results obtained with this experimental system underscore the potential of secretory pathways of MHC class I Ag presentation to elicit functional CD8+ T lymphocytes in vivo and support the hypothesis that noncytosolic processing mechanisms may compensate in vivo for the lack of proteasome participation in Ag processing in persons genetically deficient in TAP and thus contribute to pathogen control.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Apresentação de Antígeno/imunologia , Furina/fisiologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/biossíntese , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Animais , Apresentação de Antígeno/genética , Linhagem Celular Transformada , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Furina/imunologia , Furina/metabolismo , Antígenos H-2/genética , Antígenos H-2/imunologia , Antígenos H-2/metabolismo , Humanos , Células L , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T Citotóxicos/enzimologia , Vaccinia virus/genética , Vaccinia virus/imunologia , Vaccinia virus/metabolismoRESUMO
BACKGROUND/AIMS: The iron-regulatory peptide hepcidin is synthesized in the liver as an 84-aa pre-pro-hormone maturated by proteolysis through a consensus furin cleavage site to generate the bioactive 25-aa peptide secreted in the circulation. This peptide regulates iron export from enterocytes and macrophages by binding the membrane iron exporter, ferroportin, leading to its degradation. Whether pro-hepcidin could be secreted and reflect hepcidin levels remains an open question. However, the activity of the pro-peptide on ferroportin degradation has never been addressed. METHODS: To answer this question, we produced recombinant pro-hepcidin, both the wild-type form and a furin cleavage site mutant, and tested their activity on ferroportin levels in macrophagic J774 cells. Furin activity was also modulated using furin inhibitor or siRNA-mediated furin mRNA knockdown. RESULTS: We found that pro-hepcidin could fully induce ferroportin degradation, but only when processed by furin to generate the mature hepcidin-25 form. Pro-hepcidin activity was abolished in the presence of furin inhibitor and diminished after siRNA-mediated knockdown of furin mRNA. Furthermore, the mutated version of pro-hepcidin was completely inefficient at degrading ferroportin in macrophages. CONCLUSIONS: Our results demonstrate that pro-hepcidin lacks biological activity, unless fully maturated by a furin-dependent process to yield the bioactive 25-aa peptide.
Assuntos
Peptídeos Catiônicos Antimicrobianos/fisiologia , Proteínas de Transporte de Cátions/metabolismo , Furina/fisiologia , Precursores de Proteínas/fisiologia , Animais , Linhagem Celular , Hepcidinas , Macrófagos/metabolismo , CamundongosRESUMO
PURPOSE OF REVIEW: To describe recent advances in the processing of gastrointestinal hormones, and the consequences for human disease of mutations in the enzymes involved. RECENT FINDINGS: Although gastrointestinal prohormones were long regarded as devoid of biological activity, recent data indicate that the prohormones for both gastrin and gastrin-releasing peptide are bioactive, through different receptors from the mature hormones. Mutations in the family of prohormone convertases responsible for the initial steps in the processing of gastrointestinal hormones are associated with several different pathophysiological conditions in humans. SUMMARY: Human mutational studies, when taken together with the phenotypes observed in mice deficient in the prohormone convertases, emphasize the crucial importance of the processing enzymes in mammalian biology. Although the phenotypes may often be ascribed to defective production of a mature hormone or growth factor, the recognition that the precursors are independently bioactive suggests that the increased precursor concentrations may also contribute to the symptoms. The observation that the precursors often act through different receptors from the mature hormones may permit the development of precursor-selective antagonists for therapeutic use.
Assuntos
Gastroenteropatias/fisiopatologia , Hormônios Gastrointestinais/fisiologia , Mutação , Peptídeos/fisiologia , Animais , Furina/genética , Furina/fisiologia , Peptídeo Liberador de Gastrina/fisiologia , Gastrinas/fisiologia , Gastroenteropatias/enzimologia , Gastroenteropatias/genética , Hormônios/fisiologia , Humanos , Camundongos , Camundongos Knockout , Modelos Animais , Peptídeos/genética , Peptídeos/metabolismo , Pró-Proteína Convertases/fisiologia , Precursores de Proteínas/fisiologiaRESUMO
Semaphorin-3B (sema3B) and semaphorin-3F (sema3F) are secreted tumor suppressors of lung cancer. Sema3F functions as an antiangiogenic factor that repels endothelial cells and compromises their proliferation/survival. However, tumor cells expressing either endogenous or recombinant sema3B fail to repel endothelial cells efficiently. Sema3B found in the conditioned medium of such cells is almost completely cleaved by furin-like pro-protein convertases, generating inactive 61- and 22-kDa fragments. We have generated a sema3B variant that was point mutated at the cleavage site (sema3B-m), thereby conferring partial resistance to cleavage. Conditioned medium from HEK293 cells expressing sema3b-m and conditioned medium of HEK293 cells expressing sema3B contained similar concentrations of semaphorin but sema3B-m was cleaved much less than sema3B. In contrast to HEK293 cells expressing native sema3B, cells expressing sema3b-m strongly repel endothelial cells. Conditioned medium from sema3B-m-expressing cells rapidly caused disassembly of focal adhesions and a collapse of the actin cytoskeleton of endothelial cells, inhibited vascular endothelial growth factor-induced phosphorylation of extracellular signal-regulated kinase 1/2, induced apoptosis of endothelial cells, and inhibited the formation of tubes from endothelial cells in an in vitro angiogenesis assay more potently than conditioned medium from cells expressing sema3B. Furthermore, HEK293 cells expressing sema3B-m inhibited basic fibroblast growth factor-induced angiogenesis in vivo much more potently than cells expressing sema3B. Repulsion of human umbilical vascular endothelial cells by sema3B-m was mediated primarily by the neuropilin-1 (np1) receptor but sema3B-m was also able to transduce signals via neuropilin-2 (np2). These results suggest that up-regulation of furin-like pro-protein convertases in malignant cells may enable tumors to evade the antiangiogenic effects of sema3B.
Assuntos
Inibidores da Angiogênese/farmacologia , Furina/fisiologia , Glicoproteínas de Membrana/farmacologia , Semaforinas/farmacologia , Inibidores da Angiogênese/antagonistas & inibidores , Inibidores da Angiogênese/metabolismo , Sequência de Bases , Linhagem Celular , Meios de Cultivo Condicionados , Primers do DNA , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Humanos , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/metabolismo , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Semaforinas/antagonistas & inibidores , Semaforinas/metabolismo , Transdução de SinaisRESUMO
Furin is a proprotein convertase which activates a variety of regulatory proteins in the constitutive exocytic and endocytic pathway. The effect of genetic ablation of fur was studied in the endocrine pancreas to define its physiological function in the regulated secretory pathway. Pdx1-Cre/loxP furin KO mice show decreased secretion of insulin and impaired processing of known PC2 substrates like proPC2 and proinsulin II. Both secretion and PC2 activity depend on granule acidification, which was demonstrated to be significantly decreased in furin-deficient beta cells by using the acidotrophic agent 3-(2,4-dinitroanilino)-3'amino-N-methyldipropylamine (DAMP). Ac45, an accessory subunit of the proton pump V-ATPase, was investigated as a candidate substrate. Ac45 is highly expressed in islets of Langerhans and furin was able to cleave Ac45 ex vivo. Furthermore, the exact cleavage site was determined. In addition, reduced regulated secretion and proinsulin II processing could be obtained in the insulinoma cell line betaTC3 by downregulation of either furin or Ac45. Together, these data establish an important role for furin in regulated secretion, particularly in intragranular acidification most likely due to impaired processing of Ac45.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Furina/fisiologia , Ilhotas Pancreáticas/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Furina/deficiência , Furina/metabolismo , Concentração de Íons de Hidrogênio , Insulina/metabolismo , Secreção de Insulina , Insulinoma , Ilhotas Pancreáticas/ultraestrutura , Camundongos , Camundongos Knockout , Especificidade por SubstratoRESUMO
A longstanding enigmatic feature of the group 1 coronaviruses is the uncleaved phenotype of their spike protein, an exceptional property among class I fusion proteins. Here, however, we show that some group 1 coronavirus spike proteins carry a furin enzyme recognition motif and can actually be cleaved, as demonstrated for a feline coronavirus. Interestingly, this feature can be lost during cell culture adaptation by a single mutation in the cleavage motif; this, however, preserves a heparan sulfate binding motif and renders infection by the virus heparan sulfate dependent. We identified a similar cell culture adaptation for the human coronavirus OC43.