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Inflammation leads to functional iron deficiency by increasing the expression of the hepatic iron regulatory peptide hepcidin. Inflammation also stimulates fibroblast growth factor 23 (FGF23) production by increasing both Fgf23 transcription and FGF23 cleavage, which paradoxically leads to excess in C-terminal FGF23 peptides (Cter-FGF23), rather than intact FGF23 (iFGF23) hormone. We determined that the major source of Cter-FGF23 is osteocytes and investigated whether Cter-FGF23 peptides play a direct role in the regulation of hepcidin and iron metabolism in response to acute inflammation. Mice harboring an osteocyte-specific deletion of Fgf23 showed a â¼90% reduction in Cter-FGF23 levels during acute inflammation. Reduction in Cter-FGF23 led to a further decrease in circulating iron in inflamed mice owing to excessive hepcidin production. We observed similar results in mice showing impaired FGF23 cleavage owing to osteocyte-specific deletion of Furin. We next showed that Cter-FGF23 peptides bind members of the bone morphogenetic protein (BMP) family, BMP2 and BMP9, which are established inducers of hepcidin. Coadministration of Cter-FGF23 and BMP2 or BMP9 prevented the increase in Hamp messenger RNA and circulating hepcidin levels induced by BMP2/9, resulting in normal serum iron levels. Finally, injection of Cter-FGF23 in inflamed Fgf23KO mice and genetic overexpression of Cter-Fgf23 in wild type mice also resulted in lower hepcidin and higher circulating iron levels. In conclusion, during inflammation, bone is the major source of Cter-FGF23 secretion, and independently of iFGF23, Cter-FGF23 reduces BMP-induced hepcidin secretion in the liver.
Assuntos
Fatores de Crescimento de Fibroblastos , Hepcidinas , Ferro , Animais , Camundongos , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Hepcidinas/genética , Hepcidinas/metabolismo , Inflamação/genética , PeptídeosRESUMO
Although leucine zipper tumour suppressor 1 (LZTS1) has been considered a potential tumour suppressor, accumulating evidence suggests that LZTS1 is highly expressed in many cancer types. To unravel the exact role of LZTS1 in colorectal carcinogenesis, we performed the bioinformatic analysis of LZTS1, including expression differences, correlations between expression levels and survival, methylation status of LZTS1 promoter and related cellular pathways based on TCGA dataset, GEO databases and our own CRC patient cohort. Furthermore, we confirmed the oncogenic function of LZTS1 in human mammalian cells by employing a series of assays including tissue microarray, immunoblotting, cell proliferation and migration assay. We found that the expression of LZTS1 is higher in tumour samples compared to paired normal tissue in CRC cancer and its different clinical subtypes, which is, at least in part, due to the low methylation status of LZTS1 promoter in CRC tumour samples. Functional analysis identified the close relationship between high expression of LZTS1 and PI3K-AKT pathway and the epithelial-mesenchymal transition (EMT) process. Consistently, we found that the expression of LZTS1 positively correlated with the expression PIK3CD, N-cadherin in CRC tumour samples, while the expression of LZTS1 negatively correlated with the expression of E-cadherin and PTEN in CRC tumour samples. Experimental data further confirmed that overexpression of LZTS1 upregulated activity of AKT and promoted EMT process. Furthermore, depletion of LZTS1 repressed the proliferation and migration rate of CRC cells. Thus, this study indicates that LZTS1 plays an oncogenic role in colorectal carcinogenesis.
Assuntos
Neoplasias Colorretais , Metilação de DNA , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Regiões Promotoras Genéticas , Humanos , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismo , Metilação de DNA/genética , Transição Epitelial-Mesenquimal/genética , Oncogenes/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
The insulin receptor (IR) is critically involved in maintaining glucose homeostasis. It undergoes proteolytic cleavage by proprotein convertases, which is an essential step for its activation. The importance of the insulin receptor in liver is well established, but its role in pancreatic ß cells is still controversial. In this study, we investigated the cleavage of the IR by the proprotein convertase FURIN in ß cells and hepatocytes, and the contribution of the IR in pancreatic ß cells and liver to glucose homeostasis. ß-cell-specific Furin knockout (ßFurKO) mice were glucose intolerant, but liver-specific Furin knockout (LFurKO) mice were normoglycemic. Processing of the IR was blocked in ßFurKO cells, but unaffected in LFurKO mice. Most strikingly, glucose homeostasis in ß-cell-specific IR knockout (ßIRKO) mice was normal in younger mice (up to 20 weeks), and only mildly affected in older mice (24 weeks). In conclusion, FURIN cleaves the IR non-redundantly in ß cells, but redundantly in liver. Furthermore, we demonstrated that the IR in ß cells plays a limited role in glucose homeostasis.
Assuntos
Furina/deficiência , Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Fígado/metabolismo , Receptor de Insulina/metabolismo , Animais , Furina/metabolismo , Intolerância à Glucose/metabolismo , Intolerância à Glucose/patologia , Homeostase , Camundongos Knockout , Proteólise , Receptor de Insulina/deficiência , Transdução de SinaisRESUMO
PurposePREPL deficiency causes neonatal hypotonia, ptosis, neonatal feeding difficulties, childhood obesity, xerostomia, and growth hormone deficiency. Different recessive contiguous gene deletion syndromes involving PREPL and a variable combination of SLC3A1 (hypotonia-cystinuria syndrome), CAMKMT (atypical hypotonia-cystinuria syndrome), and PPM1B (2p21 deletion syndrome) have been described. In isolated PREPL deficiency, previously described only once, the absence of cystinuria complicates the diagnosis. Therefore, we developed a PREPL blood assay and further delineated the phenotype.MethodsClinical features of new subjects with PREPL deficiency were recorded. The presence of PREPL in lymphocytes and its reactivity with an activity-based probe were evaluated by western blot.ResultsFive subjects with isolated PREPL deficiency, three with hypotonia-cystinuria syndrome, and two with atypical hypotonia-cystinuria syndrome had nine novel alleles. Their IQs ranged from 64 to 112. Adult neuromuscular signs included ptosis, nasal dysarthria, facial weakness, and variable proximal and neck flexor weakness. Autonomic features are prevalent. PREPL protein and reactivity were absent in lymphocytes from subjects with PREPL deficiency, but normal in the clinically similar Prader-Willi syndrome.ConclusionPREPL deficiency causes neuromuscular, autonomic, cognitive, endocrine, and dysmorphic clinical features. PREPL is not deficient in Prader-Willi syndrome. The novel blood test should facilitate the confirmation of PREPL deficiency.
Assuntos
Estudos de Associação Genética , Predisposição Genética para Doença , Fenótipo , Serina Endopeptidases/deficiência , Adolescente , Adulto , Criança , Pré-Escolar , Aberrações Cromossômicas , Hibridização Genômica Comparativa , Ativação Enzimática , Fácies , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Prolil Oligopeptidases , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Adulto JovemRESUMO
Gaining the full activity of the insulin receptor (IR) requires the proteolytic cleavage of its proform by intra-Golgi furin-like activity. In mammalian cells, IR is expressed as two isoforms (IRB and IRA) that are responsible for insulin action. However, only IRA transmits the growth-promoting and mitogenic effects of insulin-like growth factor 2. Here we demonstrate that the two IR isoforms are similarly cleaved by furin, but when this furin-dependent maturation is inefficient, IR proforms move to the cell surface where the proprotein convertase PACE4 selectively supports IRB maturation. Therefore, in situations of impaired furin activity, the proteolytic maturation of IRB is greater than that of IRA, and accordingly, the amount of phosphorylated IRB is also greater than that of IRA. We highlight the ability of a particular proprotein convertase inhibitor to effectively reduce the maturation of IRA and its associated mitogenic signaling without altering the signals emanating from IRB. In conclusion, the selective PACE4-dependent maturation of IRB occurs when furin activity is reduced; accordingly, the pharmacological inhibition of furin reduces IRA maturation and its mitogenic potential without altering the insulin effects.
Assuntos
Fator de Crescimento Insulin-Like II/metabolismo , Pró-Proteína Convertases/metabolismo , Receptor de Insulina/metabolismo , Serina Endopeptidases/metabolismo , Células 3T3-L1 , Animais , Proliferação de Células , Furina/genética , Furina/metabolismo , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Camundongos , Pró-Proteína Convertases/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptor de Insulina/genética , Serina Endopeptidases/genéticaRESUMO
The AP-1 complex recycles between membranes and the cytoplasm and dissociates from membranes during clathrin-coated-vesicle uncoating, but also independently of vesicular transport. The µ1A N-terminal 70 amino acids are involved in regulating AP-1 recycling. In a yeast two-hybrid library screen we identified the cytoplasmic prolyl-oligopeptidase-like protein PREPL as an interaction partner of this domain. PREPL overexpression leads to reduced AP-1 membrane binding, whereas reduced PREPL expression increases membrane binding and impairs AP-1 recycling. Altered AP-1 membrane binding in PREPL-deficient cells mirrors the membrane binding of the mutant AP-1* complex, which is not able to bind PREPL. Colocalisation of PREPL with residual membrane-bound AP-1 can be demonstrated. Patient cell lines deficient in PREPL have an expanded trans-Golgi network, which could be rescued by PREPL expression. These data demonstrate PREPL as an AP-1 effector that takes part in the regulation of AP-1 membrane binding. PREPL is highly expressed in brain and at lower levels in muscle and kidney. Its deficiency causes hypotonia and growth hormone hyposecretion, supporting essential PREPL functions in AP-1-dependent secretory pathways.
Assuntos
Serina Endopeptidases/metabolismo , Fator de Transcrição AP-1/metabolismo , Rede trans-Golgi/metabolismo , Subunidades do Complexo de Proteínas Adaptadoras/metabolismo , Animais , Encéfalo/metabolismo , Linhagem Celular , Clatrina/metabolismo , Humanos , Imunoprecipitação , Rim/metabolismo , Camundongos , Músculos/metabolismo , Prolil Oligopeptidases , Ligação ProteicaRESUMO
Streptozotocin (STZ) is widely used as diabetogenic agent in animal models for diabetic nephropathy (DN). However, it is also directly cytotoxic to kidneys, making it difficult to distinguish between DN-related and STZ-induced nephropathy. Therefore, an improved protocol to generate mice for DN studies, with a quick and robust achievement of the diabetic state, without direct kidney toxicity is required. To investigate the mechanism leading to STZ-induced nephropathy, kidney damage was induced with a high dose of STZ. This resulted in delayed gastric emptying, at least partially caused by impaired desacyl ghrelin clearance. STZ uptake in the kidneys is to a large extent mediated by the sodium/glucose cotransporters (Sglts) because the Sglt inhibitor phlorizin could reduce STZ uptake in the kidneys. Consequently, the direct toxic effects in the kidney and the gastric dilatation were resolved without interfering with the ß-cell toxicity. Furthermore, pancreatic STZ uptake was increased, hereby decreasing the threshold for ß-cell toxicity, allowing for single low non-nephrotoxic STZ doses (70 mg/kg). In conclusion, this study provides novel insights into the mechanism of STZ toxicity in kidneys and suggests a more efficient regime to induce DN with little or no toxic side effects.
Assuntos
Nefropatias Diabéticas/prevenção & controle , Células Secretoras de Insulina/metabolismo , Rim/metabolismo , Florizina/farmacologia , Transportador 1 de Glucose-Sódio/antagonistas & inibidores , Animais , Antibióticos Antineoplásicos/efeitos adversos , Antibióticos Antineoplásicos/farmacocinética , Antibióticos Antineoplásicos/farmacologia , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/induzido quimicamente , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Relação Dose-Resposta a Droga , Células Secretoras de Insulina/patologia , Rim/lesões , Rim/patologia , Masculino , Camundongos , Transportador 1 de Glucose-Sódio/metabolismo , Estreptozocina/efeitos adversos , Estreptozocina/farmacocinética , Estreptozocina/farmacologiaRESUMO
Proprotein convertases (PCs) form a group of serine endoproteases that are essential for the activation of proproteins into their active form. Some PCs have been proposed to be potential therapeutic targets for cancer intervention because elevated PC activity has been observed in many different cancer types and because many of the PC substrates, such as pro-IGF-1R, pro-TGF-beta, pro-VEGF, are involved in signaling pathways related to tumor development. Curcumin, reported to possess anticancer activity, also affects many of these pathways. We therefore investigated the effect of curcumin on PC activity. Our results show that curcumin inhibits PC activity in a cell lysate-based assay but not in vitro. PC zymogen maturation in the endoplasmic reticulum appears to be inhibited by curcumin. Treating cells with thapsigargin or cyclopiazonic acid, two structurally unrelated inhibitors of the sarco- and endoplasmic reticulum Ca(2+)ATPase (SERCA), also hampered both the PC zymogen maturation and the PC activity. Importantly, curcumin, like the SERCA inhibitors, impaired ATP-driven (45)Ca(2+) uptake in the endoplasmic reticulum. These results indicate that curcumin likely restrains PC activity by inhibiting SERCA-mediated Ca(2+)-uptake activity. Experiments in three colon cancer cell lines confirm that curcumin inhibits both the (45)Ca(2+) uptake and PC activity, notably the processing of pro-IGF-1R. Both curcumin and thapsigargin inhibit the anchorage-independent growth of these three colon carcinoma cell lines. In conclusion, our findings indicate that curcumin inhibits PC zymogen maturation and consequently PC activity and that its inhibitory effect on Ca(2+) uptake into the ER allows and is sufficient to explain this phenomenon.
Assuntos
Curcumina/farmacologia , Pró-Proteína Convertases/metabolismo , Animais , Células CHO , Células CACO-2 , Cálcio/metabolismo , Carcinoma/tratamento farmacológico , Carcinoma/enzimologia , Carcinoma/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Cricetinae , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/metabolismo , Células HT29 , Humanos , Indóis/farmacologia , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Congenital deficiency of the proprotein convertase subtilisine/kexin type 1 gene (PCSK1), which encodes proprotein convertase 1/3, causes a severe multihormonal disorder marked by early-onset obesity. The single nucleotide polymorphisms (SNPs) rs6232 and rs6234-rs6235 in PCSK1 have been associated with obesity. However, case-control studies carried out in populations of different ethnicities have only partly replicated this association. Moreover, these SNPs have only weakly been associated with body mass index (weight (kg)/height (m)(2)) at a genome-wide level of significance. To investigate this discrepancy, we conducted a systematic search for studies published before December 2013 and extracted relevant data. Pooled estimates were calculated for overall and subgroup analyses. This meta-analysis confirmed the association of PCSK1 SNPs with obesity and provides the first evidence that the association between PCSK1 rs6232 and obesity is stronger for childhood obesity than for adult obesity. Moreover, we identified weak associations with body mass index and significantly stronger associations with waist circumference for rs6234-rs6235. No difference was found in the association with different obesity grades, and no association of PCSK1 rs6234-rs6235 with obesity was identified in Asian populations. This systematic Human Genome Epidemiology (HuGE) review showed convincingly that the SNPs rs6232, rs6234, and rs6235 in PCSK1 are associated with obesity in Caucasians.
Assuntos
Obesidade/genética , Pró-Proteína Convertase 1/genética , Índice de Massa Corporal , Humanos , Polimorfismo de Nucleotídeo Único , Circunferência da Cintura/genéticaRESUMO
Loss of prolyl endopeptidase-like (PREPL) encoding a serine hydrolase with (thio)esterase activity leads to the recessive metabolic disorder Congenital Myasthenic Syndrome-22 (CMS22). It is characterized by severe neonatal hypotonia, feeding problems, growth retardation, and hyperphagia leading to rapid weight gain later in childhood. The phenotypic similarities with Prader-Willi syndrome (PWS) are striking, suggesting that similar pathways are affected. The aim of this study was to identify changes in the hypothalamic-pituitary axis in mouse models for both disorders and to examine mitochondrial function in skin fibroblasts of patients and knockout cell lines. We have demonstrated that Prepl is downregulated in the brains of neonatal PWS-IC-p/+m mice. In addition, the hypothalamic-pituitary axis is similarly affected in both Prepl-/- and PWS-IC-p/+m mice resulting in defective orexigenic signaling and growth retardation. Furthermore, we demonstrated that mitochondrial function is altered in PREPL knockout HEK293T cells and can be rescued with the supplementation of coenzyme Q10. Finally, PREPL-deficient and PWS patient skin fibroblasts display defective mitochondrial bioenergetics. The mitochondrial dysfunction in PWS fibroblasts can be rescued by overexpression of PREPL. In conclusion, we provide the first molecular parallels between CMS22 and PWS, raising the possibility that PREPL substrates might become therapeutic targets for treating both disorders.
Assuntos
Camundongos Knockout , Síndromes Miastênicas Congênitas , Síndrome de Prader-Willi , Prolil Oligopeptidases , Animais , Humanos , Síndrome de Prader-Willi/metabolismo , Síndrome de Prader-Willi/genética , Síndrome de Prader-Willi/patologia , Camundongos , Síndromes Miastênicas Congênitas/genética , Síndromes Miastênicas Congênitas/metabolismo , Síndromes Miastênicas Congênitas/patologia , Células HEK293 , Prolil Oligopeptidases/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Mitocôndrias/genética , Redes e Vias Metabólicas/genética , Modelos Animais de Doenças , Ubiquinona/análogos & derivados , Ubiquinona/metabolismo , Serina Endopeptidases/metabolismo , Serina Endopeptidases/genética , Masculino , FemininoRESUMO
AIM: Aortic aneurysms (AA) frequently involve dysregulation of transforming growth factor ß (TGF-ß)-signaling in the aorta. Here, FURIN was tested as aneurysm predisposition gene given its role as proprotein convertase in pro-TGF-ß maturation. METHODS AND RESULTS: Rare FURIN variants were detected by whole-exome sequencing of 781 unrelated aortic aneurysm patients and affected relatives. Thirteen rare heterozygous FURIN variants occurred in 3.7% (29) unrelated index AA patients, of which 72% had multiple aneurysms or a dissection.FURIN maturation and activity of these variants were decreased in vitro. Patient-derived fibroblasts showed decreased pro-TGF-ß processing, phosphorylation of downstream effector SMAD2 and kinases ERK1/2, and steady-state mRNA levels of the TGF-ß-responsive ACTA2 gene. In aortic tissue, collagen and fibrillin fibers were affected. One variant (R745Q), observed in 10 unrelated cases, affected TGF-ß signaling variably, indicating effect modification by individual genetic backgrounds. CONCLUSION: FURIN is a novel, frequent genetic predisposition for abdominal-, thoracic-, and multiple aortic or middle sized artery aneurysms in older patients, by affecting intracellular TGF-ß signaling, depending on individual genetic backgrounds.
RESUMO
Neurobeachin (Nbea) is implicated in vesicle trafficking in the regulatory secretory pathway, but details on its molecular function are currently unknown. We have used Drosophila melanogaster mutants for rugose (rg), the Drosophila homolog of Nbea, to further elucidate the function of this multidomain protein. Rg is expressed in a granular pattern reminiscent of the Golgi network in neuronal cell bodies and colocalizes with transgenic Nbea, suggesting a function in secretory regulation. In contrast to Nbea(-/-) mice, rg null mutants are viable and fertile and exhibit aberrant associative odor learning, changes in gross brain morphology, and synaptic architecture as determined at the larval neuromuscular junction. At the same time, basal synaptic transmission is essentially unaffected, suggesting that structural and functional aspects are separable. Rg phenotypes can be rescued by a Drosophila rg+ transgene, whereas a mouse Nbea transgene rescues aversive odor learning and synaptic architecture; it fails to rescue brain morphology and appetitive odor learning. This dissociation between the functional redundancy of either the mouse or the fly transgene suggests that their complex composition of numerous functional and highly conserved domains support independent functions. We propose that the detailed compendium of phenotypes exhibited by the Drosophila rg null mutant provided here will serve as a test bed for dissecting the different functional domains of BEACH (for beige and human Chediak-Higashi syndrome) proteins, such as Rugose, mouse Nbea, or Nbea orthologs in other species, such as human.
Assuntos
Proteínas de Ancoragem à Quinase A/fisiologia , Aprendizagem por Associação/fisiologia , Encéfalo/citologia , Proteínas de Drosophila/fisiologia , Sinapses/fisiologia , Proteínas de Ancoragem à Quinase A/deficiência , Proteínas de Ancoragem à Quinase A/genética , Análise de Variância , Animais , Animais Geneticamente Modificados , Encéfalo/fisiologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Drosophila , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Peroxidase do Rábano Silvestre/metabolismo , Masculino , Potenciais da Membrana/genética , Mutação/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Junção Neuromuscular/citologia , Junção Neuromuscular/genética , Neurônios/citologia , Odorantes , Neurônios Receptores Olfatórios/citologia , Técnicas de Patch-Clamp , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , RNA Mensageiro/metabolismo , Estatísticas não Paramétricas , Sinapses/genéticaRESUMO
Proprotein convertase 7 (PC7) is a member of the subtilisin-like proprotein convertase family, which is involved in the endoproteolysis of a variety of precursor proteins. Under steady state conditions, PC7 is mainly localized in the trans-Golgi network, but a small fraction is found at the cell surface. So far, no sorting signals for membrane trafficking have been identified in PC7. In this study, we have examined the internalization of PC7 from the plasma membrane. Our results show that internalization of PC7 is mediated by clathrin-coated vesicles. After inhibition of clathrin-mediated endocytosis using hypertonic conditions or the small molecule inhibitor, Pitstop 2, PC7 accumulated at the plasma membrane. Furthermore, PC7 was present in isolated clathrin-coated vesicles. To determine the internalization motif, constructs were generated in which parts of the N and C terminus of the cytoplasmic tail of PC7 were deleted, and chimeric proteins were constructed consisting of the luminal and transmembrane domains of Tac (CD25) and parts of the cytoplasmic domain of PC7. Antibody uptake experiments as well as surface biotinylation experiments demonstrated that the region between Ala(713) and Cys(726) in the cytoplasmic domain of PC7 is essential and sufficient for the internalization of PC7 but not for trans-Golgi network localization. Individual amino acids in this region were substituted with alanine, which identified Pro, Leu, and Cys as the essential amino acids. In conclusion, internalization of PC7 depends on a short transferable sequence in the cytoplasmic tail, which contains the three crucial amino acids PLC.
Assuntos
Membrana Celular/enzimologia , Endocitose , Subtilisinas/química , Subtilisinas/metabolismo , Rede trans-Golgi/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Linhagem Celular , Membrana Celular/química , Membrana Celular/genética , Humanos , Dados de Sequência Molecular , Transporte Proteico , Subtilisinas/genética , Rede trans-Golgi/química , Rede trans-Golgi/metabolismoRESUMO
Neurobeachin (NBEA), a brain-enriched multidomain scaffolding protein involved in neurotransmitter release and synaptic functioning, has been identified as a candidate gene for autism spectrum disorder (ASD) in four unrelated patients haploinsufficient for NBEA. The aim of this study was to map the behavioral phenotype of Nbea(+/-) mice in order to understand its contribution to the pathogenesis of ASD. ASD-like behavioral variables of Nbea(+/-) mice were related to basal neuronal activity in different brain regions by in situ hybridizations and extracellular field recordings of synaptic plasticity in hippocampal cornu ammonis 1 (CA1) region. Levels of BDNF and phosphorylated cAMP response element-binding protein (CREB) were measured in an attempt to investigate putatively underlying changes in these neuromolecules. Nbea(+/-) mice exhibit several ASD-like features, including changes in self-grooming behavior, social behaviors, conditioned fear responses, and spatial learning and memory, which coincided with enhanced long-term potentiation (LTP) in their CA1 region. The observed alterations in learning and memory and hippocampal LTP are concomitant with decreased expression of the immediate early gene zif268 in dorsomedial striatum and hippocampal CA1 region, increased CREB phosphorylation, and increased hippocampal BDNF expression. These findings indicate that Nbea haploinsufficiency leads to various molecular and cellular changes that affect neuroplasticity and behavioral functions in mice, and could thus underlie the ASD symptomatology in NBEA deficient humans.
Assuntos
Transtorno Autístico/genética , Comportamento Animal/fisiologia , Encéfalo/fisiopatologia , Proteínas de Transporte/genética , Proteínas do Tecido Nervoso/genética , Plasticidade Neuronal/genética , Animais , Criança , Feminino , Haploinsuficiência , Humanos , Immunoblotting , Hibridização In Situ , Aprendizagem/fisiologia , Potenciação de Longa Duração/fisiologia , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Transmissão Sináptica/genéticaRESUMO
The PC (proprotein convertase) furin cleaves a large variety of proproteins and hence plays a major role in many pathologies. Therefore furin inhibition might be a good strategy for therapeutic intervention, and several furin inhibitors have been generated, although none are entirely furin-specific. To reduce potential side effects caused by cross-reactivity with other proteases, dromedary heavy-chain-derived nanobodies against catalytically active furin were developed as specific furin inhibitors. The nanobodies bound only to furin but not to other PCs. Upon overexpression in cell lines, they inhibited the cleavage of two different furin substrates, TGFß (transforming growth factor ß) and GPC3 (glypican 3). Purified nanobodies could inhibit the cleavage of diphtheria toxin into its enzymatically active A fragment, but did not inhibit cleavage of a small synthetic peptide-based substrate, suggesting a mode-of-action based on steric hindrance. The dissociation constant of purified nanobody 14 is in the nanomolar range. The nanobodies were non-competitive inhibitors with an inhibitory constant in the micromolar range as demonstrated by Dixon plot. Furthermore, anti-furin nanobodies could protect HEK (human embryonic kidney)-293T cells from diphtheria-toxin-induced cytotoxicity as efficiently as the PC inhibitor nona-D-arginine. In conclusion, these antibody-based single-domain nanobodies represent the first generation of highly specific non-competitive furin inhibitors.
Assuntos
Furina/antagonistas & inibidores , Anticorpos de Domínio Único/farmacologia , Animais , Especificidade de Anticorpos , Camelus , Catálise/efeitos dos fármacos , Cumarínicos/metabolismo , Toxina Diftérica/metabolismo , Endocitose , Furina/química , Furina/imunologia , Furina/metabolismo , Glipicanas/metabolismo , Células HEK293/metabolismo , Humanos , Cinética , Camundongos , Oligopeptídeos/metabolismo , Fragmentos de Peptídeos/metabolismo , Pró-Proteína Convertases/metabolismo , Ligação Proteica/efeitos dos fármacos , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia , Especificidade por Substrato , Fator de Crescimento Transformador beta/metabolismoRESUMO
The dysfunction of immune cell development often impairs immunological homeostasis, thus causing various human diseases. Accumulating evidence shows that the development of different immune cells from hematopoietic stem cells are highly fine-tuned by different epigenetic mechanisms including DNA methylation, histone modifications, chromatin remodeling and RNA-related regulations. Understanding how epigenetic regulators modulate normal development of immune cells contributes to the identification of new strategies for various diseases. Here, we review recent advances suggesting that epigenetic modulations can orchestrate immune cell development and functions through their impact on critical gene expression. We also discuss the aberrations of epigenetic modulations in immune cells that influence tumor progression, and the fact that underlying mechanisms affect how epigenetic drugs interfere with tumor progression in the clinic.
Assuntos
Histonas , Neoplasias , Humanos , Histonas/metabolismo , Metilação de DNA/genética , Epigênese Genética/genética , Cromatina , Neoplasias/genética , Neoplasias/metabolismoRESUMO
Autism is a neurodevelopmental disorder characterized by impaired social reciprocity, impaired communication and stereotypical behaviors. Despite strong evidence for a genetic basis, few susceptibility genes have been identified. Here, we describe the positional cloning of SCAMP5, CLIC4 and PPCDC as candidate genes for autism, starting from a person with idiopathic, sporadic autism carrying a de novo chromosomal translocation. One of these genes, SCAMP5 is silenced on the derivative chromosome, and encodes a brain-enriched protein involved in membrane trafficking, similar to the previously identified candidate genes NBEA and AMISYN. Gene silencing of Nbea, Amisyn and Scamp5 in mouse beta-TC3 cells resulted in a 2-fold increase in stimulated secretion of large dense-core vesicles (LDCVs), while overexpression suppressed secretion. Moreover, ultrastructural analysis of blood platelets from the patients with haploinsufficieny of one of the three candidate genes, showed morphological abnormalities of dense-core granules, which closely resemble LDCVs. Taken together, this study shows that in three independent patients with autism three different negative regulators of LDCV secretion are affected, respectively, suggesting that in at least a subgroup of patients the regulation of neuronal vesicle trafficking may be involved in the pathogenesis of autism.
Assuntos
Transtorno Autístico/genética , Proteínas de Transporte/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Vesículas Secretórias/metabolismo , Adulto , Animais , Transtorno Autístico/sangue , Plaquetas/patologia , Proteínas de Transporte/fisiologia , Linhagem Celular , Cromossomos Humanos Par 15 , Inativação Gênica , Humanos , Masculino , Proteínas de Membrana/fisiologia , Camundongos , Translocação GenéticaRESUMO
Type 2 diabetes is a disorder of hyperglycemia resulting from failure of beta cells to produce adequate insulin to accommodate an increased metabolic demand. Here we show that regulation of mRNA translation through phosphorylation of eukaryotic initiation factor 2 (eIF2alpha) is essential to preserve the integrity of the endoplasmic reticulum (ER) and to increase insulin production to meet the demand imposed by a high-fat diet. Accumulation of unfolded proteins in the ER activates phosphorylation of eIF2alpha at Ser51 and inhibits translation. To elucidate the role of this pathway in beta-cell function we studied glucose homeostasis in Eif2s1(tm1Rjk) mutant mice, which have an alanine substitution at Ser51. Heterozygous (Eif2s1(+/tm1Rjk)) mice became obese and diabetic on a high-fat diet. Profound glucose intolerance resulted from reduced insulin secretion accompanied by abnormal distension of the ER lumen, defective trafficking of proinsulin, and a reduced number of insulin granules in beta cells. We propose that translational control couples insulin synthesis with folding capacity to maintain ER integrity and that this signal is essential to prevent diet-induced type 2 diabetes.
Assuntos
Retículo Endoplasmático/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Glucose/metabolismo , Ilhotas Pancreáticas/metabolismo , Biossíntese de Proteínas/genética , Substituição de Aminoácidos , Animais , Células Cultivadas , Diabetes Mellitus Tipo 2/etiologia , Dietas da Moda , Dieta com Restrição de Gorduras , Retículo Endoplasmático/genética , Retículo Endoplasmático/patologia , Chaperona BiP do Retículo Endoplasmático , Fator de Iniciação 2 em Eucariotos/genética , Intolerância à Glucose/genética , Proteínas de Choque Térmico/metabolismo , Homeostase/genética , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/patologia , Camundongos , Camundongos Mutantes , Chaperonas Moleculares/metabolismo , Obesidade/genética , Fosforilação , Proinsulina/metabolismoRESUMO
Furin is the first discovered proprotein convertase member and is present in almost all mammalian cells. Therefore, by regulating the maturation of a wide range of proproteins, Furin expression and/or activity is involved in various physiological and pathophysiological processes ranging from embryonic development to carcinogenesis. Since many of these protein precursors are involved in initiating and maintaining the hallmarks of cancer, Furin has been proposed as a potential target for treating several human cancers. In contrast, other studies have revealed that some types of cancer do not benefit from Furin inhibition. Therefore, understanding the heterogeneous functions of Furin in cancer will provide important insights into the design of effective strategies targeting Furin in cancer treatment. Here, we present recent advances in understanding how Furin expression and activity are regulated in cancer cells and their influences on the activity of Furin substrates in carcinogenesis. Furthermore, we discuss how Furin represses tumorigenic properties of several cancer cells and why Furin inhibition leads to aggressive phenotypes in other tumors. Finally, we summarize the clinical applications of Furin inhibition in treating human cancers.
Assuntos
FurinaRESUMO
The generation of mature T cells and establishment of central tolerance is predominantly orchestrated by thymic epithelial cells (TECs). Proprotein convertases are responsible for the proteolysis of proproteins into their mature bioactive counterparts. Here, we found that Furin, a member of the subtilisin/kexin-like PCs family, is highly expressed in TECs compared with other members of this family. TEC-specific deletion of Furin caused severe thymic atrophy and predominantly reduced the number of medullary TECs and thymic tuft cells, and to a less degree, cortical TECs. Furin deletion attenuated the proliferation of TECs, impaired thymopoiesis, and led to autoimmune disorders in mice. Furin promotes the development of TECs via cleavage of proIGF1 receptor and pro-Insulin receptor and the activation of downstream ERK/MAPK and Akt signaling pathways. Thus, this study uncovered the role of furin in TEC development and function and highlighted the importance of post-translational modification of immature proproteins in TEC biology.