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1.
Nat Commun ; 11(1): 4818, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32968060

RESUMO

Migrating cells move across diverse assemblies of extracellular matrix (ECM) that can be separated by micron-scale gaps. For membranes to protrude and reattach across a gap, actin filaments, which are relatively weak as single filaments, must polymerize outward from adhesion sites to push membranes towards distant sites of new adhesion. Here, using micropatterned ECMs, we identify T-Plastin, one of the most ancient actin bundling proteins, as an actin stabilizer that promotes membrane protrusions and enables bridging of ECM gaps. We show that T-Plastin widens and lengthens protrusions and is specifically enriched in active protrusions where F-actin is devoid of non-muscle myosin II activity. Together, our study uncovers critical roles of the actin bundler T-Plastin to promote protrusions and migration when adhesion is spatially-gapped.


Assuntos
Movimento Celular/fisiologia , Extensões da Superfície Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Sistemas CRISPR-Cas , Adesão Celular , Linhagem Celular , Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , Técnicas de Inativação de Genes , Humanos , Cinética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/ultraestrutura , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/ultraestrutura , Miosinas/metabolismo , Pseudópodes/metabolismo , Receptor EphB2
2.
J Pharmacol Sci ; 144(2): 89-93, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32763057

RESUMO

l-3,4-dihydroxyphenylalanine (l-DOPA) is a candidate neurotransmitter. l-DOPA is released by nicotine through nicotinic receptors. Recently, G-protein coupled receptor GPR143, was identified as a receptor for l-DOPA. In this study, genetic association studies between GPR143 genetic polymorphisms and smoking behaviors revealed that the single-nucleotide polymorphism rs6640499, in the GPR143 gene, was associated with traits of smoking behaviors in Japanese individuals. In Gpr143 gene-deficient mice, nicotine-induced hypolocomotion and rewarding effect were attenuated compared to those in wild-type mice. Our findings suggest the involvement of GPR143 in the smoking behaviors.


Assuntos
Proteínas do Olho/genética , Deleção de Genes , Estudos de Associação Genética , Glicoproteínas de Membrana/genética , Nicotina/efeitos adversos , Polimorfismo de Nucleotídeo Único , Receptores Acoplados a Proteínas-G/genética , Receptores de Neurotransmissores/genética , Reforço Psicológico , Transtornos Relacionados ao Uso de Substâncias/genética , Animais , Grupo com Ancestrais do Continente Asiático , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Índice de Gravidade de Doença
3.
Am J Hum Genet ; 107(3): 473-486, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32781046

RESUMO

Africa contains more human genetic variation than any other continent, but the majority of the population-scale analyses of the African peoples have focused on just two of the four major linguistic groups, the Niger-Congo and Afro-Asiatic, leaving the Nilo-Saharan and Khoisan populations under-represented. In order to assess genetic variation and signatures of selection within a Nilo-Saharan population and between the Nilo-Saharan and Niger-Congo and Afro-Asiatic, we sequenced 50 genomes from the Nilo-Saharan Lugbara population of North-West Uganda and 250 genomes from 6 previously unsequenced Niger-Congo populations. We compared these data to data from a further 16 Eurasian and African populations including the Gumuz, another putative Nilo-Saharan population from Ethiopia. Of the 21 million variants identified in the Nilo-Saharan population, 3.57 million (17%) were not represented in dbSNP and included predicted non-synonymous mutations with possible phenotypic effects. We found greater genetic differentiation between the Nilo-Saharan Lugbara and Gumuz populations than between any two Afro-Asiatic or Niger-Congo populations. F3 tests showed that Gumuz contributed a genetic component to most Niger-Congo B populations whereas Lugabara did not. We scanned the genomes of the Lugbara for evidence of selective sweeps. We found selective sweeps at four loci (SLC24A5, SNX13, TYRP1, and UVRAG) associated with skin pigmentation, three of which already have been reported to be under selection. These selective sweeps point toward adaptations to the intense UV radiation of the Sahel.


Assuntos
Adaptação Fisiológica/genética , Variação Genética/genética , Seleção Genética/genética , Pigmentação da Pele/genética , Grupo com Ancestrais do Continente Africano/genética , Antiporters/genética , Gerenciamento de Dados , Etiópia/epidemiologia , Feminino , Genética Populacional , Genoma Humano/genética , Haplótipos/genética , Humanos , Masculino , Glicoproteínas de Membrana/genética , Oxirredutases/genética , Polimorfismo de Nucleotídeo Único/genética , Nexinas de Classificação/genética , Proteínas Supressoras de Tumor/genética , Uganda/epidemiologia
4.
Virology ; 548: 17-24, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32838939

RESUMO

The demyelinating disease progressive multifocal leukoencephalopathy (PML) is caused by the human polyomavirus, JCPyV, under conditions of prolonged immunosuppression. Initial infection is asymptomatic, and the virus establishes lifelong persistence in the host. Following the loss of immune surveillance, the virus can traffic to the central nervous system and infect oligodendrocytes to cause demyelination and PML. The mechanisms involved in glial cell infection are not completely understood. In a screen for N-glycosylated proteins that influence JCPyV pathology, we identified Adipocyte Plasma Membrane Associated Protein (APMAP) as a host cell modulator of JCPyV infection. The removal of APMAP by small interfering siRNA as well as by CRISPR-Cas9 gene editing resulted in a significant decrease in JCPyV infection. Exogenous expression of APMAP in APMAP knockout cell lines rescued susceptibility to infection. These data suggest that virus infection of glial cells is dependent on APMAP.


Assuntos
Vírus JC/fisiologia , Neuroglia/metabolismo , Infecções por Polyomavirus/metabolismo , Linhagem Celular , Interações Hospedeiro-Patógeno , Humanos , Vírus JC/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana , Neuroglia/virologia , Oligodendroglia/metabolismo , Oligodendroglia/virologia , Infecções por Polyomavirus/genética , Infecções por Polyomavirus/virologia
5.
Infect Genet Evol ; 84: 104498, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32771700

RESUMO

New coronavirus SARS-CoV-2 is capable to infect humans and cause a novel disease COVID-19. Aiming to understand a host genetic component of COVID-19, we focused on variants in genes encoding proteases and genes involved in innate immunity that could be important for susceptibility and resistance to SARS-CoV-2 infection. Analysis of sequence data of coding regions of FURIN, PLG, PRSS1, TMPRSS11a, MBL2 and OAS1 genes in 143 unrelated individuals from Serbian population identified 22 variants with potential functional effect. In silico analyses (PolyPhen-2, SIFT, MutPred2 and Swiss-Pdb Viewer) predicted that 10 variants could impact the structure and/or function of proteins. These protein-altering variants (p.Gly146Ser in FURIN; p.Arg261His and p.Ala494Val in PLG; p.Asn54Lys in PRSS1; p.Arg52Cys, p.Gly54Asp and p.Gly57Glu in MBL2; p.Arg47Gln, p.Ile99Val and p.Arg130His in OAS1) may have predictive value for inter-individual differences in the response to the SARS-CoV-2 infection. Next, we performed comparative population analysis for the same variants using extracted data from the 1000 Genomes project. Population genetic variability was assessed using delta MAF and Fst statistics. Our study pointed to 7 variants in PLG, TMPRSS11a, MBL2 and OAS1 genes with noticeable divergence in allelic frequencies between populations worldwide. Three of them, all in MBL2 gene, were predicted to be damaging, making them the most promising population-specific markers related to SARS-CoV-2 infection. Comparing allelic frequencies between Serbian and other populations, we found that the highest level of genetic divergence related to selected loci was observed with African, followed by East Asian, Central and South American and South Asian populations. When compared with European populations, the highest divergence was observed with Italian population. In conclusion, we identified 4 variants in genes encoding proteases (FURIN, PLG and PRSS1) and 6 in genes involved in the innate immunity (MBL2 and OAS1) that might be relevant for the host response to SARS-CoV-2 infection.


Assuntos
Infecções por Coronavirus/genética , Resistência à Doença/genética , Predisposição Genética para Doença , Interações Hospedeiro-Patógeno/genética , Metagenômica , Peptidil Dipeptidase A/genética , Pneumonia Viral/genética , Glicoproteína da Espícula de Coronavírus/genética , Alelos , Betacoronavirus/imunologia , Betacoronavirus/patogenicidade , Infecções por Coronavirus/imunologia , Proteínas do Olho/genética , Proteínas do Olho/imunologia , Furina/genética , Furina/imunologia , Frequência do Gene , Variação Genética , Genoma Humano , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Lectina de Ligação a Manose/genética , Lectina de Ligação a Manose/imunologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Pandemias , Peptidil Dipeptidase A/imunologia , Plasminogênio/genética , Plasminogênio/imunologia , Pneumonia Viral/imunologia , Ligação Proteica , Glicoproteína da Espícula de Coronavírus/imunologia , Tripsina/genética , Tripsina/imunologia
6.
PLoS One ; 15(8): e0237577, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790741

RESUMO

Abnormal skin melanin homeostasis results in refractory pigmentary diseases. Melanogenesis is influenced by gene regulation, ultraviolet radiation, and host epigenetic responses. Steroid receptor RNA activator (SRA), a long noncoding RNA, is known to regulate steroidogenesis and tumorigenesis. However, how SRA contributes to melanogenesis remains unknown. Using RNA interference against SRA in B16 and A375 melanoma cells, we observed increased pigmentation and increased expression of TRP1 and TRP2 at transcriptional and translational levels only in B16 cells. The constitutive phosphorylation of p38 in B16-shCtrl cells was inhibited in cells with knocked down SRAi. Moreover, the melanin content of control B16 cells was increased by SB202190, a p38 inhibitor. Furthermore, reduced p38 phosphorylation, enhanced TRP1 expression, and hypermelanosis were observed in A375 cells with RNA interference. These results indicate that SRA-p38-TRP1 axis has a regulatory role in melanin homeostasis and that SRA might be a potential therapeutic target for treating pigmentary diseases.


Assuntos
Oxirredutases Intramoleculares/metabolismo , Melaninas/metabolismo , Melanoma Experimental/patologia , Glicoproteínas de Membrana/metabolismo , Oxirredutases/metabolismo , RNA Longo não Codificante/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Regulação Neoplásica da Expressão Gênica , Oxirredutases Intramoleculares/genética , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Glicoproteínas de Membrana/genética , Camundongos , Oxirredutases/genética , Fosforilação , RNA Longo não Codificante/genética , RNA Interferente Pequeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética
7.
Nat Commun ; 11(1): 3569, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32678083

RESUMO

The clinically important MAM blood group antigen is present on haematopoietic cells of all humans except rare MAM-negative individuals. Its molecular basis is unknown. By whole-exome sequencing we identify EMP3, encoding epithelial membrane protein 3 (EMP3), as a candidate gene, then demonstrate inactivating mutations in ten known MAM-negative individuals. We show that EMP3, a purported tumour suppressor in various solid tumours, is expressed in erythroid cells. Disruption of EMP3 by CRISPR/Cas9 gene editing in an immortalised human erythroid cell line (BEL-A2) abolishes MAM expression. We find EMP3 to associate with, and stabilise, CD44 in the plasma membrane. Furthermore, cultured erythroid progenitor cells from MAM-negative individuals show markedly increased proliferation and higher reticulocyte yields, suggesting an important regulatory role for EMP3 in erythropoiesis and control of cell production. Our data establish MAM as a new blood group system and demonstrate an interaction of EMP3 with the cell surface signalling molecule CD44.


Assuntos
Antígenos de Grupos Sanguíneos/genética , Proliferação de Células , Células Eritroides/citologia , Glicoproteínas de Membrana/genética , Antígenos de Grupos Sanguíneos/química , Antígenos de Grupos Sanguíneos/metabolismo , Plaquetas/metabolismo , Células Cultivadas , Membrana Eritrocítica/metabolismo , Células Eritroides/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Mutação , Fenótipo , Ligação Proteica , Sequenciamento Completo do Exoma
8.
Mol Cell Biol ; 40(19)2020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-32719109

RESUMO

Recent studies have demonstrated the existence of a discrete pool of cholesterol in the plasma membranes (PM) of mammalian cells-referred to as the accessible cholesterol pool-that can be detected by the binding of modified versions of bacterial cytolysins (e.g., anthrolysin O). When the amount of accessible cholesterol in the PM exceeds a threshold level, the excess cholesterol moves to the endoplasmic reticulum (ER), where it regulates the SREBP2 pathway and undergoes esterification. We reported previously that the Aster/Gramd1 family of sterol transporters mediates nonvesicular movement of cholesterol from the PM to the ER in multiple mammalian cell types. Here, we investigated the PM pool of accessible cholesterol in cholesterol-loaded fibroblasts with a knockdown of Aster-A and in mouse macrophages from Aster-B and Aster-A/B-deficient mice. Nanoscale secondary ion mass spectrometry (NanoSIMS) analyses revealed expansion of the accessible cholesterol pool in cells lacking Aster expression. The increased accessible cholesterol pool in the PM was accompanied by reduced cholesterol movement to the ER, evidenced by increased expression of SREBP2-regulated genes. Cosedimentation experiments with liposomes revealed that the Aster-B GRAM domain binds to membranes in a cholesterol concentration-dependent manner and that the binding is facilitated by the presence of phosphatidylserine. These studies revealed that the Aster-mediated nonvesicular cholesterol transport pathway controls levels of accessible cholesterol in the PM, as well as the activity of the SREBP pathway.


Assuntos
Membrana Celular/metabolismo , Colesterol/metabolismo , Células 3T3-L1 , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Retículo Endoplasmático/metabolismo , Fibroblastos/metabolismo , Lipossomos/metabolismo , Macrófagos Peritoneais/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espectrometria de Massa de Íon Secundário/métodos , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo
9.
Cell Prolif ; 53(7): e12833, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32525231

RESUMO

OBJECTIVES: The current study aimed to investigate the mechanism by which exosomes secreted by CHB patients with PNALT and liver inflammation grade (≥A2) affected the development of liver cancer. MATERIALS AND METHODS: Gene expression was assessed by RT-PCR, Western blotting and immunohistochemistry. CCK-8, colony formation, transwell, scratch-wound and flow cytometry assays were used to detect cell viability, proliferation, apoptosis and metastasis. The interaction of TCF21 and HHIP was assessed by co-immunoprecipitation assay. Luciferase reporter was used to detect the combination of TCF21/HHIP and miR-25-3p. Xenograft studies in nude mice manifested tumour growth ability of miR-25-3p. Bioinformatics analyses were conducted using TargetScan, EVmiRNA, TCGA, GEO, DAVID, COEXPEDIA, UALCAN, UCSC and the Human Protein Atlas databases. RESULTS: CHB-PNALT-Exo (≥A2) promoted the proliferation and metastasis of HepG2.2.15 cells. miR-25-3p was upregulated in CHB-PNALT-Exo (≥A2). miR-25-3p overexpression promoted cell proliferation and metastasis and was related to poor survival in patients with CHB-PNALT (≥A2). The cell proliferation- and metastasis-promoting functions of CHB-PNALT-Exo (≥A2) were abolished by miR-25-3p inhibitors. TCF21 directly interacted with HHIP. Inhibition of TCF21 or HHIP promoted cell proliferation and metastasis. Knockdown of TCF21 or HHIP counteracted the effects of CHB-PNALT-Exo (≥A2) containing miR-25-3p inhibitor on cell proliferation, metastasis and the expression of Ki67, E-cadherin and caspase-3/-9. CONCLUSIONS: Transfer of miR-25-3p by CHB-PNALT-Exo promoted the development of liver cancer by inhibiting the co-expression of TCF21 and HHIP.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Transporte/genética , Exossomos/genética , Hepatite B Crônica/genética , Neoplasias Hepáticas/genética , Fígado/patologia , Glicoproteínas de Membrana/genética , MicroRNAs/genética , Adulto , Animais , Apoptose/genética , Proliferação de Células/genética , Sobrevivência Celular , Progressão da Doença , Regulação para Baixo/genética , Exossomos/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Hepatite B Crônica/patologia , Humanos , Inflamação/genética , Inflamação/patologia , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Regulação para Cima/genética , Adulto Jovem
10.
Science ; 368(6497)2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32554568

RESUMO

Meissner corpuscles are mechanosensory end organs that densely occupy mammalian glabrous skin. We generated mice that selectively lacked Meissner corpuscles and found them to be deficient in both perceiving the gentlest detectable forces acting on glabrous skin and fine sensorimotor control. We found that Meissner corpuscles are innervated by two mechanoreceptor subtypes that exhibit distinct responses to tactile stimuli. The anatomical receptive fields of these two mechanoreceptor subtypes homotypically tile glabrous skin in a manner that is offset with respect to one another. Electron microscopic analysis of the two Meissner afferents within the corpuscle supports a model in which the extent of lamellar cell wrappings of mechanoreceptor endings determines their force sensitivity thresholds and kinetic properties.


Assuntos
Epiderme/inervação , Células de Merkel/fisiologia , Células de Merkel/ultraestrutura , Percepção do Tato/fisiologia , Tato/fisiologia , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Feminino , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Proteínas Tirosina Quinases/genética , Transdução de Sinais
11.
Am J Physiol Gastrointest Liver Physiol ; 319(2): G109-G120, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32508154

RESUMO

Crohn's disease (CD) is a complex and multifactorial illness. There are still considerable gaps in our knowledge regarding its pathophysiology. A transcriptomic approach could shed some light on little-known biological alterations of the disease. We therefore aimed to explore the ileal transcriptome to gain knowledge about CD. We performed whole transcriptome gene expression analysis on ileocecal resections from CD patients and inflammatory bowel disease-free controls, as well as on a CD-independent cohort to replicate selected results. Normalized data were hierarchically clustered, and gene ontology and the molecular network were studied. Cell cultures and molecular methods were used for further evaluations. Genome-wide expression data analysis identified a robust transmembrane immunoglobulin domain-containing 1 (TMIGD1) gene underexpression in CD tissue, which was even more marked in inflamed ileum, and which was replicated in the validation cohort. Immunofluorescence showed TMIGD1 to be located in the apical microvilli of well-differentiated enterocytes but not in intestinal crypt. This apical TMIGD1 was lower in the noninflamed tissue and almost disappeared in the inflamed mucosa of surgical resections. In vitro studies showed hypoxic-dependent TMIGD1 decreased its expression in enterocyte-like cells. The gene enrichment analysis linked TMIGD1 with cell recovery and tissue remodeling in CD settings, involving guanylate cyclase activities. Transcriptomics may be useful for finding new targets that facilitate studies of the CD pathology. This is how TMIGD1 was identified in CD patients, which was related to multiciliate ileal epithelial cell differentiation.NEW & NOTEWORTHY This is a single-center translational research study that aimed to look for key targets involved in Crohn's disease and define molecular pathways through different functional analysis strategies. With this approach, we have identified and described a novel target, the almost unknown TMIGD1 gene, which may be key in the recovery of injured mucosa involving intestinal epithelial cell differentiation.


Assuntos
Doença de Crohn/genética , Células Epiteliais/fisiologia , Íleo/citologia , Glicoproteínas de Membrana/metabolismo , Transcriptoma , Adulto , Células CACO-2 , Estudos de Casos e Controles , Diferenciação Celular , Doença de Crohn/metabolismo , Regulação da Expressão Gênica , Humanos , Inflamação/metabolismo , Glicoproteínas de Membrana/genética , Consumo de Oxigênio
12.
Nat Commun ; 11(1): 2977, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32532995

RESUMO

Independent scientific achievements have led to the discovery of aberrant splicing patterns in oncogenesis, while more recent advances have uncovered novel gene fusions involving neurotrophic tyrosine receptor kinases (NTRKs) in gliomas. The exploration of NTRK splice variants in normal and neoplastic brain provides an intersection of these two rapidly evolving fields. Tropomyosin receptor kinase B (TrkB), encoded NTRK2, is known for critical roles in neuronal survival, differentiation, molecular properties associated with memory, and exhibits intricate splicing patterns and post-translational modifications. Here, we show a role for a truncated NTRK2 splice variant, TrkB.T1, in human glioma. TrkB.T1 enhances PDGF-driven gliomas in vivo, augments PDGF-induced Akt and STAT3 signaling in vitro, while next generation sequencing broadly implicates TrkB.T1 in the PI3K signaling cascades in a ligand-independent fashion. These TrkB.T1 findings highlight the importance of expanding upon whole gene and gene fusion analyses to include splice variants in basic and translational neuro-oncology research.


Assuntos
Neoplasias Encefálicas/genética , Glioma/genética , Glicoproteínas de Membrana/genética , Oncogenes/genética , Isoformas de RNA/genética , Processamento de RNA , Receptor trkB/genética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Carcinogênese/genética , Células Cultivadas , Perfilação da Expressão Gênica , Ontologia Genética , Glioma/metabolismo , Glioma/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Glicoproteínas de Membrana/metabolismo , Camundongos , Células NIH 3T3 , Células-Tronco Neurais/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Isoformas de RNA/metabolismo , Receptor trkB/metabolismo , Transdução de Sinais/genética
13.
PLoS Pathog ; 16(6): e1008621, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32544188

RESUMO

During tuberculosis, lung myeloid cells have two opposing roles: they are an intracellular niche occupied by Mycobacterium tuberculosis, and they restrict bacterial replication. Lung myeloid cells from mice infected with yellow-fluorescent protein expressing M. tuberculosis were analyzed by flow cytometry and transcriptional profiling to identify the cell types infected and their response to infection. CD14, CD38, and Abca1 were expressed more highly by infected alveolar macrophages and CD11cHi monocyte-derived cells compared to uninfected cells. CD14, CD38, and Abca1 "triple positive" (TP) cells had not only the highest infection rates and bacterial loads, but also a strong interferon-γ signature and nitric oxide synthetase-2 production indicating recognition by T cells. Despite evidence of T cell recognition and appropriate activation, these TP macrophages are a cellular compartment occupied by M. tuberculosis long-term. Defining the niche where M. tuberculosis resists elimination promises to provide insight into why inducing sterilizing immunity is a formidable challenge.


Assuntos
Antígenos CD11/imunologia , Macrófagos Alveolares , Monócitos , Mycobacterium tuberculosis/imunologia , Tuberculose/imunologia , ADP-Ribosil Ciclase 1/genética , ADP-Ribosil Ciclase 1/imunologia , Transportador 1 de Cassete de Ligação de ATP/genética , Transportador 1 de Cassete de Ligação de ATP/imunologia , Animais , Antígenos CD11/genética , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/microbiologia , Macrófagos Alveolares/patologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Knockout , Monócitos/imunologia , Monócitos/microbiologia , Monócitos/patologia , Mycobacterium tuberculosis/genética , Linfócitos T/imunologia , Linfócitos T/microbiologia , Linfócitos T/patologia , Tuberculose/genética , Tuberculose/patologia
14.
Nat Commun ; 11(1): 2140, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358586

RESUMO

The trans-synaptic interaction of the cell-adhesion molecules teneurins (TENs) with latrophilins (LPHNs/ADGRLs) promotes excitatory synapse formation when LPHNs simultaneously interact with FLRTs. Insertion of a short alternatively-spliced region within TENs abolishes the TEN-LPHN interaction and switches TEN function to specify inhibitory synapses. How alternative-splicing regulates TEN-LPHN interaction remains unclear. Here, we report the 2.9 Å resolution cryo-EM structure of the TEN2-LPHN3 complex, and describe the trimeric TEN2-LPHN3-FLRT3 complex. The structure reveals that the N-terminal lectin domain of LPHN3 binds to the TEN2 barrel at a site far away from the alternatively spliced region. Alternative-splicing regulates the TEN2-LPHN3 interaction by hindering access to the LPHN-binding surface rather than altering it. Strikingly, mutagenesis of the LPHN-binding surface of TEN2 abolishes the LPHN3 interaction and impairs excitatory but not inhibitory synapse formation. These results suggest that a multi-level coincident binding mechanism mediated by a cryptic adhesion complex between TENs and LPHNs regulates synapse specificity.


Assuntos
Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Sinapses/metabolismo , Processamento Alternativo/genética , Processamento Alternativo/fisiologia , Sítios de Ligação/genética , Células HEK293 , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mutação/genética , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Ligação Proteica/genética , Estrutura Secundária de Proteína , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/genética , Receptores de Peptídeos/química , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Sinapses/fisiologia
15.
J Neurosci ; 40(23): 4586-4595, 2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32341095

RESUMO

The epilepsy-linked gene SV2A, has a number of potential roles in the synaptic vesicle (SV) life cycle. However, how loss of SV2A function translates into presynaptic dysfunction and ultimately seizure activity is still undetermined. In this study, we examined whether the first SV2A mutation identified in human disease (R383Q) could provide information regarding which SV2A-dependent events are critical in the translation to epilepsy. We utilized a molecular replacement strategy in which exogenous SV2A was expressed in mouse neuronal cultures of either sex, which had been depleted of endogenous SV2A to mimic the homozygous human condition. We found that the R383Q mutation resulted in a mislocalization of SV2A from SVs to the plasma membrane, but had no effect on its activity-dependent trafficking. This SV2A mutant displayed reduced mobility when stranded on the plasma membrane and reduced binding to its interaction partner synaptotagmin-1 (Syt1). Furthermore, the R383Q mutant failed to rescue reduced expression and dysfunctional activity-dependent trafficking of Syt1 in the absence of endogenous SV2A. This suggests that the inability to control Syt1 expression and trafficking at the presynapse may be key in the transition from loss of SV2A function to seizure activity.SIGNIFICANCE STATEMENT SV2A is a synaptic vesicle (SV) protein, the absence or dysfunction of which is linked to epilepsy. However, the series of molecular events that result in this neurological disorder is still undetermined. We demonstrate here that the first human mutation in SV2A identified in an individual with epilepsy displays reduced binding to synaptotagmin-1 (Syt1), an SV protein essential for synchronous neurotransmitter release. Furthermore, this mutant cannot correct alterations in both Syt1 expression and trafficking when expressed in the absence of endogenous SV2A (to mimic the homozygous human condition). This suggests that the inability to control Syt1 expression and trafficking may be key in the transition from loss of SV2A function to seizure activity.


Assuntos
Epilepsia/genética , Glicoproteínas de Membrana/genética , Mutação de Sentido Incorreto/fisiologia , Proteínas do Tecido Nervoso/genética , Transporte Proteico/fisiologia , Sinaptotagmina I/biossíntese , Sinaptotagmina I/genética , Animais , Células Cultivadas , Epilepsia/metabolismo , Feminino , Expressão Gênica , Células HEK293 , Humanos , Masculino , Glicoproteínas de Membrana/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/deficiência
16.
Proc Natl Acad Sci U S A ; 117(17): 9413-9422, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32291340

RESUMO

Astrogenesis is repressed in the early embryonic period and occurs in the late embryonic period. A variety of external and internal signals contribute to the sequential differentiation of neural stem cells. Here, we discovered that immune-related CD93 plays a critical negative role in the regulation of astrogenesis in the mouse cerebral cortex. We show that CD93 expression is detected in neural stem cells and neurons but not in astrocytes and declines as differentiation proceeds. Cd93 knockout increases astrogenesis at the expense of neuron production during the late embryonic period. CD93 responds to the extracellular matrix protein Multimerin 2 (MMRN2) to trigger the repression of astrogenesis. Mechanistically, CD93 delivers signals to ß-Catenin through a series of phosphorylation cascades, and then ß-Catenin transduces these signals to the nucleus to activate Zfp503 transcription. The transcriptional repressor ZFP503 inhibits the transcription of glial fibrillary acidic protein (Gfap) by binding to the Gfap promoter with the assistance of Grg5. Furthermore, Cd93 knockout mice exhibit autism-like behaviors. Taken together, our results reveal that CD93 is a negative regulator of the onset of astrogenesis and provide insight into therapy for psychiatric disorders.


Assuntos
Astrócitos/fisiologia , Proteínas da Matriz Extracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Glicoproteínas de Membrana/metabolismo , Subfamília D de Receptores Semelhantes a Lectina de Células NK/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Transtorno Autístico , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Eletroporação , Proteínas da Matriz Extracelular/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Inflamação , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos ICR , Subfamília D de Receptores Semelhantes a Lectina de Células NK/genética , Proteínas do Tecido Nervoso/genética , Neurogênese , Neuroglia , Gravidez
17.
Am J Physiol Endocrinol Metab ; 318(6): E892-E900, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32255680

RESUMO

Proinsulin is a misfolding-prone protein, and its efficient breakdown is critical when ß-cells are confronted with high-insulin biosynthetic demands, to prevent endoplasmic reticulum stress, a key trigger of secretory dysfunction and, if uncompensated, apoptosis. Proinsulin degradation is thought to be performed by the constitutively expressed standard proteasome, while the roles of other proteasomes are unknown. We recently demonstrated that deficiency of the proinsulin chaperone glucose-regulated protein 94 (GRP94) causes impaired proinsulin handling and defective insulin secretion associated with a compensated endoplasmic reticulum stress response. Taking advantage of this model of restricted folding capacity, we investigated the role of different proteasomes in proinsulin degradation, reasoning that insulin secretory dynamics require an inducible protein degradation system. We show that the expression of only one enzymatically active proteasome subunit, namely, the inducible ß5i-subunit, was increased in GRP94 CRISPR/Cas9 knockout (KO) cells. Additionally, the level of ß5i-containing intermediate proteasomes was significantly increased in these cells, as was ß5i-related chymotrypsin-like activity. Moreover, proinsulin levels were restored in GRP94 KO upon ß5i small interfering RNA-mediated knockdown. Finally, the fraction of ß-cells expressing the ß5i-subunit is increased in human islets from type 2 diabetes patients. We conclude that ß5i is an inducible proteasome subunit dedicated to the degradation of mishandled proinsulin.


Assuntos
Diabetes Mellitus Tipo 2/genética , Estresse do Retículo Endoplasmático/genética , Degradação Associada com o Retículo Endoplasmático/genética , Secreção de Insulina/genética , Células Secretoras de Insulina/metabolismo , Proinsulina/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Animais , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Técnicas de Inativação de Genes , Humanos , Ilhotas Pancreáticas/metabolismo , Glicoproteínas de Membrana/genética , Pessoa de Meia-Idade , Complexo de Endopeptidases do Proteassoma/metabolismo , Dobramento de Proteína , Ratos
18.
Nat Commun ; 11(1): 1729, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32265438

RESUMO

The TrkB receptor is critical for the control of energy balance, as mutations in its gene (NTRK2) lead to hyperphagia and severe obesity. The main neural substrate mediating the appetite-suppressing activity of TrkB, however, remains unknown. Here, we demonstrate that selective Ntrk2 deletion within paraventricular hypothalamus (PVH) leads to severe hyperphagic obesity. Furthermore, chemogenetic activation or inhibition of TrkB-expressing PVH (PVHTrkB) neurons suppresses or increases food intake, respectively. PVHTrkB neurons project to multiple brain regions, including ventromedial hypothalamus (VMH) and lateral parabrachial nucleus (LPBN). We find that PVHTrkB neurons projecting to LPBN are distinct from those to VMH, yet Ntrk2 deletion in PVH neurons projecting to either VMH or LPBN results in hyperphagia and obesity. Additionally, TrkB activation with BDNF increases firing of these PVH neurons. Therefore, TrkB signaling is a key regulator of a previously uncharacterized neuronal population within the PVH that impinges upon multiple circuits to govern appetite.


Assuntos
Hiperfagia/metabolismo , Glicoproteínas de Membrana/metabolismo , Neurônios/metabolismo , Obesidade/metabolismo , Núcleo Hipotalâmico Paraventricular/citologia , Núcleo Hipotalâmico Paraventricular/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Apetite/genética , Comportamento Alimentar/fisiologia , Feminino , Hiperfagia/genética , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Obesidade/genética , Núcleos Parabraquiais/citologia , Núcleos Parabraquiais/metabolismo , Núcleos Parabraquiais/fisiopatologia , Proteínas Tirosina Quinases/genética , Núcleo Hipotalâmico Ventromedial/citologia , Núcleo Hipotalâmico Ventromedial/metabolismo
19.
Neuron ; 106(5): 727-742.e6, 2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32199103

RESUMO

Evidence suggests interplay among the three major risk factors for Alzheimer's disease (AD): age, APOE genotype, and sex. Here, we present comprehensive datasets and analyses of brain transcriptomes and blood metabolomes from human apoE2-, apoE3-, and apoE4-targeted replacement mice across young, middle, and old ages with both sexes. We found that age had the greatest impact on brain transcriptomes highlighted by an immune module led by Trem2 and Tyrobp, whereas APOE4 was associated with upregulation of multiple Serpina3 genes. Importantly, these networks and gene expression changes were mostly conserved in human brains. Finally, we observed a significant interaction between age, APOE genotype, and sex on unfolded protein response pathway. In the periphery, APOE2 drove distinct blood metabolome profile highlighted by the upregulation of lipid metabolites. Our work identifies unique and interactive molecular pathways underlying AD risk factors providing valuable resources for discovery and validation research in model systems and humans.


Assuntos
Envelhecimento/genética , Doença de Alzheimer/genética , Apolipoproteínas E/genética , Encéfalo/metabolismo , Serpinas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Fatores Etários , Doença de Alzheimer/metabolismo , Animais , Apolipoproteína E2/genética , Apolipoproteína E3/genética , Apolipoproteína E4/genética , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Genótipo , Humanos , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Metaboloma , Camundongos , Camundongos Transgênicos , Fatores de Proteção , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Fatores de Risco , Fatores Sexuais , Resposta a Proteínas não Dobradas/genética
20.
Nat Commun ; 11(1): 1128, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111832

RESUMO

The sterol-regulatory element binding proteins (SREBP) are central transcriptional regulators of lipid metabolism. Using haploid genetic screens we identify the SREBP Regulating Gene (SPRING/C12ORF49) as a determinant of the SREBP pathway. SPRING is a glycosylated Golgi-resident membrane protein and its ablation in Hap1 cells, Hepa1-6 hepatoma cells, and primary murine hepatocytes reduces SREBP signaling. In mice, Spring deletion is embryonic lethal yet silencing of hepatic Spring expression also attenuates the SREBP response. Mechanistically, attenuated SREBP signaling in SPRINGKO cells results from reduced SREBP cleavage-activating protein (SCAP) and its mislocalization to the Golgi irrespective of the cellular sterol status. Consistent with limited functional SCAP in SPRINGKO cells, reintroducing SCAP restores SREBP-dependent signaling and function. Moreover, in line with the role of SREBP in tumor growth, a wide range of tumor cell lines display dependency on SPRING expression. In conclusion, we identify SPRING as a previously unrecognized modulator of SREBP signaling.


Assuntos
Colesterol/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Transdução de Sinais , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , Animais , Linhagem Celular , Desenvolvimento Embrionário/genética , Retículo Endoplasmático/metabolismo , Expressão Gênica , Complexo de Golgi/metabolismo , Haploidia , Hepatócitos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metabolismo dos Lipídeos/genética , Fígado/metabolismo , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Ligação a Elemento Regulador de Esterol/genética
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