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1.
Nat Commun ; 11(1): 4930, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33004804

RESUMO

Inference of causality between gene expression and complex traits using Mendelian randomization (MR) is confounded by pleiotropy and linkage disequilibrium (LD) of gene-expression quantitative trait loci (eQTL). Here, we propose an MR method, MR-link, that accounts for unobserved pleiotropy and LD by leveraging information from individual-level data, even when only one eQTL variant is present. In simulations, MR-link shows false-positive rates close to expectation (median 0.05) and high power (up to 0.89), outperforming all other tested MR methods and coloc. Application of MR-link to low-density lipoprotein cholesterol (LDL-C) measurements in 12,449 individuals with expression and protein QTL summary statistics from blood and liver identifies 25 genes causally linked to LDL-C. These include the known SORT1 and ApoE genes as well as PVRL2, located in the APOE locus, for which a causal role in liver was not known. Our results showcase the strength of MR-link for transcriptome-wide causal inferences.


Assuntos
LDL-Colesterol/sangue , Regulação da Expressão Gênica , Predisposição Genética para Doença , Modelos Genéticos , Locos de Características Quantitativas , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , LDL-Colesterol/metabolismo , Simulação por Computador , Conjuntos de Dados como Assunto , Pleiotropia Genética , Humanos , Desequilíbrio de Ligação , Metabolismo dos Lipídeos/genética , Análise da Randomização Mendeliana , Redes e Vias Metabólicas/genética , Herança Multifatorial , Nectinas/genética , Nectinas/metabolismo , Países Baixos , Proteômica , RNA-Seq
2.
PLoS Pathog ; 16(8): e1008639, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32790743

RESUMO

Leptospirosis is a worldwide re-emerging zoonosis caused by pathogenic Leptospira spp. All vertebrate species can be infected; humans are sensitive hosts whereas other species, such as rodents, may become long-term renal carrier reservoirs. Upon infection, innate immune responses are initiated by recognition of Microbial Associated Molecular Patterns (MAMPs) by Pattern Recognition Receptors (PRRs). Among MAMPs, the lipopolysaccharide (LPS) is recognized by the Toll-Like-Receptor 4 (TLR4) and activates both the MyD88-dependent pathway at the plasma membrane and the TRIF-dependent pathway after TLR4 internalization. We previously showed that leptospiral LPS is not recognized by the human-TLR4, whereas it signals through mouse-TLR4 (mTLR4), which mediates mouse resistance to acute leptospirosis. However, although resistant, mice are known to be chronically infected by leptospires. Interestingly, the leptospiral LPS has low endotoxicity in mouse cells and is an agonist of TLR2, the sensor for bacterial lipoproteins. Here, we investigated the signaling properties of the leptospiral LPS in mouse macrophages. Using confocal microscopy and flow cytometry, we showed that the LPS of L. interrogans did not induce internalization of mTLR4, unlike the LPS of Escherichia coli. Consequently, the LPS failed to induce the production of the TRIF-dependent nitric oxide and RANTES, both important antimicrobial responses. Using shorter LPS and LPS devoid of TLR2 activity, we further found this mTLR4-TRIF escape to be dependent on both the co-purifying lipoproteins and the full-length O antigen. Furthermore, our data suggest that the O antigen could alter the binding of the leptospiral LPS to the co-receptor CD14 that is essential for TLR4-TRIF activation. Overall, we describe here a novel leptospiral immune escape mechanism from mouse macrophages and hypothesize that the LPS altered signaling could contribute to the stealthiness and chronicity of the leptospires in mice.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Leptospira/imunologia , Leptospirose/imunologia , Lipopolissacarídeos/metabolismo , Lipoproteínas/metabolismo , Antígenos O/metabolismo , Receptor 4 Toll-Like/fisiologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Citocinas/metabolismo , Feminino , Leptospirose/metabolismo , Leptospirose/microbiologia , Leptospirose/patologia , Receptores de Lipopolissacarídeos/genética , Receptores de Lipopolissacarídeos/metabolismo , Lipoproteínas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/fisiologia , Antígenos O/genética , Transdução de Sinais , Receptor 2 Toll-Like/fisiologia
3.
Rinsho Shinkeigaku ; 60(8): 543-548, 2020 Aug 07.
Artigo em Japonês | MEDLINE | ID: mdl-32641631

RESUMO

We describe an additional patient with spastic paraplegia 48 (SPG48). A 52-year-old woman with gradually increasing gait disturbance was admitted to our hospital. When she was 47 years old, acquaintances noted a shuffling gait. Gait worsening was evident at 48 years. Spastic gait was apparent at 50, and she required a walking stick at 54. Her elder brother had similar gait disturbance. No consanguinity was known. Neurologic examination at 52 disclosed spasticity and moderate weakness in the lower limbs. Spasticity and brisk reflexes in all limbs. Laboratory studies including HTLV-1 titer detected no abnormalities. MRI demonstrated mild corpus callosum narrowing and prominent anterior periventricular hyperintensities in fluid attenuation inversion recovery images. In limb muscles, electromyography (EMG) showed a chronic neurogenic pattern including reduced interference. Gene analysis identified compound homozygosity in exon 7 of adaptor-related protein complex 5 subunit zeta 1 (AP5Z1), including a novel frameshift mutation, c.1662_1672del;p.Glu554Hfs*15 in the patient, and a heterozygous missense mutation in asymptomatic family members, including her mother, two siblings, and a daughter. The frameshift mutation is considered a pathogenic variant according to American College of Medical Genetics and Genomics standards and guidelines. Based on clinical features, imaging findings and genetic abnormalities, we diagnosed this patient with SPG48. Mutations in AP5Z1, which encodes the ζ subunit of AP-5, underlie SPG48. The AP-5 adaptor protein complex, which is mutated in SPG48, binds to both spastizin and spatacsin. While hereditary spastic paraplegias generally are clinically and genetically heterogenous, SPG48, SPG11, and SPG15 are clinically similar.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Mutação da Fase de Leitura , Paraparesia Espástica/genética , Ventrículos Cerebrais/diagnóstico por imagem , Ventrículos Cerebrais/patologia , Corpo Caloso/diagnóstico por imagem , Corpo Caloso/patologia , Feminino , Transtornos Neurológicos da Marcha/etiologia , Genes Recessivos , Homozigoto , Humanos , Angiografia por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Paraparesia Espástica/complicações
4.
Nat Commun ; 11(1): 3123, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561740

RESUMO

Intracellular trafficking of organelles, driven by kinesin-1 stepping along microtubules, underpins essential cellular processes. In absence of other proteins on the microtubule surface, kinesin-1 performs micron-long runs. Under crowding conditions, however, kinesin-1 motility is drastically impeded. It is thus unclear how kinesin-1 acts as an efficient transporter in intracellular environments. Here, we demonstrate that TRAK1 (Milton), an adaptor protein essential for mitochondrial trafficking, activates kinesin-1 and increases robustness of kinesin-1 stepping on crowded microtubule surfaces. Interaction with TRAK1 i) facilitates kinesin-1 navigation around obstacles, ii) increases the probability of kinesin-1 passing through cohesive islands of tau and iii) increases the run length of kinesin-1 in cell lysate. We explain the enhanced motility by the observed direct interaction of TRAK1 with microtubules, providing an additional anchor for the kinesin-1-TRAK1 complex. Furthermore, TRAK1 enables mitochondrial transport in vitro. We propose adaptor-mediated tethering as a mechanism regulating kinesin-1 motility in various cellular environments.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Cinesina/metabolismo , Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/isolamento & purificação , Animais , Linhagem Celular Tumoral , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Cinesina/genética , Cinesina/isolamento & purificação , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Microscopia de Fluorescência , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismo
5.
Nat Commun ; 11(1): 3020, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32541686

RESUMO

The subversion of endocytic routes leads to malignant transformation and has been implicated in human cancers. However, there is scarce evidence for genetic alterations of endocytic proteins as causative in high incidence human cancers. Here, we report that Epsin 3 (EPN3) is an oncogene with prognostic and therapeutic relevance in breast cancer. Mechanistically, EPN3 drives breast tumorigenesis by increasing E-cadherin endocytosis, followed by the activation of a ß-catenin/TCF4-dependent partial epithelial-to-mesenchymal transition (EMT), followed by the establishment of a TGFß-dependent autocrine loop that sustains EMT. EPN3-induced partial EMT is instrumental for the transition from in situ to invasive breast carcinoma, and, accordingly, high EPN3 levels are detected at the invasive front of human breast cancers and independently predict metastatic rather than loco-regional recurrence. Thus, we uncover an endocytic-based mechanism able to generate TGFß-dependent regulatory loops conferring cellular plasticity and invasive behavior.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Neoplasias da Mama/fisiopatologia , Endocitose , Proteínas Adaptadoras de Transporte Vesicular/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Caderinas/genética , Caderinas/metabolismo , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Invasividade Neoplásica , Metástase Neoplásica , Transdução de Sinais , Fator de Transcrição 4/genética , Fator de Transcrição 4/metabolismo , Fator de Crescimento Transformador beta/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
6.
Cytogenet Genome Res ; 160(6): 316-320, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32575107

RESUMO

Based on a literature review and our database, we report on the smallest 14q deletion identified in a brain tumor characterized by 1p/19q codeletion low-grade oligodendroglioma. In 2013, array-comparative genomic hybridization of the brain tumor revealed 1p/19q codeletion as a sole abnormality. In 2019, the patient relapsed showing additional abnormalities including a 14q deletion of 16.5 Mb at 14q24.2q31.3. This region overlaps with 2 previously identified minimal regions, 14q21.2q24.3 and 14q31.3q32.1, based on 142 cases of glioma. The authors reported no correlation between these 2 regions and survival. By extracting these 2 regions from our patient's deletion and comparing it to 12 other cases of 1p/19q codeletion oligodendrogliomas reported in the literature, we narrowed down the 14q loss possible critical region to 5.6 Mb mapping at 14q31.1q31.2. This region contains 2 potential relapse-related genes: SEL1L and STON2.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Deleção Cromossômica , Cromossomos Humanos Par 14/genética , Cromossomos Humanos Par 19/genética , Cromossomos Humanos Par 1/genética , Recidiva Local de Neoplasia/genética , Oligodendroglioma/genética , Proteínas/genética , Feminino , Humanos , Pessoa de Meia-Idade , Recidiva , Estudos Retrospectivos
7.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 37(5): 509-513, 2020 May 10.
Artigo em Chinês | MEDLINE | ID: mdl-32335874

RESUMO

OBJECTIVE: To identify pathogenic variants in two families with patients suspected for Joubert syndrome(UBST) by cerebellar vermis hypoplasia. METHODS: Clinical data and peripheral venous blood and skin tissue samples were collected for the extraction of genomic DNA. Potential variants were screened by using targeted capture and next generation sequencing. Suspected variants were validated by PCR and Sanger sequencing. The frequency of the variants in the population was calculated. Pathogenicity of the variants was predicted by following the guidelines of the American College of Medical Genetics and Genomics (ACMG). Prenatal diagnosis was provided to these families upon subsequent pregnancy. RESULTS: The proband of family 1 was found to harbor homozygous c.2072delT (p.F691S*fs19) frameshift variant of the AHI1 gene, which may cause premature termination of translation of the Abelson helper integration site 1 after the 691st amino acid. The proband of family 2 was found to harbor compound heterozygous variants of the CPLANE1 gene, namely c.7243dupA (p.T2415Nfs*7) and c.8001delG (p.K2667Nfs*31), which can respectively lead to premature termination of translation of ciliogenesis and planar polarity effector 1 after the 2145th and 2667th amino acids. All of the three variants were previously unreported, and were predicted to be pathogenic by bioinformatic analysis. CONCLUSION: The AHI1 c.2072delT and CPLANE1 c.7243dupA and c.8001delG variants probably underlay JBTS3 in family 1 and JBTS17 in family 2, respectively. Based on above results, prenatal diagnosis may be offered to the affected families upon their subsequent pregnancies.


Assuntos
Anormalidades Múltiplas , Proteínas Adaptadoras de Transporte Vesicular , Cerebelo/anormalidades , Anormalidades do Olho , Testes Genéticos , Doenças Renais Císticas , Proteínas de Membrana , Diagnóstico Pré-Natal , Retina/anormalidades , Anormalidades Múltiplas/diagnóstico , Anormalidades Múltiplas/genética , Proteínas Adaptadoras de Transporte Vesicular/genética , Anormalidades do Olho/diagnóstico , Anormalidades do Olho/genética , Feminino , Variação Genética , Humanos , Doenças Renais Císticas/diagnóstico , Doenças Renais Císticas/genética , Proteínas de Membrana/genética , Mutação , Gravidez
8.
Am J Chin Med ; 48(2): 341-356, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32138537

RESUMO

MicroRNA 145 (miR-145) is a critical modulator of cardiovascular diseases. The downregulation of myocardial miR-145 is followed by an increase in disabled-2 (Dab2) expression in cardiomyocytes. (-)-epigallocatechin gallate (EGCG) is a flavonoid that has been evaluated extensively due to its diverse pharmacological properties including anti-inflammatory effects. The aim of this study was to investigate the cardioprotective effects of EGCG under hypoxia-induced stress in vitro and in vivo. The hypoxic insult led to the suppression of miR-145 expression in cultured rat cardiomyocytes in a concentration-dependent manner. Western blotting and real-time PCR were performed. In rat myocardial infarction study, in situ hybridization, and immunofluorescent analyses were adopted. The western blot and real-time PCR data revealed that hypoxic stress with 2.5% O2 suppressed the expression of miR-145 and Wnt3a/ß-catenin in cultured rat cardiomyocytes but augmented Dab2. Treatment with EGCG attenuated Dab2 expression, but increased Wnt3a and ß-catenin in hypoxic cultured cardiomyocytes. Following in vivo myocardial infarction (MI) study, the data revealed the myocardial infarct area reduced by 48.5%, 44.6%, and 48.5% in EGCG (50mg/kg) or miR-145 dominant or Dab2 siRNA groups after myocardial infarction for 28 days, respectively. This study demonstrated that EGCG increased miR-145, Wnt3a, and ß-catenin expression but attenuated Dab2 expression. Moreover, EGCG ameliorated myocardial ischemia in vivo. The novel suppressive effect was mediated through the miR-145 and Dab2/Wnt3a/ß-catenin pathways.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Catequina/análogos & derivados , Expressão Gênica/efeitos dos fármacos , MicroRNAs/genética , MicroRNAs/metabolismo , Isquemia Miocárdica/tratamento farmacológico , Isquemia Miocárdica/genética , Miócitos Cardíacos/metabolismo , Fitoterapia , Proteína Wnt3A/genética , Proteína Wnt3A/metabolismo , beta Catenina/genética , beta Catenina/metabolismo , Animais , Catequina/farmacologia , Catequina/uso terapêutico , Células Cultivadas , Relação Dose-Resposta a Droga , Ratos
9.
J Cell Biol ; 219(3)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32045479

RESUMO

Regulated secretion is a fundamental cellular process in which biologically active molecules stored in long-lasting secretory granules (SGs) are secreted in response to external stimuli. Many studies have described mechanisms responsible for biogenesis and secretion of SGs, but how SGs mature remains poorly understood. In a genetic screen, we discovered a large number of endolysosomal trafficking genes required for proper SG maturation, indicating that maturation of SGs might occur in a manner similar to lysosome-related organelles (LROs). CD63, a tetraspanin known to decorate LROs, also decorates SG membranes and facilitates SG maturation. Moreover, CD63-mediated SG maturation requires type II phosphatidylinositol 4 kinase (PI4KII)-dependent early endosomal sorting and accumulation of phosphatidylinositol 4-phosphate (PI4P) on SG membranes. In addition, the PI4P effector Past1 is needed for formation of stable PI4KII-containing endosomal tubules associated with this process. Our results reveal that maturation of post-Golgi-derived SGs requires trafficking via the endosomal system, similar to mechanisms employed by LROs.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Endossomos/metabolismo , Glândulas Salivares/metabolismo , Vesículas Secretórias/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Endossomos/genética , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Transporte Proteico , Glândulas Salivares/embriologia , Vesículas Secretórias/genética , Tetraspanina 30/genética , Tetraspanina 30/metabolismo , Fatores de Tempo
10.
Nat Cell Biol ; 22(2): 187-199, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31932738

RESUMO

Traditionally viewed as an autodigestive pathway, autophagy also facilitates cellular secretion; however, the mechanisms underlying these processes remain unclear. Here, we demonstrate that components of the autophagy machinery specify secretion within extracellular vesicles (EVs). Using a proximity-dependent biotinylation proteomics strategy, we identify 200 putative targets of LC3-dependent secretion. This secretome consists of a highly interconnected network enriched in RNA-binding proteins (RBPs) and EV cargoes. Proteomic and RNA profiling of EVs identifies diverse RBPs and small non-coding RNAs requiring the LC3-conjugation machinery for packaging and secretion. Focusing on two RBPs, heterogeneous nuclear ribonucleoprotein K (HNRNPK) and scaffold-attachment factor B (SAFB), we demonstrate that these proteins interact with LC3 and are secreted within EVs enriched with lipidated LC3. Furthermore, their secretion requires the LC3-conjugation machinery, neutral sphingomyelinase 2 (nSMase2) and LC3-dependent recruitment of factor associated with nSMase2 activity (FAN). Hence, the LC3-conjugation pathway controls EV cargo loading and secretion.


Assuntos
Autofagossomos/metabolismo , Autofagia/genética , Vesículas Extracelulares/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Ligação a RNA/genética , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Autofagossomos/química , Proteína 7 Relacionada à Autofagia/deficiência , Proteína 7 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/deficiência , Proteínas Relacionadas à Autofagia/genética , Transporte Biológico , Biotinilação , Vesículas Extracelulares/química , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisossomos/química , Lisossomos/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/genética , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteômica/métodos , Células RAW 264.7 , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/classificação , Proteínas de Ligação a RNA/metabolismo , Receptores Estrogênicos/genética , Receptores Estrogênicos/metabolismo , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismo
11.
PLoS One ; 15(1): e0227855, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31999741

RESUMO

The Src substrate Tks5 helps scaffold matrix-remodeling invadopodia in invasive cancer cells. Focus was directed here on how the five SH3 domains of Tks5 impact that activity. Mutations designed to inhibit protein-protein interactions were created in the individual SH3 domains of Tks5, and the constructs were introduced into the LNCaP prostate carcinoma cell line, a model system with intrinsically low Tks5 expression and which our lab had previously showed the dependence of Src-dependent Tks5 phosphorylation on invadopodia development. In LNCaP cells, acute increases in wild-type Tks5 led to increased gelatin matrix degradation. A similar result was observed when Tks5 was mutated in its 4th or 5th SH3 domains. This was in contrast to the 1st, 2nd, and 3rd SH3 domain mutations of Tks5 where each had a remarkable accentuating effect on gelatin degradation. Conversely, in the invadopodia-competent Src-3T3 model system, mutations in any one of the first three SH3 domains had a dominant negative effect that largely eliminated the presence of invadopodia, inhibited gelatin degradation activity, and redistributed both Src, cortactin, and Tks5 to what are likely endosomal compartments. A hypothesis involving Tks5 conformational states and the regulation of endosomal trafficking is presented as an explanation for these seemingly disparate results.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Carcinoma/genética , Neoplasias da Próstata/genética , Quinases da Família src/genética , Proteínas Adaptadoras de Transporte Vesicular/química , Carcinoma/metabolismo , Carcinoma/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Cortactina/genética , Fibroblastos/metabolismo , Fibroblastos/patologia , Gelatina/genética , Gelatina/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Mutação/genética , Fosforilação , Podossomos/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Domínios e Motivos de Interação entre Proteínas/genética , Domínios de Homologia de src/genética
12.
J Cell Biol ; 219(2)2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-31865373

RESUMO

Podosomes are compartmentalized actin-rich adhesions, defined by their ability to locally secrete proteases and remodel extracellular matrix. Matrix remodeling by endothelial podosomes facilitates invasion and thereby vessel formation. However, the mechanisms underlying endothelial podosome formation and function remain unclear. Here, we demonstrate that Septin2, Septin6, and Septin7 are required for maturation of nascent endothelial podosomes into matrix-degrading organelles. We show that podosome development occurs through initial mobilization of the scaffolding protein Tks5 and F-actin accumulation, followed by later recruitment of Septin2. Septin2 localizes around the perimeter of podosomes in close proximity to the basolateral plasma membrane, and phosphoinositide-binding residues of Septin2 are required for podosome function. Combined, our results suggest that the septin cytoskeleton forms a diffusive barrier around nascent podosomes to promote their maturation. Finally, we show that Septin2-mediated regulation of podosomes is critical for endothelial cell invasion associated with angiogenesis. Therefore, targeting of Septin2-mediated podosome formation is a potentially attractive anti-angiogenesis strategy.


Assuntos
Proteínas de Ciclo Celular/genética , Neovascularização Fisiológica/genética , Septinas/genética , Citoesqueleto de Actina/genética , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Movimento Celular/genética , Células Cultivadas , Células Endoteliais/metabolismo , Matriz Extracelular/genética , Humanos , Morfogênese/genética , Podossomos/genética
13.
Fish Shellfish Immunol ; 97: 114-124, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31841694

RESUMO

As a member of tumor necrosis factor receptor (TNFR)-associated factor (TRAF) family, TRAF3 is an important regulator of NF-κB and type I interferon (IFN) activation, especially in Toll-like receptors (TLRs)- and retinoic acid inducible gene I (RIG-I)-like receptors (RLRs)-mediated signaling pathway. In the present study, a TRAF3 homologue named Lc-TRAF3 was characterized in large yellow croaker (Larimichthys crocea). The open reading frame (ORF) of Lc-TRAF3 contains 1788 bp encoding a protein of 595 amino acids (aa). Sequence analysis indicated that Lc-TRAF3 is conserved in vertebrates, constituted with a N-terminal RING finger, two TRAF-type zinc fingers, and a C-terminal TRAF-MATH domain. The genome organization of Lc-TRAF3 is conserved in fish, with 13 exons and 12 introns, but different from that in birds or mammals, which contains 10 exons and 9 introns. Lc-TRAF3 was identified as cytosolic protein base on fluorescence microscopy analysis. Expression analysis revealed that Lc-TRAF3 was broadly distributed in examined organs/tissues, with the highest expression level in gill and weakest in brain, and could be up-regulated under poly I:C, LPS, PGN, and Pseudomonas plecoglossicida stimulation in vivo. Interestingly, overexpression Lc-TRAF3 could induce the activation of NF-κB, and Lc-TRAF3 co-transfected with Lc-TRIF induced a significantly higher level of NF-κB and IRF3 promoter activity, implying that Lc-TRAF3 may function as an enhancer in Lc-TRIF-mediated signaling pathway.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Fatores Reguladores de Interferon/genética , NF-kappa B/metabolismo , Perciformes/imunologia , Transdução de Sinais , Fator 3 Associado a Receptor de TNF/genética , Proteínas Adaptadoras de Transporte Vesicular/imunologia , Animais , Bactérias/imunologia , Fatores Reguladores de Interferon/imunologia , NF-kappa B/imunologia , Perciformes/genética , Perciformes/microbiologia , Fator 3 Associado a Receptor de TNF/imunologia
14.
Biomed Pharmacother ; 121: 109580, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31704614

RESUMO

Colorectal cancer (CRC) is a malignant tumor with a high incidence and death rate in the world. Molecular interactions inside cells or tissues during tumor occurrence, development, and drug resistance are important for disease prevention and treatment. The long non-coding RNA SNHG14 has been proven to exert its oncogenic function in multiple cancers. However, there is no study regarding the role of SNHG14 in CRC research. In the present study, we applied RT-qPCR and western blot to determine the gene expression levels. MTT and TUNEL assays were used to detect cell proliferation and apoptosis rate. Cell migration and invasion abilities were determined by wound healing and transwell assays, respectively. StarBase was used to predict the potential binding sites and luciferase reporter assay was applied to confirm the direct interactions. Besides, we conducted a xenograft experiment to detect tumor growth rate in vivo. Our results showed that SNHG14 and ATG14 were both significantly higher in CRC tumor tissues than the normal ones, while miR-186 was decreased. The similar results were also observed in CRC cell lines. We confirmed that SNHG14 could directly interact with miR-186 and inhibited its expression. Meanwhile, miR-186 could directly bind ATG14 to inhibit its expression level. In vitro experiments showed that higher expression of SNHG14 led to higher cell proliferation, migration and invasion, while miR-186 significantly inhibited these tumor phenotypes. Furthermore, overexpression of ATG14 could strongly recover the CRC phenotypes attenuated by shSNHG14 or miR-186 mimics. Interestingly, we constructed cisplatin-resistant CRC cells and found that overexpression of ATG14 significantly enhanced the cell proliferation rate and inhibited cell apoptosis. Our research indicated that the novel axis of SNHG14/miR-186/ATG14 could play a vital role in regulating CRC cell progression. Moreover, this axis showed its clinical potential in regulating cisplatin resistance during CRC treatment.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Relacionadas à Autofagia/genética , Cisplatino/farmacologia , Neoplasias Colorretais/tratamento farmacológico , MicroRNAs/genética , RNA Longo não Codificante/genética , Animais , Antineoplásicos/farmacologia , Apoptose/genética , Autofagia/genética , Movimento Celular/genética , Proliferação de Células/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos , Humanos , Camundongos , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Toxicol Lett ; 321: 54-60, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31862508

RESUMO

Ricin toxin (RT) is a natural plant-derived protein toxin from the seed of castor beans that belongs to a family of type II ribosome-inactivating proteins (RIPs). In addition to its main toxic mechanism of inhibiting the synthesis of cellular proteins, RT can induce the production of inflammatory cytokines and cause inflammatory injury. Macrophages play a crucial role in innate immunity and the adaptive immune response as the first line of host defense against bacterial infections and various types of invading pathogens. Upon activation, macrophages release types of cytokines to remove pathogens. However, the effect of RT on the immune response and its mechanism are not well characterized. In the current study, we investigated the activation of the TLR4-mediated signaling pathway by low-dose RT treatment and its interaction with signaling molecules in the transduction pathway. We found that low-dose RT can activate MyD88- and TRIF-dependent signaling pathways, revealing a possible mechanism by which low-dose RT-activates TLR4-mediated signaling pathways. We also confirmed that the TLR4-induced activation of the inflammatory signaling pathways was produced via its binding to RT. This study may help to identify the most important target molecules and clarify the mechanism of inflammatory injury of ricin.


Assuntos
Inflamação/induzido quimicamente , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Ricina/toxicidade , Receptor 4 Toll-Like/agonistas , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Substâncias para a Guerra Química , Citocinas/metabolismo , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Células RAW 264.7 , Transdução de Sinais , Células THP-1 , Receptor 4 Toll-Like/metabolismo
16.
Genes (Basel) ; 10(12)2019 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-31817908

RESUMO

Non-syndromic cleft lip with or without cleft palate (nsCL/P) ranks among the most common human congenital malformations, and has a multifactorial background in which both exogenous and genetic risk factors act in concert. The present report describes a genome-wide association study (GWAS) involving a total of 285 nsCL/P patients and 1212 controls from the Netherlands and Belgium. Twenty of the 40 previously reported nsC/LP susceptibility loci were replicated, which underlined the validity of this sample. SNV-based analysis of the data identified an as yet unreported suggestive locus at chromosome 16p12.1 (p-value of the lead SNV: 4.17 × 10-7). This association was replicated in two of three patient/control replication series (Central European and Yemeni). Gene analysis of the GWAS data prioritized SH3PXD2A at chromosome 10q24.33 as a candidate gene for nsCL/P. To date, support for this gene as a cleft gene has been restricted to data from zebrafish and a knockout mouse model. The present GWAS was the first to implicate SH3PXD2A in non-syndromic cleft formation in humans. In summary, although performed in a relatively small sample, the present GWAS generated novel insights into nsCL/P etiology.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Cromossomos Humanos Par 16/genética , Fenda Labial/genética , Fissura Palatina/genética , Animais , Bélgica , Cromossomos Humanos Par 10/genética , Feminino , Estudo de Associação Genômica Ampla , Humanos , Masculino , Camundongos , Camundongos Knockout , Países Baixos , Fatores de Risco , Peixe-Zebra
17.
Immunity ; 51(6): 997-1011.e7, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31851905

RESUMO

Toll-like receptor (TLR) activation induces inflammatory responses in macrophages by activating temporally defined transcriptional cascades. Whether concurrent changes in the cellular metabolism that occur upon TLR activation influence the quality of the transcriptional responses remains unknown. Here, we investigated how macrophages adopt their metabolism early after activation to regulate TLR-inducible gene induction. Shortly after TLR4 activation, macrophages increased glycolysis and tricarboxylic acid (TCA) cycle volume. Metabolic tracing studies revealed that TLR signaling redirected metabolic fluxes to generate acetyl-Coenzyme A (CoA) from glucose resulting in augmented histone acetylation. Signaling through the adaptor proteins MyD88 and TRIF resulted in activation of ATP-citrate lyase, which in turn facilitated the induction of distinct LPS-inducible gene sets. We postulate that metabolic licensing of histone acetylation provides another layer of control that serves to fine-tune transcriptional responses downstream of TLR activation. Our work highlights the potential of targeting the metabolic-epigenetic axis in inflammatory settings.


Assuntos
ATP Citrato (pro-S)-Liase/metabolismo , Acetilcoenzima A/metabolismo , Histonas/metabolismo , Macrófagos/metabolismo , Receptor 4 Toll-Like/metabolismo , Acetilação , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Ciclo do Ácido Cítrico/fisiologia , Glicólise/fisiologia , Humanos , Lipopolissacarídeos/metabolismo , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Transdução de Sinais , Transcrição Genética/genética
18.
Int J Mol Sci ; 20(23)2019 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-31766750

RESUMO

Cytoplasmic aggregates and nuclear depletion of the ubiquitous RNA-binding protein TDP-43 have been described in the autoptic brain tissues of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTLD) patients and both TDP-43 loss-of-function and gain-of-function mechanisms seem to contribute to the neurodegenerative process. Among the wide array of RNA targets, TDP-43 regulates progranulin (GRN) mRNA stability and sortilin (SORT1) splicing. Progranulin is a secreted neurotrophic and neuro-immunomodulatory factor whose endocytosis and delivery to the lysosomes are regulated by the neuronal receptor sortilin. Moreover, GRN loss-of-function mutations are causative of a subset of FTLD cases showing TDP-43 pathological aggregates. Here we show that TDP-43 loss-of-function differently affects the progranulin-sortilin axis in murine and human neuronal cell models. We demonstrated that although TDP-43 binding to GRN mRNA occurs similarly in human and murine cells, upon TDP-43 depletion, a different control of sortilin splicing and protein content may determine changes in extracellular progranulin uptake that account for increased or unchanged secreted protein in murine and human cells, respectively. As targeting the progranulin-sortilin axis has been proposed as a therapeutic approach for GRN-FTLD patients, the inter-species differences in TDP-43-mediated regulation of this pathway must be considered when translating studies from animal models to patients.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Modelos Biológicos , Doenças Neurodegenerativas/metabolismo , Progranulinas/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Humanos , Camundongos , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/terapia , Progranulinas/genética , Especificidade da Espécie
19.
Immun Inflamm Dis ; 7(4): 318-325, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31691534

RESUMO

INTRODUCTION: Complexins (CPLXs), initially identified in neuronal presynaptic terminals, are cytoplasmic proteins that interact with the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) complex to regulate the fusion of vesicles to the plasma membrane. Although much is known about CPLX function in neuronal synaptic vesicle exocytosis, their distribution and role in immune cells are still unclear. In this study, we investigated CPLX2 knockout (KO) mice to reveal the role of CPLXs in exocytosis of lymphocytes. METHODS: We examined the expression of CPLXs and SNAREs in lymphocytes. To study the effect of CPLXs on the immune system in vivo, we analyzed the immune phenotype of CPLX2 KO mice. Furthermore, antibodies secretion from the peritoneal cavity, spleen, and bone marrow cells of wild-type (WT) and CPLX2 KO mice were determined. RESULTS: CPLX2 was detected in B cells but not in T cells, while other CPLXs and SNAREs were expressed at a similar level in both B and T cells. To clarify the function of CPLX2 in B lymphocytes, serum concentrations of immunoglobulin G (IgG), IgA, IgM, and IgE were measured in WT and CPLX2 KO mice using enzyme-linked immunosorbent assay. The level of IgM, which mainly consists of natural antibodies, was higher in KO mice than that in WT mice, while the levels of other antibodies were similar in both types of mice. Additionally, we found that spontaneous secretion of IgM and IgG1 was enhanced from the splenic antibody-secreting cells (ASCs) of CPLX2 KO mice. CONCLUSION: Our data suggest that CPLX2 inhibits spontaneous secretion of IgM and IgG1 from splenic ASCs. This study provides new insight into the mechanism of antibody secretion of ASCs.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/imunologia , Linfócitos B/imunologia , Imunoglobulinas/imunologia , Proteínas do Tecido Nervoso/imunologia , Baço/imunologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Linfócitos B/citologia , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas SNARE/genética , Proteínas SNARE/imunologia , Baço/citologia
20.
Psychiatry Res Neuroimaging ; 294: 110991, 2019 12 30.
Artigo em Inglês | MEDLINE | ID: mdl-31683112

RESUMO

Schizophrenia patients have a higher probability of altered structural and functional differences between the left and right hemisphere. Schizotypy as its nonclinical manifestation has been related to a higher incidence of non-right-handedness and atypical right-hemispheric language dominance. It has been suggested that genes involved in cilia function might link brain asymmetry and neurodevelopmental disorders. We assessed DNA methylation in the promoter regions of seven candidate genes involved in cilia function and psychiatric disorders from buccal cells and investigated their association with schizotypy and language lateralization in 60 healthy adults. Moreover, we determined microstructural properties of the planum temporale in a subsample of 52 subjects using neurite orientation dispersion and density imaging (NODDI). We found a significant association between schizotypy and DNA methylation in the AHI1 promoter region. Moreover, AHI1 DNA methylation significantly predicted language lateralization and asymmetry in estimated planum temporale neurite density. Finally, stronger leftward asymmetry in estimated neurite density was associated with a more pronounced right ear advantage (left hemisphere dominance) in the forced-right condition of the dichotic listening task, measuring attentional modulation of language lateralization. Our results are in line with a shared molecular basis of schizotypy and functional hemispheric asymmetries that is based on cilia function.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Encéfalo/fisiopatologia , Cílios/genética , Transtorno da Personalidade Esquizotípica/genética , Transtorno da Personalidade Esquizotípica/fisiopatologia , Atenção , Percepção Auditiva , Mapeamento Encefálico , Metilação de DNA , Lateralidade Funcional , Humanos , Idioma , Imagem por Ressonância Magnética , Masculino , Mucosa Bucal , Esquizofrenia/fisiopatologia , Lobo Temporal/fisiopatologia
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