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1.
Mol Metab ; 53: 101305, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34303022

RESUMO

OBJECTIVE: Recent studies indicate that brown adipose tissue, in addition to its role in thermogenesis, has a role in the regulation of whole-body metabolism. Here we characterize the metabolic effects of deleting Rab10, a protein key for insulin stimulation of glucose uptake into white adipocytes, solely from brown adipocytes. METHODS: We used a murine brown adipocyte cell line and stromal vascular fraction-derived in vitro differentiated brown adipocytes to study the role of Rab10 in insulin-stimulated GLUT4 translocation to the plasma membrane and insulin-stimulated glucose uptake. We generated a brown adipocyte-specific Rab10 knockout for in vivo studies of metabolism and thermoregulation. RESULTS: We demonstrate that deletion of Rab10 from brown adipocytes results in a two-fold reduction of insulin-stimulated glucose transport by reducing translocation of the GLUT4 glucose transporter to the plasma membrane, an effect linked to whole-body glucose intolerance and insulin resistance in female mice. This effect on metabolism is independent of the thermogenic function of brown adipocytes, thereby revealing a metabolism-specific role for brown adipocytes in female mice. The reduced glucose uptake induced by Rab10 deletion disrupts ChREBP regulation of de novo lipogenesis (DNL) genes, providing a potential link between DNL in brown adipocytes and whole-body metabolic regulation in female mice. However, deletion of Rab10 from male mice does not induce systemic insulin resistance, although ChREBP regulation is disrupted. CONCLUSIONS: Our studies of Rab10 reveal the role of insulin-regulated glucose transport into brown adipocytes in whole-body metabolic homeostasis of female mice. Importantly, the contribution of brown adipocytes to whole-body metabolic regulation is independent of its role in thermogenesis. It is unclear whether the whole-body metabolic sexual dimorphism is because female mice are permissive to the effects of Rab10 deletion from brown adipocytes or because male mice are resistant to the effect.


Assuntos
Adipócitos Marrons/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Glucose/metabolismo , Homeostase , Termogênese , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas rab de Ligação ao GTP/deficiência
2.
Elife ; 102021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33666175

RESUMO

Rab GTPases are molecular switches that regulate membrane trafficking in all cells. Neurons have particular demands on membrane trafficking and express numerous Rab GTPases of unknown function. Here, we report the generation and characterization of molecularly defined null mutants for all 26 rab genes in Drosophila. In flies, all rab genes are expressed in the nervous system where at least half exhibit particularly high levels compared to other tissues. Surprisingly, loss of any of these 13 nervous system-enriched Rabs yielded viable and fertile flies without obvious morphological defects. However, all 13 mutants differentially affected development when challenged with different temperatures, or neuronal function when challenged with continuous stimulation. We identified a synaptic maintenance defect following continuous stimulation for six mutants, including an autophagy-independent role of rab26. The complete mutant collection generated in this study provides a basis for further comprehensive studies of Rab GTPases during development and function in vivo.


Assuntos
Drosophila melanogaster/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/fisiologia , Técnicas de Introdução de Genes , Imidazóis , Neurônios/fisiologia , Temperatura , Proteínas rab de Ligação ao GTP/deficiência
3.
Am J Med Genet A ; 185(3): 986-989, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33368989

RESUMO

Carpenter syndrome (acrocephalopolysyndactyly type II) is a rare autosomal recessive disorder. It was clinically diagnosed in a female baby with polysyndactyly and craniosynostosis in a referral clinic in Northern Tanzania. In the RAB23 gene, a previously described homozygous variant c.82C>T p.(Arg28*) was detected that results in a premature stop codon. Both parents were demonstrated to be heterozygous carriers of this variant. Herewith, its pathogenicity is proved. A literature search suggests this is the first molecularly confirmed case of Carpenter syndrome in continental Africa.


Assuntos
Anormalidades Múltiplas/genética , Acrocefalossindactilia/genética , Códon sem Sentido , Mutação Puntual , Proteínas rab de Ligação ao GTP/genética , Anormalidades Múltiplas/epidemiologia , Acrocefalossindactilia/diagnóstico por imagem , Acrocefalossindactilia/epidemiologia , Feminino , Deformidades Congênitas do Pé/diagnóstico por imagem , Deformidades Congênitas do Pé/genética , Deformidades Congênitas da Mão/diagnóstico por imagem , Deformidades Congênitas da Mão/genética , Homozigoto , Humanos , Processamento de Imagem Assistida por Computador , Lactente , Masculino , Fenótipo , Exame Físico , Tanzânia/epidemiologia , Tomografia Computadorizada por Raios X , Proteínas rab de Ligação ao GTP/deficiência
4.
FEBS J ; 288(1): 190-211, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32248620

RESUMO

Warburg micro syndrome (WMS) is a hereditary autosomal neuromuscular disorder in humans caused by mutations in Rab18, Rab3GAP1, or Rab3GAP2 genes. Rab3GAP1/2 forms a heterodimeric complex, which acts as a guanosine nucleotide exchange factor and activates Rab18. Although the genetic causes of WMS are known, it is still unclear whether loss of the Rab3GAP-Rab18 module affects neuronal or muscle cell physiology or both, and how. In this work, we characterize a Rab3GAP2 mutant Drosophila line to establish a novel animal model for WMS. Similarly to symptoms of WMS, loss of Rab3GAP2 leads to highly decreased motility in Drosophila that becomes more serious with age. We demonstrate that these mutant flies are defective for autophagic degradation in multiple tissues including fat cells and muscles. Loss of Rab3GAP-Rab18 module members leads to perturbed autolysosome morphology due to destabilization of Rab7-positive autophagosomal and late endosomal compartments and perturbation of lysosomal biosynthetic transport. Importantly, overexpression of UVRAG or loss of Atg14, two alternative subunits of the Vps34/PI3K (vacuole protein sorting 34/phosphatidylinositol 3-kinase) complexes in fat cells, mimics the autophagic phenotype of Rab3GAP-Rab18 module loss. We find that GTP-bound Rab18 binds to Atg6/Beclin1, a permanent subunit of Vps34 complexes. Finally, we show that Rab3GAP2 and Rab18 are present on autophagosomal and autolysosomal membranes and colocalize with Vps34 Complex I subunits. Our data suggest that the Rab3GAP-Rab18 module regulates autolysosomal maturation through its interaction with the Vps34 Complex I, and perturbed autophagy due to loss of the Rab3GAP-Rab18 module may contribute to the development of WMS.


Assuntos
Anormalidades Múltiplas/genética , Catarata/congênito , Classe III de Fosfatidilinositol 3-Quinases/genética , Córnea/anormalidades , Proteínas de Drosophila/genética , Hipogonadismo/genética , Deficiência Intelectual/genética , Lisossomos/metabolismo , Microcefalia/genética , Atrofia Óptica/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab3 de Ligação ao GTP/genética , Anormalidades Múltiplas/metabolismo , Anormalidades Múltiplas/patologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Adipócitos/metabolismo , Adipócitos/patologia , Animais , Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Catarata/genética , Catarata/metabolismo , Catarata/patologia , Classe III de Fosfatidilinositol 3-Quinases/deficiência , Córnea/metabolismo , Córnea/patologia , Modelos Animais de Doenças , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Regulação da Expressão Gênica , Humanos , Hipogonadismo/metabolismo , Hipogonadismo/patologia , Deficiência Intelectual/metabolismo , Deficiência Intelectual/patologia , Lisossomos/patologia , Microcefalia/metabolismo , Microcefalia/patologia , Músculos/metabolismo , Músculos/patologia , Neurônios/metabolismo , Neurônios/patologia , Atrofia Óptica/metabolismo , Atrofia Óptica/patologia , Ligação Proteica , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas rab de Ligação ao GTP/deficiência , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab3 de Ligação ao GTP/deficiência , proteínas de unión al GTP Rab7
5.
Cell Death Dis ; 11(10): 827, 2020 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-33012781

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease characterized by chronic non-specific inflammation of the interstitial lung and extensive deposition of collagen fibers leading to destruction of lung function. Studies have demonstrated that exposure to fine particulate matter (PM2.5) increases the risk of IPF. In order to recover from PM2.5-induced lung injury, alveolar epithelial cells need to be repaired and regenerated to maintain lung function. Type 2 alveolar epithelial cells (AEC2) are stem cells in the adult lung that contribute to the lung repair process through complex signaling. Our previous studies demonstrated that RAB6, a RAS family member lowly expressed in lung cancer, inhibited lung cancer stem cell self-renewal, but it is unclear whether or not and how RAB6 may regulate AEC2 cell proliferation and self-renewal in PM2.5-induced pulmonary fibrosis. Here, we demonstrated that knockout of RAB6 inhibited pulmonary fibrosis, oxidative stress, and AEC2 cell death in PM2.5-injured mice. In addition, knockout of RAB6 decreased Dickkopf 1(DKK1) autocrine and activated proliferation, self-renewal, and wnt/ß-catenin signaling of PM2.5-injured AEC2 cells. RAB6 overexpression increased DKK1 autocrine and inhibited proliferation, self-renewal and wnt/ß-catenin signaling in AEC2 cells in vitro. Furthermore, DKK1 inhibitors promoted proliferation, self-renewal and wnt/ß-catenin signaling of RAB6 overexpressing AEC2 cells, and attenuated PM2.5-induced pulmonary fibrosis in mice. These data establish RAB6 as a regulator of DKK1 autocrine and wnt/ß-catenin signal that serves to regulate AEC2 cell proliferation and self-renewal, and suggest a mechanism that RAB6 disruption may promote AEC2 cell proliferation and self-renewal to enhance lung repair following PM2.5 injury.


Assuntos
Autorrenovação Celular/genética , Fibrose/genética , Lesão Pulmonar/metabolismo , Material Particulado/farmacologia , Proteínas rab de Ligação ao GTP/deficiência , Animais , Proliferação de Células/genética , Proliferação de Células/fisiologia , Fibrose/induzido quimicamente , Humanos , Fibrose Pulmonar Idiopática/patologia , Pulmão/metabolismo , Lesão Pulmonar/induzido quimicamente , Lesão Pulmonar/patologia , Camundongos Endogâmicos C57BL , Material Particulado/metabolismo , Células-Tronco/metabolismo , Via de Sinalização Wnt/genética , Via de Sinalização Wnt/fisiologia
6.
Cell Rep ; 31(10): 107733, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32521258

RESUMO

Endocytosis has been proposed to modulate cell signaling activities. However, the role of endocytosis in embryogenesis, which requires coordination of multiple signaling inputs, has remained less understood. We previously showed that mouse embryos lacking a small guanosine triphosphate (GTP)-binding protein Rab7 implicated in endocytic flow are defective in gastrulation. Here, we investigate how subcellular defects associated with Rab7 deficiency are related to the observed developmental defects. Rab7-deficient embryos fail to organize mesodermal tissues due to defects in Wnt-ß-catenin signaling. Visceral endoderm (VE)-specific ablation of Rab7 results in patterning defects similar to systemic Rab7 deletion. Rab7 mutants accumulate the Wnt antagonist Dkk1 in the extracellular space and in intracellular compartments throughout the VE epithelium. These data indicate that Rab7-dependent endocytosis regulates the concentration and availability of extracellular Dkk1, thereby relieving the epiblast of antagonism. This intercellular mechanism therefore organizes distinct spatiotemporal patterns of canonical Wnt activity during the peri-gastrulation stages of embryonic development.


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas Wnt/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Desenvolvimento Embrionário , Endocitose , Endoderma/metabolismo , Feminino , Gastrulação , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Gravidez , Proteínas Wnt/antagonistas & inibidores , Via de Sinalização Wnt , Proteínas rab de Ligação ao GTP/deficiência , proteínas de unión al GTP Rab7
7.
Technol Cancer Res Treat ; 19: 1533033820915958, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32301398

RESUMO

Rabs have been reported to be involved in the carcinogenesis process and in the progression of cancer. However, it is unclear whether or not Rab9 is associated with the development of cancer. In the present study, we aimed to investigate the role of Rab9 in the biological functions of gastric cancer cells. The gastric cancer cell lines AGS and MKN45 were transfected with siRNA-Rab9 to block the expression of Rab9. The cell viability, proliferation, migration, invasion, and apoptosis were examined using Cell Count Kit-8, colony formation, wound healing, Transwell, and flow cytometry assays, respectively. Our data showed that silencing of Rab9 significantly inhibited the viability, proliferation, migration, and invasion abilities of AGS and MKN45 cells. Moreover, transfection with siRab9 promoted the rate of apoptosis in AGS and MKN45 cells through regulating the Bcl-2-Bax axis and the Caspase cascade. We also found that silencing of Rab9 inhibited activation of the Akt signaling pathway by downregulating the phosphorylation level of Akt. In conclusion, our data suggest that Rab9 plays an oncogenic role in the progression of gastric cancer, providing a potential target for the treatment of gastric cancer.


Assuntos
Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias Gástricas/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Apoptose/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Progressão da Doença , Técnicas de Silenciamento de Genes/métodos , Humanos , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Proteínas rab de Ligação ao GTP/deficiência , Proteínas rab de Ligação ao GTP/genética
8.
Mol Brain ; 13(1): 13, 2020 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-31996256

RESUMO

Presynaptic active zone cytomatrix proteins are essential elements of neurotransmitter release machinery that govern neural transmission. Among active zone proteins, cytomatrix at the active zone-associated structural protein (CAST) is known to regulate active zone size in retinal photoreceptors and neurotransmitter release by recruiting Ca2+ channels at various synapses. However, the role of ELKS-a protein from the same family as CAST-and the synergistic roles of CAST/ELKS have not been thoroughly investigated, particularly with regard to mouse behavior. Here, we generated ELKS conditional KO in mouse forebrain synapses by crossing ELKS flox mice with a CaMKII promoter-induced Cre line. Results showed that CAST is dominant at these synapses and that ELKS can support CAST function, but is less effective in the ELKS single KO. Pups of CAST/ELKS double KO in the forebrain were born in Mendelian rations but resulted in eventual death right after the birth. Anatomically, the forebrain neuronal compositions of CAST KO and CAST/ELKS double KO mice were indistinguishable, and the sensory neural network from whiskers on the face was identified as barrelette-like patches in the spinal trigeminal nucleus. Therefore, depletion of CAST and ELKS disrupts neurotransmission from sensory to motor networks, which can lead to deficits in exploration and failure to suckle.


Assuntos
Proteínas do Citoesqueleto/deficiência , Comportamento Exploratório/fisiologia , Hipocampo/metabolismo , Proteínas do Tecido Nervoso/deficiência , Comportamento de Sucção/fisiologia , Proteínas rab de Ligação ao GTP/deficiência , Animais , Animais Recém-Nascidos , Animais Lactentes , Peso Corporal , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/fisiologia , Feminino , Hipocampo/anormalidades , Masculino , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Teste de Campo Aberto , Sinapses/fisiologia , Núcleos do Trigêmeo/anormalidades , Vibrissas/anormalidades , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/fisiologia
9.
Biochem Biophys Res Commun ; 519(1): 113-120, 2019 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-31474334

RESUMO

Gastric cancer (GC) is still a major lethal gastrointestinal tumor. In this study, we clarified that RAB13, which is a member of Rab GTPase family and responsible for cargos delivery between the Golgi and the plasma membrane, plays critical roles in the proliferation and the chemotherapeutic resistance in GC cells. Analyzing RAB13 expression in GC specimens, we found that its mRNA level was higher in cancerous tissues compared with normal counterparts and this increase was further associated with malignant progression of GC. Moreover, increased RAB13 indicated poor overall survival (OS) and progression free survival (PFS) in GC patients. We then found that deletion of RAB13 inhibited the proliferation and promoted the apoptosis in AGS and NCI-N87 cells, the impairments of viability which was due to reduced amount of RAB13 anchoring the plasma membrane and attenuated cellular response to EGF treatment and the activation of downstream Akt/ERK/mTOR signaling pathways accordingly. Moreover, in vitro experiments showed that RAB13 deletion enhanced the sensitization of AGS and NCI-N87 cells toward cisplatin (CDDP) and 5-fluorouracil (5-FU) treatment respectively. Together, these data demonstrate that RAB13 promotes the proliferation and confers CDDP and 5-FU resistance to GC cells, which provides experimental support to target this protein in future clinical practice.


Assuntos
Antineoplásicos/farmacologia , Biomarcadores Tumorais/metabolismo , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fluoruracila/farmacologia , Neoplasias Gástricas/tratamento farmacológico , Proteínas rab de Ligação ao GTP/metabolismo , Biomarcadores Tumorais/análise , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Biologia Computacional , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Células Tumorais Cultivadas , Proteínas rab de Ligação ao GTP/análise , Proteínas rab de Ligação ao GTP/deficiência
10.
J Pathol ; 249(2): 227-240, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31144312

RESUMO

Rab25 can function as both a tumor suppressor and a tumor promoter across different tissues. This study sought to clarify the role of Rab25 as a tumor suppressor in skin squamous cell carcinoma (SCC). Rab25 loss was closely associated with neoplastic transition in both humans and mice. Rab25 loss was well correlated with increased cell proliferation and poor differentiation in human SCC. While Rab25 knockout (KO) in mice did not induce spontaneous tumor formation, it did significantly accelerate tumor generation and promote malignant transformation in a mouse two-stage skin carcinogenesis model. Xenografting of a Rab25-deficient human keratinocyte cell line, HaCaT, also elicited neoplastic transformation. Notably, Rab25 deficiency led to dysregulation of integrins ß1, ß4, and α6, which matched well with increased epidermal proliferation and impaired desmosome-tight junction formation. Rab25 deficiency induced impairment of integrin recycling, leading to the improper expression of integrins. In line with this, significant attenuation of integrin ß1, ß4, and α6 expression was identified in human SCCs where Rab25 was deficient. Collectively, these results suggest that loss of Rab25 promotes the development and neoplastic transition of SCC through dysregulation of integrin trafficking. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Integrinas/metabolismo , Queratinócitos/metabolismo , Proteínas/metabolismo , Neoplasias Cutâneas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , Integrinas/genética , Queratinócitos/patologia , Camundongos da Linhagem 129 , Camundongos Knockout , Transporte Proteico , Proteínas/genética , Transdução de Sinais , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Carga Tumoral , Proteínas Supressoras de Tumor/genética , Proteínas rab de Ligação ao GTP/deficiência , Proteínas rab de Ligação ao GTP/genética
11.
Cell Rep ; 26(5): 1213-1226.e7, 2019 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-30699350

RESUMO

Pancreatic ß cells secrete insulin by Ca2+-triggered exocytosis. However, there is no apparent secretory site similar to the neuronal active zones, and the cellular and molecular localization mechanism underlying polarized exocytosis remains elusive. Here, we report that ELKS, a vertebrate active zone protein, is used in ß cells to regulate Ca2+ influx for insulin secretion. ß cell-specific ELKS-knockout (KO) mice showed impaired glucose-stimulated first-phase insulin secretion and reduced L-type voltage-dependent Ca2+ channel (VDCC) current density. In situ Ca2+ imaging of ß cells within islets expressing a membrane-bound G-CaMP8b Ca2+ sensor demonstrated initial local Ca2+ signals at the ELKS-localized vascular side of the ß cell plasma membrane, which were markedly decreased in ELKS-KO ß cells. Mechanistically, ELKS directly interacted with the VDCC-ß subunit via the GK domain. These findings suggest that ELKS and VDCCs form a potent insulin secretion complex at the vascular side of the ß cell plasma membrane for polarized Ca2+ influx and first-phase insulin secretion from pancreatic islets.


Assuntos
Cálcio/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Subunidades Proteicas/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/metabolismo , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Glucose/farmacologia , Humanos , Insulina/metabolismo , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Proteínas do Tecido Nervoso/deficiência , Ligação Proteica/efeitos dos fármacos , Proteínas rab de Ligação ao GTP/deficiência
12.
J Cell Sci ; 131(22)2018 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-30333141

RESUMO

Cytokine receptors, such as tumor necrosis factor receptor I (TNFRI, also known as TNFRSF1A) and lymphotoxin ß receptor (LTßR), activate inflammatory nuclear factor (NF)-κB signaling upon stimulation. We have previously demonstrated that depletion of ESCRT components leads to endosomal accumulation of TNFRI and LTßR, and their ligand-independent signaling to NF-κB. Here, we studied whether other perturbations of the endolysosomal system could trigger intracellular accumulation and signaling of ligand-free LTßR. While depletion of the CORVET components had no effect, knockdown of Rab7a or HOPS components, or pharmacological inhibition of lysosomal degradation, caused endosomal accumulation of LTßR and increased its interaction with the TRAF2 and TRAF3 signaling adaptors. However, the NF-κB pathway was not activated under these conditions. We found that knockdown of Rab7a or HOPS components led to sequestration of LTßR in intraluminal vesicles of endosomes, thus precluding NF-κB signaling. This was in contrast to the LTßR localization on the outer endosomal membrane that was seen after ESCRT depletion and was permissive for signaling. We propose that the inflammatory response induced by intracellular accumulation of endocytosed cytokine receptors critically depends on the precise receptor topology within endosomal compartments.


Assuntos
Receptor beta de Linfotoxina/metabolismo , NF-kappa B/metabolismo , Endossomos/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Lisossomos/metabolismo , Transporte Proteico , Transdução de Sinais , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismo , Proteínas de Transporte Vesicular/deficiência , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/deficiência , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7
13.
Cell Rep ; 23(2): 415-428, 2018 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-29642001

RESUMO

Axonal regrowth is crucial for recovery from CNS injury but is severely restricted in adult mammals. We used a genome-wide loss-of-function screen for factors limiting axonal regeneration from cerebral cortical neurons in vitro. Knockdown of 16,007 individual genes identified 580 significant phenotypes. These molecules share no significant overlap with those suggested by previous expression profiles. There is enrichment for genes in pathways related to transport, receptor binding, and cytokine signaling, including Socs4 and Ship2. Among transport-regulating proteins, Rab GTPases are prominent. In vivo assessment with C. elegans validates a cell-autonomous restriction of regeneration by Rab27. Mice lacking Rab27b show enhanced retinal ganglion cell axon regeneration after optic nerve crush and greater motor function and raphespinal sprouting after spinal cord trauma. Thus, a comprehensive functional screen reveals multiple pathways restricting axonal regeneration and neurological recovery after injury.


Assuntos
Axônios/metabolismo , Sistema Nervoso Central/fisiologia , Genoma , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/antagonistas & inibidores , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Feminino , Redes Reguladoras de Genes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regeneração Nervosa , Nervo Óptico/fisiologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Recuperação de Função Fisiológica , Células Ganglionares da Retina/metabolismo , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Proteínas Supressoras da Sinalização de Citocina/antagonistas & inibidores , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Proteínas rab de Ligação ao GTP/antagonistas & inibidores , Proteínas rab de Ligação ao GTP/deficiência , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
14.
PLoS One ; 13(12): e0203100, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30596653

RESUMO

During vertebrate cardiac development NOTCH signaling activity in the endocardium is essential for the crosstalk between endocardium and myocardium that initiates ventricular trabeculation and valve primordium formation. This crosstalk leads later to the maturation and compaction of the ventricular chambers and the morphogenesis of the cardiac valves, and its alteration may lead to disease. Although endocardial NOTCH signaling has been shown to be crucial for heart development, its physiological role in the myocardium has not been clearly established. Here we have used mouse genetics to evaluate the role of NOTCH in myocardial development. We have inactivated the unique and ubiquitous NOTCH effector RBPJ in early cardiomyocytes progenitors, and examined its consequences in cardiac development and function. Our results show that mice with Tnnt2-Cre-mediated myocardial-specific deletion of Rbpj develop to term, with homozygous mutant animals showing normal expression of cardiac development markers, and normal adult heart function. Similar observations have been obtained after Notch1 deletion with Tnnt2-Cre. We have also deleted Rbpj in both myocardial and endocardial progenitor cells, using the Nkx2.5-Cre driver, resulting in ventricular septal defect (VSD), double outlet right ventricle (DORV), and bicuspid aortic valve (BAV), due to NOTCH signaling abrogation in the endocardium of cardiac valves. Our data demonstrate that NOTCH-RBPJ inactivation in the myocardium does not affect heart development or adult cardiac function.


Assuntos
Deleção de Genes , Cardiopatias Congênitas , Coração/embriologia , Miocárdio/metabolismo , Receptor Notch1/deficiência , Transdução de Sinais , Proteínas rab de Ligação ao GTP/deficiência , Animais , Cardiopatias Congênitas/embriologia , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/metabolismo , Camundongos , Camundongos Knockout , Miocárdio/patologia
15.
Mol Autism ; 8: 59, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29152164

RESUMO

Background: Autism spectrum disorder (ASD), a developmental disorder of early childhood onset, affects males four times more frequently than females, suggesting a role for the sex chromosomes. In this study, we describe a family with ASD in which a predicted pathogenic nonsense mutation in the X-chromosome gene RAB39B segregates with ASD phenotype. Methods: Clinical phenotyping, microarray, and whole genome sequencing (WGS) were performed on the five members of this family. Maternal and female sibling X inactivation ratio was calculated, and phase was investigated. Mutant-induced pluripotent stem cells engineered for an exon 2 nonsense mutation were generated and differentiated into cortical neurons for expression and pathway analyses. Results: Two males with an inherited RAB39B mutation both presented with macrocephaly, intellectual disability (ID), and ASD. Their female sibling with the same mutation presented with ID and a broad autism phenotype. In contrast, their transmitting mother has no neurodevelopmental diagnosis. Our investigation of phase indicated maternal preferential inactivation of the mutated allele, with no such bias observed in the female sibling. We offer the explanation that this bias in X inactivation may explain the absence of a neurocognitive phenotype in the mother. Our cellular knockout model of RAB39B revealed an impact on expression in differentiated neurons for several genes implicated in brain development and function, supported by our pathway enrichment analysis. Conclusions: Penetrance for ASD is high among males but more variable among females with RAB39B mutations. A critical role for this gene in brain development and function is demonstrated.


Assuntos
Transtorno do Espectro Autista/genética , Deficiência Intelectual/genética , Megalencefalia/genética , Proteínas rab de Ligação ao GTP/genética , Alelos , Transtorno do Espectro Autista/complicações , Transtorno do Espectro Autista/diagnóstico , Sistemas CRISPR-Cas/genética , Células Cultivadas , Criança , Códon sem Sentido , Feminino , Fibroblastos/citologia , Genótipo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Deficiência Intelectual/complicações , Deficiência Intelectual/diagnóstico , Masculino , Megalencefalia/complicações , Megalencefalia/diagnóstico , Linhagem , Fenótipo , Sequenciamento Completo do Genoma , Proteínas rab de Ligação ao GTP/deficiência
16.
Am J Hum Genet ; 101(5): 824-832, 2017 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-29106825

RESUMO

The Rab GTPase family comprises ∼70 GTP-binding proteins, functioning in vesicle formation, transport and fusion. They are activated by a conformational change induced by GTP-binding, allowing interactions with downstream effectors. Here, we report five individuals with two recurrent de novo missense mutations in RAB11B; c.64G>A; p.Val22Met in three individuals and c.202G>A; p.Ala68Thr in two individuals. An overlapping neurodevelopmental phenotype, including severe intellectual disability with absent speech, epilepsy, and hypotonia was observed in all affected individuals. Additionally, visual problems, musculoskeletal abnormalities, and microcephaly were present in the majority of cases. Re-evaluation of brain MRI images of four individuals showed a shared distinct brain phenotype, consisting of abnormal white matter (severely decreased volume and abnormal signal), thin corpus callosum, cerebellar vermis hypoplasia, optic nerve hypoplasia and mild ventriculomegaly. To compare the effects of both variants with known inactive GDP- and active GTP-bound RAB11B mutants, we modeled the variants on the three-dimensional protein structure and performed subcellular localization studies. We predicted that both variants alter the GTP/GDP binding pocket and show that they both have localization patterns similar to inactive RAB11B. Evaluation of their influence on the affinity of RAB11B to a series of binary interactors, both effectors and guanine nucleotide exchange factors (GEFs), showed induction of RAB11B binding to the GEF SH3BP5, again similar to inactive RAB11B. In conclusion, we report two recurrent dominant mutations in RAB11B leading to a neurodevelopmental syndrome, likely caused by altered GDP/GTP binding that inactivate the protein and induce GEF binding and protein mislocalization.


Assuntos
Epilepsia/genética , Deficiência Intelectual/genética , Hipotonia Muscular/genética , Mutação , Doenças do Nervo Óptico/congênito , Proteínas rab de Ligação ao GTP/genética , Adolescente , Sequência de Aminoácidos , Sítios de Ligação , Vermis Cerebelar/diagnóstico por imagem , Vermis Cerebelar/metabolismo , Vermis Cerebelar/patologia , Criança , Pré-Escolar , Corpo Caloso/diagnóstico por imagem , Corpo Caloso/metabolismo , Corpo Caloso/patologia , Epilepsia/diagnóstico por imagem , Epilepsia/patologia , Feminino , Expressão Gênica , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Humanos , Deficiência Intelectual/diagnóstico por imagem , Deficiência Intelectual/patologia , Imageamento por Ressonância Magnética , Masculino , Modelos Moleculares , Hipotonia Muscular/diagnóstico por imagem , Hipotonia Muscular/patologia , Doenças do Nervo Óptico/diagnóstico por imagem , Doenças do Nervo Óptico/genética , Doenças do Nervo Óptico/patologia , Fenótipo , Ligação Proteica , Substância Branca/diagnóstico por imagem , Substância Branca/metabolismo , Substância Branca/patologia , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/deficiência
17.
EMBO Rep ; 18(10): 1727-1739, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28835545

RESUMO

Autophagy (macroautophagy) is a highly conserved eukaryotic degradation pathway in which cytosolic components and organelles are sequestered by specialized autophagic membranes and degraded through the lysosomal system. The autophagic pathway maintains basal cellular homeostasis and helps cells adapt during stress; thus, defects in autophagy can cause detrimental effects. It is therefore crucial that autophagy is properly regulated. In this study, we show that the cysteine protease Atg4B, a key enzyme in autophagy that cleaves LC3, is an interactor of the small GTPase Rab7b. Indeed, Atg4B interacts and co-localizes with Rab7b on vesicles. Depletion of Rab7b increases autophagic flux as indicated by the increased size of autophagic structures as well as the magnitude of macroautophagic sequestration and degradation. Importantly, we demonstrate that Rab7b regulates LC3 processing by modulating Atg4B activity. Taken together, our findings reveal Rab7b as a novel negative regulator of autophagy through its interaction with Atg4B.


Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Cisteína Endopeptidases/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Relacionadas à Autofagia/genética , Cisteína Endopeptidases/genética , Regulação da Expressão Gênica , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas rab de Ligação ao GTP/deficiência , Proteínas rab de Ligação ao GTP/genética , proteínas de unión al GTP Rab7
18.
J Cell Sci ; 130(15): 2491-2505, 2017 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-28596241

RESUMO

Misplaced formation of microvilli to basolateral domains and intracellular inclusions in enterocytes are pathognomonic features in congenital enteropathy associated with mutation of the apical plasma membrane receptor syntaxin 3 (STX3). Although the demonstrated binding of Myo5b to the Rab8a and Rab11a small GTPases in vitro implicates cytoskeleton-dependent membrane sorting, the mechanisms underlying the microvillar location defect remain unclear. By selective or combinatory disruption of Rab8a and Rab11a membrane traffic in vivo, we demonstrate that transport of distinct cargo to the apical brush border rely on either individual or both Rab regulators, whereas certain basolateral cargos are redundantly transported by both factors. Enterocyte-specific Rab8a and Rab11a double-knockout mouse neonates showed immediate postnatal lethality and more severe enteropathy than single knockouts, with extensive formation of microvilli along basolateral surfaces. Notably, following an inducible Rab11a deletion from neonatal enterocytes, basolateral microvilli were induced within 3 days. These data identify a potentially important and distinct mechanism for a characteristic microvillus defect exhibited by enterocytes of patients with neonatal enteropathy.


Assuntos
Enterócitos/metabolismo , Doenças Genéticas Inatas/metabolismo , Enteropatias/metabolismo , Microvilosidades/metabolismo , Proteínas rab de Ligação ao GTP/deficiência , Animais , Enterócitos/patologia , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/patologia , Enteropatias/congênito , Enteropatias/patologia , Camundongos , Camundongos Knockout , Microvilosidades/genética , Microvilosidades/patologia , Miosina Tipo V/genética , Miosina Tipo V/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo
19.
Dev Cell ; 42(1): 22-36.e12, 2017 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-28625565

RESUMO

Highly conserved intraflagellar transport (IFT) protein complexes direct both the assembly of primary cilia and the trafficking of signaling molecules. IFT complexes initially accumulate at the base of the cilium and periodically enter the cilium, suggesting an as-yet-unidentified mechanism that triggers ciliary entry of IFT complexes. Using affinity-purification and mass spectrometry of interactors of the centrosomal and ciliopathy protein, CEP19, we identify CEP350, FOP, and the RABL2B GTPase as proteins organizing the first known mechanism directing ciliary entry of IFT complexes. We discover that CEP19 is recruited to the ciliary base by the centriolar CEP350/FOP complex and then specifically captures GTP-bound RABL2B, which is activated via its intrinsic nucleotide exchange. Activated RABL2B then captures and releases its single effector, the intraflagellar transport B holocomplex, from the large pool of pre-docked IFT-B complexes, and thus initiates ciliary entry of IFT.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cílios/metabolismo , Flagelos/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Centríolos/metabolismo , Ciliopatias , Técnicas de Inativação de Genes , Guanosina Trifosfato/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nucleotídeos/metabolismo , Fenótipo , Ligação Proteica , Estabilidade Proteica , Transporte Proteico , Reprodutibilidade dos Testes , Proteínas rab de Ligação ao GTP/deficiência
20.
Cell Mol Neurobiol ; 37(7): 1303-1310, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28132130

RESUMO

Oligodendrocyte precursor cell (OPC) maturation requires membrane addition for myelin sheath formation. Since the Rab system has been shown to contribute to membrane addition in other cell types, in this study, we explored the role of Rab in OPC maturation. SiRNA and shRNA techniques and conditional knockout mice provided in vitro and in vivo evidence that Rab10 is involved in OPC maturation and may affect myelination during OPC development.


Assuntos
Células Precursoras de Oligodendrócitos/patologia , Oligodendroglia/fisiologia , Proteínas rab de Ligação ao GTP/deficiência , Animais , Animais Recém-Nascidos , Células Cultivadas , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Ratos , Ratos Sprague-Dawley , Proteínas rab de Ligação ao GTP/genética
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