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1.
Sheng Li Xue Bao ; 71(4): 555-561, 2019 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-31440752

RESUMO

The aim of the present study was to establish a cell model of volume-regulated anion channel subunit LRRC8A and investigate the physiological characteristics of LRRC8A. The eukaryotic expression vectors of LRRC8A and YFP-H148Q/I152L were constructed and transfected into Fischer rat thyroid (FRT) cells by Lipofectamine 2000. The FRT cell lines co-expressing LRRC8A and YFP-H148Q/I152L were obtained by antibiotic screening. The expression of LRRC8A and YFP-H148Q/I152L in FRT cells was detected by the inverted fluorescence microscope. The fluorescence quenching kinetic experiment was done to verify the function and effectiveness of the cell model. Then the cell model was utilized to study the physiological characteristics of LRRC8A, such as the characteristics of anion transport, the opening of LRRC8A by osmotic pressure, the effect of anion transport velocity, and the effect of chloride channel inhibitors on LRRC8A anion channel. The results of the inverted fluorescence microscope showed that LRRC8A was expressed on the cell membrane and YFP-H148Q/I152L was expressed in the cytoplasm. The results of fluorescence quenching kinetic test showed that under the condition of low osmotic state, LRRC8A could transport some kinds of anions, such as iodine and chloride ions. Osmotic pressure played a key role in the regulation of LRRC8A volume-regulated anion channel opening. Chloride channel inhibitors inhibited ion transport of LRRC8A channel in a dose-dependent manner. It is suggested that LRRC8A has the characteristics of classic volume-regulated anion channels by using the cell model of FRT cells co-expressing LRRC8A and YFP-H148Q/I152L.


Assuntos
Canais de Cloreto/antagonistas & inibidores , Transporte de Íons , Proteínas de Membrana/fisiologia , Animais , Ânions , Células Cultivadas , Microscopia de Fluorescência , Ratos , Ratos Endogâmicos F344 , Glândula Tireoide/citologia , Transfecção
2.
Yi Chuan ; 41(6): 451-468, 2019 Jun 20.
Artigo em Chinês | MEDLINE | ID: mdl-31257195

RESUMO

Endocytic transport is imperative for the exchange of information between cells and the external environment. Specifically, the process of endocytic transport comprises precise regulation of uptake and sorting of extracellular macromolecules, phospholipids, and membrane proteins. In the endocytic transport system, the recycling pathways are responsible for delivering membrane proteins and phospholipids back to the plasma membrane. Thus, endocytic recycling plays critical roles in various biological processes, including nutrient absorption, cell polarity establishment, cell migration, cell division, synaptic plasticity, immune response, and growth factor receptor regulation. There are two essential types of recycling pathways in eukaryotic cells, recycling of clathrin-dependent endocytic cargos (CDE recycling) and recycling of clathrin-independent endocytic cargos (CIE recycling). The transferrin receptor TfR and the low-density lipoprotein receptor LDLR, which have essential physiological roles in vivo, are representative membrane proteins of the CDE recycling transport. In recent years, various membrane proteins governed by CIE recycling transport have been identified, including IL2 receptor α-subunit, major histocompatibility complex MHC Class I, and glucose transporter GLUT4. Therefore, the investigation of the regulatory mechanisms of CIE recycling has drawn notable attention in the field. Moreover, CIE recycling research presents fundamental significance in cell biology, which also provides scientific evidence and potential therapeutic clues for the diagnosis and treatment strategies of diseases such as type 2 diabetes and cancer. Compared with the CDE recycling, the study on CIE recycling started later, and there is much to be learned of its regulatory mechanisms. To this end, this review summarizes the features of endocytic recycling pathways, focuses on the molecular basis of CIE recycling regulation and elaborates on the latest progress and newly developed research model systems in the field of CIE recycling.


Assuntos
Endocitose , Proteínas de Membrana/fisiologia , Transporte Proteico , Clatrina , Diabetes Mellitus Tipo 2 , Endossomos , Humanos
3.
Nat Neurosci ; 22(7): 1075-1088, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31209379

RESUMO

Microglia rapidly respond to changes in neural activity and inflammation to regulate synaptic connectivity. The extracellular signals, particularly neuron-derived molecules, that drive these microglial functions at synapses remain a key open question. Here we show that whisker lesioning, known to dampen cortical activity, induces microglia-mediated synapse elimination. This synapse elimination is dependent on signaling by CX3CR1, the receptor for microglial fractalkine (also known as CXCL1), but not complement receptor 3. Furthermore, mice deficient in CX3CL1 have profound defects in synapse elimination. Single-cell RNA sequencing revealed that Cx3cl1 is derived from cortical neurons, and ADAM10, a metalloprotease that cleaves CX3CL1 into a secreted form, is upregulated specifically in layer IV neurons and in microglia following whisker lesioning. Finally, inhibition of ADAM10 phenocopies Cx3cr1-/- and Cx3cl1-/- synapse elimination defects. Together, these results identify neuron-to-microglia signaling necessary for cortical synaptic remodeling and reveal that context-dependent immune mechanisms are utilized to remodel synapses in the mammalian brain.


Assuntos
Proteína ADAM10/fisiologia , Secretases da Proteína Precursora do Amiloide/fisiologia , Receptor 1 de Quimiocina CX3C/fisiologia , Quimiocina CX3CL1/fisiologia , Proteínas de Membrana/fisiologia , Microglia/fisiologia , Córtex Sensório-Motor/fisiopatologia , Tato/fisiologia , Vibrissas/lesões , Proteína ADAM10/antagonistas & inibidores , Proteína ADAM10/genética , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/genética , Animais , Receptor 1 de Quimiocina CX3C/deficiência , Receptor 1 de Quimiocina CX3C/genética , Contagem de Células , Feminino , Regulação da Expressão Gênica , Masculino , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Técnicas Analíticas Microfluídicas , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Córtex Sensório-Motor/metabolismo , Córtex Sensório-Motor/patologia , Transdução de Sinais/fisiologia , Análise de Célula Única , Transcriptoma , Vibrissas/fisiologia
4.
Nat Commun ; 10(1): 2602, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197136

RESUMO

Temperature is a key factor for determining the lifespan of both poikilotherms and homeotherms. It is believed that animals live longer at lower body temperatures. However, the precise mechanism remains largely unknown. Here, we report that autophagy serves as a boost mechanism for longevity at low temperature in the nematode Caenorhabditis elegans. The adiponectin receptor AdipoR2 homolog PAQR-2 signaling detects temperature drop and augments the biosynthesis of two ω-6 polyunsaturated fatty acids, γ-linolenic acid and arachidonic acid. These two polyunsaturated fatty acids in turn initiate autophagy in the epidermis, delaying an age-dependent decline in collagen contents, and extending the lifespan. Our findings reveal that the adiponectin receptor PAQR-2 signaling acts as a regulator linking low temperature with autophagy to extend lifespan, and suggest that such a mechanism may be evolutionally conserved among diverse organisms.


Assuntos
Autofagia/fisiologia , Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/fisiologia , Longevidade/fisiologia , Proteínas de Membrana/fisiologia , Animais , Animais Geneticamente Modificados , Ácido Araquidônico/biossíntese , Temperatura Baixa , Colágeno/metabolismo , Epiderme/metabolismo , Ácidos Graxos Ômega-6/biossíntese , Interferência de RNA , Transdução de Sinais/fisiologia
5.
Physiol Rev ; 99(3): 1433-1466, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31066629

RESUMO

FMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase that is expressed almost exclusively in the hematopoietic compartment. Its ligand, FLT3 ligand (FL), induces dimerization and activation of its intrinsic tyrosine kinase activity. Activation of FLT3 leads to its autophosphorylation and initiation of several signal transduction cascades. Signaling is initiated by the recruitment of signal transduction molecules to activated FLT3 through binding to specific phosphorylated tyrosine residues in the intracellular region of FLT3. Activation of FLT3 mediates cell survival, cell proliferation, and differentiation of hematopoietic progenitor cells. It acts in synergy with several other cytokines to promote its biological effects. Deregulated FLT3 activity has been implicated in several diseases, most prominently in acute myeloid leukemia where around one-third of patients carry an activating mutant of FLT3 which drives the disease and is correlated with poor prognosis. Overactivity of FLT3 has also been implicated in autoimmune diseases, such as rheumatoid arthritis. The observation that gain-of-function mutations of FLT3 can promote leukemogenesis has stimulated the development of inhibitors that target this receptor. Many of these are in clinical trials, and some have been approved for clinical use. However, problems with acquired resistance to these inhibitors are common and, furthermore, only a fraction of patients respond to these selective treatments. This review provides a summary of our current knowledge regarding structural and functional aspects of FLT3 signaling, both under normal and pathological conditions, and discusses challenges for the future regarding the use of targeted inhibition of these pathways for the treatment of patients.


Assuntos
Tirosina Quinase 3 Semelhante a fms/fisiologia , Animais , Antineoplásicos/farmacologia , Células-Tronco Hematopoéticas/fisiologia , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Transdução de Sinais , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/genética
6.
Nat Plants ; 5(5): 539-550, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31076735

RESUMO

Post-transcriptional gene silencing (PTGS) is a major mechanism regulating gene expression in higher eukaryotes. To identify novel players in PTGS, a forward genetics screen was performed on an Arabidopsis thaliana line overexpressing a strong growth-repressive gene, ETHYLENE RESPONSE FACTOR6 (ERF6). We identified six independent ethyl-methanesulfonate mutants rescuing the dwarfism of ERF6-overexpressing plants as a result of transgene silencing. Among the causative genes, ETHYLENE-INSENSITIVE5, SUPERKILLER2 and HASTY1 have previously been reported to inhibit PTGS. Notably, the three other causative genes have not, to date, been related to PTGS: UTP:RNA-URIDYLYLTRANSFERASE1 (URT1), C-TERMINAL DOMAIN PHOSPHATASE-LIKE3 (CPL3) and RESURRECTION1 (RST1). We show that these genes may participate in protecting the 3' end of transgene transcripts. We present a model in which URT1, CPL3 and RST1 are classified as PTGS suppressors, as compromisation of these genes provokes the accumulation of aberrant transcripts which, in turn, trigger the production of small interfering RNAs, initiating RNA silencing.


Assuntos
Proteínas de Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas/genética , Proteínas de Membrana/fisiologia , Fosfoproteínas Fosfatases/fisiologia , Interferência de RNA , RNA Nucleotidiltransferases/fisiologia , RNA de Plantas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação/genética , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , RNA Nucleotidiltransferases/genética , RNA Nucleotidiltransferases/metabolismo , RNA de Plantas/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transgenes/genética
7.
J Microbiol ; 57(7): 626-636, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31054134

RESUMO

Beta haemolytic Group A streptococcus (GAS) or Streptococcus pyogenes are strict human pathogens responsible for mild to severe fatal invasive infections. Even with enormous number of reports exploring the role of S. pyogenes exotoxins in its pathogenesis, inadequate knowledge on the biofilm process and the potential role of exotoxins in bacterial dissemination from matured biofilms has been a hindrance in development of effective and targeted treatments. Therefore, the present study was aimed in investigating the uncharted role of these exotoxins in biofilm process. Through our study the putative role of ciaRH in the SpeA dependent ablation of biofilm formation could be speculated and thus helping in bacterial dissemination. The seed-dispersal effect of SpeA was time and concentration dependent and seen to be consistent within various streptococcal species. Transcriptome analysis of SpeA treated S. pyogenes biofilms revealed the involvement of many transcriptional regulators (ciaRH) and response genes (luxS, shr, shp, SPy_0572), hinting towards specific mechanisms underlying the dispersal effect by SpeA. This finding opens up a discussion towards understanding a new mechanism involved in the pathogenesis of Streptococcus pyogenes and might help in understanding the bacterial infections in a better way.


Assuntos
Proteínas de Bactérias , Biofilmes/efeitos dos fármacos , Exotoxinas , Proteínas de Membrana , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes , Proteínas de Bactérias/farmacologia , Proteínas de Bactérias/fisiologia , Exotoxinas/farmacologia , Exotoxinas/fisiologia , Perfilação da Expressão Gênica/métodos , Humanos , Proteínas de Membrana/farmacologia , Proteínas de Membrana/fisiologia , Streptococcus pyogenes/efeitos dos fármacos , Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidade , Superantígenos/fisiologia
8.
Nat Commun ; 10(1): 2024, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-31048699

RESUMO

Mutations in the polycystins cause autosomal dominant polycystic kidney disease (ADPKD). Here we show that transmembrane protein 33 (TMEM33) interacts with the ion channel polycystin-2 (PC2) at the endoplasmic reticulum (ER) membrane, enhancing its opening over the whole physiological calcium range in ER liposomes fused to planar bilayers. Consequently, TMEM33 reduces intracellular calcium content in a PC2-dependent manner, impairs lysosomal calcium refilling, causes cathepsins translocation, inhibition of autophagic flux upon ER stress, as well as sensitization to apoptosis. Invalidation of TMEM33 in the mouse exerts a potent protection against renal ER stress. By contrast, TMEM33 does not influence pkd2-dependent renal cystogenesis in the zebrafish. Together, our results identify a key role for TMEM33 in the regulation of intracellular calcium homeostasis of renal proximal convoluted tubule cells and establish a causal link between TMEM33 and acute kidney injury.


Assuntos
Lesão Renal Aguda/patologia , Cálcio/metabolismo , Túbulos Renais Proximais/metabolismo , Proteínas de Membrana/metabolismo , Canais de Cátion TRPP/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Lesão Renal Aguda/genética , Animais , Membrana Celular/metabolismo , Modelos Animais de Doenças , Embrião não Mamífero , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Túbulos Renais Proximais/citologia , Lisossomos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Knockout , Mutação , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/patologia , RNA Interferente Pequeno/metabolismo , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/fisiologia
9.
Prog Retin Eye Res ; 69: 137-158, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30982505

RESUMO

ELOngation of Very Long chain fatty acids-4 (ELOVL4) is an elongase responsible for the biosynthesis of very long chain (VLC, ≥C28) saturated (VLC-SFA) and polyunsaturated (VLC-PUFA) fatty acids in brain, retina, skin, Meibomian glands, and testes. Fascinatingly, different mutations in this gene have been reported to cause vastly different phenotypes in humans. Heterozygous inheritance of seven different mutations in the coding sequence and 5' untranslated region of ELOVL4 causes autosomal dominant Stargardt-like macular dystrophy (STGD3), while homozygous inheritance of three more mutant variants causes severe seizures with ichthyosis, hypertonia, and even death. Some recent studies have described heterozygous inheritance in yet another three mutant ELOVL4 variants, two that cause spinocerebellar ataxia-34 (SCA34) with erythrokeratodermia (EKV) and one that causes SCA34 without EKV. We identified the specific enzymatic reactions catalyzed by ELOVL4 and, using a variety of genetically engineered mouse models, have actively searched for the mechanisms by which ELOVL4 impacts neural function and health. In this review, we critically compare and contrast the various animal model and case studies involving ELOVL4 deficiency via either mutation or deletion, and the resulting consequences on neuronal health and function in both the retina and central nervous system.


Assuntos
Fenômenos Fisiológicos Celulares/fisiologia , Doenças do Sistema Nervoso Central/fisiopatologia , Proteínas do Olho/fisiologia , Mamíferos/fisiologia , Proteínas de Membrana/fisiologia , Células Fotorreceptoras de Vertebrados/fisiologia , Doenças Retinianas/fisiopatologia , Animais , Doenças do Sistema Nervoso Central/genética , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Doenças Retinianas/genética , Doenças Retinianas/metabolismo
10.
Life Sci ; 226: 149-155, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30981764

RESUMO

AIMS: To investigate the effects and mechanisms of DLL3 in inflammation-mediated A2058 melanoma cell invasion and metastasis. MATERIALS AND METHODS: Melanoma A2058 cells was stimulated with lipopolysaccharide (LPS), with or without transfection of DLL3 siRNA, or DLL3 overexpression vector, or Twist1 siRNA. Cell migration and invasion were detected by wound healing and transwell invasion assay. The production of inflammatory factors TNF-α and IL-6 was measured by ELISA. The expression of Notch signaling-related molecules was detected by PCR and western blot. The protein expression of MMP1, MMP9, VEGF, DLL3, and EMT-related molecules was tested by western blot. KEY FINDINGS: LPS treatment increased migration and invasion of A2058 cells, accompanied by increased expression of TNF-α and IL-6. DLL3 was both upregulated in the LPS- or TNF-α-stimulated A2058 cells, and DLL3 knockdown inhibited LPS-induced inflammation, migration and invasion of A2058 cells, accompanied by down-regulation of MMP1, MMP9 and VEGF. Besides, DLL3 knockdown inhibits the expression of Twist1, a key EMT regulating factor, as well as the EMT hallmarks slug, N-cadherin and vimentin. Moreover, Twist1 silence inhibited EMT, and limited LPS-induced migration and invasion of A2058 cells, with decreased expression of MMP1, MMP9 and VEGF and reduced production of TNF-α and IL-6 in LPS-stimulated A2058 cells. SIGNIFICANCE: Knockdown of DLL3 restricts LPS-induced inflammation, migration and invasion of A2058 melanoma cells via blocking Twist1-mediated EMT. Therefore, targeting DLL3 may be a promising therapeutic strategy against inflammation-aggravated melanoma progression.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Melanoma/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Caderinas , Linhagem Celular Tumoral , Movimento Celular/genética , Regulação para Baixo , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Humanos , Inflamação/genética , Interleucina-6 , Lipopolissacarídeos/farmacologia , Metaloproteinase 1 da Matriz , Metaloproteinase 9 da Matriz , Melanoma/genética , Invasividade Neoplásica/genética , Proteínas Nucleares/fisiologia , Transdução de Sinais , Ativação Transcricional , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Proteína 1 Relacionada a Twist/fisiologia , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular , Vimentina
11.
Nat Commun ; 10(1): 1871, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015410

RESUMO

The size of the membrane-bound nucleus scales with cell size in a wide range of cell types but the mechanisms determining overall nuclear size remain largely unknown. Here we investigate the role of fission yeast inner nuclear membrane proteins in determining nuclear size, and propose that the Lap2-Emerin-Man1 domain protein Lem2 acts as a barrier to membrane flow between the nucleus and other parts of the cellular membrane system. Lem2 deletion increases membrane flow into and out of the nuclear envelope in response to changes in membrane synthesis and nucleocytoplasmic transport, altering nuclear size. The endoplasmic reticulum protein Lnp1 acts as a secondary barrier to membrane flow, functionally compensating for lack of Lem2. We propose that this is part of the mechanism that maintains nuclear size proportional to cellular membrane content and thus to cell size. Similar regulatory principles may apply to other organelles in the eukaryotic subcellular membrane network.


Assuntos
Núcleo Celular/fisiologia , Proteínas de Ligação a DNA/fisiologia , Proteínas de Membrana/fisiologia , Membrana Nuclear/metabolismo , Proteínas Nucleares/fisiologia , Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/fisiologia , Transporte Ativo do Núcleo Celular/fisiologia , Retículo Endoplasmático/metabolismo , Domínios Proteicos/fisiologia
12.
J Biochem ; 165(5): 411-414, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30873542

RESUMO

Prostate transmembrane protein androgen-induced 1 (PMEPA1)/transmembrane prostate androgen-induced protein (TMEPAI), a direct target and a negative regulator of transforming growth factor beta signalling, has an oncogenic role in many cancers. We observed that knockout (KO) of PMEPA1 in human breast cancer cell line MDA-MB-231 using a CRISPR-Cas9 system resulted in reduction of in vivo tumour growth and lung metastasis but not of in vitro monolayer growth capacity of these KO cell lines. This phenomenon was associated with PMEPA1 KO-mediated downregulation of the key proangiogenic factors vascular endothelial growth factor alpha (VEGFA) and interleukin-8 (IL8) that are essential for in vivo but not in vitro growing cells and are also substantial for initiation of lung metastasis.


Assuntos
Neoplasias da Mama/patologia , Proliferação de Células/genética , Técnicas de Silenciamento de Genes , Neoplasias Pulmonares/secundário , Proteínas de Membrana/fisiologia , Animais , Linhagem Celular Tumoral , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Regulação para Baixo , Feminino , Xenoenxertos , Humanos , Interleucina-8/metabolismo , Neoplasias Pulmonares/prevenção & controle , Proteínas de Membrana/genética , Camundongos , Fator A de Crescimento do Endotélio Vascular/metabolismo
13.
Cell Commun Signal ; 17(1): 28, 2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-30902093

RESUMO

BACKGROUND: Macroautophagy (hereafter autophagy) is a tightly regulated process that delivers cellular components to lysosomes for degradation. Damage-regulated autophagy modulator 1 (DRAM1) induces autophagy and is necessary for p53-mediated apoptosis. However, the signalling pathways regulated by DRAM1 are not fully understood. METHODS: HEK293T cells were transfected with FLAG-DRAM1 plasmid. Autophagic proteins (LC3 and p62), phosphorylated p53 and the phosphorylated proteins of the class I PI3K-Akt-mTOR-ribosomal protein S6 (rpS6) signalling pathway were detected with Western blot analysis. Cellular distribution of DRAM1 was determined with immunostaining. DRAM1 was knocked down in HEK293T cells using siRNA oligos which is confirmed by quantitative RT-PCR. Cells were serum starved for 18 h after overexpression or knockdown of DRAM1 to decrease the rpS6 activity to the basal level, and then the cells were stimulated with insulin growth factor, epidermal growth factor or serum. rpS6 phosphorylation and rpS6 were detected with Western blotting. Similarly, after overexpression or knockdown of DRAM1, phosphorylation of IGF-1Rß and IGF-1R were examined with Western blotting. Cell viability was determined with CCK-8 assay and colony formation assay. Finally, human cancer cells Hela, SW480, and HCT116 were transfected with the FLAG-DRAM1 plasmid and phosphorylated rpS6 and rpS6 were detected with Western blot analysis. RESULTS: DRAM1 induced autophagy and inhibited rpS6 phosphorylation in an mTORC1-dependent manner in HEK293T cells. DRAM1 didn't affect the phosphorylated and total levels of p53. Furthermore, DRAM1 inhibited the activation of the PI3K-Akt pathway stimulated with growth factors or serum. DRAM1 was localized at the plasma membrane and regulate the phosphorylation of IGF-1 receptor. DRAM1 decreased cell viability and colony numbers upon serum starvation. Additionally, DRAM1 inhibited rpS6 phosphorylation in several human cancer cells. CONCLUSIONS: Here we provided evidence that DRAM1 inhibited rpS6 phosphorylation in multiple cell types. DRAM1 inhibited the phosphorylation of Akt and the activation of Akt-rpS6 pathway stimulated with growth factors and serum. Furthermore, DRAM1 regulated the activation of IGF-1 receptor. Thus, our results identify that the class I PI3K-Akt-rpS6 pathway is regulated by DRAM1 and may provide new insight into the potential role of DRAM1 in human cancers.


Assuntos
Autofagia/fisiologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas de Membrana , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor IGF Tipo 1/metabolismo , Proteína S6 Ribossômica/metabolismo , Apoptose , Proliferação de Células , Sobrevivência Celular , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Fosforilação , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo
15.
Proc Natl Acad Sci U S A ; 116(12): 5775-5784, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30819882

RESUMO

Close appositions between the endoplasmic reticulum (ER) and the plasma membrane (PM) are a general feature of all cells and are abundant in neurons. A function of these appositions is lipid transport between the two adjacent bilayers via tethering proteins that also contain lipid transport modules. However, little is known about the properties and dynamics of these proteins in neurons. Here we focused on TMEM24/C2CD2L, an ER-localized SMP domain containing phospholipid transporter expressed at high levels in the brain, previously shown to be a component of ER-PM contacts in pancreatic ß-cells. TMEM24 is enriched in neurons versus glial cells and its levels increase in parallel with neuronal differentiation. It populates ER-PM contacts in resting neurons, but elevations of cytosolic Ca2+ mediated by experimental manipulations or spontaneous activity induce its transient redistribution throughout the entire ER. Dissociation of TMEM24 from the plasma membrane is mediated by phosphorylation of an array of sites in the C-terminal region of the protein. These sites are only partially conserved in C2CD2, the paralogue of TMEM24 primarily expressed in nonneuronal tissues, which correspondingly display a much lower sensitivity to Ca2+ elevations. ER-PM contacts in neurons are also sites where Kv2 (the major delayed rectifier K+ channels in brain) and other PM and ER ion channels are concentrated, raising the possibility of a regulatory feedback mechanism between neuronal excitability and lipid exchange between the ER and the PM.


Assuntos
Sinalização do Cálcio/fisiologia , Proteínas de Membrana/metabolismo , Neurônios/fisiologia , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Lipídeos , Mamíferos/metabolismo , Proteínas de Membrana/fisiologia , Camundongos , Neurônios/metabolismo , Fosfolipídeos/metabolismo , Fosforilação , Cultura Primária de Células , Sinaptotagminas/metabolismo
16.
PLoS Pathog ; 15(3): e1007684, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30883606

RESUMO

Phagocytosis is a complex process that eliminates microbes and is performed by specialised cells such as macrophages. Toll-like receptor 4 (TLR4) is expressed on the surface of macrophages and recognizes Gram-negative bacteria. Moreover, TLR4 has been suggested to play a role in the phagocytosis of Gram-negative bacteria, but the mechanisms remain unclear. Here we have used primary human macrophages and engineered THP-1 monocytes to show that the TLR4 sorting adapter, TRAM, is instrumental for phagocytosis of Escherichia coli as well as Staphylococcus aureus. We find that TRAM forms a complex with Rab11 family interacting protein 2 (FIP2) that is recruited to the phagocytic cups of E. coli. This promotes activation of the actin-regulatory GTPases Rac1 and Cdc42. Our results show that FIP2 guided TRAM recruitment orchestrates actin remodelling and IRF3 activation, two events that are both required for phagocytosis of Gram-negative bacteria.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Fagocitose/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Transporte/fisiologia , Endocitose , Endossomos , Escherichia coli/patogenicidade , Células HEK293 , Humanos , Fator Regulador 3 de Interferon , Lipopolissacarídeos , Macrófagos/imunologia , Macrófagos/metabolismo , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide , Cultura Primária de Células , Transporte Proteico , Transdução de Sinais , Staphylococcus aureus/patogenicidade , Células THP-1 , Receptor 4 Toll-Like/metabolismo , Proteína cdc42 de Ligação ao GTP , Proteínas rac1 de Ligação ao GTP
17.
Chemosphere ; 223: 1-11, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30763911

RESUMO

Fluoride is capable of inducing immunotoxicity, but its molecular mechanisms remain elusive. This study aimed to explore the roles of Protein kinase receptor-like ER kinase (PERK) and inositol requiring enzyme 1 (IRE1) signaling pathways in excessive fluoride-induced immunotoxicity, focusing on the regulatory roles of these two pathways in cell division and apoptosis. Firstly, we assessed the changes in cell division and apoptosis in rats exposed to 0, 50, or 100 mg/L fluoride, and detected the expression of PERK and IRE1 signaling-related proteins in spleen. Additionally, to validate the role of these two pathways, we evaluated the changes in cell division and apoptosis of primary lymphocytes from rat's spleen to 4 mM fluoride after knockdown of PERK and IRE1 in vitro. In vivo results confirmed that fluoride inhibited cell division, promoted the apoptosis and resulted in histological and ultrastructural abnormalities of rat spleen. In addition, fluoride induced activation of the PERK and IRE1 signalings and the associated apoptosis. Moreover, the in vitro results further verified the findings in vivo that fluoride activated these two signalings in B lymphocytes. Importantly, after knockdown of PERK and IRE1 in lymphocytes, the cell division ability was restored, and apoptosis decreased in fluoride-treated lymphocytes; the results correlated well with the expression of PERK and IRE1 signaling-related proteins, thus confirming the pivotal role of these pathways in immunosuppression by excessive fluoride. This study indicates that the mechanisms underlying the deleterious effects of fluoride on immune system are related to activation of the PERK and IRE1 signaling pathways.


Assuntos
Fluoretos/toxicidade , Linfócitos/metabolismo , Proteínas de Membrana/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Transdução de Sinais , Baço/patologia , eIF-2 Quinase/fisiologia , Animais , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linfócitos/patologia , Ratos , Baço/efeitos dos fármacos
18.
G3 (Bethesda) ; 9(4): 1085-1102, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30733383

RESUMO

Wsc1p and Mid2p are transmembrane signaling proteins of cell wall stress in the budding yeast Saccharomyces cerevisiae When an environmental stress compromises cell wall integrity, they activate a cell response through the Cell Wall Integrity (CWI) pathway. Studies have shown that the cytoplasmic domain of Wsc1p initiates the CWI signaling cascade by interacting with Rom2p, a Rho1-GDP-GTP exchange factor. Binding of Rom2p to the cytoplasmic tail of Wsc1p requires dephosphorylation of specific serine residues but the mechanism by which the sensor is dephosphorylated and how it subsequently interacts with Rom2p remains unclear. We hypothesize that Wsc1p and Mid2p must be physically associated with interacting proteins other than Rom2p that facilitate its interaction and regulate the activation of CWI pathway. To address this, a cDNA plasmid library of yeast proteins was expressed in bait strains bearing membrane yeast two-hybrid (MYTH) reporter modules of Wsc1p and Mid2p, and their interacting preys were recovered and sequenced. 14 previously unreported interactors were confirmed for Wsc1p and 29 for Mid2p The interactors' functionality were assessed by cell growth assays and CWI pathway activation by western blot analysis of Slt2p/Mpk1p phosphorylation in null mutants of each interactor under defined stress conditions. The susceptibility of these strains to different stresses were tested against antifungal agents and chemicals. This study reports important novel protein interactions of Wsc1p and Mid2p that are associated with the cellular response to oxidative stress induced by Hydrogen Peroxide and cell wall stress induced by Caspofungin.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Glicoproteínas de Membrana/fisiologia , Proteínas de Membrana/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico , Caspofungina/farmacologia , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Peróxido de Hidrogênio/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Espectrometria de Massas , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Estresse Oxidativo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Purificação por Afinidade em Tandem , Proteínas ras/genética , Proteínas ras/metabolismo , Proteínas ras/fisiologia
19.
Genet Test Mol Biomarkers ; 23(3): 204-208, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30758234

RESUMO

AIMS: Autosomal recessive nonsyndromic hearing loss (ARNSHL) is the most common form of hereditary deafness. Despite its frequency, the diagnosis of this disorder continues to be a challenging task given its extreme genetic heterogeneity. The purpose of this study was to identify the causative mutation in a consanguineous United Arab Emirates (UAE) family with ARNSHL. MATERIALS AND METHODS: Clinical exome sequencing (CES) followed by segregation analysis via Sanger sequencing was used to identify the causative mutation. In addition, 109 deaf individuals and 50 deafness-free controls from the UAE population were screened for the identified mutation. RESULTS AND DISCUSSION: CES identified the STRC frameshift mutation c.4510del (p.Glu1504Argfs*32) as the causative mutation in this family. Moreover, segregation analysis confirmed the above finding. In addition, the absence of this variant in 109 unrelated deaf individuals and 50 healthy controls indicates that it is rare in the UAE population. CONCLUSION: The present study represents the first STRC mutation reported in the UAE population. It also reinforces the power of next-generation sequencing in the diagnosis of heterogenous disorders such as nonsyndromic hearing loss.


Assuntos
Surdez/genética , Proteínas de Membrana/genética , Adulto , Exoma/genética , Feminino , Mutação da Fase de Leitura/genética , Genes Recessivos/genética , Perda Auditiva Neurossensorial/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Masculino , Proteínas de Membrana/fisiologia , Mutação , Linhagem , Emirados Árabes Unidos , Sequenciamento Completo do Exoma
20.
J Biochem ; 165(5): 391-400, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30726905

RESUMO

VAP (VAMP-associated protein) is a type II integral membrane protein of the endoplasmic reticulum (ER), and its N-terminal major sperm protein (MSP) domain faces the cytoplasmic side. VAP functions as a tethering molecule at the membrane contact sites between the ER and intracellular organelles and regulates a wide variety of cellular functions, including lipid transport, membrane trafficking, microtubule reorganization and unfolded protein response. VAP-point mutations in human vapb are strongly associated with amyotrophic lateral sclerosis. Importantly, the MSP domain of VAP is cleaved, secreted and interacts with the axon growth cone guidance receptors (Eph, Robo, Lar), suggesting that VAP could function as a circulating hormone similar to the Caenorhabditis elegans MSP protein. In this review, we discuss not only the intracellular functions of VAP but also the recently discovered extracellular functions and their implications for neurodegenerative disease.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Esclerose Amiotrófica Lateral/metabolismo , Animais , Transporte Biológico , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Citosol/metabolismo , Humanos , Metabolismo dos Lipídeos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Microtúbulos/metabolismo , Mutação Puntual , Homologia de Sequência de Aminoácidos , Resposta a Proteínas não Dobradas
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