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1.
Invest Ophthalmol Vis Sci ; 61(1): 1, 2020 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-31995153

RESUMO

Purpose: Vacuolar protein sorting 35 (Vps35) mutations and protein dysfunction have been linked to the hyperphosphorylation and accumulation of tau protein in a number of central neurodegenerative disorders. The aims of the present study were to investigate the mechanism underlying the tau hyperphosphorylation caused by Vps35 deficiency. Methods: The cells used in this study were primary retinal ganglion cells (RGCs). The rat retinal glutamate excitotoxicity model was used in vivo. Fresh retinal tissues or eyeballs were collected at different time points. The expression and interactions of Vps35, Cdk5/p35, tau hyperphosphorylation, LAMP1, EEA1 and UBE1 in RGCs were studied by immunofluorescence staining, Western blotting, and immunoprecipitation. Results: The downregulation and overexpression of Vps35 increased and decreased the expression of p35 and tau hyperphosphorylation, respectively. More important, roscovitine, a Cdk5 inhibitor, could effectively decrease the hyperphosphorylated tau level induced by Vps35 deficiency. Furthermore, this study confirmed that the inhibition of Vps35 could increase the activity of Cdk5/p35 by affecting the lysosomal degradation of p35 and lead to the degeneration of RGCs. Conclusions: These findings demonstrate the possibility that Cdk5/p35 acts as a "cargo" of Vps35 and provide new insights into the pathogenesis of RGC degeneration caused by hyperphosphorylated tau protein. Vps35 is a potential target for basic research and clinical treatment of RGC degeneration in many ocular diseases such as glaucoma.


Assuntos
Quinase 5 Dependente de Ciclina/metabolismo , Fosfotransferases/metabolismo , Células Ganglionares da Retina/metabolismo , Proteínas de Transporte Vesicular/deficiência , Proteínas tau/metabolismo , Animais , Western Blotting , Células Cultivadas , Quinase 5 Dependente de Ciclina/antagonistas & inibidores , Regulação para Baixo , Técnica Indireta de Fluorescência para Anticorpo , Ácido Glutâmico/toxicidade , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Masculino , Camundongos , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Degeneração Retiniana/induzido quimicamente , Degeneração Retiniana/metabolismo , Roscovitina/farmacologia , Transfecção , Enzimas Ativadoras de Ubiquitina/metabolismo , Proteínas de Transporte Vesicular/metabolismo
2.
Cancer Sci ; 111(3): 857-868, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31930596

RESUMO

Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cell-to-cell communication. The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1), which is closely associated with nasopharyngeal carcinoma (NPC) pathogenesis, can trigger multiple cell signaling pathways that affect cell progression. Several reports have shown that LMP1 promotes EV secretion, and LMP1 trafficking by EVs can enhances cancer progression and metastasis. However, the molecular mechanism by which LMP1 promotes EV secretion is not well understood. In the present study, we found that LMP1 promotes EV secretion by upregulated syndecan-2 (SDC2) and synaptotagmin-like-4 (SYTL4) through nuclear factor (NF)-κB signaling in NPC cells. Further study indicated that SDC2 interacted with syntenin, which promoted the formation of the EVs, and SYTL4 is associated with the release of EVs. Moreover, we found that stimulation of EV secretion by LMP1 can enhance the proliferation and invasion ability of recipient NPC cells and tumor growth in vivo. In summary, we found a new mechanism by which LMP1 upregulates SDC2 and SYTL4 through NF-κB signaling to promote EV secretion, and further enhance cancer progression of NPC.


Assuntos
Vesículas Extracelulares/metabolismo , Herpesvirus Humano 4/metabolismo , Carcinoma Nasofaríngeo/metabolismo , Neoplasias Nasofaríngeas/metabolismo , Sindecana-2/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas da Matriz Viral/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , NF-kappa B/metabolismo , Transdução de Sinais/fisiologia , Regulação para Cima/fisiologia
3.
Mol Cell Biochem ; 465(1-2): 115-123, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31893334

RESUMO

Increasing studies have indicated that hypoxia serves as a pivotal microenvironmental factor that facilitates activation of hepatic stellate cells (HSCs). However, the mechanism by which hypoxia activates HSCs is not clear. Here, we demonstrated that plasmacytoma variant translocation 1 (PVT1) and autophagy were overexpressed in liver fibrotic specimens. In primary mouse HSCs, both PVT1 and autophagy were induced by hypoxia. Further study showed that hypoxia-induced autophagy depended on expression of PVT1 and miR-152 in HSCs. Luciferase reporter assay indicated that autophagy-related gene 14 (ATG14) was a direct target of miR-152. In addition, inhibition of autophagy by 3-methyladenine and Beclin-1 siRNA impeded activation of HSCs cultured in 1% O2. Taken together, autophagy induction via the PVT1-miR-152-ATG14 signaling pathway contributes to activation of HSCs under hypoxia condition.


Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Células Estreladas do Fígado/metabolismo , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Transdução de Sinais , Proteínas de Transporte Vesicular/metabolismo , Animais , Hipóxia Celular , Células Estreladas do Fígado/patologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Camundongos
4.
Cell Mol Life Sci ; 77(3): 511-529, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31218450

RESUMO

The sperm acrosome is a lysosome-related organelle that develops using membrane trafficking from the Golgi apparatus as well as the endolysosomal compartment. How vesicular trafficking is regulated in spermatids to form the acrosome remains to be elucidated. VPS13B, a RAB6-interactor, was recently shown involved in endomembrane trafficking. Here, we report the generation of the first Vps13b-knockout mouse model and show that male mutant mice are infertile due to oligoasthenoteratozoospermia. This phenotype was explained by a failure of Vps13b deficient spermatids to form an acrosome. In wild-type spermatids, immunostaining of Vps13b and Rab6 revealed that they transiently locate to the acrosomal inner membrane. Spermatids lacking Vps13b did not present with the Golgi structure that characterizes wild-type spermatids and showed abnormal targeting of PNA- and Rab6-positive Golgi-derived vesicles to Eea1- and Lamp2-positive structures. Altogether, our results uncover a function of Vps13b in the regulation of the vesicular transport between Golgi apparatus, acrosome, and endolysosome.


Assuntos
Acrossomo/metabolismo , Transporte Biológico/fisiologia , Complexo de Golgi/metabolismo , Espermatogênese/fisiologia , Proteínas de Transporte Vesicular/metabolismo , Animais , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Transporte Proteico/fisiologia , Espermátides/metabolismo , Espermatozoides/metabolismo
5.
PLoS Genet ; 15(12): e1008557, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31869332

RESUMO

TRAnsport Protein Particle complexes (TRAPPs) are ubiquitous regulators of membrane traffic mediating nucleotide exchange on the Golgi regulatory GTPases RAB1 and RAB11. In S. cerevisiae and metazoans TRAPPs consist of two large oligomeric complexes: RAB11-activating TRAPPII and RAB1-activating TRAPPIII. These share a common core TRAPPI hetero-heptamer, absent in metazoans but detected in minor proportions in yeast, likely originating from in vitro-destabilized TRAPPII/III. Despite overall TRAPP conservation, the budding yeast genome has undergone extensive loss of genes, and lacks homologues of some metazoan TRAPP subunits. With nearly twice the total number of genes of S. cerevisiae, another ascomycete Aspergillus nidulans has also been used for studies on TRAPPs. We combined size-fractionation chromatography with single-step purification coupled to mass-spectrometry and negative-stain electron microscopy to establish the relative abundance, composition and architecture of Aspergillus TRAPPs, which consist of TRAPPII and TRAPPIII in a 2:1 proportion, plus a minor amount of TRAPPI. We show that Aspergillus TRAPPIII contains homologues of metazoan TRAPPC11, TRAPPC12 and TRAPPC13 subunits, absent in S. cerevisiae, and establish that these subunits are recruited to the complex by Tca17/TRAPPC2L, which itself binds to the 'Trs33 side' of the complex. Thus Aspergillus TRAPPs compositionally resemble mammalian TRAPPs to a greater extent than those in budding yeast. Exploiting the ability of constitutively-active (GEF-independent, due to accelerated GDP release) RAB1* and RAB11* alleles to rescue viability of null mutants lacking essential TRAPP subunits, we establish that the only essential role of TRAPPs is activating RAB1 and RAB11, and genetically classify each essential subunit according to their role(s) in TRAPPII (TRAPPII-specific subunits) or TRAPPII and TRAPPIII (core TRAPP subunits). Constitutively-active RAB mutant combinations allowed examination of TRAPP composition in mutants lacking essential subunits, which led to the discovery of a stable Trs120/Trs130/Trs65/Tca17 TRAPPII-specific subcomplex whose Trs20- and Trs33-dependent assembly onto core TRAPP generates TRAPPII.


Assuntos
Aspergillus nidulans/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Cromatografia em Gel , Proteínas Fúngicas/metabolismo , Humanos , Mamíferos/metabolismo , Espectrometria de Massas , Saccharomyces cerevisiae/metabolismo
6.
Life Sci ; 237: 116902, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31610195

RESUMO

AIMS: Insulin-like growth factor binding protein-related protein 1 (IGFBPrP1) promotes hepatic stellate cell (HSC) autophagy and activation. However, the underlying mechanism remains unknown. Noncoding RNAs (ncRNAs) including long noncoding RNAs (lncRNAs) and microRNAs (miRNAs), have received increasing attention. We aimed to investigate the roles of the lncRNA nuclear enriched abundant transcript 1 (NEAT1), miR-29b, and autophagy related protein 9a (Atg9a), and their relationships with each other during IGFBPrP1-induced HSC autophagy and activation. MAIN METHODS: Levels of NEAT1, miR-29b, Atg9a, and autophagy were detected in adenovirus-mediated IGFBPrP1 (AdIGFBPrP1)-treated mouse liver tissue and immortalized mouse hepatic stellate cell line JS1 transfected with either AdIGFBPrP1 or siIGFBPrP1. In AdIGFBPrP1-treated JS1 cells, autophagy and activation were detected after altering NEAT1, miR-29b, or Atg9a levels. In AdIGFBPrP1-treated JS1 cells, relationships among NEAT1, miR-29b, and Atg9a were explored using dual-luciferase reporter assays, Western blot, qRT-PCR, and immunofluorescence. KEY FINDINGS: IGFBPrP1 increased levels of NEAT1, Atg9a, and autophagy while decreasing the level of miR-29b in mouse liver tissues and mouse HSCs. Moreover, NEAT1 increased HSC autophagy and activation while miR-29b decreased both processes. Atg9a also participated in IGFBPrP1-induced HSC autophagy and activation. Importantly, NEAT1, miR-29b, and Atg9a formed a NEAT1/miR-29b/Atg9a regulatory axis for IGFBPrP1-induced HSC autophagy and activation. SIGNIFICANCE: Our study unveiled the new NEAT1/miR-29b/Atg9a regulatory axis involved in IGFBPrP1-induced mouse HSC autophagy and activation. The study thus provides new insights in the pathogenesis and potential therapeutic strategies of liver fibrosis.


Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Células Estreladas do Fígado/patologia , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Cirrose Hepática/patologia , Proteínas de Membrana/metabolismo , MicroRNAs/genética , RNA Longo não Codificante/genética , Proteínas de Transporte Vesicular/metabolismo , Adenoviridae/genética , Animais , Proteínas Relacionadas à Autofagia/genética , Células Cultivadas , Células Estreladas do Fígado/metabolismo , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Cirrose Hepática/etiologia , Cirrose Hepática/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Transporte Vesicular/genética
7.
Nat Cell Biol ; 21(10): 1234-1247, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31570833

RESUMO

Phosphoinositides have a pivotal role in the maturation of nascent phagosomes into microbicidal phagolysosomes. Following degradation of their contents, mature phagolysosomes undergo resolution, a process that remains largely uninvestigated. Here we studied the role of phosphoinositides in phagolysosome resolution. Phosphatidylinositol-4-phosphate (PtdIns(4)P), which is abundant in maturing phagolysosomes, was depleted as they tubulated and resorbed. Depletion was caused, in part, by transfer of phagolysosomal PtdIns(4)P to the endoplasmic reticulum, a process mediated by oxysterol-binding protein-related protein 1L (ORP1L), a RAB7 effector. ORP1L formed discrete tethers between the phagolysosome and the endoplasmic reticulum, resulting in distinct regions with alternating PtdIns(4)P depletion and enrichment. Tubules emerged from PtdIns(4)P-rich regions, where ADP-ribosylation factor-like protein 8B (ARL8B) and SifA- and kinesin-interacting protein/pleckstrin homology domain-containing family M member 2 (SKIP/PLEKHM2) accumulated. SKIP binds preferentially to monophosphorylated phosphoinositides, of which PtdIns(4)P is most abundant in phagolysosomes, contributing to their tubulation. Accordingly, premature hydrolysis of PtdIns(4)P impaired SKIP recruitment and phagosome resolution. Thus, resolution involves phosphoinositides and tethering of phagolysosomes to the endoplasmic reticulum.


Assuntos
Retículo Endoplasmático/metabolismo , Monócitos/metabolismo , Fagossomos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Receptores de Esteroides/genética , Transdução de Sinais , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Animais , Sistemas CRISPR-Cas , Retículo Endoplasmático/ultraestrutura , Edição de Genes , Regulação da Expressão Gênica , Humanos , Camundongos , Monócitos/ultraestrutura , Fagocitose , Fagossomos/ultraestrutura , Cultura Primária de Células , Proteólise , Células RAW 264.7 , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Esteroides/antagonistas & inibidores , Receptores de Esteroides/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
8.
Mol Cell ; 76(5): 811-825.e14, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31628041

RESUMO

Physical contact between organelles is vital to the function of eukaryotic cells. Lipid droplets (LDs) are dynamic organelles specialized in lipid storage that interact physically with mitochondria in several cell types. The mechanisms coupling these organelles are, however, poorly understood, and the cell-biological function of their interaction remains largely unknown. Here, we discover in adipocytes that the outer mitochondrial membrane protein MIGA2 links mitochondria to LDs. We identify an amphipathic LD-targeting motif and reveal that MIGA2 binds to the membrane proteins VAP-A or VAP-B in the endoplasmic reticulum (ER). We find that in adipocytes MIGA2 is involved in promoting triglyceride (TAG) synthesis from non-lipid precursors. Our data indicate that MIGA2 links reactions of de novo lipogenesis in mitochondria to TAG production in the ER, thereby facilitating efficient lipid storage in LDs. Based on its presence in many tissues, MIGA2 is likely critical for lipid and energy homeostasis in a wide spectrum of cell types.


Assuntos
Adipócitos/metabolismo , Lipogênese/fisiologia , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Células 3T3 , Adipócitos/fisiologia , Animais , Células COS , Diferenciação Celular/fisiologia , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Gotículas Lipídicas/metabolismo , Lipogênese/genética , Proteínas de Membrana/fisiologia , Camundongos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/fisiologia , Triglicerídeos/biossíntese , Proteínas de Transporte Vesicular/metabolismo
9.
Nat Cell Biol ; 21(10): 1206-1218, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31548609

RESUMO

Cholesterol activates the master growth regulator, mTORC1 kinase, by promoting its recruitment to the surface of lysosomes by the Rag guanosine triphosphatases (GTPases). The mechanisms that regulate lysosomal cholesterol content to enable mTORC1 signalling are unknown. Here, we show that oxysterol binding protein (OSBP) and its anchors at the endoplasmic reticulum (ER), VAPA and VAPB, deliver cholesterol across ER-lysosome contacts to activate mTORC1. In cells lacking OSBP, but not other VAP-interacting cholesterol carriers, the recruitment of mTORC1 by the Rag GTPases is inhibited owing to impaired transport of cholesterol to lysosomes. By contrast, OSBP-mediated cholesterol trafficking drives constitutive mTORC1 activation in a disease model caused by the loss of the lysosomal cholesterol transporter, Niemann-Pick C1 (NPC1). Chemical and genetic inactivation of OSBP suppresses aberrant mTORC1 signalling and restores autophagic function in cellular models of Niemann-Pick type C (NPC). Thus, ER-lysosome contacts are signalling hubs that enable cholesterol sensing by mTORC1, and targeting the sterol-transfer activity of these signalling hubs could be beneficial in patients with NPC.


Assuntos
Colesterol/metabolismo , Retículo Endoplasmático/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Doenças de Niemann-Pick/metabolismo , Receptores de Esteroides/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células HEK293 , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Receptores de Esteroides/genética , Transdução de Sinais , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
10.
Nat Commun ; 10(1): 4271, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31537807

RESUMO

Protein recycling through the endolysosomal system relies on molecular assemblies that interact with cargo proteins, membranes, and effector molecules. Among them, the COMMD/CCDC22/CCDC93 (CCC) complex plays a critical role in recycling events. While CCC is closely associated with retriever, a cargo recognition complex, its mechanism of action remains unexplained. Herein we show that CCC and retriever are closely linked through sharing a common subunit (VPS35L), yet the integrity of CCC, but not retriever, is required to maintain normal endosomal levels of phosphatidylinositol-3-phosphate (PI(3)P). CCC complex depletion leads to elevated PI(3)P levels, enhanced recruitment and activation of WASH (an actin nucleation promoting factor), excess endosomal F-actin and trapping of internalized receptors. Mechanistically, we find that CCC regulates the phosphorylation and endosomal recruitment of the PI(3)P phosphatase MTMR2. Taken together, we show that the regulation of PI(3)P levels by the CCC complex is critical to protein recycling in the endosomal compartment.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Endossomos/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Actinas/metabolismo , Animais , Linhagem Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Fosforilação , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética
11.
Genes (Basel) ; 10(10)2019 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-31557940

RESUMO

The autophagy-lysosome pathway, which involves many crucial genes and proteins, plays crucial roles in the maintenance of intracellular homeostasis by the degradation of damaged components. At present, some of these genes and proteins have been identified but their specific functions are largely unknown. This study was performed to clone and characterize the full-length cDNA sequences of nine key autolysosome-related genes (vps11, vps16, vps18, vps33b, vps41, lamp1, mcoln1, ctsd1 and tfeb) from yellow catfish Pelteobagrus fulvidraco. The expression of these genes and the transcriptional responses to a high-fat diet and fatty acids (FAs) (palmitic acid (PA) and oleic acid (OA)) were investigated. The mRNAs of these genes could be detected in heart, liver, muscle, spleen, brain, mesenteric adipose tissue, intestine, kidney and ovary, but varied with the tissues. In the liver, the mRNA levels of the nine autolysosome-related genes were lower in fish fed a high-fat diet than those fed the control, indicating that a high-fat diet inhibited formation of autolysosomes. Palmitic acid (a saturated FA) significantly inhibited the formation of autolysosomes at 12 h, 24 h and 48 h incubation. In contrast, oleic acid (an unsaturated FA) significantly induced the formation of autolysosomes at 12 h, but inhibited them at 24 h. At 48 h, the effects of OA incubation on autolysosomes were OA concentration-dependent in primary hepatocytes of P. fulvidraco. The results of flow cytometry and laser confocal observations confirmed these results. PA and OA incubation also increased intracellular non-esterified fatty acid (NEFA) concentration at 12 h, 24 h and 48 h, and influenced mRNA levels of fatty acid binding protein (fabp) and fatty acid transport protein 4 (fatp4) which facilitate FA transport in primary hepatocytes of P. fulvidraco. The present study demonstrated the molecular characterization of the nine autolysosome-related genes and their transcriptional responses to fat and FAs in fish, which provides the basis for further exploring their regulatory mechanism in vertebrates.


Assuntos
Autofagossomos/metabolismo , Peixes-Gato/metabolismo , Gorduras na Dieta/farmacologia , Ácidos Graxos/farmacologia , Fígado/metabolismo , Lisossomos/metabolismo , Animais , Gorduras na Dieta/metabolismo , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Ácidos Graxos/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Fígado/efeitos dos fármacos , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
12.
Cancer Sci ; 110(11): 3424-3433, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31495050

RESUMO

Tongue squamous cell carcinoma (TSCC) has a poor prognosis due to its early metastasis through blood and lymphatic vessels. We undertook a systematic review to investigate the prognostic significance of blood microvessel density (MVD) and lymphatic vessel density (LVD) in TSCC patients. We carried out a systematic search in Ovid Medline, Scopus, and Cochrane libraries. All studies that evaluated the prognostic significance of MVD/LVD markers in TSCC were systematically retrieved. Our results showed that MVD/LVD markers, CD31, CD34, CD105, factor VIII, lymphatic vessel endothelial hyaluronan receptor-1, and D2-40 were evaluated in TSCC patients until 28 June 2018. Six out of 13 studies reported markers that were associated with poor prognosis in TSCC. Two out of three studies suggested that a high number of D2-40+ vessels predicated low overall survival (OS); the third study reported that the ratio of D2-40+ over factor VIII+ vessels is associated with low OS. Most of the other markers had controversial results for prognostication. We found higher expression of MVD/LVD markers were commonly, but not always, associated with shorter survival in TSCC patients. It is therefore not currently possible to recommend implementation of these markers as reliable prognosticators in clinical practice. More studies (especially for D2-40) with larger patient cohorts are needed.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/metabolismo , Vasos Linfáticos/metabolismo , Microvasos/metabolismo , Neoplasias da Língua/metabolismo , Anticorpos Monoclonais Murinos/metabolismo , Antígenos CD34/metabolismo , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/patologia , Endoglina/metabolismo , Fator VIII/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Linfangiogênese , Neovascularização Patológica , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Prognóstico , Neoplasias da Língua/mortalidade , Neoplasias da Língua/patologia , Proteínas de Transporte Vesicular/metabolismo
13.
Nat Commun ; 10(1): 4307, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31541088

RESUMO

To facilitate proper mitotic cell partitioning, the Golgi disassembles by suppressing vesicle fusion. However, the underlying mechanism has not been characterized previously. Here, we report a Ran pathway-independent attenuation mechanism that allows Importin-α (a nuclear transport factor) to suppress the vesicle fusion mediated by p115 (a vesicular tethering factor) and is required for mitotic Golgi disassembly. We demonstrate that Importin-α directly competes with p115 for interaction with the Golgi protein GM130. This interaction, promoted by a phosphate moiety on GM130, is independent of Importin-ß and Ran. A GM130 K34A mutant, in which the Importin-α-GM130 interaction is specifically disrupted, exhibited abundant Golgi puncta during metaphase. Importantly, a mutant showing enhanced p115-GM130 interaction presented proliferative defects and G2/M arrest, demonstrating that Importin-α-GM130 binding modulates the Golgi disassembly that governs mitotic progression. Our findings illuminate that the Ran and kinase-phosphatase pathways regulate multiple aspects of mitosis coordinated by Importin-α (e.g. spindle assembly, Golgi disassembly).


Assuntos
Autoantígenos/metabolismo , Complexo de Golgi/metabolismo , Proteínas da Matriz do Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Metáfase/fisiologia , Proteínas de Transporte Vesicular/metabolismo , alfa Carioferinas/metabolismo , Autoantígenos/genética , Cristalografia por Raios X , Pontos de Checagem da Fase G2 do Ciclo Celular , Células HEK293 , Humanos , Fusão de Membrana , Proteínas de Membrana/genética , Mitose/fisiologia , Fosforilação , Ligação Proteica , beta Carioferinas/metabolismo , Proteína ran de Ligação ao GTP/metabolismo
14.
Nat Commun ; 10(1): 4320, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31541095

RESUMO

OTULIN (OTU Deubiquitinase With Linear Linkage Specificity) specifically hydrolyzes methionine1 (Met1)-linked ubiquitin chains conjugated by LUBAC (linear ubiquitin chain assembly complex). Here we report on the mass spectrometric identification of the OTULIN interactor SNX27 (sorting nexin 27), an adaptor of the endosomal retromer complex responsible for protein recycling to the cell surface. The C-terminal PDZ-binding motif (PDZbm) in OTULIN associates with the cargo-binding site in the PDZ domain of SNX27. By solving the structure of the OTU domain in complex with the PDZ domain, we demonstrate that a second interface contributes to the selective, high affinity interaction of OTULIN and SNX27. SNX27 does not affect OTULIN catalytic activity, OTULIN-LUBAC binding or Met1-linked ubiquitin chain homeostasis. However, via association, OTULIN antagonizes SNX27-dependent cargo loading, binding of SNX27 to the VPS26A-retromer subunit and endosome-to-plasma membrane trafficking. Thus, we define an additional, non-catalytic function of OTULIN in the regulation of SNX27-retromer assembly and recycling to the cell surface.


Assuntos
Endopeptidases/metabolismo , Endossomos/metabolismo , Nexinas de Classificação/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Endopeptidases/química , Técnicas de Inativação de Genes , Transportador de Glucose Tipo 1/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Domínios PDZ , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Nexinas de Classificação/química , Nexinas de Classificação/genética , Ubiquitinação , Proteínas de Transporte Vesicular/metabolismo
15.
Histochem Cell Biol ; 152(5): 333-343, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31410570

RESUMO

The membrane skeletal complex, protein 4.1G-membrane palmitoylated protein 6 (MPP6), is localized in spermatogonia and early spermatocytes of mouse seminiferous tubules. In this study, we investigated the Lin7 family of scaffolding proteins, which interact with MPP6. By immunohistochemistry, Lin7a and Lin7c were localized in germ cells, and Lin7c had especially strong staining in spermatogonia and early spermatocytes, characterized by staging of seminiferous tubules. By immunoelectron microscopy, Lin7 localization appeared under cell membranes in germ cells. The Lin7 staining pattern in seminiferous tubules was partially similar to that of 4.1G, cell adhesion molecule 1 (CADM1), and melanoma cell adhesion molecule (MCAM). Lin7-positive cells included type A spermatogonia, as revealed by double staining for Lin28a. Lin7 staining became weaker in MPP6-deficient mice by immunohistochemistry and western blotting, indicating that MPP6 transports and maintains Lin7 in germ cells. The histology of seminiferous tubules was unchanged in MPP6-deficient mice compared to that of wild-type mice. In cultured spermatogonial stem cells maintained with glial cell line-derived neurotropic factor (GDNF), Lin7 was clearly expressed and immunolocalized along cell membranes, especially at cell-cell junctions. Thus, Lin7 protein is expressed in germ cells, and Lin7, particularly Lin7c, is a useful marker for early spermatogenesis.


Assuntos
Guanilato Quinases/análise , Proteínas Ligadas a Lipídeos/análise , Túbulos Seminíferos/química , Proteínas de Transporte Vesicular/análise , Animais , Células Cultivadas , Guanilato Quinases/deficiência , Guanilato Quinases/metabolismo , Proteínas Ligadas a Lipídeos/deficiência , Proteínas Ligadas a Lipídeos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Túbulos Seminíferos/metabolismo , Proteínas de Transporte Vesicular/metabolismo
16.
Proc Jpn Acad Ser B Phys Biol Sci ; 95(7): 312-320, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31406056

RESUMO

Intracellular organelles were long viewed as isolated compartments floating in the cytosol. However, this view has been radically changed within the last decade by the discovery that most organelles communicate with the endoplasmic reticulum (ER) network via membrane contact sites (MCSs) that are essential for intracellular homeostasis. Protrudin is an ER resident protein that was originally shown to regulate neurite formation by promoting endosome trafficking. More recently, however, protrudin has been found to serve as a tethering factor at MCSs. The roles performed by protrudin at MCSs are mediated by its various domains, including inactivation of the small GTPase Rab11, bending of the ER membrane, and functional interactions with other molecules such as the motor protein KIF5 and the ER protein VAP. Mutations in the protrudin gene (ZFYVE27) are associated with hereditary spastic paraplegia, an axonopathy that results from defective ER structure. This review, examines the pleiotropic molecular functions of protrudin and its role in interorganellar communication.


Assuntos
Membranas Intracelulares/metabolismo , Organelas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Sequência de Bases , Humanos , Mutação , Domínios Proteicos , Transporte Proteico , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética
17.
Cells ; 8(7)2019 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-31262095

RESUMO

The yeast vacuole is a vital organelle, which is required for the degradation of aberrant intracellular or extracellular substrates and the recycling of the resulting nutrients as newly available building blocks for the cellular metabolism. Like the plant vacuole or the mammalian lysosome, the yeast vacuole is the destination of biosynthetic trafficking pathways that transport the vacuolar enzymes required for its functions. Moreover, substrates destined for degradation, like extracellular endocytosed cargoes that are transported by endosomes/multivesicular bodies as well as intracellular substrates that are transported via different forms of autophagosomes, have the vacuole as destination. We found that non-selective bulk autophagy of cytosolic proteins as well as the selective autophagic degradation of peroxisomes (pexophagy) and ribosomes (ribophagy) was dependent on the armadillo repeat protein Vac8 in Saccharomyces cerevisiae. Moreover, we showed that pexophagy and ribophagy depended on the palmitoylation of Vac8. In contrast, we described that Vac8 was not involved in the acidification of the vacuole nor in the targeting and maturation of certain biosynthetic cargoes, like the aspartyl-protease Pep4 (PrA) and the carboxy-peptidase Y (CPY), indicating a role of Vac8 in the uptake of selected cargoes. In addition, we found that the hallmark phenotype of the vac8 strain, namely the characteristic appearance of fragmented and clustered vacuoles, depended on the growth conditions. This fusion defect observed in standard glucose medium can be complemented by the replacement with oleic acid or glycerol medium. This complementation of vacuolar morphology also partially restores the degradation of peroxisomes. In summary, we found that Vac8 controlled vacuolar morphology and activity in a context- and cargo-dependent manner.


Assuntos
Autofagia , Membranas Intracelulares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Lipoilação , Peroxissomos/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Deleção de Sequência , Proteínas de Transporte Vesicular/genética
18.
Int J Mol Sci ; 20(13)2019 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-31323913

RESUMO

We describe a 7-year-old male with high functioning autism spectrum disorder (ASD) and maternally-inherited rare missense variant of Synaptotagmin-like protein 4 (SYTL4) gene (Xq22.1; c.835C>T; p.Arg279Cys) and an unknown missense variant of Transmembrane protein 187 (TMEM187) gene (Xq28; c.708G>T; p. Gln236His). Multiple in-silico predictions described in our study indicate a potentially damaging status for both X-linked genes. Analysis of predicted atomic threading models of the mutant and the native SYTL4 proteins suggest a potential structural change induced by the R279C variant which eliminates the stabilizing Arg279-Asp60 salt bridge in the N-terminal half of the SYTL4, affecting the functionality of the protein's critical RAB-Binding Domain. In the European (Non-Finnish) population, the allele frequency for this variant is 0.00042. The SYTL4 gene is known to directly interact with several members of the RAB family of genes, such as, RAB27A, RAB27B, RAB8A, and RAB3A which are known autism spectrum disorder genes. The SYTL4 gene also directly interacts with three known autism genes: STX1A, SNAP25 and STXBP1. Through a literature-based analytical approach, we identified three of five (60%) autism-associated serum microRNAs (miRs) with high predictive power among the total of 298 mouse Sytl4 associated/predicted microRNA interactions. Five of 13 (38%) miRs were differentially expressed in serum from ASD individuals which were predicted to interact with the mouse equivalent Sytl4 gene. TMEM187 gene, like SYTL4, is a protein-coding gene that belongs to a group of genes which host microRNA genes in their introns or exons. The novel Q236H amino acid variant in the TMEM187 in our patient is near the terminal end region of the protein which is represented by multiple sequence alignments and hidden Markov models, preventing comparative structural analysis of the variant harboring region. Like SYTL4, the TMEM187 gene is expressed in the brain and interacts with four known ASD genes, namely, HCFC1; TMLHE; MECP2; and GPHN. TMM187 is in linkage with MECP2, which is a well-known determinant of brain structure and size and is a well-known autism gene. Other members of the TMEM gene family, TMEM132E and TMEM132D genes are associated with bipolar and panic disorders, respectively, while TMEM231 is a known syndromic autism gene. Together, TMEM187 and SYTL4 genes directly interact with recognized important ASD genes, and their mRNAs are found in extracellular vesicles in the nervous system and stimulate target cells to translate into active protein. Our evidence shows that both these genes should be considered as candidate genes for autism. Additional biological testing is warranted to further determine the pathogenicity of these gene variants in the causation of autism.


Assuntos
Proteínas de Membrana/metabolismo , MicroRNAs/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Encéfalo/metabolismo , Predisposição Genética para Doença/genética , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos , MicroRNAs/genética , Mutação de Sentido Incorreto/genética , Ligação Proteica , Proteínas de Transporte Vesicular/genética
19.
Nat Commun ; 10(1): 3105, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31308371

RESUMO

Fas plays a major role in regulating ligand-induced apoptosis in many cell types. It is well known that several cancers demonstrate reduced cell surface levels of Fas and thus escape a potential control system via ligand-induced apoptosis, although underlying mechanisms are unclear. Here we report that the endosome associated trafficking regulator 1 (ENTR1), controls cell surface levels of Fas and Fas-mediated apoptotic signalling. ENTR1 regulates, via binding to the coiled coil domain protein Dysbindin, the delivery of Fas from endosomes to lysosomes thereby controlling termination of Fas signal transduction. We demonstrate that ENTR1 is cleaved during Fas-induced apoptosis in a caspase-dependent manner revealing an unexpected interplay of apoptotic signalling and regulation of endolysosomal trafficking resulting in a positive feedback signalling-loop. Our data provide insights into the molecular mechanism of Fas post-endocytic trafficking and signalling, opening possible explanations on how cancer cells regulate cell surface levels of death receptors.


Assuntos
Antígenos de Neoplasias/fisiologia , Endocitose/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas de Transporte Vesicular/fisiologia , Antígenos de Neoplasias/análise , Antígenos de Neoplasias/metabolismo , Apoptose , Disbindina/metabolismo , Proteína Ligante Fas/análise , Proteína Ligante Fas/metabolismo , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/análise , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 13/análise , Proteína Tirosina Fosfatase não Receptora Tipo 13/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 13/fisiologia , Transdução de Sinais , Proteínas de Transporte Vesicular/análise , Proteínas de Transporte Vesicular/metabolismo , Receptor fas/análise , Receptor fas/metabolismo
20.
Cell Mol Neurobiol ; 39(8): 1207-1215, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31342232

RESUMO

The KDEL (Lys-Asp-Glu-Leu) receptors (KDELRs), proteins with seven transmembrane domains, are primarily responsible for endoplasmic reticulum (ER) homeostasis. Recent studies have found additional function of KDELRs in growth, cellular secretory traffic, immune response, and autophagy; however, its role in tumorigenesis is still poorly understood. Here, we showed that KDELR2 is highly expressed in glioblastoma (GBM) tissues. Reviewing the expression of KDELR2 in TCGA and REMBRANDT database, we found that higher expression of KDELR2 is associated with shorter survival of GBM patients. We explored the effect of KDELR2 on tumorigenesis in GBM cells and animal model (nude mice), and identified KDELR2 as oncogene promoting cell proliferation. Additionally, KDELR2 expression in GBM cells correlated positively with HIF1alpha (HIF1α) expression, and we demonstrated by ChIP-qPCR and luciferase reporter assay that the upstream region of the KDELR2 gene is directly targeted by HIF1alpha. Taken together, our data suggest that KDELR2 is a target gene downstream of HIF1-alpha driving the malignancy of GBM and could eventually serve as a therapeutic target for the treatment of GBM patients.


Assuntos
Neoplasias Encefálicas/patologia , Carcinogênese/patologia , Glioblastoma/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Carcinogênese/genética , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos Nus , Análise de Sobrevida , Transcrição Genética , Regulação para Cima/genética , Proteínas de Transporte Vesicular/genética
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