RESUMO
The biocide chlorpyrifos (CPF) was shown to produce cognition impairment following single and long-term exposure. The complete mechanisms that lead to the CPF induced cognitive disorders remain to be discovered. Aß and tau proteins production was induced in basal forebrain SN56 cholinergic cells, by CPF, through proteasome 20S inhibition and Rab5 overexpression, leading to cell death both after acute and repeated administration, which was related with cognitive disorders induction. The results obtained in our study procure novel information related to the mechanisms involved in CPF neurodegeneration, which could be responsible for cognitive dysfunction and may lead to a promising alternative treatment of these effects.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Morte Celular/efeitos dos fármacos , Clorpirifos/farmacologia , Inseticidas/farmacologia , Neurônios/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas rab5 de Ligação ao GTP/biossíntese , Proteínas tau/metabolismo , Animais , Linhagem Celular , Camundongos , Neurônios/patologiaRESUMO
The primate lentiviral accessory protein Nef downregulates CD4 and major histocompatibility complex class I (MHC-I) from the cell surface via independent endosomal trafficking pathways to promote viral pathogenesis. In addition, Nef antagonizes a novel restriction factor, SERINC5 (Ser5), to increase viral infectivity. To explore the molecular mechanism of Ser5 antagonism by Nef, we determined how Nef affects Ser5 expression and intracellular trafficking in comparison to CD4 and MHC-I. We confirm that Nef excludes Ser5 from human immunodeficiency virus type 1 (HIV-1) virions by downregulating its cell surface expression via similar functional motifs required for CD4 downregulation. We find that Nef decreases both Ser5 and CD4 expression at steady-state levels, which are rescued by NH4Cl or bafilomycin A1 treatment. Nef binding to Ser5 was detected in living cells using a bimolecular fluorescence complementation assay, where Nef membrane association is required for interaction. In addition, Nef triggers rapid Ser5 internalization via receptor-mediated endocytosis and relocalizes Ser5 to Rab5+ early, Rab7+ late, and Rab11+ recycling endosomes. Manipulation of AP-2, Rab5, Rab7, and Rab11 expression levels affects the Nef-dependent Ser5 and CD4 downregulation. Moreover, although Nef does not promote Ser5 polyubiquitination, Ser5 downregulation relies on the ubiquitination pathway, and both K48- and K63-specific ubiquitin linkages are required for the downregulation. Finally, Nef promotes Ser5 colocalization with LAMP1, which is enhanced by bafilomycin A1 treatment, suggesting that Ser5 is targeted to lysosomes for destruction. We conclude that Nef uses a similar mechanism to downregulate Ser5 and CD4, which sorts Ser5 into a point-of-no-return degradative pathway to counteract its restriction.IMPORTANCE Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) express an accessory protein called Nef to promote viral pathogenesis. Nef drives immune escape in vivo through downregulation of CD4 and MHC-I from the host cell surface. Recently, Nef was reported to counteract a novel host restriction factor, Ser5, to increase viral infectivity. Nef downregulates cell surface Ser5, thus preventing its incorporation into virus particles, resulting in disruption of its antiviral activity. Here, we report mechanistic studies of Nef-mediated Ser5 downregulation in comparison to CD4 and MHC-I. We demonstrate that Nef binds directly to Ser5 in living cells and that Nef-Ser5 interaction requires Nef association with the plasma membrane. Subsequently, Nef internalizes Ser5 from the plasma membrane via receptor-mediated endocytosis, and targets ubiquitinated Ser5 to endosomes and lysosomes for destruction. Collectively, these results provide new insights into our ongoing understanding of the Nef-Ser5 arms race in HIV-1 infection.
Assuntos
Antígenos CD4/biossíntese , Endocitose/imunologia , HIV-1/patogenicidade , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Complexo 2 de Proteínas Adaptadoras/biossíntese , Linhagem Celular Tumoral , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Células HEK293 , Antígenos HLA-A/biossíntese , Células HeLa , Humanos , Células Jurkat , Proteínas de Membrana Lisossomal/metabolismo , Macrolídeos/farmacologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Transporte Proteico/fisiologia , Ubiquitinação/fisiologia , Proteínas rab de Ligação ao GTP/biossíntese , Proteínas rab5 de Ligação ao GTP/biossíntese , proteínas de unión al GTP Rab7RESUMO
OBJECTIVE: This study is to investigate the role of EMC-6 in the pathogenesis of cervical cancer, especially concerning its relationship with autophagy. PATIENTS AND METHODS: Totally 100 invasive cervical cancer, 80 cervical intraepithelial neoplasia (CIN), and 80 normal cervical tissue samples were obtained. Expression levels of EMC-6, Beclin1, and Rab5a in the tissues were detected by immunohistochemistry. RESULTS: Our results showed that positive staining of EMC-6 was mainly located in the nucleus. Compared with the normal cervical tissue, the positive rates of EMC-6 were significantly increased in the CIN and cervical cancer tissues. Moreover, the EMC-6 positive rate in the CIN tissue was higher than the cervical cancer tissue. No significant association was observed between the expression levels of EMC-6 and the clinicopathological features of cervical cancer, including age, FIGO staging, tumor size, tumor type, histological type, cell differentiation, and lymph node metastasis. Compared with the normal cervical tissue, the positive rate of Beclin1 in the CIN tissue was significantly declined, which was further significantly down-regulated in the cervical cancer tissue. However, the positive rate of Rab5a in the CIN tissue was significantly higher than the normal cervical tissue. Moreover, compared with the normal cervical and CIN tissues, the positive rate of Rab5a in the cervical cancer tissue was further significantly increased. EMC-6 was not associated with Beclin1 and Rab5a. CONCLUSIONS: The expression level of EMC-6 is significantly elevated in cervical cancer, without significant correlation with Beclin1 and Rab5a. These findings might contribute to the understanding of the pathogenesis of cervical cancer and the involved role of EMC-6.
Assuntos
Proteína Beclina-1/biossíntese , Proteína Beclina-1/fisiologia , Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana/biossíntese , Proteínas de Membrana/fisiologia , Neoplasias do Colo do Útero/metabolismo , Proteínas rab5 de Ligação ao GTP/biossíntese , Proteínas rab5 de Ligação ao GTP/fisiologia , Adulto , Idoso , Carcinoma de Células Escamosas/patologia , Estudos de Casos e Controles , Feminino , Humanos , Imuno-Histoquímica , Metástase Linfática , Pessoa de Meia-Idade , Displasia do Colo do Útero/patologiaRESUMO
Rab family protein Rab5a has been implicated in cancer progression. To date, its expression pattern in human pancreatic cancer has not been investigated. This study aims to examine clinical significance, biological role, and potential mechanism of action of mRab5a in human pancreatic cancer. We analyzed Rab5a protein in cancer tissue of 111 cases of pancreatic cancer using immunohistochemistry. The results show that Rab5a overexpression correlates with high T stage, positive nodal status, and advanced TNM stage. We performed knockdown of Rab5a through transfection of Rab5a-specific siRNA in the Capan-2 cell line, which shows high endogenous expression, and of Rab5a plasmid in the CFPAC-1 cell line, which shows low endogenous expression. Rab5a knockdown inhibited cell proliferation and invasion while its overexpression promoted cell proliferation and invasion. In addition, overexpression of Rab5a induced resistance to 5-FU and gemcitabine while its knockdown reduced resistance to 5-FU and gemcitabine. Furthermore, our results show that Rab5a overexpression upregulates Wnt signaling and expression of Wnt target genes including c-myc and MMP7. Blocking Wnt signaling abolished the effects of Rab5a on Wnt targets and on cancer cell proliferation. In summary, our results show that Rab5a is overexpressed in pancreatic cancer and promotes aggressive biological behavior through regulation of the Wnt/ß-catenin signaling pathway.
Assuntos
Biomarcadores Tumorais/análise , Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/patologia , Proteínas rab5 de Ligação ao GTP/biossíntese , Western Blotting , Progressão da Doença , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , Via de Sinalização Wnt/fisiologia , Proteínas rab5 de Ligação ao GTP/análise , Neoplasias PancreáticasRESUMO
Signals derived from the microenvironment contribute greatly to tumorigenesis . The underlying mechanism requires thorough investigation. Here, we use Drosophila testis as a model system to address this question, taking the advantage of the ease to distinguish germline and somatic cells and to track the cell numbers. In an EMS mutagenesis screen, we identified Rab5, a key factor in endocytosis, for its nonautonomous role in germline proliferation. The disruption of Rab5 in somatic cyst cells, which escort the development of germline lineage, induced the overproliferation of underdifferentiated but genetically wild-type germ cells. We demonstrated that this nonautonomous effect was mediated by the transcriptional activation of Dpp [the fly homolog of bone morphogenetic protein (BMP)] by examining the Dpp-reporter expression and knocking down Dpp to block germline overgrowth. Consistently, the protein levels of Bam, the germline prodifferentiation factor normally accumulated in the absence of BMP/Dpp signaling, decreased in the overproliferating germ cells. Further, we discovered that the JNK signaling pathway operated between Rab5 and Dpp, because simultaneously inhibiting the JNK pathway and Rab5 in cyst cells prevented both dpp transcription and germline tumor growth. Additionally, we found that multiple endocytic genes, such as avl, TSG101, Vps25, or Cdc42, were required in the somatic cyst cells to restrict germline amplification. These findings indicate that when the endocytic state of the surrounding cells is impaired, genetically wild-type germ cells overgrow. This nonautonomous model of tumorigenesis provides a simple system to dissect the relation between tumor and its niche.
Assuntos
Proteínas Morfogenéticas Ósseas/genética , Proliferação de Células/genética , Proteínas de Drosophila/genética , MAP Quinase Quinase 4/genética , Proteínas rab5 de Ligação ao GTP/genética , Animais , Carcinogênese/genética , Diferenciação Celular/genética , Microambiente Celular/genética , Modelos Animais de Doenças , Proteínas de Drosophila/biossíntese , Drosophila melanogaster/genética , Endocitose/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/crescimento & desenvolvimento , Humanos , Masculino , Neoplasias/genética , Transdução de Sinais , Proteínas rab5 de Ligação ao GTP/biossínteseRESUMO
Pancreatic cancer is a common type of cancer with poor prognosis worldwide. Postoperative survival depends on the existence of metastasis. Elucidation of the mechanism underlying cancer progression is important to improve prognosis. The RAS-associated protein RAB5 activates intracellular membrane trafficking, and RAB5 expression is correlated to progression and epithelial mesenchymal transition in various cancers.The expression of RAB5 and E-cadherin in 111 pancreatic cancer samples was investigated by immunohistochemical staining, and the relationship among RAB5 expression, clinicopathological factors, and E-cadherin expression was assessed. Furthermore, RAB5 suppression analysis by siRNA was performed to determine the roles of RAB5 in morphological change, proliferation potency, cell migration ability, and invasiveness of the pancreatic cancer cell line.High RAB5 expression correlated with the presence of lymphatic invasion and venous invasion and low E-cadherin expression. Patients with high RAB5 expression had a poorer prognosis than those with low RAB5 expression. RAB5 suppression in pancreatic cancer cells enhanced E-cadherin expression; changed cell morphology from spindle to round; and inhibited proliferation, invasion, and cell migration.RAB5 contributes to poor prognosis and progression in pancreatic cancer patients. It may be a promising candidate for individualized therapy in refractory pancreatic cancer.
Assuntos
Caderinas/biossíntese , Pâncreas/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas rab5 de Ligação ao GTP/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD , Western Blotting , Caderinas/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Microscopia de Fluorescência , Pessoa de Meia-Idade , Pâncreas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Prognóstico , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas rab5 de Ligação ao GTP/genéticaRESUMO
Rab5a, a key member of the Rab family of GTPases, was determined to be a regulator of vascular smooth muscle cell (VSMC) proliferation and migration. However, the exact regulatory mechanism remains unclear. As Rab5a has been shown to be associated with autophagy, which is essential for the conversion of VSMCs from a contractile to a synthetic phenotype in order to prevent cell death due to oxidative stress. The present study hypothesized that autophagy may be responsible for the proliferation and migration of VSMCs via the Rab5a protein. The aim of the present study was to evaluate the effect of Rab5a on autophagy in VSMCs. The human aorta vascular smooth muscle cell line, T/G HAVSMCs, was treated with small interfering (si)RNA against Rab5a and/or plateletderived growth factor (PDGF). Following treatment, the phenotype transition of the VSMCs was evaluated by detecting the mRNA and protien expression levels of VSMC molecular markers using reverse transcriptionquantitative polymerase chain reaction and western blotting, respectively. In addition, autophagy in VSMCs was evaluated by western blotting for autophagyassociated proteins, flow cytometry of acidic vesicular organelles, punctate fluorescence of microtubule associated protein light chain 3 and transmission electron microscopy of typical scattered doublemembrane vacuolar structures. Additionally, the proliferation, migration, cell cycle and apoptotic response of VSMCs were detected by sulforhodamine B assay, transwell assay and flow cytometry, respectively. The results revealed that transfection with siRNA against Rab5a led to a significant decrease in Rab5a protein expression, while the reduced expression trend of Rab5a was rescued by intervention with PDGF. Furthermore, cells transfected with siRNA against Rab5a inhibited the autophagy of VSMCs. Downregulated Rab5a inhibited the phenotype transition of VSMCs. Additionally, downregulated Rab5a led to slowed cell growth, decreased numbers of migrated cells, decreased numbers of cells at the G0G1 phase and a higher apoptosis rate. However, PDGF significantly rescued these phenomena caused by siRNA against Rab5a. These results indicated that Rab5amediated autophagy may regulate the phenotype transition and cell behavior of VSMCs through the activation of the extracellularregulated kinase 1/2 signaling pathway.
Assuntos
Autofagia/genética , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Fator de Crescimento Derivado de Plaquetas/genética , Proteínas rab5 de Ligação ao GTP/biossíntese , Aorta/citologia , Aorta/metabolismo , Linhagem Celular , Movimento Celular/genética , Proliferação de Células/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Estresse Oxidativo , Fator de Crescimento Derivado de Plaquetas/metabolismo , RNA Interferente Pequeno/genética , Proteínas rab5 de Ligação ao GTP/antagonistas & inibidores , Proteínas rab5 de Ligação ao GTP/genéticaRESUMO
MiR-130a has been demonstrated to play important roles in many types of cancers. Nevertheless, its biological function in breast cancer remains largely unknown. In this study, we found that the expression level of miR-130a was down-regulated in breast cancer tissues and cells. Overexpression of miR-130a was able to inhibit cell proliferation, invasion and migration in MCF7 and MDA-MB-435 cells. With the bioinformatics analysis, we further identified that RAB5A was a directly target of miR-130a, and its mRNA and protein level was negatively regulated by miR-130a. Immunohistochemistry verified RAB5A was upregulated in breast cancer tissues. Therefore, the data reported here demonstrate that miR-130a is an important tumor suppressor in breast cancer, and imply that miR-130a/RAB5A axis have potential as therapeutic targets for breast cancer.
Assuntos
Neoplasias da Mama/genética , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica/genética , MicroRNAs/genética , Proteínas rab5 de Ligação ao GTP/biossíntese , Western Blotting , Neoplasias da Mama/patologia , Movimento Celular/genética , Proliferação de Células/genética , Regulação para Baixo , Feminino , Genes Supressores de Tumor , Humanos , Imuno-Histoquímica , Invasividade Neoplásica/genética , Reação em Cadeia da Polimerase em Tempo Real , Transfecção , Proteínas rab5 de Ligação ao GTP/genéticaRESUMO
Actin protrusion at the cell periphery is central to the formation of invadopodia during tumor cell migration and invasion. Although RUFY3 (RUN and FYVE domain containing 3)/SINGAR1 (single axon-related1)/RIPX (Rap2 interacting protein X) has an important role in neuronal development, its pathophysiologic role and relevance to cancer are still largely unknown. The purpose of this study was to elucidate the molecular mechanisms by which RUFY3 involves in gastric cancer cell migration and invasion. Here, our data show that overexpression of RUFY3 leads to the formation of F-actin-enriched protrusive structures at the cell periphery and induces gastric cancer cell migration. Furthermore, P21-activated kinase-1 (PAK1) interacts with RUFY3, and promotes RUFY3 expression and RUFY3-induced gastric cancer cell migration; inhibition of PAK1 attenuates RUFY3-induced SGC-7901 cell migration and invasion. Importantly, we found that the inhibitory effect of cell migration and invasion is significantly enhanced by knockdown of both PAK1 and RUFY3 compared with knockdown of RUFY3 alone or PAK1 alone. Strikingly, we found significant upregulation of RUFY3 in gastric cancer samples with invasive carcinoma at pathologic TNM III and TNM IV stages, compared with their non-tumor counterparts. Moreover, an obvious positive correlation was observed between the protein expression of RUFY3 and PAK1 in 40 pairs of gastric cancer samples. Therefore, these findings provide important evidence that PAK1 can positively regulate RUFY3 expression, which contribute to the metastatic potential of gastric cancer cells, maybe blocking PAK1-RUFY3 signaling would become a potential metastasis therapeutic strategy for gastric cancer.
Assuntos
Carcinoma/genética , Neoplasias Gástricas/genética , Quinases Ativadas por p21/biossíntese , Proteínas rab5 de Ligação ao GTP/biossíntese , Actinas/genética , Carcinoma/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Proteínas do Citoesqueleto , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Invasividade Neoplásica/genética , Metástase Neoplásica , Transdução de Sinais/genética , Neoplasias Gástricas/patologia , Quinases Ativadas por p21/genética , Proteínas rab5 de Ligação ao GTP/genéticaRESUMO
Synaptic demise and accumulation of dysfunctional proteins are thought of as common features in neurodegeneration. However, the mechanisms by which synaptic proteins turn over remain elusive. In this paper, we study Drosophila melanogaster lacking active TBC1D24/Skywalker (Sky), a protein that in humans causes severe neurodegeneration, epilepsy, and DOOR (deafness, onychdystrophy, osteodystrophy, and mental retardation) syndrome, and identify endosome-to-lysosome trafficking as a mechanism for degradation of synaptic vesicle-associated proteins. In fly sky mutants, synaptic vesicles traveled excessively to endosomes. Using chimeric fluorescent timers, we show that synaptic vesicle-associated proteins were younger on average, suggesting that older proteins are more efficiently degraded. Using a genetic screen, we find that reducing endosomal-to-lysosomal trafficking, controlled by the homotypic fusion and vacuole protein sorting (HOPS) complex, rescued the neurotransmission and neurodegeneration defects in sky mutants. Consistently, synaptic vesicle proteins were older in HOPS complex mutants, and these mutants also showed reduced neurotransmission. Our findings define a mechanism in which synaptic transmission is facilitated by efficient protein turnover at lysosomes and identify a potential strategy to suppress defects arising from TBC1D24 mutations in humans.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Doenças Neurodegenerativas/metabolismo , Transmissão Sináptica/fisiologia , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Anormalidades Craniofaciais/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Endossomos/metabolismo , Proteínas Ativadoras de GTPase , Deformidades Congênitas da Mão/genética , Perda Auditiva Neurossensorial/genética , Humanos , Deficiência Intelectual/genética , Lisossomos/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana , Mutação , Unhas Malformadas/genética , Proteínas do Tecido Nervoso , Doenças Neurodegenerativas/genética , Junção Neuromuscular/metabolismo , Transporte Proteico , Proteólise , Compostos de Piridínio/farmacologia , Compostos de Amônio Quaternário/farmacologia , Proteínas R-SNARE/biossíntese , Proteínas R-SNARE/genética , Vesículas Sinápticas/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/biossínteseRESUMO
Drug resistance poses a major challenge to ovarian cancer treatment. Understanding mechanisms of drug resistance is important for finding new therapeutic targets. In the present work, a cisplatin-resistant ovarian cancer cell line A2780-DR was established with a resistance index of 6.64. The cellular accumulation of cisplatin was significantly reduced in A2780-DR cells as compared with A2780 cells consistent with the general character of drug resistance. Quantitative proteomic analysis identified 340 differentially expressed proteins between A2780 and A2780-DR cells, which involve in diverse cellular processes, including metabolic process, cellular component biogenesis, cellular processes, and stress responses. Expression levels of Ras-related proteins Rab 5C and Rab 11B in A2780-DR cells were lower than those in A2780 cells as confirmed by real-time quantitative PCR and Western blotting. The short hairpin (sh)RNA-mediated knockdown of Rab 5C in A2780 cells resulted in markedly increased resistance to cisplatin whereas overexpression of Rab 5C in A2780-DR cells increases sensitivity to cisplatin, demonstrating that Rab 5C-dependent endocytosis plays an important role in cisplatin resistance. Our results also showed that expressions of glycolytic enzymes pyruvate kinase, glucose-6-phosphate isomerase, fructose-bisphosphate aldolase, lactate dehydrogenase, and phosphoglycerate kinase 1 were down-regulated in drug resistant cells, indicating drug resistance in ovarian cancer is directly associated with a decrease in glycolysis. Furthermore, it was found that glutathione reductase were up-regulated in A2780-DR, whereas vimentin, HSP90, and Annexin A1 and A2 were down-regulated. Taken together, our results suggest that drug resistance in ovarian cancer cell line A2780 is caused by multifactorial traits, including the down-regulation of Rab 5C-dependent endocytosis of cisplatin, glycolytic enzymes, and vimentin, and up-regulation of antioxidant proteins, suggesting Rab 5C is a potential target for treatment of drug-resistant ovarian cancer. This constitutes a further step toward a comprehensive understanding of drug resistance in ovarian cancer.
Assuntos
Cisplatino/farmacologia , Endocitose/genética , Glicólise/genética , Neoplasias Ovarianas/tratamento farmacológico , Proteínas rab5 de Ligação ao GTP/genética , Anexina A1/biossíntese , Anexina A2/biossíntese , Antineoplásicos/farmacologia , Antioxidantes/metabolismo , Transporte Biológico/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/metabolismo , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos , Feminino , Perfilação da Expressão Gênica , Glutationa Redutase/biossíntese , Proteínas de Choque Térmico HSP90/biossíntese , Humanos , Neoplasias Ovarianas/patologia , Proteômica , Interferência de RNA , RNA Interferente Pequeno , Espécies Reativas de Oxigênio/metabolismo , Vimentina/biossíntese , Proteínas rab5 de Ligação ao GTP/biossínteseRESUMO
Rab family small GTPases regulate membrane trafficking by spatiotemporal recruitment of various effectors. However, it remains largely unclear how the expression and functions of Rab proteins are regulated in response to extracellular or intracellular stimuli. Here we show that Ypt53, one isoform of Rab5 in Saccharomyces cerevisiae, is up-regulated significantly under nutrient stress. Under non-stress conditions, Vps21, a constitutively expressed Rab5 isoform, is crucial to Golgi-vacuole trafficking and to vacuolar hydrolase activity. However, when cells are exposed to nutrient stress for an extended period of time, the up-regulated Ypt53 and the constitutive Vps21 function redundantly to maintain these activities, which, in turn, prevent the accumulation of reactive oxygen species and maintain mitochondrial respiration. Together, our results clarify the relative roles of these constitutive and nutrient stress-inducible Rab5 proteins that ensure adaptable vesicle trafficking and vacuolar hydrolase activity, thereby allowing cells to adapt to environmental changes.
Assuntos
Proteínas de Saccharomyces cerevisiae/biossíntese , Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico/fisiologia , Vacúolos/metabolismo , Proteínas rab de Ligação ao GTP/biossíntese , Proteínas rab5 de Ligação ao GTP/biossíntese , Transporte Biológico Ativo/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação Fúngica da Expressão Gênica/fisiologia , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Consumo de Oxigênio/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Regulação para Cima/fisiologia , Vacúolos/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/genéticaRESUMO
BACKGROUND: Integrin-mediated adhesion of cells to the extracellular matrix (ECM) relies on the dynamic formation of focal adhesions (FAs), which are biochemical and mechanosensitive platforms composed of a large variety of cytosolic and transmembrane proteins. During migration, there is a constant turnover of ECM contacts that initially form as nascent adhesions at the leading edge, mature into FAs as actomyosin tension builds up, and are then disassembled at the cell rear, thus allowing for cell detachment. Although the mechanisms of FA assembly have largely been defined, the molecular circuitry that regulates their disassembly still remains elusive. RESULTS: Here, we show that RN-tre, a GTPase-activating protein (GAP) for Rabs including Rab5 and Rab43, is a novel regulator of FA dynamics and cell migration. RN-tre localizes to FAs and to a pool of Rab5-positive vesicles mainly associated with FAs undergoing rapid remodeling. We found that RN-tre inhibits endocytosis of ß1, but not ß3, integrins and delays the turnover of FAs, ultimately impairing ß1-dependent, but not ß3-dependent, chemotactic cell migration. All of these effects are mediated by its GAP activity and rely on Rab5. CONCLUSIONS: Our findings identify RN-tre as the Rab5-GAP that spatiotemporally controls FA remodeling during chemotactic cell migration.
Assuntos
Movimento Celular/genética , Endocitose/genética , Proteínas Ativadoras de GTPase/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Adesão Celular/fisiologia , Células Cultivadas , Doxiciclina/farmacologia , Endocitose/fisiologia , Adesões Focais/metabolismo , Proteínas Ativadoras de GTPase/genética , Integrina beta1/metabolismo , Integrina beta3/metabolismo , Camundongos , Camundongos Knockout , Interferência de RNA , RNA Interferente Pequeno , Transdução de Sinais/fisiologia , Proteínas rab de Ligação ao GTP/biossíntese , Proteínas rab de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/biossínteseRESUMO
An outstanding question is how cells control the number and size of membrane organelles. The small GTPase Rab5 has been proposed to be a master regulator of endosome biogenesis. Here, to test this hypothesis, we developed a mathematical model of endosome dependency on Rab5 and validated it by titrating down all three Rab5 isoforms in adult mouse liver using state-of-the-art RNA interference technology. Unexpectedly, the endocytic system was resilient to depletion of Rab5 and collapsed only when Rab5 decreased to a critical level. Loss of Rab5 below this threshold caused a marked reduction in the number of early endosomes, late endosomes and lysosomes, associated with a block of low-density lipoprotein endocytosis. Loss of endosomes caused failure to deliver apical proteins to the bile canaliculi, suggesting a requirement for polarized cargo sorting. Our results demonstrate for the first time, to our knowledge, the role of Rab5 as an endosome organizer in vivo and reveal the resilience mechanisms of the endocytic system.
Assuntos
Endossomos/metabolismo , Lisossomos/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Animais , Polaridade Celular , Células Cultivadas , Endocitose , Técnicas de Silenciamento de Genes , Hepatócitos/citologia , Hepatócitos/metabolismo , Isoenzimas/biossíntese , Isoenzimas/deficiência , Isoenzimas/genética , Isoenzimas/metabolismo , Lipoproteínas LDL/metabolismo , Fígado/citologia , Fígado/enzimologia , Fígado/metabolismo , Camundongos , Corpos Multivesiculares/metabolismo , Especificidade de Órgãos , Biossíntese de Proteínas , Interferência de RNA , RNA Mensageiro/análise , RNA Mensageiro/genética , Fatores de Tempo , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab5 de Ligação ao GTP/biossíntese , Proteínas rab5 de Ligação ao GTP/deficiência , Proteínas rab5 de Ligação ao GTP/genéticaRESUMO
The expression of Rab proteins has been associated with cancer. However, few data are available on Rab5A expression in human breast cancer or its impact on disease progression. First, we examined the functional role of Rab5A in breast cancer cells. The expression of Rab5A in MDA-MB-231 cells can be stimulated by epidermal growth factor in a dose-dependent manner. The epidermal growth factor-induced increase of Rab5A expression correlated well with enhanced migration in wound healing migration assays in these cells. Furthermore, we evaluated the expression of Rab5A in breast cancer specimens using immunohistochemical staining, then analyzed the relationship between the expression of Rab5A and clinicopathological parameters. The increased expression of Rab5A protein in 123 breast cancer samples was associated with higher histological grade (P = 0.004), more lymphovascular invasion (P = 0.027), more axillary lymph node (LN) metastasis (P = 0.008), and a higher number of axillary LN metastases (P = 0.043). Among 218 axillary LNs of more than 10 breast cancer patients with node metastases, 167 metastatic LNs were found to have increased Rab5A expression. Rab5A is associated with axillary LN metastasis in breast cancer patients.
Assuntos
Neoplasias da Mama/patologia , Metástase Linfática/patologia , Proteínas rab5 de Ligação ao GTP/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Progressão da Doença , Fator de Crescimento Epidérmico/farmacologia , Feminino , Humanos , Linfonodos/patologia , Pessoa de Meia-Idade , Proteínas rab5 de Ligação ao GTP/biossínteseRESUMO
Endocytic system dysfunction is one of the earliest disturbances that occur in Alzheimer's disease (AD), and may underlie the selective vulnerability of cholinergic basal forebrain (CBF) neurons during the progression of dementia. Herein we report that genes regulating early and late endosomes are selectively upregulated within CBF neurons in mild cognitive impairment (MCI) and AD. Specifically, upregulation of rab4, rab5, rab7, and rab27 was observed in CBF neurons microdissected from postmortem brains of individuals with MCI and AD compared to age-matched control subjects with no cognitive impairment (NCI). Upregulated expression of rab4, rab5, rab7, and rab27 correlated with antemortem measures of cognitive decline in individuals with MCI and AD. qPCR validated upregulation of these select rab GTPases within microdissected samples of the basal forebrain. Moreover, quantitative immunoblot analysis demonstrated upregulation of rab5 protein expression in the basal forebrain of subjects with MCI and AD. The elevation of rab4, rab5, and rab7 expression is consistent with our recent observations in CA1 pyramidal neurons in MCI and AD. These findings provide further support that endosomal pathology accelerates endocytosis and endosome recycling, which may promote aberrant endosomal signaling and neurodegeneration throughout the progression of AD.
Assuntos
Doença de Alzheimer/enzimologia , Núcleo Basal de Meynert/enzimologia , Neurônios Colinérgicos/enzimologia , Disfunção Cognitiva/enzimologia , Regulação para Cima/fisiologia , Proteínas rab de Ligação ao GTP/biossíntese , Proteínas rab4 de Ligação ao GTP/biossíntese , Proteínas rab5 de Ligação ao GTP/biossíntese , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/fisiopatologia , Núcleo Basal de Meynert/fisiopatologia , Disfunção Cognitiva/fisiopatologia , Feminino , Humanos , Masculino , Proteínas rab de Ligação ao GTP/genética , Proteínas rab27 de Ligação ao GTP , Proteínas rab4 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/genética , proteínas de unión al GTP Rab7RESUMO
Sporadic motor neuron disease (MND) is characterized by progressive degeneration of motor neurons and intraneuronal cytoplasmic translocation and deposition of the nuclear protein TDP-43. There is a paucity of data on the subcellular mechanisms of the nuclear-cytoplasmic trafficking of TDP-43, particularly about the precise role of the endosomal-lysosomal system (ELS). In the present study, using a neuron-specific morphometric approach, we examined the expression of the early endosomal marker Rab5 and lysosomal cathepsins B, D, F, and L as well as PAS-stained structures in the anterior horn cells in 11 individuals affected by sporadic MND and 5 age-matched controls. This was compared with the expression of ubiquitin, p62 and TDP-43 and its phosphorylated form. The principal finding was the increased expression of the endosomal marker Rab5 and lysosomal cathepsin D, and of PAS-positive structures in motor neurons of MND cases. Furthermore, the area-portion of Rab5 immunoreactivity correlated well with the intracellular accumulation of ubiquitin, p62 and (phosphorylated) TDP-43. However, double immunolabelling and immunogold electron microscopy excluded colocalization of phosphorylated TDP-43 with the ELS. These data contrast with observations on neuronal cytopathology in Alzheimer's or prion diseases where the disease-specific proteins are processed within endosomes, and suggest a distinct role of the ELS in MND.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Endossomos/metabolismo , Endossomos/patologia , Doença dos Neurônios Motores/metabolismo , Doença dos Neurônios Motores/patologia , Proteínas rab5 de Ligação ao GTP/biossíntese , Transporte Ativo do Núcleo Celular/genética , Idoso , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/ultraestrutura , Endossomos/ultraestrutura , Feminino , Humanos , Líquido Intracelular/química , Líquido Intracelular/metabolismo , Lisossomos/metabolismo , Lisossomos/patologia , Lisossomos/ultraestrutura , Masculino , Pessoa de Meia-Idade , Fosforilação/genética , Transporte Proteico/genética , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/ultraestruturaRESUMO
Persistent mitochondrial hyperpolarization (MHP) and enhanced calcium fluxing underlie aberrant T cell activation and death pathway selection in systemic lupus erythematosus. Treatment with rapamycin, which effectively controls disease activity, normalizes CD3/CD28-induced calcium fluxing but fails to influence MHP, suggesting that altered calcium fluxing is downstream or independent of mitochondrial dysfunction. In this article, we show that activity of the mammalian target of rapamycin (mTOR), which is a sensor of the mitochondrial transmembrane potential, is increased in lupus T cells. Activation of mTOR was inducible by NO, a key trigger of MHP, which in turn enhanced the expression of HRES-1/Rab4, a small GTPase that regulates recycling of surface receptors through early endosomes. Expression of HRES-1/Rab4 was increased in CD4(+) lupus T cells, and in accordance with its dominant impact on the endocytic recycling of CD4, it was inversely correlated with diminished CD4 expression. HRES-1/Rab4 overexpression was also inversely correlated with diminished TCRzeta protein levels. Pull-down studies revealed a direct interaction of HRES-1/Rab4 with CD4 and TCRzeta. Importantly, the deficiency of the TCRzeta chain and of Lck and the compensatory up-regulation of FcepsilonRIgamma and Syk, which mediate enhanced calcium fluxing in lupus T cells, were reversed in patients treated with rapamcyin in vivo. Knockdown of HRES-1/Rab4 by small interfering RNA and inhibitors of lysosomal function augmented TCRzeta protein levels in vitro. The results suggest that activation of mTOR causes the loss of TCRzeta in lupus T cells through HRES-1/Rab4-dependent lysosomal degradation.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Proteínas Quinases/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas rab4 de Ligação ao GTP/imunologia , Adolescente , Adulto , Western Blotting , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Feminino , Citometria de Fluxo , Expressão Gênica/efeitos dos fármacos , Humanos , Imunossupressores/uso terapêutico , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/metabolismo , Microscopia Confocal , Pessoa de Meia-Idade , Óxido Nítrico/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Quinases/metabolismo , RNA Interferente Pequeno , Receptores de Antígenos de Linfócitos T/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sirolimo/uso terapêutico , Serina-Treonina Quinases TOR , Transfecção , Proteínas rab4 de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/biossínteseRESUMO
After endocytosis, most membrane proteins and lipids return to the plasma membrane (recycling pathway), but some membrane components are delivered to lysosomes (degradation pathway). These two pathways diverge in early endosomes. The recycling pathway involves recycling endosomes and the degradation pathway incorporates late endosomes and lysosomes. In many cell lines, these organelles often are located in the perinuclear region where they visually intermix. The present study, by tracking specific ligands (epidermal growth factor and transferrin) and expression of Rab proteins (Rab5, Rab7, and Rab11), demonstrated that, in COS-1 cells, the two pathways were spatially segregated. Recycling endosomes were mostly confined within the ring-shaped structure of the Golgi complex ("the Golgi ring"), whereas late endosomes and lysosomes were excluded from inside the Golgi ring. Thus, the unique organization of endocytic organelles in COS-1 cells can be utilized to visualize endocytic trafficking pathways in detail.
Assuntos
Endocitose/fisiologia , Endossomos/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Complexo de Golgi/metabolismo , Rim/metabolismo , Transferrina/metabolismo , Animais , Células COS , Chlorocebus aethiops , Rim/citologia , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Transporte Proteico/fisiologia , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab5 de Ligação ao GTP/biossíntese , Proteínas rab5 de Ligação ao GTP/genéticaRESUMO
Chagas disease is an incurable illness caused by the protozoan Trypanosoma cruzi. Cardiomyocytes represent important targets for the parasite infection and alterations in their physiology were reported. Because endocytosis is involved in different cellular events and guanosine triphosphatase (GTPase) Rab proteins play important roles in various aspects of the membrane traffic, our aim was to characterize the expression of Rab proteins in T. cruzi-infected cardiomyocytes, which displayed a downregulation of Rab7 and Rab11, whereas the expression of Rab5a was maintained in the infected cultures even after longer periods of parasite internalization, but early endosome antigen 1 was partially downregulated. The parasite infection also decreased the uptake of fluid phase ligands by the cardiac cultures. The regulation of GTPase proteins and effector molecules can contribute to the altered physiology of the host cells by modifying the normal incoming of nutrients as well as interfering with other important events related to the endocytic pathway.