RESUMO
Docosahexaenoic acid (DHA, C22:6 ω3) may be involved in various neuroprotective mechanisms that could prevent Alzheimer's disease (AD). Its influence has still been little explored regarding the dysfunction of the endolysosomal pathway, known as an early key event in the physiopathological continuum triggering AD. This dysfunction could result from the accumulation of degradation products of the precursor protein of AD, in particular the C99 fragment, capable of interacting with endosomal proteins and thus contributing to altering this pathway from the early stages of AD. This study aims to evaluate whether neuroprotection mediated by DHA can also preserve the endolysosomal function. AD-typical endolysosomal abnormalities were recorded in differentiated human SH-SY5Y neuroblastoma cells expressing the Swedish form of human amyloid precursor protein. This altered phenotype included endosome enlargement, the reduced secretion of exosomes, and a higher level of apoptosis, which confirmed the relevance of the cellular model chosen for studying the associated deleterious mechanisms. Second, neuroprotection mediated by DHA was associated with a reduced interaction of C99 with the Rab5 GTPase, lower endosome size, restored exosome production, and reduced neuronal apoptosis. Our data reveal that DHA may influence protein localization and interactions in the neuronal membrane environment, thereby correcting the dysfunction of endocytosis and vesicular trafficking associated with AD.
Assuntos
Doença de Alzheimer , Ácidos Docosa-Hexaenoicos , Endossomos , Lisossomos , Neurônios , Proteínas rab5 de Ligação ao GTP , Humanos , Ácidos Docosa-Hexaenoicos/farmacologia , Ácidos Docosa-Hexaenoicos/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Proteínas rab5 de Ligação ao GTP/metabolismo , Endossomos/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Neurônios/efeitos dos fármacos , Lisossomos/metabolismo , Linhagem Celular Tumoral , Precursor de Proteína beta-Amiloide/metabolismo , Apoptose , Fármacos Neuroprotetores/farmacologia , Sobrevivência Celular/efeitos dos fármacosRESUMO
BACKGROUND: Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by exacerbated synovial inflammation and joint destruction. Recent studies suggest toll-like receptor 4 (TLR4) internalization facilitate inflammatory response of macrophage. The role of TLR4 internalization in the pathogenesis of RA is unknown. PURPOSE: To investigate the role and mechanism of TLR4 internalization in macrophage inflammatory response of RA and explore whether TLR4 internalization mediates the anti-arthritic effect of Xiaowugui (XWG) decoction, a patented herbal formula used in China. METHODS: The co-expression of TLR4 and the internalization marker, early endosome antigen 1 (EEA1), in the synovial samples of RA patients and joint tissue of collagen-induced arthritis (CIA) mice, were evaluated using immunofluorescence. The effect of Rab5a-mediated early internalization of TLR4 on the activation induced by lipopolysaccharide (LPS) in RAW264.7 cells was investigated using small interfering RNAs that act against Rab5a. CIA was induced in Rab5a-/- mice to evaluate the role of Rab5a in vivo. The disease progression and expression of Rab5a and TLR4 in the joint tissue were evaluated in CIA mice treated with XWG. Inflammatory factors production, TLR4 internalization, and activation of downstream signaling pathways were examined in RAW264.7 cells treated with XWG in vitro. RESULTS: The co-expression and co-localization of TLR4 and EEA1 were elevated in the synovial samples of RA patients and joint tissue of CIA mice. Pharmaceutical inhibition of TLR4 internalization reduced macrophages inflammatory responses induced by LPS. The co-expression and co-localization of Rab5a and TLR4 were significantly increased in macrophages treated with LPS. Silencing Rab5a reduced LPS-induced TLR4 internalization, inflammatory factors production, and phosphorylation of Jun N-terminal kinases (JNK) and p65. Genetic deletion of Rab5a inhibited TLR4 internalization and the development of arthritis in vivo. The co-expression of TLR4 and Rab5a was also elevated in the synovial samples of RA patients. XWG treatment of mice with CIA alleviated arthritis and reduced the co-expression of Rab5a and TLR4 in the joint tissue. XWG treatment of macrophage inhibited LPS-induced IL-6 and TNF-α production, co-expression of Rab5a and TLR4, and phosphorylation of JNK and p65. CONCLUSIONS: Our findings highlight the pathogenic role of TLR4 internalization in patients with RA and identify a novel Rab5a-dependent internalization pathway that promotes macrophage inflammatory response. XWG treatment demonstrated outstanding therapeutic effects in experimental arthritis, and targeting the Rab5a-mediated internalization of TLR4 may be the main underlying mechanism.
Assuntos
Artrite Experimental , Artrite Reumatoide , Medicamentos de Ervas Chinesas , Macrófagos , Receptor 4 Toll-Like , Proteínas rab5 de Ligação ao GTP , Animais , Receptor 4 Toll-Like/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Camundongos , Humanos , Artrite Experimental/tratamento farmacológico , Artrite Experimental/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Artrite Reumatoide/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Células RAW 264.7 , Masculino , Camundongos Endogâmicos DBA , Lipopolissacarídeos , Membrana Sinovial/efeitos dos fármacos , FemininoRESUMO
Natural killer cells (NK) are the "professional killer" of tumors and play a crucial role in anti-tumor immunotherapy. NK cell desensitization is a key mechanism of tumor immune escape. Dysregulated NKG2D-NKG2DL signaling is a primary driver of this desensitization process. However, the factors that regulate NK cell desensitization remain largely uncharacterized. Here, we present the first report that circular RNA circARAP2 (hsa_circ_0069396) is involved in the soluble MICA (sMICA)-induced NKG2D endocytosis in the NK cell desensitization model. CircARAP2 was upregulated during NK cell desensitization and the loss of circARAP2 alleviated NKG2D endocytosis and NK cell desensitization. Using Chromatin isolation by RNA purification (ChIRP) and RNA pull-down approaches, we identified that RAB5A, a molecular marker of early endosomes, was its downstream target. Notably, transcription factor CTCF was an intermediate functional partner of circARAP2. Mechanistically, we discovered that circARAP2 interacted with CTCF and inhibited the recruitment of CTCF-Polycomb Repressive Complex 2 (PRC2) to the promoter region of RAB5A, thereby erasing histone H3K27 and H3K9 methylation suppression to enhance RAB5A transcription. These data demonstrate that inhibition of circARAP2 effectively alleviates sMICA-induced NKG2D endocytosis and NK cell desensitization, providing a novel target for therapeutic intervention in tumor immune evasion.
Assuntos
Fator de Ligação a CCCTC , Antígenos de Histocompatibilidade Classe I , Células Matadoras Naturais , RNA Circular , Proteínas rab5 de Ligação ao GTP , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Humanos , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , RNA Circular/genética , RNA Circular/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Endocitose , Endossomos/metabolismo , Camundongos , AnimaisRESUMO
CD36 is a type 2 cell surface scavenger receptor expressed in various tissues. In macrophages, CD36 recognizes oxidized low-density lipoprotein (ox-LDL), which promotes the formation of foam cells, the first step toward an atherosclerotic arterial lesion. CD36 possesses a variety of posttranslational modifications, among them N-glycosylation and O-GlcNAc modification. Some of the roles of these modifications on CD36 are known, such as N-linked glycosylation, which provides proper folding and trafficking to the plasma membrane in the human embryonic kidney. This study aimed to determine whether variations in the availability of UDP-GlcNAc could impact Rab-5-mediated endocytic trafficking and, therefore, the cellular localization of CD36. These preliminary results suggest that the availability of the substrate UDP-GlcNAc, modulated in response to treatment with Thiamet G (TMG), OSMI-1 (O-GlcNAcylation enzymes modulators) or Azaserine (HBP modulator), influences the localization of CD36 in J774 macrophages, and the endocytic trafficking as evidenced by the regulatory protein Rab-5, between the plasma membrane and the cytoplasm.
Assuntos
Antígenos CD36 , Macrófagos , Antígenos CD36/metabolismo , Macrófagos/metabolismo , Animais , Camundongos , Linhagem Celular , Glicosilação , Membrana Celular/metabolismo , Humanos , Lipoproteínas LDL/metabolismo , Hexosaminas/metabolismo , Hexosaminas/biossíntese , Proteínas rab5 de Ligação ao GTP/metabolismo , Transporte Proteico , Vias Biossintéticas , Processamento de Proteína Pós-TraducionalRESUMO
Cells depend on their endolysosomal system for nutrient uptake and downregulation of plasma membrane proteins. These processes rely on endosomal maturation, which requires multiple membrane fusion steps. Early endosome fusion is promoted by the Rab5 GTPase and its effector, the hexameric CORVET tethering complex, which is homologous to the lysosomal HOPS. How these related complexes recognize their specific target membranes remains entirely elusive. Here, we solve the structure of CORVET by cryo-electron microscopy and revealed its minimal requirements for membrane tethering. As expected, the core of CORVET and HOPS resembles each other. However, the function-defining subunits show marked structural differences. Notably, we discover that unlike HOPS, CORVET depends not only on Rab5 but also on phosphatidylinositol-3-phosphate (PI3P) and membrane lipid packing defects for tethering, implying that an organelle-specific membrane code enables fusion. Our data suggest that both shape and membrane interactions of CORVET and HOPS are conserved in metazoans, thus providing a paradigm how tethering complexes function.
Assuntos
Microscopia Crioeletrônica , Endossomos , Fosfatos de Fosfatidilinositol , Endossomos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fusão de Membrana , Proteínas rab5 de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Humanos , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/genética , Membrana Celular/metabolismo , Animais , Lisossomos/metabolismoRESUMO
Tumor hypoxia has been associated with cancer progression, angiogenesis, and metastasis via modifications in the release and cargo composition of extracellular vesicles secreted by tumor cells. Indeed, hypoxic extracellular vesicles are known to trigger a variety of angiogenic responses via different mechanisms. We recently showed that hypoxia promotes endosomal signaling in tumor cells via HIF-1α-dependent induction of the guanine exchange factor ALS2, which activates Rab5, leading to downstream events involved in cell migration and invasion. Since Rab5-dependent signaling is required for endothelial cell migration and angiogenesis, we explored the possibility that hypoxia promotes the release of small extracellular vesicles containing ALS2, which in turn activate Rab5 in recipient endothelial cells leading to pro-angiogenic properties. In doing so, we found that hypoxia promoted ALS2 expression and incorporation as cargo within small extracellular vesicles, leading to subsequent transfer to recipient endothelial cells and promoting cell migration, tube formation, and downstream Rab5 activation. Consequently, ALS2-containing small extracellular vesicles increased early endosome size and number in recipient endothelial cells, which was followed by subsequent sequestration of components of the ß-catenin destruction complex within endosomal compartments, leading to stabilization and nuclear localization of ß-catenin. These events converged in the expression of ß-catenin target genes involved in angiogenesis. Knockdown of ALS2 in donor tumor cells precluded its incorporation into small extracellular vesicles, preventing Rab5-downstream events and endothelial cell responses, which depended on Rab5 activity and guanine exchange factor activity of ALS2. These findings indicate that vesicular ALS2, secreted in hypoxia, promotes endothelial cell events leading to angiogenesis. Finally, these events might explain how tumor angiogenesis proceeds in hypoxic conditions.
Assuntos
Movimento Celular , Vesículas Extracelulares , Fatores de Troca do Nucleotídeo Guanina , Transdução de Sinais , beta Catenina , Proteínas rab5 de Ligação ao GTP , Humanos , Proteínas rab5 de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , beta Catenina/metabolismo , Vesículas Extracelulares/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Linhagem Celular TumoralRESUMO
ZFYVE21 is an ancient, endosome-associated protein that is highly expressed in endothelial cells (ECs) but whose function(s) in vivo are undefined. Here, we identified ZFYVE21 as an essential regulator of vascular barrier function in the aging kidney. ZFYVE21 levels significantly decline in ECs in aged human and mouse kidneys. To investigate attendant effects, we generated EC-specific Zfyve21-/- reporter mice. These knockout mice developed accelerated aging phenotypes including reduced endothelial nitric oxide (ENOS) activity, failure to thrive, and kidney insufficiency. Kidneys from Zfyve21 EC-/- mice showed interstitial edema and glomerular EC injury. ZFYVE21-mediated phenotypes were not programmed developmentally as loss of ZFYVE21 in ECs during adulthood phenocopied its loss prenatally, and a nitric oxide donor normalized kidney function in adult hosts. Using live cell imaging and human kidney organ cultures, we found that in a GTPase Rab5- and protein kinase Akt-dependent manner, ZFYVE21 reduced vesicular levels of inhibitory caveolin-1 and promoted transfer of Golgi-derived ENOS to a perinuclear Rab5+ vesicular population to functionally sustain ENOS activity. Thus, our work defines a ZFYVE21- mediated trafficking mechanism sustaining ENOS activity and demonstrates the relevance of this pathway for maintaining kidney function with aging.
Assuntos
Envelhecimento , Caveolina 1 , Células Endoteliais , Rim , Camundongos Knockout , Óxido Nítrico Sintase Tipo III , Óxido Nítrico , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Animais , Humanos , Envelhecimento/metabolismo , Envelhecimento/fisiologia , Células Endoteliais/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Rim/metabolismo , Caveolina 1/metabolismo , Caveolina 1/genética , Óxido Nítrico/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Camundongos , Masculino , Complexo de Golgi/metabolismo , Insuficiência Renal/metabolismo , Insuficiência Renal/fisiopatologia , Insuficiência Renal/genética , Doadores de Óxido Nítrico/farmacologia , Fenótipo , Camundongos Endogâmicos C57BLRESUMO
AIMS: Nerve growth factor (NGF) loss is a potential factor for the degeneration of basal forebrain cholinergic neurons (BFCNs) in Alzheimer's disease (AD), and Rab5a is a key regulatory molecule of NGF signaling transduction. Here, we investigated the changes of Rab5a in 5 × FAD mice and further explored the mechanism of Electroacupuncture (EA) treatment in improving cognition in the early stage of AD. METHODS: The total Rab5a and Rab5a-GTP in 5-month-old 5 × FAD mice and wild-type mice were detected using WB and IP technologies. 5 × FAD mice were treated with EA at the Bai hui (DU20) and Shen ting (DU24) acupoints for 4 weeks and CRE/LOXP technology was used to confirm the role of Rab5a in AD mediated by EA stimulation. The Novel Object Recognition and Morris water maze tests were used to evaluate the cognitive function of 5 × FAD mice. The Nissl, immunohistochemistry, and Thioflavin S staining were used to observe pathological morphological changes in the basal forebrain circuit. The Golgi staining was used to investigate the synaptic plasticity of the basal forebrain circuit and WB technology was used to detect the expression levels of cholinergic-related and NGF signal-related proteins. RESULTS: The total Rab5a was unaltered, but Rab5a-GTP increased and the rab5a-positive early endosomes appeared enlarged in the hippocampus of 5 × FAD mice. Notably, EA reduced Rab5a-GTP in the hippocampus in the early stage of 5 × FAD mice. EA could improve object recognition memory and spatial learning memory by reducing Rab5a activity in the early stage of 5 × FAD mice. Moreover, EA could reduce Rab5a activity to increase NGF transduction and increase the levels of phosphorylated TrkA, AKT, and ERK in the basal forebrain and hippocampus, and increase the expression of cholinergic-related proteins, such as ChAT, vAchT, ChT1, m1AchR, and m2AchR in the basal forebrain and ChAT, m1AchR, and m2AchR in the hippocampus, improving synaptic plasticity in the basal forebrain hippocampal circuit in the early stage of 5 × FAD mice. CONCLUSIONS: Rab5a hyperactivation is an early pathological manifestation of 5 × FAD mice. EA could suppress Rab5a-GTP to promote the transduction of NGF signaling, and enhance the synaptic plasticity of the basal forebrain hippocampal circuit improving cognitive impairment in the early stage of 5 × FAD mice.
Assuntos
Doença de Alzheimer , Eletroacupuntura , Fator de Crescimento Neural , Transdução de Sinais , Proteínas rab5 de Ligação ao GTP , Animais , Masculino , Camundongos , Doença de Alzheimer/terapia , Doença de Alzheimer/metabolismo , Aprendizagem/fisiologia , Aprendizagem em Labirinto/fisiologia , Memória/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fator de Crescimento Neural/metabolismo , Plasticidade Neuronal/fisiologia , Proteínas rab5 de Ligação ao GTP/metabolismo , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: Previous reports suggest that lipid droplets (LDs) in the hepatocyte can be catabolized by a direct engulfment from nearby endolysosomes (microlipophagy). Further, it is likely that this process is compromised by chronic ethanol (EtOH) exposure leading to hepatic steatosis. This study investigates the hepatocellular machinery supporting microlipophagy and EtOH-induced alterations in this process with a focus on the small, endosome-associated, GTPase Rab5. METHODS AND RESULTS: Here we report that this small Ras-related GTPase is a resident component of LDs, and its activity is important for hepatocellular LD-lysosome proximity and physical interactions. We find that Rab5 siRNA knockdown causes an accumulation of LDs in hepatocytes by inhibiting lysosome dependent LD catabolism. Importantly, Rab5 appears to support this process by mediating the recruitment of early endosomal and or multivesicular body compartments to the LD surface before lysosome fusion. Interestingly, while wild-type or a constituently active GTPase form (Q79L) of Rab5 supports LD-lysosome transport, this process is markedly reduced in cells expressing a GTPase dead (S34N) Rab5 protein or in hepatocytes exposed to chronic EtOH. CONCLUSIONS: These findings support the novel premise of an early endosomal/multivesicular body intermediate compartment on the LD surface that provides a "docking" site for lysosomal trafficking, not unlike the process that occurs during the hepatocellular degradation of endocytosed ligands that is also known to be compromised by EtOH exposure.
Assuntos
Etanol , Hepatócitos , Lisossomos , Proteínas rab5 de Ligação ao GTP , Proteínas rab5 de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Lisossomos/metabolismo , Lisossomos/efeitos dos fármacos , Etanol/farmacologia , Hepatócitos/metabolismo , Hepatócitos/efeitos dos fármacos , Humanos , Gotículas Lipídicas/metabolismo , Autofagia/efeitos dos fármacos , Animais , Endossomos/metabolismoRESUMO
Vesicular trafficking, including secretion and endocytosis, plays fundamental roles in the unique biology of Plasmodium falciparum blood-stage parasites. Endocytosis of host cell cytosol (HCC) provides nutrients and room for parasite growth and is critical for the action of antimalarial drugs and parasite drug resistance. Previous work showed that PfVPS45 functions in endosomal transport of HCC to the parasite's food vacuole, raising the possibility that malaria parasites possess a canonical endolysosomal system. However, the seeming absence of VPS45-typical functional interactors such as rabenosyn 5 (Rbsn5) and the repurposing of Rab5 isoforms and other endolysosomal proteins for secretion in apicomplexans question this idea. Here, we identified a parasite Rbsn5-like protein and show that it functions with VPS45 in the endosomal transport of HCC. We also show that PfRab5b but not PfRab5a is involved in the same process. Inactivation of PfRbsn5L resulted in PI3P and PfRab5b decorated HCC-filled vesicles, typical for endosomal compartments. Overall, this indicates that despite the low sequence conservation of PfRbsn5L and the unusual N-terminal modification of PfRab5b, principles of endosomal transport in malaria parasite are similar to that of model organisms. Using a conditional double protein inactivation system, we further provide evidence that the PfKelch13 compartment, an unusual apicomplexa-specific endocytosis structure at the parasite plasma membrane, is connected upstream of the Rbsn5L/VPS45/Rab5b-dependent endosomal route. Altogether, this work indicates that HCC uptake consists of a highly parasite-specific part that feeds endocytosed material into an endosomal system containing more canonical elements, leading to the delivery of HCC to the food vacuole.
Assuntos
Citosol , Endossomos , Plasmodium falciparum , Proteínas de Protozoários , Proteínas rab5 de Ligação ao GTP , Proteínas rab5 de Ligação ao GTP/metabolismo , Endossomos/metabolismo , Citosol/metabolismo , Plasmodium falciparum/metabolismo , Plasmodium falciparum/genética , Humanos , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Endocitose , Malária Falciparum/parasitologia , Malária Falciparum/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/genética , Animais , Interações Hospedeiro-Parasita , Vacúolos/metabolismo , Eritrócitos/parasitologia , Eritrócitos/metabolismo , Transporte ProteicoRESUMO
Extracellular vesicles-mediated exchange of miRNA cargos between diverse types of mammalian cells is a major mechanism of controlling cellular miRNA levels and activity, thus regulating the expression of miRNA-target genes in both donor and recipient cells. Despite tremendous excitement related to extracellular vesicles-associated miRNAs as biomarkers or having therapeutic potential, the mechanism of selective packaging of miRNAs into endosomes and multivesicular bodies for subsequent extracellular export is poorly studied due to the lack of an in vitro assay system. Here, we have developed an in vitro assay with endosomes isolated from mammalian macrophage cells to follow miRNA packaging into endocytic organelles. The synthetic miRNAs, used in the assay, get imported inside the isolated endosomes during the in vitro reaction and become protected from RNase in a time- and concentration-dependent manner. The selective miRNA accumulation inside endosomes requires both ATP and GTP hydrolysis and the miRNA-binding protein HuR. The HuR-miRNA complex binds and stimulates the endosomal RalA GTPase to facilitate the import of miRNAs into endosomes and their subsequent export as part of the extracellular vesicles. The endosomal targeting of miRNAs is also very much dependent on the endosome maturation process that is controlled by Rab5 protein and ATP. In summary, we provide an in vitro method to aid in the investigation of the mechanism of miRNA packaging process for its export from mammalian macrophage cells.
Assuntos
Proteína Semelhante a ELAV 1 , Endossomos , Macrófagos , MicroRNAs , Proteínas ral de Ligação ao GTP , Trifosfato de Adenosina/metabolismo , Endossomos/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismo , Humanos , Proteínas ral de Ligação ao GTP/metabolismo , Proteína Semelhante a ELAV 1/metabolismo , Macrófagos/metabolismo , Células HEK293RESUMO
While the role of endocytosis in focal adhesion turnover-coupled cell migration has been established in addition to its conventional role in cellular functions, the molecular regulators and precise molecular mechanisms that underlie this process remain largely unknown. In this study, we report that proto-oncoprotein hematopoietic PBX-interacting protein (HPIP) localizes to focal adhesions as well as endosomal compartments along with RUN FYVE domain-containing protein 3 (RUFY3) and Rab5, an early endosomal protein. HPIP contains two coiled-coil domains (CC1 and CC2) that are necessary for its association with Rab5 and RUFY3 as CC domain double mutant, that is, mtHPIPΔCC1-2 failed to support it. Furthermore, we show that HPIP and RUFY3 activate Rab5 by serving as noncanonical guanine nucleotide exchange factors of Rab5. In support of this, either deletion of coiled-coil domains or silencing of HPIP or RUFY3 impairs Rab5 activation and Rab5-dependent cell migration. Mechanistic studies further revealed that loss of HPIP or RUFY3 expression severely impairs Rab5-mediated focal adhesion disassembly, FAK activation, fibronectin-associated-ß1 integrin trafficking, and thus cell migration. Together, this study underscores the importance of HPIP and RUFY3 as noncanonical guanine nucleotide exchange factors of Rab5 and in integrin trafficking and focal adhesion turnover, which implicates in cell migration.
Assuntos
Adesões Focais , Fatores de Troca do Nucleotídeo Guanina , Movimento Celular , Endocitose , Adesões Focais/genética , Adesões Focais/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismo , Humanos , Linhagem Celular , Linhagem Celular TumoralRESUMO
IMPORTANCE: Although Micropterus salmoides rhabdovirus (MSRV) causes serious fish epidemics worldwide, the detailed mechanism of MSRV entry into host cells remains unknown. Here, we comprehensively investigated the mechanism of MSRV entry into epithelioma papulosum cyprinid (EPC) cells. This study demonstrated that MSRV enters EPC cells via a low pH, dynamin-dependent, microtubule-dependent, and clathrin-mediated endocytosis. Subsequently, MSRV transports from early endosomes to late endosomes and further into lysosomes in a microtubule-dependent manner. The characterization of MSRV entry will further advance the understanding of rhabdovirus cellular entry pathways and provide novel targets for antiviral drug against MSRV infection.
Assuntos
Bass , Rhabdoviridae , Animais , Rhabdoviridae/metabolismo , Bass/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Endocitose , Dinaminas/metabolismo , Microtúbulos/metabolismo , Clatrina/metabolismo , Concentração de Íons de Hidrogênio , Internalização do VírusRESUMO
Rab GTPases are important regulators of intracellular vesicular trafficking. RAB5C is a member of the Rab GTPase family that plays an important role in the endocytic pathway, membrane protein recycling and signaling. Here we report on 12 individuals with nine different heterozygous de novo variants in RAB5C. All but one patient with missense variants (n = 9) exhibited macrocephaly, combined with mild-to-moderate developmental delay. Patients with loss of function variants (n = 2) had an apparently more severe clinical phenotype with refractory epilepsy and intellectual disability but a normal head circumference. Four missense variants were investigated experimentally. In vitro biochemical studies revealed that all four variants were damaging, resulting in increased nucleotide exchange rate, attenuated responsivity to guanine exchange factors and heterogeneous effects on interactions with effector proteins. Studies in C. elegans confirmed that all four variants were damaging in vivo and showed defects in endocytic pathway function. The variant heterozygotes displayed phenotypes that were not observed in null heterozygotes, with two shown to be through a dominant negative mechanism. Expression of the human RAB5C variants in zebrafish embryos resulted in defective development, further underscoring the damaging effects of the RAB5C variants. Our combined bioinformatic, in vitro and in vivo experimental studies and clinical data support the association of RAB5C missense variants with a neurodevelopmental disorder characterized by macrocephaly and mild-to-moderate developmental delay through disruption of the endocytic pathway.
Assuntos
Deficiência Intelectual , Megalencefalia , Transtornos do Neurodesenvolvimento , Animais , Humanos , Criança , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Caenorhabditis elegans/metabolismo , Transtornos do Neurodesenvolvimento/genética , Deficiência Intelectual/genética , Fenótipo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Megalencefalia/genética , Deficiências do Desenvolvimento/genética , Mutação de Sentido Incorreto/genética , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismoRESUMO
Maturation from early to late endosomes depends on the exchange of their marker proteins Rab5 to Rab7. This requires Rab7 activation by its specific guanine nucleotide exchange factor (GEF) Mon1-Ccz1. Efficient GEF activity of this complex on membranes depends on Rab5, thus driving Rab-GTPase exchange on endosomes. However, molecular details on the role of Rab5 in Mon1-Ccz1 activation are unclear. Here, we identify key features in Mon1 involved in GEF regulation. We show that the intrinsically disordered N-terminal domain of Mon1 autoinhibits Rab5-dependent GEF activity on membranes. Consequently, Mon1 truncations result in higher GEF activity in vitro and alterations in early endosomal structures in Drosophila nephrocytes. A shift from Rab5 to more Rab7-positive structures in yeast suggests faster endosomal maturation. Using modeling, we further identify a conserved Rab5-binding site in Mon1. Mutations impairing Rab5 interaction result in poor GEF activity on membranes and growth defects in vivo. Our analysis provides a framework to understand the mechanism of Ras-related in brain (Rab) conversion and organelle maturation along the endomembrane system.
Assuntos
Proteínas de Drosophila , Proteínas de Saccharomyces cerevisiae , Animais , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Transporte Proteico , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Endossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Drosophila/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismoRESUMO
Despite their significance in receptor-mediated internalization and continued signal transduction in cells, early/sorting endosomes (EE/SE) remain incompletely characterized, with many outstanding questions that surround the dynamics of their size and number. While several studies have reported increases in EE/SE size and number resulting from endocytic events, few studies have addressed such dynamics in a methodological and quantitative manner. Herein we apply quantitative fluorescence microscopy to measure the size and number of EE/SE upon internalization of two different ligands: transferrin and epidermal growth factor. Additionally, we used siRNA knock-down to determine the involvement of 5 different endosomal RAB proteins (RAB4, RAB5, RAB8A, RAB10 and RAB11A) in EE/SE dynamics. Our study provides new information on the dynamics of endosomes during endocytosis, an important reference for researchers studying receptor-mediated internalization and endocytic events.
Assuntos
Proteínas rab4 de Ligação ao GTP , Proteínas rab5 de Ligação ao GTP , Endocitose/fisiologia , Endossomos/metabolismo , Transporte Proteico/fisiologia , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab4 de Ligação ao GTP/genética , Proteínas rab4 de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismo , Humanos , Linhagem Celular TumoralRESUMO
Localized translation is vital to polarized cells and requires precise and robust distribution of different mRNAs and ribosomes across the cell. However, the underlying molecular mechanisms are poorly understood and important players are lacking. Here, we discovered a Rab5 effector, the five-subunit endosomal Rab5 and RNA/ribosome intermediary (FERRY) complex, that recruits mRNAs and ribosomes to early endosomes through direct mRNA-interaction. FERRY displays preferential binding to certain groups of transcripts, including mRNAs encoding mitochondrial proteins. Deletion of FERRY subunits reduces the endosomal localization of transcripts in cells and has a significant impact on mRNA levels. Clinical studies show that genetic disruption of FERRY causes severe brain damage. We found that, in neurons, FERRY co-localizes with mRNA on early endosomes, and mRNA loaded FERRY-positive endosomes are in close proximity of mitochondria. FERRY thus transforms endosomes into mRNA carriers and plays a key role in regulating mRNA distribution and transport.
Assuntos
Endossomos , Proteínas rab5 de Ligação ao GTP , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Endossomos/metabolismo , Transporte Biológico , Endocitose/fisiologiaRESUMO
The pentameric FERRY Rab5 effector complex is a molecular link between mRNA and early endosomes in mRNA intracellular distribution. Here, we determine the cryo-EM structure of human FERRY. It reveals a unique clamp-like architecture that bears no resemblance to any known structure of Rab effectors. A combination of functional and mutational studies reveals that while the Fy-2 C-terminal coiled-coil acts as binding region for Fy-1/3 and Rab5, both coiled-coils and Fy-5 concur to bind mRNA. Mutations causing truncations of Fy-2 in patients with neurological disorders impair Rab5 binding or FERRY complex assembly. Thus, Fy-2 serves as a binding hub connecting all five complex subunits and mediating the binding to mRNA and early endosomes via Rab5. Our study provides mechanistic insights into long-distance mRNA transport and demonstrates that the particular architecture of FERRY is closely linked to a previously undescribed mode of RNA binding, involving coiled-coil domains.
Assuntos
Proteínas de Transporte Vesicular , Proteínas rab5 de Ligação ao GTP , Humanos , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/análise , Proteínas rab5 de Ligação ao GTP/metabolismo , Endossomos/genética , Endossomos/metabolismoRESUMO
The objective of this research was to analyze the miR-145 function in thyroid papillary carcinoma cells and explore its possible mechanism. For this purpose, the TPC-1 cell line was selected, miR-145 overexpression and rab5c shRNA lentiviral vector were constructed, and transfected into PTC cells. Luciferase reporter gene was performed to determine the relationship between miR-145 and rab5c, Western blot and qPCR were performed to detach the expression of the related genes, CCK-8 cell proliferation assay and Transwell cell invasion assay were used to determine the proliferation and invasion ability of PTC-1 cells. Results showed that MiR-145 overexpression inhibited the wt-rab5c (wild-type rab5c)luciferase activity, decreased the expression of rab5c mRNA and protein levels in the TPC-1 cell line, inhibited the proliferation and invasion of PTC cell line TPC-1(P < 0.05). In TPC-1 cells, both miR-145 overexpression and RNA interference with rab5c could increase the expression of the p-ERK protein (P < 0.05). In conclusion, MiR-145 inhibits the proliferation and invasion of PTC cells by downregulating rab5c and activating MAPK/ERK pathway in vitro.
Assuntos
Carcinoma Papilar , MicroRNAs , Neoplasias da Glândula Tireoide , Humanos , Sistema de Sinalização das MAP Quinases/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Carcinoma Papilar/genética , Proliferação de Células/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Câncer Papilífero da Tireoide/genética , Câncer Papilífero da Tireoide/patologia , Transdução de Sinais , Regulação Neoplásica da Expressão Gênica , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismoRESUMO
Complement activation and Rab GTPase trafficking are commonly observed in inflammatory responses. Recruitment of innate immune cells to sites of infection or injury and secretion of inflammatory chemokines are promoted by complement component 5a (C5a) that activates the cell surface protein C5a receptor1 (C5aR1). Persistent activation can lead to a myriad of inflammatory and autoimmune diseases. Here, we demonstrate that the mechanism of C5a induced chemotaxis of human monocyte-derived macrophages (HMDMs) and their secretion of inflammatory chemokines are controlled by Rab5a. We find that C5a activation of the G protein coupled receptor C5aR1 expressed on the surface of HMDMs, recruits ß-arrestin2 via Rab5a trafficking, then activates downstream phosphatidylinositol 3-kinase (PI3K)/Akt signaling that culminates in chemotaxis and secretion of pro-inflammatory chemokines from HMDMs. High-resolution lattice light-sheet microscopy on live cells showed that C5a activates C5aR1-GFP internalization and colocalization with Rab5a-tdTomato but not with dominant negative mutant Rab5a-S34N-tdTomato in HEK293 cells. We found that Rab5a is significantly upregulated in differentiated HMDMs and internalization of C5aR1 is dependent on Rab5a. Interestingly, while knockdown of Rab5a inhibited C5aR1-mediated Akt phosphorylation, it did not affect C5aR1-mediated ERK1/2 phosphorylation or intracellular calcium mobilization in HMDMs. Functional analysis using transwell migration and µ-slide chemotaxis assays indicated that Rab5a regulates C5a-induced chemotaxis of HMDMs. Further, C5aR1 was found to mediate interaction of Rab5a with ß-arrestin2 but not with G proteins in HMDMs. Furthermore, C5a-induced secretion of pro-inflammatory chemokines (CCL2, CCL3) from HMDMs was attenuated by Rab5a or ß-arrestin2 knockdown or by pharmacological inhibition with a C5aR1 antagonist or a PI3K inhibitor. These findings reveal a C5a-C5aR1-ß-arrestin2-Rab5a-PI3K signaling pathway that regulates chemotaxis and pro-inflammatory chemokine secretion in HMDMs and suggests new ways of selectively modulating C5a-induced inflammatory outputs.