RESUMO
E-cadherin, a transmembrane protein involved in epithelial cell-cell adhesion and signaling, is found in exosomal fractions isolated from human body fluids. A cellular mechanism for recruitment of E-cadherin into extracellular vesicles (EVs) has not yet been defined. Here, we show that E-cadherin is incorporated into the membrane of EVs with the extracellular domain exposed at the vesicle surface. This recruitment depends on the endosomal sorting complex required for transport I (ESCRT-I) component Tsg101 and a highly conserved tetrapeptide P(S/T)AP late domain motif in the cytoplasmic tail of E-cadherin that mediates interaction with Tsg101. Mutation of this motif results in a loss of interaction and a dramatic decrease in exosomal E-cadherin secretion. We conclude, that the process of late domain mediated exosomal recruitment is exerted by this endogenous non-ESCRT transmembrane protein.
RESUMO
Banfer and Jacob introduce us to galectins - ß-galactoside-binding lectins - and their range of functions in multicellular organisms.
Assuntos
GalectinasRESUMO
Cells use unconventional secretion to deliver the ß-galactoside binding lectin galectin-3 from the cell interior into the extracellular milieu. This process starts with galectin-3 recruitment into intraluminal vesicles (ILVs), which are later released at the plasma membrane as exosomes. Electron microscopy is utilized to determine the location of GFP-tagged galectin-3 in pelleted exosomes. We also describe how these vesicles are harvested from cell culture media to determine their composition. The fluorescent protein GFP was fused with the exosomal sorting motif of galectin-3 to direct GFP into exosomes. Recruitment of this fusion construct into the lumen of exosomes can be assessed by proteinase K accessibility analysis.
Assuntos
Exossomos , Corpos Multivesiculares , Exocitose , Exossomos/metabolismo , Galectina 3/genética , Galectina 3/metabolismo , Galectinas/metabolismo , Corpos Multivesiculares/metabolismoRESUMO
Due to its increasing production, durability and multiple applications, plastic is a material we encounter every day. Small plastic particles from the µm to the mm range are classified as microplastics and produced for cosmetic and medical products, but are also a result of natural erosion and decomposition of macroplastics. Although being omnipresent in our environment and already detected in various organisms, less is known about the effects of microplastics on humans in general, or on vascular biology in particular. Here we investigated the effects of carboxylated polystyrene microplastic particles (PS, 1 µm) on murine endothelial and immune cells, which are both crucially involved in vascular inflammation, using in vitro and in vivo approaches. In vitro, PS induced adhesion molecule expression in endothelial cells with subsequent adhesion of leukocytes both under static and flow conditions. In monocytic cells, PS enhanced pro-inflammatory cytokine expression and release. Accordingly, administering mice with PS led to enhanced aortic expression of cytokines and adhesion molecules. Furthermore, we identified neutrophils as the PS-clearing blood leukocyte population. The findings from this study for the first time indicate polystyrene microplastic as a new environmental risk factor for endothelial inflammation.
Assuntos
Células Endoteliais/efeitos dos fármacos , Microplásticos/efeitos adversos , Plásticos/efeitos adversos , Poliestirenos/efeitos adversos , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Ácidos Carboxílicos/efeitos adversos , Linhagem Celular , Citocinas/metabolismo , Células Endoteliais/metabolismo , Humanos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismoRESUMO
Carbohydrate-binding galectins are expressed in various tissues of multicellular organisms. They are involved in autophagy, cell migration, immune response, inflammation, intracellular transport, and signaling. In recent years, novel roles of galectin-interaction with membrane components have been characterized, which lead to the formation of vesicles with diverse functions. These vesicles are part of intracellular transport pathways, belong to the cellular degradation machinery, or can be released for cell-to-cell communication. Several characteristics of galectins in the lumen or at the membrane of newly formed vesicular structures are discussed in this review and illustrate the need to fully elucidate their contributions at the molecular and structural level.
Assuntos
Galectinas/metabolismo , Animais , Transporte Biológico Ativo , Comunicação Celular , Vesículas Citoplasmáticas/metabolismo , Exossomos/metabolismo , Vesículas Extracelulares/metabolismo , Galectinas/química , Humanos , Modelos Biológicos , Transdução de Sinais , Vesículas Transportadoras/metabolismoRESUMO
Maturation of iron-sulfur (Fe-S) proteins in eukaryotes requires complex machineries in mitochondria and cytosol. Initially, Fe-S clusters are assembled on dedicated scaffold proteins and then are trafficked to target apoproteins. Within the cytosolic Fe-S protein assembly (CIA) machinery, the conserved P-loop nucleoside triphosphatase Nbp35 performs a scaffold function. In yeast, Nbp35 cooperates with the related Cfd1, which is evolutionary less conserved and is absent in plants. Here, we investigated the potential scaffold function of human CFD1 (NUBP2) in CFD1-depleted HeLa cells by measuring Fe-S enzyme activities or 55Fe incorporation into Fe-S target proteins. We show that CFD1, in complex with NBP35 (NUBP1), performs a crucial role in the maturation of all tested cytosolic and nuclear Fe-S proteins, including essential ones involved in protein translation and DNA maintenance. CFD1 also matures iron regulatory protein 1 and thus is critical for cellular iron homeostasis. To better understand the scaffold function of CFD1-NBP35, we resolved the crystal structure of Chaetomium thermophilum holo-Cfd1 (ctCfd1) at 2.6-Å resolution as a model Cfd1 protein. Importantly, two ctCfd1 monomers coordinate a bridging [4Fe-4S] cluster via two conserved cysteine residues. The surface-exposed topology of the cluster is ideally suited for both de novo assembly and facile transfer to Fe-S apoproteins mediated by other CIA factors. ctCfd1 specifically interacted with ATP, which presumably associates with a pocket near the Cfd1 dimer interface formed by the conserved Walker motif. In contrast, ctNbp35 preferentially bound GTP, implying differential regulation of the two fungal scaffold components during Fe-S cluster assembly and/or release.
Assuntos
Apoproteínas/química , Chaetomium/química , Proteínas Fúngicas/química , Proteínas de Ligação ao GTP/química , Proteína 1 Reguladora do Ferro/química , Proteínas Ferro-Enxofre/química , Motivos de Aminoácidos , Apoproteínas/genética , Apoproteínas/metabolismo , Domínio Catalítico , Chaetomium/genética , Chaetomium/metabolismo , Cristalografia por Raios X , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Células HeLa , Humanos , Proteína 1 Reguladora do Ferro/genética , Proteína 1 Reguladora do Ferro/metabolismo , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismoRESUMO
The beta-galactoside binding lectin galectin-3 (Gal3) is found intracellularly and in the extracellular space. Secretion of this lectin is mediated independently of the secretory pathway by a not yet defined nonclassical mechanism. Here, we found Gal3 in the lumen of exosomes. Superresolution and electron microscopy studies visualized Gal3 recruitment and sorting into intraluminal vesicles. Exosomal Gal3 release depends on the endosomal sorting complex required for transport I (ESCRT-I) component Tsg101 and functional Vps4a. Either Tsg101 knockdown or expression of dominant-negative Vps4aE228Q causes an intracellular Gal3 accumulation at multivesicular body formation sites. In addition, we identified a highly conserved tetrapeptide P(S/T)AP motif in the amino terminus of Gal3 that mediates a direct interaction with Tsg101. Mutation of the P(S/T)AP motif results in a loss of interaction and a dramatic decrease in exosomal Gal3 secretion. We conclude that Gal3 is a member of endogenous non-ESCRT proteins which are P(S/T)AP tagged for exosomal release.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Exossomos/metabolismo , Galectina 3/metabolismo , Corpos Multivesiculares/metabolismo , Fatores de Transcrição/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Cães , Endossomos/ultraestrutura , Exossomos/ultraestrutura , Células Madin Darby de Rim Canino , Microscopia Eletrônica , Corpos Multivesiculares/ultraestruturaRESUMO
Ca(2+)-activated K(+) channels (KCa) play a pivotal role in the endothelium-dependent hyperpolarization and regulation of vascular tone and blood pressure. For activation, KCa depend on an increase of intracellular calcium which is substantially mediated by Ca(2+)-permeable cation channels including the transient receptor potential V4 (TRPV4). It has been proposed that KCa and Ca(2+)-permeable cation channels may be clustered in localized positions within the cell membrane to form functional units and that caveolae may constitute the scaffolding for such microcompartmental organization. Here, we sought to elucidate the composition and functional relevance of these microcompartments in vitro and in vivo. We show that TRPV4 and small-conductance KCa2.3 are enriched in caveolae of human microvascular endothelial cells. Using immunoprecipitation, immunocytology and superresolution microscopy, we found a caveolae-dependent association between caveolin-1, TRPV4 and small conductance KCa2.3, but not intermediate conductance KCa3.1, in endothelial cells under static condition. Mechanical stimulation of cells via exposure to shear stress led to a partial de-novo colocalization of KCa3.1 with Cav-1 and TRPV4. In a mouse model of genetic Cav-1 deficiency, we found significantly reduced KCa-mediated currents as determined by patch-clamping in carotid artery endothelial cells (CAEC) from Cav-1(-/-) mice compared to wildtype. Functionally, Cav-1 deficiency was associated with impaired endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilation in response to shear stress and acetylcholine. In summary, our findings provide evidence for a dynamic microcompartmentation of TRPV4/KCa in caveolae of endothelial cells and highlight the importance of Cav-1 for endothelial KCa functions and flow-induced vasodilation.
Assuntos
Cavéolas/fisiologia , Caveolina 1/metabolismo , Células Endoteliais/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Cálcio/metabolismo , Caveolina 1/genética , Compartimento Celular , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Camundongos , Camundongos Knockout , Técnicas de Patch-Clamp , Vasodilatação/fisiologiaRESUMO
The cytoplasmic RNA helicase RIG-I mediates innate sensing of RNA viruses. The genomes of influenza A virus (FLUAV) are encapsidated by the nucleoprotein and associated with RNA polymerase, posing potential barriers to RIG-I sensing. We show that RIG-I recognizes the 5'-triphosphorylated dsRNA on FLUAV nucleocapsids but that polymorphisms at position 627 of the viral polymerase subunit PB2 modulate RIG-I sensing. Compared to mammalian-adapted PB2-627K, avian FLUAV nucleocapsids possessing PB2-627E are prone to increased RIG-I recognition, and RIG-I-deficiency partially restores PB2-627E virus infection of mammalian cells. Heightened RIG-I sensing of PB2-627E nucleocapsids correlates with previously established lower affinity of 627E-containing PB2 for nucleoprotein and is increased by further nucleocapsid instability. The effect of RIG-I on PB2-627E nucleocapsids is independent of antiviral signaling, suggesting that RIG-I-nucleocapsid binding alone can inhibit infection. These results indicate that RIG-I is a direct avian FLUAV restriction factor and highlight nucleocapsid disruption as an antiviral strategy.
Assuntos
RNA Helicases DEAD-box/metabolismo , Interações Hospedeiro-Patógeno , Vírus da Influenza A/imunologia , Nucleocapsídeo/imunologia , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Animais , Linhagem Celular , Proteína DEAD-box 58 , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/fisiologia , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Nucleocapsídeo/genética , Nucleocapsídeo/fisiologia , Orthomyxoviridae , Ligação Proteica , RNA de Cadeia Dupla/metabolismo , RNA Viral/metabolismo , Receptores Imunológicos , Replicação ViralRESUMO
Suppressor of fused (SUFU) is an essential negative regulator of the mammalian Hedgehog (HH) signaling pathway and its loss is associated with cancer development. On a cellular level, endogenous SUFU can mainly be detected in the cytoplasm and the nucleus. However, immunostaining of pancreatic cancer specimen revealed the existence of cell types showing selective enrichment of endogenous SUFU in the nucleus. Following up on this observation, we found that a SUFU construct which was experimentally tethered exclusively to the nucleus was unable to antagonize endogenous HH signaling, in contrast to control SUFU. These data suggest that alterations in the normal subcellular distribution of SUFU might interfere with its established negative role on the HH pathway. Performing a multi-well kinase screen in human cells identified RIO kinase 3 (RIOK3) as a novel modulator of SUFU subcellular distribution. Functionally, RIOK3 acts as a SUFU-dependent positive regulator of HH signaling. Taken together, we propose that factors modulating the nucleo-cytoplasmic distribution of SUFU impact on the normal function of this tumor suppressing protein.
Assuntos
Proteínas Hedgehog/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Animais , Células COS , Nucléolo Celular/metabolismo , Chlorocebus aethiops , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Proteínas Repressoras/análiseRESUMO
The role of post-translational tubulin modifications in the development and maintenance of a polarized epithelium is not well understood. We studied the balance between detyrosinated (detyr-) and tyrosinated (tyr-) tubulin in the formation of MDCK cell monolayers. Increased quantities of detyrosinated microtubules were detected during assembly into confluent cell sheets. These tubules were composed of alternating stretches of detyr- and tyr-tubulin. Constant induction of tubulin tyrosination, which decreased the levels of detyr-tubulin by overexpression of tubulin tyrosine ligase (TTL), disrupted monolayer establishment. Detyr-tubulin-depleted cells assembled into isolated islands and developed a prematurely polarized architecture. Thus, tubulin detyrosination is required for the morphological differentiation from non-polarized cells into an epithelial monolayer. Moreover, membrane trafficking, in particular to the apical domain, was slowed down in TTL-overexpressing cells. This effect could be reversed by TTL knockdown, which suggests that detyr-tubulin-enriched microtubules serve as cytoskeletal tracks to guide membrane cargo in polarized MDCK cells.