RESUMO
Selective retrograde transport from endosomes back to the trans-Golgi network (TGN) is important for maintaining protein homeostasis, recycling receptors, and returning molecules that were transported to the wrong compartments. Two important transmembrane proteins directed to this pathway are the Cation-Independent Mannose-6-phosphate receptor (CI-MPR) and the ATP7B copper transporter. Among CI-MPR functions is the delivery of acid hydrolases to lysosomes, while ATP7B facilitates the transport of cytosolic copper ions into organelles or the extracellular space. Precise subcellular localization of CI-MPR and ATP7B is essential for the proper functioning of these proteins. This study shows that both CI-MPR and ATP7B interact with a variant of the clathrin adaptor 1 (AP-1) complex that contains a specific isoform of the γ-adaptin subunit called γ2. Through synchronized anterograde trafficking and cell-surface uptake assays, we demonstrated that AP-1γ2 is dispensable for ATP7B and CI-MPR exit from the TGN while being critically required for ATP7B and CI-MPR retrieval from endosomes to the TGN. Moreover, AP-1γ2 depletion leads to the retention of endocytosed CI-MPR in endosomes enriched in retromer complex subunits. These data underscore the importance of AP-1γ2 as a key component in the sorting and trafficking machinery of CI-MPR and ATP7B, highlighting its essential role in the transport of proteins from endosomes.
Assuntos
Complexo 1 de Proteínas Adaptadoras , ATPases Transportadoras de Cobre , Endossomos , Transporte Proteico , Receptor IGF Tipo 2 , Rede trans-Golgi , Humanos , Endossomos/metabolismo , Células HeLa , Transporte Proteico/genética , Receptor IGF Tipo 2/genética , Receptor IGF Tipo 2/metabolismo , Rede trans-Golgi/genética , Rede trans-Golgi/metabolismo , ATPases Transportadoras de Cobre/genética , ATPases Transportadoras de Cobre/metabolismo , Complexo 1 de Proteínas Adaptadoras/genética , Complexo 1 de Proteínas Adaptadoras/metabolismo , Subunidades gama do Complexo de Proteínas Adaptadoras/metabolismoRESUMO
Studying the evolutionary history of gene families is a challenging and exciting task with a wide range of implications. In addition to exploring fundamental questions about the origin and evolution of genes, disentangling their evolution is also critical to those who do functional/structural studies to allow a deeper and more precise interpretation of their results in an evolutionary context. The sirtuin gene family is a group of genes that are involved in a variety of biological functions mostly related to aging. Their duplicative history is an open question, as well as the definition of the repertoire of sirtuin genes among vertebrates. Our results show a well-resolved phylogeny that represents an improvement in our understanding of the duplicative history of the sirtuin gene family. We identified a new sirtuin gene family member (SIRT3.2) that was apparently lost in the last common ancestor of amniotes but retained in all other groups of jawed vertebrates. According to our experimental analyses, elephant shark SIRT3.2 protein is located in mitochondria, the overexpression of which leads to an increase in cellular levels of ATP. Moreover, in vitro analysis demonstrated that it has deacetylase activity being modulated in a similar way to mammalian SIRT3. Our results indicate that there are at least eight sirtuin paralogs among vertebrates and that all of them can be traced back to the last common ancestor of the group that existed between 676 and 615 millions of years ago.
Assuntos
Sirtuína 3 , Sirtuínas , Animais , Sirtuínas/genética , Sirtuína 3/genética , Evolução Molecular , Vertebrados/genética , Filogenia , MamíferosRESUMO
Viviana A. Cavieres and Gonzalo A. Mardones were not included as authors in the original publication [...].
RESUMO
Glycolipid glycosylation is an intricate process that mainly takes place in the Golgi by the complex interplay between glycosyltransferases. Several features such as the organization, stoichiometry and composition of these complexes may modify their sorting properties, sub-Golgi localization, enzymatic activity and in consequence, the pattern of glycosylation at the plasma membrane. In spite of the advance in our comprehension about physiological and pathological cellular states of glycosylation, the molecular basis underlying the metabolism of glycolipids and the players involved in this process remain not fully understood. In the present work, using biochemical and fluorescence microscopy approaches, we demonstrate the existence of a physical association between two ganglioside glycosyltransferases, namely, ST3Gal-II (GD1a synthase) and ß3GalT-IV (GM1 synthase) with Golgi phosphoprotein 3 (GOLPH3) in mammalian cultured cells. After GOLPH3 knockdown, the localization of both enzymes was not affected, but the fomation of ST3Gal-II/ß3GalT-IV complex was compromised and glycolipid expression pattern changed. Our results suggest a novel control mechanism of glycolipid expression through the regulation of the physical association between glycolipid glycosyltransferases mediated by GOLPH3.
Assuntos
Glicolipídeos , Glicosiltransferases , Animais , Gangliosídeo G(M1)/metabolismo , Gangliosídeos/metabolismo , Glicolipídeos/metabolismo , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Complexo de Golgi/metabolismo , Mamíferos/metabolismo , Fosfoproteínas/metabolismoRESUMO
Protein trafficking is altered when normal cells acquire a tumor phenotype. A key subcellular compartment in regulating protein trafficking is the Golgi apparatus, but its role in carcinogenesis is still not well defined. Golgi phosphoprotein 3 (GOLPH3), a peripheral membrane protein mostly localized at the trans-Golgi network, is overexpressed in several tumor types including glioblastoma multiforme (GBM), the most lethal primary brain tumor. Moreover, GOLPH3 is currently considered an oncoprotein, however its precise function in GBM is not fully understood. Here, we analyzed in T98G cells of GBM, which express high levels of epidermal growth factor receptor (EGFR), the effect of stable RNAi-mediated knockdown of GOLPH3. We found that silencing GOLPH3 caused a significant reduction in the proliferation of T98G cells and an unexpected increase in total EGFR levels, even at the cell surface, which was however less prone to ligand-induced autophosphorylation. Furthermore, silencing GOLPH3 decreased EGFR sialylation and fucosylation, which correlated with delayed ligand-induced EGFR downregulation and its accumulation at endo-lysosomal compartments. Finally, we found that EGF failed at promoting EGFR ubiquitylation when the levels of GOLPH3 were reduced. Altogether, our results show that GOLPH3 in T98G cells regulates the endocytic trafficking and activation of EGFR likely by affecting its extent of glycosylation and ubiquitylation.
Assuntos
Carcinogênese/genética , Glioblastoma/genética , Proteínas de Membrana/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Receptores ErbB/genética , Regulação Neoplásica da Expressão Gênica/genética , Glioblastoma/patologia , Glicosilação , Complexo de Golgi/genética , Humanos , Proteínas de Membrana/antagonistas & inibidores , Transporte Proteico/genética , Ubiquitinação/genética , Rede trans-Golgi/genéticaRESUMO
Lipid droplets (LD) are dynamically-regulated organelles that originate from the endoplasmic reticulum (ER), and function in the storage, trafficking and metabolism of neutral lipids. In mammary epithelial cells (MEC) of lactating animals, intact LD are secreted intact into milk to form milk lipids by a novel apocrine mechanism. The secretion of intact LD and the relatively large amounts of lipid secreted by lactating MEC increase demands on the cellular processes responsible for lipid synthesis and LD formation. As yet these processes are poorly defined due to limited understanding of LD-ER interactions. To overcome these limitations, we used rapid-freezing and freeze-substitution methods in conjunction with 3D electron tomography and high resolution immunolocalization to define interactions between LD with ER in MEC of pregnant and lactating rats. Using these approaches, we identified distinct ER domains that contribute to lipid droplet formation and stabilization and which possess unique features previously unrecognized or not fully appreciated. Our results show nascent lipid droplets within the ER lumen and the association of both forming and mature droplets with structurally unique regions of ER cisternae, characterized by the presence of perilipin-2, a protein implicated in lipid droplet formation, and enzymes involved in lipid synthesis. These data demonstrate that milk lipids originate from LD-ER domains with novel structural features and suggest a mechanism for initial droplet formation in the ER lumen and subsequent maturation of the droplets in association with ER cisternae.
Assuntos
Tomografia com Microscopia Eletrônica/métodos , Retículo Endoplasmático/metabolismo , Gotículas Lipídicas/metabolismo , Lipídeos/análise , Glândulas Mamárias Animais/ultraestrutura , Leite/química , Animais , Retículo Endoplasmático/ultraestrutura , Feminino , Lactação , Gotículas Lipídicas/ultraestrutura , Glândulas Mamárias Animais/metabolismo , Perilipina-1/metabolismo , Gravidez , RatosRESUMO
The permeability of endothelial cells is regulated by the stability of the adherens junctions, which is highly sensitive to kinase-mediated phosphorylation and endothelial nitric oxide synthase (eNOS)-mediated S-nitrosylation of its protein components. Solid tumors can produce a variety of factors that stimulate these signaling pathways leading to endothelial cell hyperpermeability. This generates stromal conditions that facilitate tumoral growth and dissemination. Galectin-8 (Gal-8) is overexpressed in several carcinomas and has a variety of cellular effects that can contribute to tumor pathogenicity, including angiogenesis. Here we explored whether Gal-8 has also a role in endothelial permeability. We show that recombinant Gal-8 activates eNOS, induces S-nitrosylation of p120-catenin (p120) and dissociation of adherens junction, leading to hyperpermeability of the human endothelial cell line EAhy926. This pathway involves focal-adhesion kinase (FAK) activation downstream of eNOS as a requirement for eNOS-mediated p120 S-nitrosylation. This suggests a reciprocal, yet little understood, regulation of phosphorylation and S-nitrosylation events acting upon adherens junction permeability. In addition, glutathione S-transferase (GST)-Gal-8 pull-down experiments and function-blocking ß1-integrin antibodies point to ß1-integrins as cell surface components involved in Gal-8-induced hyperpermeability. Endogenous Gal-8 secreted from the breast cancer cell line MCF-7 has similar hyperpermeability and signaling effects. Furthermore, the mouse cremaster model system showed that Gal-8 also activates eNOS, induces S-nitrosylation of adherens junction components and is an effective hyperpermeability agent in vivo. These results add endothelial permeability regulation by S-nitrosylation as a new function of Gal-8 that can potentially contribute to the pathogenicity of tumors overexpressing this lectin.
Assuntos
Junções Aderentes/metabolismo , Galectinas/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Transdução de Sinais/fisiologia , Animais , Linhagem Celular Tumoral , Células Endoteliais/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Glutationa Transferase , Humanos , Células MCF-7 , Masculino , Camundongos , Fosforilação/fisiologiaRESUMO
The HIV accessory protein Nef is a major determinant of viral pathogenesis that facilitates viral particle release, prevents viral antigen presentation and increases infectivity of new virus particles. These functions of Nef involve its ability to remove specific host proteins from the surface of infected cells, including the CD4 receptor. Nef binds to the adaptor protein 2 (AP-2) and CD4 in clathrin-coated pits, forcing CD4 internalization and its subsequent targeting to lysosomes. Herein, we report that this lysosomal targeting requires a variant of AP-1 containing isoform 2 of γ-adaptin (AP1G2, hereafter γ2). Depletion of the γ2 or µ1A (AP1M1) subunits of AP-1, but not of γ1 (AP1G1), precludes Nef-mediated lysosomal degradation of CD4. In γ2-depleted cells, CD4 internalized by Nef accumulates in early endosomes and this alleviates CD4 removal from the cell surface. Depletion of γ2 also hinders EGFR-EGF-complex targeting to lysosomes, an effect that is not observed upon γ1 depletion. Taken together, our data provide evidence that the presence of γ1 or γ2 subunits delineates two distinct variants of AP-1 complexes, with different functions in protein sorting.
Assuntos
Subunidades gama do Complexo de Proteínas Adaptadoras/metabolismo , Antígenos CD4/metabolismo , Regulação para Baixo , HIV-1/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Endocitose , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Lisossomos/metabolismo , Fosfoproteínas/metabolismo , Ligação Proteica , Transporte Proteico , Proteólise , Técnicas do Sistema de Duplo-HíbridoRESUMO
Brain regions affected by Alzheimer disease (AD) display well-recognized early neuropathologic features in the endolysosomal and autophagy systems of neurons, including enlargement of endosomal compartments, progressive accumulation of autophagic vacuoles, and lysosomal dysfunction. Although the primary causes of these disturbances are still under investigation, a growing body of evidence suggests that the amyloid precursor protein (APP) intracellular C-terminal fragment ß (C99), generated by cleavage of APP by ß-site APP cleaving enzyme 1 (BACE-1), is the primary cause of the endosome enlargement in AD and the earliest initiator of synaptic plasticity and long-term memory impairment. The aim of the present study was to evaluate the possible relationship between the endolysosomal degradation pathway and autophagy on the proteolytic processing and turnover of C99. We found that pharmacologic treatments that either inhibit autophagosome formation or block the fusion of autophagosomes to endolysosomal compartments caused an increase in C99 levels. We also found that inhibition of autophagosome formation by depletion of Atg5 led to higher levels of C99 and to its massive accumulation in the lumen of enlarged perinuclear, lysosomal-associated membrane protein 1 (LAMP1)-positive organelles. In contrast, activation of autophagosome formation, either by starvation or by inhibition of the mammalian target of rapamycin, enhanced lysosomal clearance of C99. Altogether, our results indicate that autophagosomes are key organelles to help avoid C99 accumulation preventing its deleterious effects.-González, A. E., Muñoz, V. C., Cavieres, V. A., Bustamante, H. A., Cornejo, V.-H., Januário, Y. C., González, I., Hetz, C., daSilva, L. L., Rojas-Fernández, A., Hay, R. T., Mardones, G. A., Burgos, P. V. Autophagosomes cooperate in the degradation of intracellular C-terminal fragments of the amyloid precursor protein via the MVB/lysosomal pathway.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Autofagossomos/fisiologia , Lisossomos/fisiologia , Corpos Multivesiculares/fisiologia , Precursor de Proteína beta-Amiloide/genética , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Regulação da Expressão Gênica/fisiologia , Inativação Gênica , Humanos , Naftiridinas/farmacologia , Neuroglia , RNA Interferente Pequeno , Serina-Treonina Quinases TOR/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Newly synthesized envelope glycoproteins of neuroinvasive viruses can be sorted in a polarized manner to the somatodendritic and/or axonal domains of neurons. Although critical for transneuronal spread of viruses, the molecular determinants and interregulation of this process are largely unknown. We studied the polarized sorting of the attachment (NiV-G) and fusion (NiV-F) glycoproteins of Nipah virus (NiV), a paramyxovirus that causes fatal human encephalitis, in rat hippocampal neurons. When expressed individually, NiV-G exhibited a non-polarized distribution, whereas NiV-F was specifically sorted to the somatodendritic domain. Polarized sorting of NiV-F was dependent on interaction of tyrosine-based signals in its cytosolic tail with the clathrin adaptor complex AP-1. Co-expression of NiV-G with NiV-F abolished somatodendritic sorting of NiV-F due to incorporation of NiV-Gâ¢NiV-F complexes into axonal transport carriers. We propose that faster biosynthetic transport of unassembled NiV-F allows for its proteolytic activation in the somatodendritic domain prior to association with NiV-G and axonal delivery of NiV-Gâ¢NiV-F complexes. Our study reveals how interactions of viral glycoproteins with the host's transport machinery and between themselves regulate their polarized sorting in neurons.
Assuntos
Polaridade Celular/fisiologia , Neurônios/metabolismo , Proteínas do Envelope Viral/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Embrião de Mamíferos , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Neurônios/fisiologia , Vírus Nipah , Sinais Direcionadores de Proteínas , Estrutura Terciária de Proteína , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Proteínas do Envelope Viral/química , Montagem de VírusRESUMO
Autophagy is an evolutionarily conserved process by which cytoplasmic proteins and organelles are catabolized. During starvation, the protein TOR (target of rapamycin), a nutrient-responsive kinase, is inhibited, and this induces autophagy. In autophagy, double-membrane autophagosomes envelop and sequester intracellular components and then fuse with lysosomes to form autolysosomes, which degrade their contents to regenerate nutrients. Current models of autophagy terminate with the degradation of the autophagosome cargo in autolysosomes, but the regulation of autophagy in response to nutrients and the subsequent fate of the autolysosome are poorly understood. Here we show that mTOR signalling in rat kidney cells is inhibited during initiation of autophagy, but reactivated by prolonged starvation. Reactivation of mTOR is autophagy-dependent and requires the degradation of autolysosomal products. Increased mTOR activity attenuates autophagy and generates proto-lysosomal tubules and vesicles that extrude from autolysosomes and ultimately mature into functional lysosomes, thereby restoring the full complement of lysosomes in the cell-a process we identify in multiple animal species. Thus, an evolutionarily conserved cycle in autophagy governs nutrient sensing and lysosome homeostasis during starvation.
Assuntos
Autofagia/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisossomos/metabolismo , Fenômenos Fisiológicos da Nutrição , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Linhagem Celular , Chlorocebus aethiops , Células HeLa , Homeostase/fisiologia , Humanos , Lisossomos/ultraestrutura , Ratos , Transdução de Sinais , Serina-Treonina Quinases TOR , Células VeroRESUMO
Tyrosine-based signals fitting the YXXØ motif mediate sorting of transmembrane proteins to endosomes, lysosomes, the basolateral plasma membrane of polarized epithelial cells, and the somatodendritic domain of neurons through interactions with the homologous µ1, µ2, µ3, and µ4 subunits of the corresponding AP-1, AP-2, AP-3, and AP-4 complexes. Previous x-ray crystallographic analyses identified distinct binding sites for YXXØ signals on µ2 and µ4, which were located on opposite faces of the proteins. To elucidate the mode of recognition of YXXØ signals by other members of the µ family, we solved the crystal structure at 1.85 Å resolution of the C-terminal domain of the µ3 subunit of AP-3 (isoform A) in complex with a peptide encoding a YXXØ signal (SDYQRL) from the trans-Golgi network protein TGN38. The µ3A C-terminal domain consists of an immunoglobulin-like ß-sandwich organized into two subdomains, A and B. The YXXØ signal binds in an extended conformation to a site on µ3A subdomain A, at a location similar to the YXXØ-binding site on µ2 but not µ4. The binding sites on µ3A and µ2 exhibit similarities and differences that account for the ability of both proteins to bind distinct sets of YXXØ signals. Biochemical analyses confirm the identification of the µ3A site and show that this protein binds YXXØ signals with 14-19 µm affinity. The surface electrostatic potential of µ3A is less basic than that of µ2, in part explaining the association of AP-3 with intracellular membranes having less acidic phosphoinositides.
Assuntos
Complexo 3 de Proteínas Adaptadoras/química , Subunidades mu do Complexo de Proteínas Adaptadoras/química , Tirosina/química , Complexo 3 de Proteínas Adaptadoras/metabolismo , Subunidades mu do Complexo de Proteínas Adaptadoras/metabolismo , Sequência de Aminoácidos , Animais , Calorimetria/métodos , Clatrina/química , Endossomos/metabolismo , Humanos , Imunoglobulinas/química , Lisossomos/química , Camundongos , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosfatidilinositóis/química , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Ratos , Homologia de Sequência de Aminoácidos , Eletricidade Estática , Tirosina/metabolismoRESUMO
Cetaceans represent a natural experiment within the tree of life in which a lineage changed from terrestrial to aquatic habitats. This shift involved phenotypic modifications, representing an opportunity to explore the genetic bases of phenotypic diversity. Among the different molecular systems that maintain cellular homeostasis, ion channels are crucial for the proper physiological functioning of all living species. This study aims to explore the evolution of ion channels during the evolutionary history of cetaceans. To do so, we created a bioinformatic pipeline to annotate the repertoire of ion channels in the genome of the species included in our sampling. Our main results show that cetaceans have, on average, fewer protein-coding genes and a higher percentage of annotated ion channels than non-cetacean mammals. Signals of positive selection were detected in ion channels related to the heart, locomotion, visual and neurological phenotypes. Interestingly, we predict that the NaV1.5 ion channel of most toothed whales (odontocetes) is sensitive to tetrodotoxin, similar to NaV1.7, given the presence of tyrosine instead of cysteine, in a specific position of the ion channel. Finally, the gene turnover rate of the cetacean crown group is more than three times faster than that of non-cetacean mammals.
Assuntos
Cetáceos , Evolução Molecular , Canais Iônicos , Animais , Cetáceos/genética , Cetáceos/fisiologia , Canais Iônicos/genética , Canais Iônicos/metabolismo , Filogenia , Biologia Computacional/métodos , GenomaRESUMO
Induction of the adenosine receptor A2B (A2BAR) expression in diabetic glomeruli correlates with an increased abundance of its endogenous ligand adenosine and the progression of kidney dysfunction. Remarkably, A2BAR antagonism protects from proteinuria in experimental diabetic nephropathy. We found that A2BAR antagonism preserves the arrangement of podocytes on the glomerular filtration barrier, reduces diabetes-induced focal adhesion kinase (FAK) activation, and attenuates podocyte foot processes effacement. In spreading assays using human podocytes in vitro, adenosine enhanced the rate of cell body expansion on laminin-coated glass and promoted peripheral pY397-FAK subcellular distribution, while selective A2BAR antagonism impeded these effects and attenuated the migratory capability of podocytes. Increased phosphorylation of the Myosin2A light chain accompanied the effects of adenosine. Furthermore, when the A2BAR was stimulated, the cells expanded more broadly and more staining of pS19 myosin was detected which co-localized with actin cables, suggesting increased contractility potential in cells planted onto a matrix with a stiffness similar to of the glomerular basement membrane. We conclude that A2BAR is involved in adhesion dynamics and contractile actin bundle formation, leading to podocyte foot processes effacement. The antagonism of this receptor may be an alternative to the intervention of glomerular barrier deterioration and proteinuria in the diabetic kidney disease.
Assuntos
Adesão Celular , Diabetes Mellitus Experimental , Proteína-Tirosina Quinases de Adesão Focal , Podócitos , Proteinúria , Receptor A2B de Adenosina , Podócitos/metabolismo , Podócitos/efeitos dos fármacos , Podócitos/patologia , Animais , Humanos , Proteinúria/metabolismo , Ratos , Receptor A2B de Adenosina/metabolismo , Adesão Celular/efeitos dos fármacos , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Masculino , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/tratamento farmacológico , Antagonistas do Receptor A2 de Adenosina/farmacologia , Adenosina/metabolismo , Adenosina/farmacologia , Movimento Celular/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Cadeias Leves de Miosina/metabolismoRESUMO
Adaptor protein complex 4 (AP-4) is a heterotetrameric complex that promotes export of selected cargo proteins from the trans-Golgi network. Mutations in each of the AP-4 subunits cause a complicated form of Hereditary Spastic Paraplegia (HSP). Herein, we report that ApoER2, a receptor in the Reelin signaling pathway, is a cargo of the AP-4 complex. We identify the motif ISSF/Y within the ApoER2 cytosolic domain as necessary for interaction with the canonical signal-binding pocket of the µ4 (AP4M1) subunit of AP-4. AP4E1- knock-out (KO) HeLa cells and hippocampal neurons from Ap4e1-KO mice display increased co-localization of ApoER2 with Golgi markers. Furthermore, hippocampal neurons from Ap4e1-KO mice and AP4M1-KO human iPSC-derived cortical i3Neurons exhibit reduced ApoER2 protein expression. Analyses of biosynthetic transport of ApoER2 reveal differential post-Golgi trafficking of the receptor, with lower axonal distribution in KO compared to wild-type neurons, indicating a role of AP-4 and the ISSF/Y motif in the axonal localization of ApoER2. Finally, analyses of Reelin signaling in mouse hippocampal and human cortical KO neurons show that AP4 deficiency causes no changes in Reelin-dependent activation of the AKT pathway and only mild changes in Reelin-induced dendritic arborization, but reduces Reelin-induced ERK phosphorylation, CREB activation, and Golgi deployment. This work thus establishes ApoER2 as a novel cargo of the AP-4 complex, suggesting that defects in the trafficking of this receptor and in the Reelin signaling pathway could contribute to the pathogenesis of HSP caused by mutations in AP-4 subunits.
Assuntos
Complexo 4 de Proteínas Adaptadoras , Proteínas Relacionadas a Receptor de LDL , Paraplegia Espástica Hereditária , Animais , Humanos , Camundongos , Complexo 4 de Proteínas Adaptadoras/genética , Complexo 4 de Proteínas Adaptadoras/metabolismo , Células HeLa , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas Relacionadas a Receptor de LDL/metabolismo , Receptores de Superfície Celular , Paraplegia Espástica Hereditária/genética , Paraplegia Espástica Hereditária/metabolismoRESUMO
In this study, we investigated the inter-organelle communication between the Golgi apparatus (GA) and mitochondria. Previous observations suggest that GA-derived vesicles containing phosphatidylinositol 4-phosphate (PI(4)P) play a role in mitochondrial fission, colocalizing with DRP1, a key protein in this process. However, the functions of these vesicles and potentially associated proteins remain unknown. GOLPH3, a PI(4)P-interacting GA protein, is elevated in various types of solid tumors, including breast cancer, yet its precise role is unclear. Interestingly, GOLPH3 levels influence mitochondrial mass by affecting cardiolipin synthesis, an exclusive mitochondrial lipid. However, the mechanism by which GOLPH3 influences mitochondria is not fully understood. Our live-cell imaging analysis showed GFP-GOLPH3 associating with PI(4)P vesicles colocalizing with YFP-DRP1 at mitochondrial fission sites. We tested the functional significance of these observations with GOLPH3 knockout in MDA-MB-231 cells of breast cancer, resulting in a fragmented mitochondrial network and reduced bioenergetic function, including decreased mitochondrial ATP production, mitochondrial membrane potential, and oxygen consumption. Our findings suggest a potential negative regulatory role for GOLPH3 in mitochondrial fission, impacting mitochondrial function and providing insights into GA-mitochondria communication.
Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/patologia , Células MDA-MB-231 , Dinâmica Mitocondrial , Complexo de Golgi/metabolismo , Metabolismo Energético , Proteínas de Membrana/metabolismoRESUMO
The early events of HIV-1 infection involve the transport of the viral core into the nucleus. This event triggers the translocation of CPSF6 from paraspeckles into nuclear speckles forming puncta-like structures. Our investigations revealed that neither HIV-1 integration nor reverse transcription is required for the formation of puncta-like structures. Moreover, HIV-1 viruses without viral genome are competent for the induction of CPSF6 puncta-like structures. In agreement with the notion that HIV-1 induced CPSF6 puncta-like structures are biomolecular condensates, we showed that osmotic stress and 1,6-hexanediol induced the disassembly of CPSF6 condensates. Interestingly, replacing the osmotic stress by isotonic media re-assemble CPSF6 condensates in the cytoplasm of the cell. To test whether CPSF6 condensates were important for infection we utilized hypertonic stress, which prevents formation of CPSF6 condensates, during infection. Remarkably, preventing the formation of CPSF6 condensates inhibits the infection of wild type HIV-1 but not of HIV-1 viruses bearing the capsid changes N74D and A77V, which do not form CPSF6 condensates during infection1,2. We also investigated whether the functional partners of CPSF6 are recruited to the condensates upon infection. Our experiments revealed that CPSF5, but not CPSF7, co-localized with CPSF6 upon HIV-1 infection. We found condensates containing CPSF6/CPSF5 in human T cells and human primary macrophages upon HIV-1 infection. Additionally, we observed that the integration cofactor LEDGF/p75 changes distribution upon HIV-1 infection and surrounds the CPSF6/CPSF5 condensates. Overall, our work demonstrated that CPSF6 and CPSF5 are forming biomolecular condensates that are important for infection of wild type HIV-1 viruses.
Assuntos
Infecções por HIV , Soropositividade para HIV , HIV-1 , Humanos , Condensados Biomoleculares , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Núcleo Celular/metabolismo , Soropositividade para HIV/metabolismo , HIV-1/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Replicação ViralRESUMO
High-Mobility Group (HMG) proteins are involved in different processes such as transcription, replication, DNA repair, and immune response. The role of HMG proteins in the immune response of fish has been studied mainly for HMGB1, where its expression can be induced by the stimulation of viral/bacterial PAMPs and can act as a proinflammatory mediator and as a global regulator of transcription in response to temperature. However, for BbX this role remains to be discovered. In this work, we identified the BbX of E. maclovinus and evaluated the temporal expression levels after simultaneous challenge with P. salmonis and thermal stress. Phylogenetic analysis does not significantly deviate from the expected organismal relationships suggesting orthologous relationships and that BbX was present in the common ancestor of the group. BbX mRNA expression levels were very high in the intestinal tissue of E. maclovinus (foregut, midgut, and hindgut). Nevertheless, the protein levels analyzed by WB showed the highest levels of BbX protein in the liver (constitutive expression). On the other hand, the mRNA expression levels of BbX in the liver of E. maclovinus injected with P. salmonis and subjected to thermal stress showed an increase at days 16 and 20 in all treatments applied at 12 °C and 18 °C. Meanwhile, the protein levels quantified by WB showed a statistically significant increase in the HMG-Bbx at all experimental times (4, 8, 12, 16, and 20 dpi). However, at 4 dpi the HMG-Bbx protein levels were much higher than the other days evaluated. The results suggest that BbX protein may be implicated in the response mechanism to temperature and bacterial stimulation in the foregut, midgut, hindgut, and liver, according to our findings at the level of mRNA and protein. Furthermore, our WB analysis suggests an effect of P. salmonis on the expression of this protein that can be observed in condition C+ 12 °C compared to C- 12 °C. Then, there is an effect of temperature that can be evidenced in the condition AM 18 °C and SM 18 °C, compared to AB 18 °C and SB 18 °C at 4, 8, and 12 dpi. We found not differences in the levels of this protein if the thermal stress is achieved through acclimatization or shock. More research is necessary to clarify the importance of this type of HMG in the immune response and thermal tolerance in fish.
Assuntos
Perciformes , Fatores de Transcrição , Animais , Filogenia , Regulação da Expressão Gênica , Peixes , RNA MensageiroRESUMO
Endogenous viral elements (EVEs) are genomic DNA sequences derived from viruses. Some EVEs have open reading frames (ORFs) that can express proteins with physiological roles in their host. Furthermore, some EVEs exhibit a protective role against exogenous viral infection in their host. Endogenous parvoviral elements (EPVs) are highly represented in mammalian genomes, and although some of them contain ORFs, their function is unknown. We have shown that the locus EPV-Dependo.43-ODegus, an EPV with an intact ORF, is transcribed in Octodon degus (degu). Here we examine the antiviral activity of the protein encoded in this EPV, named DeRep. DeRep was produced in bacteria and used to generate antibodies that recognize DeRep in western blots of degu tissue. To test if DeRep could protect against exogenous parvovirus, we challenged cells with the minute virus of mice (MVM), a model autonomous parvovirus. We observed that MVM protein expression, DNA damage induced by replication, viral DNA, and cytopathic effects are reduced when DeRep is expressed in cells. The results of this study demonstrate that DeRep is expressed in degu and can inhibit parvovirus replication. This is the first time that an EPV has been shown to have antiviral activity against an exogenous virus.
Assuntos
Infecções por Parvoviridae , Parvovirus , Vírus , Animais , Camundongos , Antivirais/farmacologia , Parvovirus/genética , Genoma , Vírus/genética , MamíferosRESUMO
Adaptor protein complex 4 (AP-4) is a heterotetrameric complex that promotes protein export from the trans -Golgi network. Mutations in each of the AP-4 subunits cause a complicated form of Hereditary Spastic Paraplegia (HSP). Herein, we report that ApoER2, a receptor in the Reelin signaling pathway, is a cargo of the AP-4 complex. We identify the motif ISSF/Y within the ApoER2 cytosolic domain as necessary for interaction with the canonical signal-binding pocket of the µ4 (AP4M1) subunit of AP-4. AP4E1 -knock-out (KO) HeLa cells and hippocampal neurons from Ap4e1 -KO mice display increased Golgi localization of ApoER2. Furthermore, hippocampal neurons from Ap4e1 -KO mice and AP4M1 -KO human iPSC-derived cortical i3Neurons exhibit reduced ApoER2 protein expression. Analyses of biosynthetic transport of ApoER2 reveal differential post-Golgi trafficking of the receptor, with lower axonal distribution in KO compared to wild-type neurons, indicating a role of AP-4 and the ISSF/Y motif in the axonal localization of ApoER2. Finally, analyses of Reelin signaling in mouse hippocampal and human cortical KO neurons show that AP4 deficiency causes no changes in Reelin-dependent activation of the AKT pathway and only mild changes in Reelin-induced dendritic arborization, but reduces Reelin-induced ERK phosphorylation, CREB activation, and Golgi deployment. Altogether, this work establishes ApoER2 as a novel cargo of the AP-4 complex, suggesting that defects in the trafficking of this receptor and in the Reelin signaling pathway could contribute to the pathogenesis of HSP caused by mutations in AP-4 subunits.