RESUMO
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 , Animais , Humanos , Masculino , Ratos , Adenosina/metabolismo , Adenosina/farmacologia , Antagonistas do Receptor A2 de Adenosina/farmacologia , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/tratamento farmacológico , Proteína-Tirosina Quinases de Adesão Focal/efeitos dos fármacos , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Cadeias Leves de Miosina/metabolismo , Fosforilação/efeitos dos fármacos , Podócitos/metabolismo , Podócitos/efeitos dos fármacos , Podócitos/patologia , Proteinúria/metabolismo , Receptor A2B de Adenosina/efeitos dos fármacos , Receptor A2B de Adenosina/metabolismoRESUMO
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
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
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
Viviana A. Cavieres and Gonzalo A. Mardones were not included as authors in the original publication [...].
RESUMO
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
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
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
The TAR DNA Binding Protein (TARDBP) gene has become relevant after the discovery of its several pathogenic mutations. The lack of evolutionary history is in contrast to the amount of studies found in the literature. This study investigated the evolutionary dynamics associated with the retrotransposition of the TARDBP gene in primates. We identified novel retropseudogenes that likely originated in the ancestors of anthropoids, catarrhines, and lemuriformes, i.e. the strepsirrhine clade that inhabit Madagascar. We also found species-specific retropseudogenes in the Philippine tarsier, Bolivian squirrel monkey, capuchin monkey and vervet. The identification of a retropseudocopy of the TARDBP gene overlapping a lncRNA that is potentially expressed opens a new avenue to investigate TARDBP gene regulation, especially in the context of TARDBP associated pathologies.
Assuntos
Primatas , Tarsiidae , Animais , Cebus , Cercopithecidae , Proteínas de Ligação a DNA/genética , Primatas/genética , Especificidade da Espécie , Tarsiidae/genéticaRESUMO
Golgi phosphoprotein 3 (GOLPH3) was the first reported oncoprotein of the Golgi apparatus. It was identified as an evolutionarily conserved protein upon its discovery about 20 years ago, but its function remains puzzling in normal and cancer cells. The GOLPH3 gene is part of a group of genes that also includes the GOLPH3L gene. Because cancer has deep roots in multicellular evolution, studying the evolution of the GOLPH3 gene family in non-model species represents an opportunity to identify new model systems that could help better understand the biology behind this group of genes. The main goal of this study is to explore the evolution of the GOLPH3 gene family in birds as a starting point to understand the evolutionary history of this oncoprotein. We identified a repertoire of three GOLPH3 genes in birds. We found duplicated copies of the GOLPH3 gene in all main groups of birds other than paleognaths, and a single copy of the GOLPH3L gene. We suggest there were at least three independent origins for GOLPH3 duplicates. Amino acid divergence estimates show that most of the variation is located in the N-terminal region of the protein. Our transcript abundance estimations show that one paralog is highly and ubiquitously expressed, and the others were variable. Our results are an example of the significance of understanding the evolution of the GOLPH3 gene family, especially for unraveling its structural and functional attributes.
Assuntos
Aves/genética , Evolução Molecular , Complexo de Golgi/genética , Proteínas de Membrana/genética , Proteínas Oncogênicas/genética , Sequência de Aminoácidos/genética , Animais , Carcinogênese/genética , Duplicação Gênica , Humanos , Neoplasias/genética , Fosfoproteínas/genética , Alinhamento de SequênciaRESUMO
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
Golgi phosphoprotein 3 (GOLPH3) is a peripheral membrane protein localized at the trans-Golgi network that is also distributed in a large cytosolic pool. GOLPH3 has been involved in several post-Golgi protein trafficking events, but its precise function at the molecular level is not well understood. GOLPH3 is also considered the first oncoprotein of the Golgi apparatus, with important roles in several types of cancer. Yet, it is unknown how GOLPH3 is regulated to achieve its contribution in the mechanisms that lead to tumorigenesis. Binding of GOLPH3 to Golgi membranes depends on its interaction to phosphatidylinositol-4-phosphate. However, an early finding showed that GTP promotes the binding of GOLPH3 to Golgi membranes and vesicles. Nevertheless, it remains largely unknown whether this response is consequence of the function of GTP-dependent regulatory factors, such as proteins of the RAB family of small GTPases. Interestingly, in Drosophila melanogaster the ortholog of GOLPH3 interacts with- and behaves as effector of the ortholog of RAB1. However, there is no experimental evidence implicating GOLPH3 as a possible RAB1 effector in mammalian cells. Here, we show that human GOLPH3 interacted directly with either RAB1A or RAB1B, the two isoforms of RAB1 in humans. The interaction was nucleotide dependent and it was favored with GTP-locked active state variants of these GTPases, indicating that human GOLPH3 is a bona fide effector of RAB1A and RAB1B. Moreover, the expression in cultured cells of the GTP-locked variants resulted in less distribution of GOLPH3 in the Golgi apparatus, suggesting an intriguing model of GOLPH3 regulation.
Assuntos
Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Proteínas rab1 de Ligação ao GTP/metabolismo , Células HeLa , Humanos , Proteínas de Membrana/genética , Transporte Proteico , Proteínas rab1 de Ligação ao GTP/genética , Rede trans-GolgiRESUMO
Ubiquitination regulates several biological processes, however the role of specific members of the ubiquitinome on intracellular membrane trafficking is not yet fully understood. Here, we search for ubiquitin-related genes implicated in protein membrane trafficking performing a High-Content siRNA Screening including 1187 genes of the human "ubiquitinome" using amyloid precursor protein (APP) as a reporter. We identified the deubiquitinating enzyme PSMD14, a subunit of the 19S regulatory particle of the proteasome, specific for K63-Ub chains in cells, as a novel regulator of Golgi-to-endoplasmic reticulum (ER) retrograde transport. Silencing or pharmacological inhibition of PSMD14 with Capzimin (CZM) caused a robust increase in APP levels at the Golgi apparatus and the swelling of this organelle. We showed that this phenotype is the result of rapid inhibition of Golgi-to-ER retrograde transport, a pathway implicated in the early steps of the autophagosomal formation. Indeed, we observed that inhibition of PSMD14 with CZM acts as a potent blocker of macroautophagy by a mechanism related to the retention of Atg9A and Rab1A at the Golgi apparatus. As pharmacological inhibition of the proteolytic core of the 20S proteasome did not recapitulate these effects, we concluded that PSMD14, and the K63-Ub chains, act as a crucial regulatory factor for macroautophagy by controlling Golgi-to-ER retrograde transport.
Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Macroautofagia , Complexo de Endopeptidases do Proteassoma/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Células HeLa , Humanos , Proteínas de Membrana/metabolismo , Modelos Biológicos , Fenótipo , Transporte Proteico , RNA Interferente Pequeno/metabolismo , Reprodutibilidade dos Testes , Transativadores/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab1 de Ligação ao GTP/metabolismoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Golgi phosphoprotein 3 (GOLPH3) is a conserved protein of the Golgi apparatus that in humans has been implicated in tumorigenesis. However, the precise function of GOLPH3 in malignant transformation is still unknown. Nevertheless, clinicopathological data shows that in more than a dozen kinds of cancer, including gliomas, GOLPH3 could be found overexpressed, which correlates with poor prognosis. Experimental data shows that overexpression of GOLPH3 leads to transformation of primary cells and to tumor growth enhancement. Conversely, the knocking down of GOLPH3 in GOLPH3-overexpressing tumor cells reduces tumorigenic features, such as cell proliferation and cell migration and invasion. The cumulative evidence indicate that GOLPH3 is an oncoprotein that promotes tumorigenicity by a mechanism that impact at different levels in different types of cells, including the sorting of Golgi glycosyltransferases, signaling pathways, and the actin cytoskeleton. How GOLPH3 connects mechanistically these processes has not been determined yet. Further studies are important to have a more complete understanding of the role of GOLPH3 as oncoprotein. Given the genetic diversity in cancer, a still outstanding aspect is how in this inherent heterogeneity GOLPH3 could possibly exert its oncogenic function. We have aimed to evaluate the contribution of GOLPH3 overexpression in the malignant phenotype of different types of tumor cells. Here, we analyzed the effect on cell migration that resulted from stable, RNAi-mediated knocking down of GOLPH3 in T98G cells of glioblastoma multiforme, a human glioma cell line with unique features. We found that the reduction of GOLPH3 levels produced dramatic changes in cell morphology, involving rearrangements of the actin cytoskeleton and reduction in the number and dynamics of focal adhesions. These effects correlated with decreased cell migration and invasion due to affected persistence and directionality of cell motility. Moreover, the knocking down of GOLPH3 also caused a reduction in autoactivation of focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase that regulates focal adhesions. Our data support a model in which GOLPH3 in T98G cells promotes cell migration by stimulating the activity of FAK.
Assuntos
Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Adesões Focais/fisiologia , Proteínas de Membrana/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Fosforilação , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
Inter-organelle signalling has essential roles in cell physiology encompassing cell metabolism, aging and temporal adaptation to external and internal perturbations. How such signalling coordinates different organelle functions within adaptive responses remains unknown. Membrane traffic is a fundamental process in which membrane fluxes need to be sensed for the adjustment of cellular requirements and homeostasis. Studying endoplasmic reticulum-to-Golgi trafficking, we found that Golgi-based, KDEL receptor-dependent signalling promotes lysosome repositioning to the perinuclear area, involving a complex process intertwined to autophagy, lipid-droplet turnover and Golgi-mediated secretion that engages the microtubule motor protein dynein-LRB1 and the autophagy cargo receptor p62/SQSTM1. This process, here named 'traffic-induced degradation response for secretion' (TIDeRS) discloses a cellular mechanism by which nutrient and membrane sensing machineries cooperate to sustain Golgi-dependent protein secretion.
Assuntos
Autofagia , Gotículas Lipídicas/metabolismo , Lisossomos/metabolismo , Receptores de Peptídeos/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Dineínas/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Células HeLa , Humanos , Lisossomos/ultraestrutura , Microscopia Eletrônica de Transmissão , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Transporte Proteico , Proteína Sequestossoma-1/metabolismo , Transdução de SinaisRESUMO
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
Increasing evidence indicates that the Golgi apparatus plays active roles in cancer, but a comprehensive understanding of its functions in the oncogenic transformation has not yet emerged. At the same time, the Golgi is becoming well recognized as a hub that integrates its functions of protein and lipid biosynthesis to signal transduction for cell proliferation and migration in cancer cells. Nevertheless, the active function of the Golgi apparatus in cancer cells has not been fully evaluated as a target for combined treatment. Here, we analyzed the effect of perturbing the Golgi apparatus on the sensitivity of the MDA-MB-231 breast cancer cell line to the drugs Actinomycin D and Vinblastine. We disrupted the function of ARF1, a protein necessary for the homeostasis of the Golgi apparatus. We found that the expression of the ARF1-Q71L mutant increased the sensitivity of MDA-MB-231 cells to both Actinomycin D and Vinblastine, resulting in decreased cell proliferation and cell migration, as well as in increased apoptosis. Likewise, the combined treatment of cells with Actinomycin D or Vinblastine and Brefeldin A or Golgicide A, two disrupting agents of the ARF1 function, resulted in similar effects on cell proliferation, cell migration and apoptosis. Interestingly, each combined treatment had distinct effects on ERK1/2 and AKT signaling, as indicated by the decreased levels of either phospho-ERK1/2 or phospho-AKT. Our results suggest that disruption of Golgi function could be used as a strategy for the sensitization of cancer cells to chemotherapy.
Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Adenocarcinoma/tratamento farmacológico , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Dactinomicina/farmacologia , Vimblastina/farmacologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
The balance between ovarian folliculogenesis and follicular atresia is critical for female fertility and is strictly regulated by a complex network of neuroendocrine and intra-ovarian signals. Despite the numerous functions executed by granulosa cells (GCs) in ovarian physiology, the role of multifunctional proteins able to simultaneously coordinate/modulate several cellular pathways is unclear. Soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (α-SNAP) is a multifunctional protein that participates in SNARE-mediated membrane fusion events. In addition, it regulates cell-to-cell adhesion, AMPK signaling, autophagy and apoptosis in different cell types. In this study we examined the expression pattern of α-SNAP in ovarian tissue and the consequences of α-SNAP (M105I) mutation (hyh mutation) in folliculogenesis and female fertility. Our results showed that α-SNAP protein is highly expressed in GCs and its expression is modulated by gonadotropin stimuli. On the other hand, α-SNAP-mutant mice show a reduction in α-SNAP protein levels. Moreover, increased apoptosis of GCs and follicular atresia, reduced ovulation rate, and a dramatic decline in fertility is observed in α-SNAP-mutant females. In conclusion, α-SNAP plays a critical role in the balance between follicular development and atresia. Consequently, a reduction in its expression/function (M105I mutation) causes early depletion of ovarian follicles and female subfertility.