RESUMO
Fabry disease (FD) is an X-linked progressive multisystem disease due to mutations in the gene encoding the lysosomal enzyme α-galactosidase A (α-GalA). The deficiency in α-GalA activity leads to an intra-lysosomal accumulation of neutral glycosphingolipids, mainly globotriaosylceramide (Gb3), in various organs and systems. Enzyme replacement therapy is available and alternative therapeutic approaches are being explored. No diagnostic test, other than sequencing of the α-galactosidase A gene, is available, no biomarker has been proven useful to screen for and predict the disease, and underlying mechanisms are still elusive. The aim of this study is to identify FD specific biomarkers and to better understand the pathophysiological changes that occur over time in FD. We compared peripheral blood mononuclear cells (PBMC) from FD patients (n = 8) with control PBMC from healthy individuals (n = 6), by two-dimensional electrophoresis (2DE) and the detected differentially expressed proteins were then subjected to matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). In FD patients we identified, among the down-regulated proteins, Calnexin, Rho GDP-dissociation inhibitor 2, Rho GDP-dissociation inhibitor 1, Chloride intracellular channel protein 1; on the other hand γ-enolase, 14-3-3 protein theta, 14-3-3 protein zeta/delta, and galectin-1 were identified as up-regulated proteins. Calnexin and Rho GDP-dissociation inhibitor-1,2 are related to protein folding, signal transduction and cell proliferation. This is the first time that γ-enolase and galectin-1 are described to be up-regulated in Fabry patients. Levels of γ-enolase increase dramatically in cardiovascular accidents and cerebral trauma, whereas galectins are regulators of acute and chronic inflammation. These findings may improve our understanding of the molecular mechanisms underlying the pathology and provide new insight and knowledge for future studies in this field.
Assuntos
Doença de Fabry/metabolismo , Leucócitos Mononucleares/metabolismo , Proteoma/metabolismo , Proteínas 14-3-3/biossíntese , Adulto , Biomarcadores , Calnexina/biossíntese , Proliferação de Células , Canais de Cloreto/biossíntese , Regulação para Baixo , Doença de Fabry/diagnóstico , Feminino , Galectina 1/biossíntese , Expressão Gênica , Humanos , Inflamação , Masculino , Pessoa de Meia-Idade , Fosfopiruvato Hidratase/biossíntese , Dobramento de Proteína , Proteômica , Transdução de Sinais , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Regulação para Cima , alfa-Galactosidase/genética , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/biossíntese , Inibidor beta de Dissociação do Nucleotídeo Guanina rho/biossínteseRESUMO
The limitations of high-level expression of virus surface proteins in yeast are not well understood. The inefficiency of yeast to produce active human virus surface glycoproteins, as well as other mammalian glycoproteins, is usually explained by the inefficient folding of the glycoprotein into its characteristic and functional three-dimensional structure from a random coil. The endoplasmic reticulum (ER) is a highly versatile protein factory that is equipped with chaperones and folding enzymes essential for protein folding. To improve folding and solubility of viral surface glycoprotein, the genes encoding human ER resident chaperones calnexin, calreticulin, immunoglobin binding protein (BiP), protein disulfide isomerase (PDI) and foldase (ERp57) were coexpressed together with hemagglutinin gene from measles virus in the yeast Saccharomyces cerevisiae. The effect of coexpressing chaperones on the total yield of measles virus hemagglutinin (MeH) as well as the intracellular fate of the glycoprotein was determined. Our results demonstrated that coexpression of human calnexin noticeably enhanced the quantity of the soluble glycosylated form of MeH in yeast. The coexpression of human calreticulin-, PDI-, ERp57- and BiP-encoding genes did not improve the quality of recombinant MeH.
Assuntos
Calnexina/biossíntese , Calnexina/genética , Expressão Gênica , Hemaglutininas Virais/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Hemaglutininas Virais/genética , Hemaglutininas Virais/metabolismo , Humanos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , SolubilidadeRESUMO
BACKGROUND: Exosomes are nanovesicles of endocytic origin released by cells and present in human body fluids such as plasma, breast milk, and bronchoalveolar lavage fluid. These vesicles take part in communication between cells. Recently, it was shown that exosomes contain both mRNA and microRNA. This RNA can be shuttled between cells (exosomal shuttle RNA), which is a new route of communication between cells. The aim of this study was to determine whether nasal secretions harbor exosomes and furthermore, whether these exosomes contain RNA. METHODS: Human nasal lavage fluid (NLF) underwent centrifugation and filtration to discard cells and debris, followed by a final ultracentrifugation at 120,000 × g to pellet the exosomes. Exosomes were detected using electron microscopy (EM), flow cytometry, and Western blot. RNA was extracted and analyzed using a Bioanalyzer. RESULTS: Exosomes were visualized as 40-80 nm, CD63(+) vesicles using EM. Flow cytometry of exosomes using anti-major histocompatibility complex class II beads revealed exosomes positive for the tetraspanins CD9, CD63, and CD81. Western blot confirmed the presence of exosomal protein and absence of proteins from the endoplasmic reticulum (ER), because the exosomes were positive for Tsg101, but negative for the ER marker, calnexin. Bioanalyzer analysis revealed that, these exosomes contain RNA. CONCLUSION: This study shows for the first time that NLF contains exosomes and that these exosomes contain RNA. Further characterization of the exosomal RNA and proteins may provide important information about communication in the nose and potentially provide a source of biomarkers for upper airway diseases.
Assuntos
Biomarcadores/metabolismo , Secreções Corporais/metabolismo , Exossomos/metabolismo , Mucosa Nasal/metabolismo , RNA/metabolismo , Antígenos CD/biossíntese , Calnexina/biossíntese , Comunicação Celular , Separação Celular , Proteínas de Ligação a DNA/biossíntese , Complexos Endossomais de Distribuição Requeridos para Transporte/biossíntese , Exossomos/genética , Exossomos/ultraestrutura , Citometria de Fluxo , Humanos , Microscopia Eletrônica , Nariz/patologia , RNA/genética , Fatores de Transcrição/biossíntese , UltracentrifugaçãoRESUMO
Human 29IJ6 IgG was expressed in silkworm using a Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid system. The mean amounts of 296IJ6 IgG produced in larval hemolymph and whole pupae were 30.1 microg/larva and 78.0 microg/pupa, respectively. The use of molecular chaperones including calreticulin (CRT), calnexin (CNX), and immunoglobulin heavy chain binding protein (BiP, GRP78) improved the production of 296IJ6 IgG secretion in the larvae fivefold. The total yield of recombinant 29IJ6 IgG was 239 microg/mL when coexpressed with CRT. However, the overexpression of molecular chaperones had negative effects on secretion. The N-linked glycans of secreted 296IJ6 IgG in silkworm hemolymph were dominated by paucimannose structures. Small amounts of GlcNAc residues linked to the Manalpha1,3 branch were detected. When molecular chaperones were coexpressed, the compositions of N-linked glycans in the IgG1 produced were unchanged compared with those produced without them. This suggests that N-glycosylation is controlled by a regulatory function in the Golgi apparatus even though the post-translational modification of 296IJ6 IgG was assisted by the coexpression of molecular chaperones. Therefore, if the glycosylation pathways that coexpress N-acetylglucosaminyltransferase, galactosyltransferase, and sialyltransferase could be improved, silkworm larvae might prove a useful system for producing human antibodies.
Assuntos
Bombyx/metabolismo , Imunoglobulina G/biossíntese , Chaperonas Moleculares/biossíntese , Polissacarídeos/química , Animais , Bombyx/química , Calnexina/biossíntese , Calnexina/química , Calreticulina/biossíntese , Calreticulina/química , Chaperona BiP do Retículo Endoplasmático , Complexo de Golgi/metabolismo , Proteínas de Choque Térmico/biossíntese , Proteínas de Choque Térmico/química , Humanos , Imunoglobulina G/química , Chaperonas Moleculares/química , Nucleopoliedrovírus/metabolismo , Polissacarídeos/metabolismo , Proteínas Recombinantes/biossínteseRESUMO
Alpha-glucosidase I regulates trimming of the terminal alpha-1,2-glucose residue in the N-glycan processing pathway, which plays an important role in quality control systems in mammalian cells. Previously, we identified the gene encoding alpha-glucosidase I in the opportunistic human fungal pathogen Aspergillus fumigatus, namely Afcwh41. Deletion of the Afcwh41 gene results in a severe reduction of conidia formation, a temperature-sensitive deficiency of cell wall integrity, and abnormalities of polar growth and septation. An upregulation of the genes encoding Rho-type GTPases was also observed, which suggests activation of the cell wall integrity pathway in the mutant. Using 2D gel analysis, we revealed that the proteins involved in protein assembly, ubiquitin-mediated degradation and actin organization are altered in the DeltaAfcwh41 mutant. Evidence was obtained for a defect in the polarized localization of the actin cytoskeleton in the mutant. Our results suggest that blocking of the glucose trimming in A. fumigatus might induce accumulation of misfolded proteins in the endoplasmic reticulum; these misfolded proteins are probably required for cell wall synthesis and thus activate the cell wall integrity pathway, which then causes the abnormal polarity associated with the DeltaAfcwh41 mutant.
Assuntos
Aspergillus fumigatus/fisiologia , Proteoma/análise , alfa-Glucosidases/deficiência , Actinas/metabolismo , Aspergillus fumigatus/efeitos dos fármacos , Calnexina/biossíntese , Polaridade Celular , Parede Celular/metabolismo , Ditiotreitol/farmacologia , Eletroforese em Gel Bidimensional , Retículo Endoplasmático/efeitos dos fármacos , Proteínas Fúngicas/biossíntese , Proteínas dos Microfilamentos/biossíntese , Dobramento de Proteína , Esporos Fúngicos/metabolismo , Espectrometria de Massas em Tandem , Proteínas rho de Ligação ao GTP/biossínteseRESUMO
Calnexin is an endoplasmic reticulum (ER) lectin that mediates protein folding on the rough ER. Calnexin also interacts with ER calcium pumps that localize to the mitochondria-associated membrane (MAM). Depending on ER homeostasis, varying amounts of calnexin target to the plasma membrane. However, no regulated sorting mechanism is so far known for calnexin. Our results now describe how the interaction of calnexin with the cytosolic sorting protein PACS-2 distributes calnexin between the rough ER, the MAM, and the plasma membrane. Under control conditions, more than 80% of calnexin localizes to the ER, with the majority on the MAM. PACS-2 knockdown disrupts the calnexin distribution within the ER and increases its levels on the cell surface. Phosphorylation by protein kinase CK2 of two calnexin cytosolic serines (Ser554/564) reduces calnexin binding to PACS-2. Consistent with this, a Ser554/564 Asp phosphomimic mutation partially reproduces PACS-2 knockdown by increasing the calnexin signal on the cell surface and reducing it on the MAM. PACS-2 knockdown does not reduce retention of other ER markers. Therefore, our results suggest that the phosphorylation state of the calnexin cytosolic domain and its interaction with PACS-2 sort this chaperone between domains of the ER and the plasma membrane.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Calnexina/biossíntese , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica , Sequência de Aminoácidos , Calnexina/química , Calnexina/fisiologia , Citosol/química , Células HeLa , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Transporte VesicularRESUMO
High-grade glioma cells express subunits of the ENaC/Deg superfamily, including members of ASIC subfamily. Our previous work has shown that glioma cells exhibit a basally active cation current, which is not present in low-grade tumor cells or normal astrocytes, and that can be blocked by amiloride. When ASIC2 is present within the channel complex in the plasma membrane, the channel is rendered non-functional because of inherent negative effectors that require ASIC2. We have previously shown that high-grade glioma cells functionally express this current because of the lack of ASIC2 in the plasma membrane. We now hypothesize that ASIC2 trafficking in glioma cells is regulated by a specific chaperone protein, namely Hsc70. Our results demonstrated that Hsc70 co-immunoprecipitates with ASIC2 and that it is overexpressed in glioma cells as compared with normal astrocytes. In contrast, there was no difference in the expression of calnexin, which also co-immunoprecipitates with ASIC2. In addition, glycerol and sodium 4-phenylbutyrate reduced the amount of Hsc70 expressed in glioma cells to levels found in normal astrocytes. Transfection of Hsc70 siRNA inhibited the constitutively activated amiloride-sensitive current, decreased migration, and increased ASIC2 surface expression in glioma cells. These results support an association between Hsc70 and ASIC2 that may underlie the increased retention of ASIC2 in the endoplasmic reticulum of glioma cells. The data also suggest that decreasing Hsc70 expression promotes reversion of a high-grade glioma cell to a more normal astrocytic phenotype.
Assuntos
Retículo Endoplasmático/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Glioma/metabolismo , Proteínas de Choque Térmico HSC70/metabolismo , Proteínas de Membrana/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Canais de Sódio/biossíntese , Canais Iônicos Sensíveis a Ácido , Astrócitos/metabolismo , Calnexina/biossíntese , Calnexina/genética , Linhagem Celular Tumoral , Retículo Endoplasmático/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioma/genética , Proteínas de Choque Térmico HSC70/antagonistas & inibidores , Proteínas de Choque Térmico HSC70/genética , Humanos , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Canais de Sódio/genética , TransfecçãoRESUMO
The type I membrane protein calnexin is a conserved key component of the quality control mechanism in the endoplasmic reticulum. It functions as a molecular chaperone that monitors the folding state of nascent polypeptides entering the endoplasmic reticulum. Calnexin also behaves as a lectin, as its chaperoning activity involves binding of oligosaccharide moieties present on newly imported glycoproteins. We isolated the calnexin gene (HpCNE1) from the methylotrophic yeast Hansenula polymorpha, and used HpCNE1 expression plasmids for super-transformation of H. polymorpha strains secreting target proteins of biotechnological interest. The elevated dosage of HpCNE1 enhanced secretion of the four proteins tested: three glycoproteins and one unglycosylated product. Secretion of bacterial alginate epimerase AlgE1 was increased threefold on average, and secretion of both human interferon-gamma and fungal consensus phytase twofold. With phytase and AlgE1 this improvement was all the more remarkable, as the secretion level was already high in the original strains (g L(-1) range). The same approach improved secretion of human serum albumin, which lacks N-linked glycans, about twofold. Glycosylation of the pro-MFalpha1 leader may account for the effect of calnexin in this case. Our results argue that cooverexpression of calnexin can serve as a generally applicable tool for enhancing the secretion of all types of heterologous protein by H. polymorpha.
Assuntos
Calnexina/genética , Calnexina/metabolismo , Dosagem de Genes , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/metabolismo , 6-Fitase/genética , 6-Fitase/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Calnexina/biossíntese , Calnexina/química , Carboidratos Epimerases/genética , Carboidratos Epimerases/metabolismo , Clonagem Molecular , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Interferon gama/genética , Interferon gama/metabolismo , Dados de Sequência Molecular , Alinhamento de Sequência , Albumina Sérica/genética , Albumina Sérica/metabolismo , Transformação GenéticaRESUMO
Traumatic brain injury (TBI) initiates a complex genetic response that may include the expression of organelle specific stress genes. We investigated the effects of brain trauma on the expression of a number of stress genes by in situ hybridization and Western blot analysis including the endoplasmic reticulum (ER) stress gene grp78, ER protein processing enzymes calnexin and protein disulphide isomerase (PDI), the mitochondrial stress gene hsp60, and the cytoplasmic stress gene hsp70. Male Sprague-Dawley rats were subjected either to sham-surgery or moderate (1.8-2.2 atm) parasagittal fluid-percussion (F-P) brain injury followed by 30 min of either normoxic or hypoxic (30-40 mm Hg) gas levels. Expression of grp78 was increased in the ipsilateral cerebral cortex and dentate gyrus beginning 4 h after trauma plus hypoxia. Similarly, mRNA encoding the mitochondrial hsp60 was induced in the ipsilateral outer cortical layers at 4-24 h after TBI plus hypoxia. Calnexin and PDI mRNAs were not significantly altered following TBI with or without secondary hypoxia. In contrast, mRNA of the cytoplasmic hsp70 was strongly induced at 4 h after brain injury in multiple brain regions within the injured hemisphere, and this expression was greatly enhanced by secondary hypoxia. Because subcellular stress gene expression may reflect where unfolded or damaged proteins are abundant, these findings suggest that abnormal proteins are localized mainly in the cytoplasm, and to a lesser degree in the ER lumen and mitochondria after brain trauma. Thus, distinct parts of the cellular machinery respond to traumatic and metabolic stresses in specific ways.
Assuntos
Lesões Encefálicas/metabolismo , Frações Subcelulares/metabolismo , Animais , Western Blotting , Lesões Encefálicas/genética , Calnexina/biossíntese , Calnexina/genética , Chaperonina 60/biossíntese , Chaperonina 60/genética , Proteínas de Choque Térmico HSP70/biossíntese , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico/biossíntese , Proteínas de Choque Térmico/genética , Hibridização In Situ , Masculino , Chaperonas Moleculares/biossíntese , Chaperonas Moleculares/genética , Proteínas do Tecido Nervoso/biossíntese , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-DawleyRESUMO
We report the cloning of a Paracoccidioides brasiliensis cDNA, here named PbCnx, encoding the homologue of the endoplasmic reticulum molecular chaperone calnexin. Calnexin specifically recognizes monoglucosylated glycoproteins in the endoplasmic reticulum, thus being an essential component of the complex that interacts with the folded state of nascent secreted glycoproteins. The PbCnx open reading frame was found in a 1701 base pair (bp) fragment that encodes a 567 amino acid protein with an estimated mass of 62 680 Da. Northern and Southern blot hybridizations showed that PbCnx is encoded by a single, or a low number of, gene copies. PbCnx contains the hallmark KPEDWD motifs that are found in all members of the calnexin/calreticulin family proteins. A cDNA-encoding PbCnx was overexpressed as recombinant protein in Escherichia coli. The purified recombinant PbCnx was recognized by 6 out of 10 sera from PCM patients, a result that rules out its possible consideration for further use in diagnosis. Using confocal microscopy with anti-PbCnx mouse serum against yeast forms, a cytoplasmic staining pattern was observed.
Assuntos
Calnexina/genética , Paracoccidioides/genética , Paracoccidioides/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Western Blotting , Calnexina/biossíntese , Calnexina/imunologia , Clonagem Molecular , DNA Complementar/genética , DNA Fúngico/química , DNA Fúngico/genética , Epitopos/imunologia , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Dados de Sequência Molecular , Paracoccidioidomicose/sangue , Paracoccidioidomicose/imunologia , RNA Fúngico/química , RNA Fúngico/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Homologia de SequênciaRESUMO
UNLABELLED: The recognized structural proteins of the enamel matrix are amelogenin, ameloblastin, and enamelin. While a large volume of data exists showing that amelogenin self-assembles into multimeric units referred to as nanospheres, other reports of enamel matrix protein-protein interactions are scant. We believe that each of these enamel matrix proteins must interact with other organic components of ameloblasts and the enamel matrix. Likely protein partners would include integral membrane proteins and additional secreted proteins. INTRODUCTION: The purpose of this study was to identify and catalog additional proteins that play a significant role in enamel formation. MATERIALS AND METHODS: We used the yeast two-hybrid assay to identify protein partners for amelogenin, ameloblastin, and enamelin. Once identified, RT-PCR was used to assess gene transcription of these newly identified and potential "enamel" proteins in ameloblast-like LS8 cells. RESULTS: In the context of this yeast assay, we identified a number of secreted proteins and integral membrane proteins that interact with amelogenin, ameloblastin, and enamelin. Additionally, proteins whose functions range from the inhibition of soft tissue mineralization, calcium ion transport, and phosphorylation events have been identified as protein partners to these enamel matrix proteins. For each protein identified using this screening strategy, future studies are planned to confirm this physiological relationship to biomineralization in vivo. CONCLUSION: Identifying integral membrane proteins of the secretory surface of ameloblast cells (Tomes' processes) and additional enamel matrix proteins, based on their abilities to interact with the most abundant enamel matrix proteins, will better define the molecular mechanisms of enamel formation at its most rudimentary level.
Assuntos
Esmalte Dentário/metabolismo , Transcrição Gênica , Ameloblastos/metabolismo , Amelogenina , Animais , Antígenos CD/biossíntese , Biglicano , Proteínas Sanguíneas/metabolismo , Calnexina/biossíntese , Calnexina/metabolismo , Membrana Celular/metabolismo , DNA Complementar/metabolismo , Proteínas do Esmalte Dentário/química , Proteínas do Esmalte Dentário/metabolismo , Dentina/metabolismo , Proteínas da Matriz Extracelular , Camundongos , Modelos Biológicos , Fases de Leitura Aberta , Fosforilação , Glicoproteínas da Membrana de Plaquetas/biossíntese , Ligação Proteica , Proteoglicanas/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tetraspanina 30 , Fatores de Tempo , Técnicas do Sistema de Duplo-Híbrido , alfa-2-Glicoproteína-HS , alfa-Fetoproteínas/metabolismoRESUMO
We have developed a simple scheme for the isolation of parasitophorous vacuoles (PVs) that harbor Leishmania parasites. This scheme exploits the observation that PVs display endoplasmic reticulum molecules, including the transmembrane protein calnexin. The presence of calnexin at the surface of the PVs distinguishes them from late endosomal vesicles of comparable density. As a result, PVs can be isolated by calnexin affinity selection from an enriched PV fraction obtained by sucrose density fractionation.
Assuntos
Calnexina/biossíntese , Leishmania/isolamento & purificação , Macrófagos/parasitologia , Fagossomos/metabolismo , Vacúolos/parasitologia , Animais , Calnexina/química , Linhagem Celular , Centrifugação com Gradiente de Concentração , Leishmania/metabolismo , Macrófagos/ultraestrutura , Camundongos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Fagossomos/parasitologia , Fagossomos/ultraestrutura , Frações Subcelulares/metabolismo , Frações Subcelulares/parasitologia , Vacúolos/ultraestruturaRESUMO
Fractalkine is the only known member of the CX(3)C-chemokine family, and so is its receptor CX(3)CR1. Fractalkine, typically is expressed by neurons where it is inserted in the plasma membrane ("chemokine on a stalk"). It can, however, be clipped off by a specific enzyme and diffuse into the extracellular space. CX(3)CR1 is primarily expressed by microglia, the phagocytes of the brain. This study was aimed at studying gene expression changes in cultured rat microglia upon fractalkine stimulation using gene chip technology. Six genes turned out to be upregulated, amongst which milk-fat globule EGF factor-8 protein (MFG-E8) was the most surprising, but also the most revealing one. We hypothesize that it serves as a bridging molecule between apoptotic cells (neurons) and microglia. Since the docking to microglia is, in part, mediated by members of the integrin family, six of these molecules have been-post hoc-included in real-time PCR confirmations of chip results. Two of them-integrin alpha(2) and integrin beta(5)-were upregulated as well. These data provide a much closer look into molecular mechanisms involved in apoptosis of neurons and their removal by microglia.
Assuntos
Quimiocinas CX3C/fisiologia , Glicolipídeos/biossíntese , Glicoproteínas/biossíntese , Proteínas de Membrana/fisiologia , Microglia/metabolismo , Proteínas do Leite/biossíntese , Regulação para Cima , Animais , Antígenos de Superfície , Calnexina/biossíntese , Calnexina/genética , Células Cultivadas , Quimiocina CX3CL1 , Perfilação da Expressão Gênica , Glutationa Transferase/biossíntese , Glutationa Transferase/genética , Quinase 3 da Glicogênio Sintase , Glicolipídeos/genética , Glicoproteínas/genética , Ribonucleoproteínas Nucleares Heterogêneas , Integrina alfa2/biossíntese , Integrina alfa2/genética , Gotículas Lipídicas , Microglia/enzimologia , Microglia/imunologia , Proteínas do Leite/genética , Proteínas Musculares/biossíntese , Proteínas Musculares/genética , Análise de Sequência com Séries de Oligonucleotídeos , Proteína de Ligação a Regiões Ricas em Polipirimidinas , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/genética , Ratos , Ratos Wistar , Proteínas S100/biossíntese , Proteínas S100/genética , Regulação para Cima/imunologiaRESUMO
A stringent quality control process selects misfolded polypeptides generated in the endoplasmic reticulum (ER) for ER-associated degradation (ERAD). Here we assessed the maintenance of efficient glycoprotein folding in cells with defective ERAD caused by lack of adaptation of the intralumenal level of ER degradation-enhancing alpha-mannosidase-like protein (EDEM) to an increase in the ER cargo load. When these cells were converted into factories for production of high levels of human beta-secretase, maturation of this N-glycosylated aspartic protease progressed as in wild-type cells initially to gradually become less efficient. Up-regulation of EDEM to strengthen the ERAD machinery (but not up-regulation of calnexin to reinforce the folding machinery) was instrumental in maintaining folding efficiency and secretory capacity. Our data underscore the important role that the degradation machinery plays in maintaining a functional folding environment in the ER.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/fisiologia , Animais , Calnexina/biossíntese , Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Detergentes/farmacologia , Fibroblastos/metabolismo , Glicoproteínas/química , Glicosilação , Complexo de Golgi/metabolismo , Imunoprecipitação , Cinética , Proteínas de Membrana/química , Camundongos , Proteínas Nucleares/metabolismo , Desnaturação Proteica , Dobramento de Proteína , Fatores de Transcrição de Fator Regulador X , Fatores de Tempo , Fatores de Transcrição , Transfecção , Transgenes , Regulação para CimaRESUMO
MHC tetramers have become essential tools for the analysis of antigen specific responses of CD8+ and CD4+ T cells. However, the use of MHC class II tetramers is hampered by the relatively low yields of most current expression systems. We have devised an insect cell/baculovirus expression system in which yields of 50-70 mg of recombinant HLA-DR4 molecules, with or without covalently linked peptide, per liter of insect cell supernatant, are routinely obtained. These yields are rendered possible by an optimized design and use of DRalpha and DRbeta expression cassettes and by co-expression of a housekeeping chaperone of the endoplasmic reticulum, calreticulin, which, due to its co-secretion, increases secretion of HLA-DR molecules two- to threefold. A tetramer produced in the system specifically was shown to stain an HLA-DR4 restricted T cell line obtained from a healthy donor by in vitro priming, but which recognizes a type I diabetes autoantigen. Co-expression of chaperones may represent a general strategy for enhancing yields of recombinant proteins expressed in insect cells and facilitate production of MHC class II tetramers in the future.
Assuntos
Antígeno HLA-DR4/biossíntese , Proteínas Recombinantes/síntese química , Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Baculoviridae , Sequência de Bases , Calnexina/biossíntese , Calnexina/genética , Calnexina/farmacologia , Calreticulina/biossíntese , Calreticulina/genética , Calreticulina/farmacologia , Primers do DNA , Citometria de Fluxo , Vetores Genéticos , Antígeno HLA-DR4/química , Antígeno HLA-DR4/efeitos dos fármacos , Humanos , Insetos , Dados de Sequência MolecularRESUMO
Characterization of the molecular basis of tumor recognition by T cells has shown that major histocompatibility complex (MHC) class I molecules play a crucial role in presenting antigenic peptide epitopes to cytotoxic T lymphocytes. MHC class Ia downregulation has been repeatedly described on melanoma cells and is thought to be involved in the failure of the immune system to control tumor progression. Proper assembly of MHC class I molecules is dependent on several cofactors, e.g. the chaperones calnexin and calreticulin residing in the endoplasmic reticulum. Alterations in the expression of these chaperones may have important implications for MHC class I assembly, peptide loading, and presentation on the tumor cell surface and thus may contribute to the immune escape phenotype of tumor cells. In the present study, we compared melanoma lesions representing different stages of tumor progression with regard to the expression of calnexin and calreticulin in tumor cells by means of immunohistochemistry. Metastatic melanoma lesions exhibited significant downregulation of calnexin as compared to primary melanoma lesions. In contrast, chaperone calreticulin was expressed in melanoma cells of primary as well as of metastatic lesions. Our data suggest that chaperone-downregulation, particularly calnexin-downregulation, may contribute to the metastatic phenotype of melanoma cells in vivo. Consistently, conserved chaperone expression in metastatic melanoma lesions may be a useful criterion for selection of patients for treatment with T cell-based immunotherapies.
Assuntos
Calnexina/biossíntese , Calreticulina/biossíntese , Regulação para Baixo , Retículo Endoplasmático/metabolismo , Regulação Neoplásica da Expressão Gênica , Melanoma/metabolismo , Calnexina/genética , Calreticulina/genética , Progressão da Doença , Humanos , Imuno-Histoquímica , Imunoterapia/métodos , Melanoma/patologia , Metástase Neoplásica , Fenótipo , Neoplasias Cutâneas/metabolismo , Linfócitos T/metabolismoRESUMO
Voltage-gated potassium channels control the membrane potential of excitable cells. To understand their function, knowledge of their structure is essential. However, these channels are scarce in natural sources, and overexpression is necessary to generate material for structural studies. We have compared functional expression of the Drosophila Shaker H4 potassium channel in stable insect cell lines and in baculovirus-infected insect cells, using three different baculovirus promoters. Stable insect cell lines expressed correctly assembled channel, which was glycosylated and found predominantly at, or close to, the cell surface. In comparison, the majority of baculovirus-overexpressed Shaker was intracellular and incorrectly assembled. The proportion of functional Shaker increased, however, if the weaker basic protein promoter was used rather than the stronger p10 or polyhedrin promoters. In addition, co-expression of the molecular chaperone, calnexin, increased the quantity of correctly assembled channel protein, suggesting that calnexin can be used to increase the efficiency of channel expression in insect cells.
Assuntos
Calnexina/biossíntese , Canais de Potássio/biossíntese , Animais , Baculoviridae/genética , Calnexina/genética , Linhagem Celular , Proteínas de Drosophila , Regulação da Expressão Gênica/efeitos dos fármacos , Insetos , Chaperonas Moleculares/farmacologia , Canais de Potássio/genética , Regiões Promotoras Genéticas , Superfamília Shaker de Canais de Potássio , Transfecção , Regulação para CimaRESUMO
Calnexin (Cnx) has been characterized as a membrane-bound protein that transiently interacts in a unique chaperone system with newly synthesized glycoproteins in order to allow the establishment of their proper tertiary and, in most cases, quarternary structures. The aim of the study was to identify and to locate the expression of Cnx in the three major salivary glands of humans by different methods. Strong expression of Cnx protein and mRNA were generally found in serous salivary secretory units. With regard to mucous secretory units, expression of Cnx was only detectable at a low level in mucous acinar cells of sublingual glands, but not of submandibular glands. Expression of Cnx was always preserved in the surface epithelium of intralobar and interlobular duct segments. In addition, expression of Cnx was detected in sebaceous glands of parotid tissues, with a distribution pattern resembling that seen in sebaceous glands of the normal skin. In conclusion, production of saliva is associated with the expression of Cnx. Synthesis of molecules in mucous secretory units is not necessarily associated with a strong Cnx expression, whereas synthesis in serous secretory units apparently is. The tissue-specific Cnx expression is also paralleled by the observation that the secretions produced by the major salivary glands differ in their composition and amount.
Assuntos
Calnexina/biossíntese , Glândulas Salivares/metabolismo , Western Blotting , Humanos , Imuno-Histoquímica , Hibridização In Situ , Glândulas Salivares/citologia , Glândulas Sebáceas/citologia , Glândulas Sebáceas/metabolismoRESUMO
It has been suggested that the behavior and function of Paneth cells in metaplasia are different from those found in normal intestinal mucosa. In this study, we investigated whether calnexin, a protein involved in secretory pathways, might be associated with differentiation and function of Paneth cells in normal small intestine, in complete intestinal metaplasia of the stomach, and in Paneth cell-rich adenomas. Differentiation and function of Paneth cells was monitored by Ki67, lysozyme, and morphologic features. Using a newly established monoclonal antibody, we found that calnexin is regularly synthesized by Paneth cells of normal small intestine. In these cells, the staining intensity of calnexin was inversely correlated with their content of secretory granules (lysozyme). In contrast, Paneth cells of intestinal metaplasia and Paneth cell-rich adenomas showed a reduced immunostaining of both calnexin and lysozyme. Moreover, these Paneth cells synthesized the proliferation marker Ki67, a phenomenon that was never observed in Paneth cells of normal small intestine. In vitro experiments using CaCo2 cells showed that the expression of calnexin is not directly affected by the induction of mitosis. In conclusion, calnexin probably reflects the status of Paneth cell differentiation and function. The results do not necessarily indicate that calnexin has a function in Paneth cell proliferation.
Assuntos
Calnexina/biossíntese , Celulas de Paneth/citologia , Adenoma/metabolismo , Adenoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Células CACO-2/metabolismo , Células CACO-2/patologia , Calnexina/análise , Diferenciação Celular/fisiologia , Feminino , Mucosa Gástrica/metabolismo , Humanos , Imuno-Histoquímica , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Antígeno Ki-67/metabolismo , Masculino , Metaplasia/metabolismo , Metaplasia/patologia , Pessoa de Meia-Idade , Muramidase/metabolismo , Celulas de Paneth/metabolismo , Celulas de Paneth/patologia , Vesículas Secretórias/enzimologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Estômago/patologia , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologiaRESUMO
In the past, exposure of workers to mixtures of soluble and insoluble nickel compounds by inhalation during nickel refining correlated with increased incidences of lung and nasal cancers. Insoluble nickel subsulfide and nickel oxide (NiO) are carcinogenic in animals by inhalation; soluble nickel sulfate is not. Particles of insoluble nickel compounds were phagocytized by C3H/10T1/2 mouse embryo cells and induced morphological transformation in these cells with the following order of potency: NiO (black) > NiO (green) > nickel subsulfide. Foci induced by black/green NiO and nickel monosulfide developed into anchorage-independent transformed cell lines. Random arbitrarily primed-polymerase chain reaction mRNA differential display showed that nine c-DNA fragments are differentially expressed between nontransformed and nickel compound-transformed 10T1/2 cell lines in 6% of total mRNA; 130 genes would be differentially expressed in 100% of the mRNA. Fragment R3-2 was a sequence in the mouse calnexin gene, fragment R3-1 a portion of the Wdr1 gene, and fragment R2-4 a portion of the ect-2 protooncogene. These three genes were overexpressed in transformed cell lines. Fragment R1-2 was 90% homologous to a fragment of the DRIP/TRAP-80 (vitamin D receptor interacting protein/thyroid hormone receptor-activating protein 80) genes and was expressed in nontransformed but not in nickel-transformed cell lines. Specific insoluble carcinogenic nickel compounds are phagocytized into 10T1/2 cells and likely generate oxygen radicals, which would cause mutations in protooncogenes, and chromosome breakage, and mutations in tumor suppressor genes, inactivating them. These compounds also induce methylation of promoters of tumor suppressor genes, inactivating them. This could lead to permanent overexpresssion of the ect-2, calnexin, and Wdr1 genes and suppression of expression of the DRIP/TRAP-80 gene that we observed, which likely contribute to induction and maintenance of transformed phenotypes.