RESUMO
The lumen of the endoplasmic reticulum (ER) is usually considered an oxidative environment; however, oxidized thiol-disulfides and reduced pyridine nucleotides occur there parallelly, indicating that the ER lumen lacks components which connect the two systems. Here, we investigated the luminal presence of the thioredoxin (Trx)/thioredoxin reductase (TrxR) proteins, capable of linking the protein thiol and pyridine nucleotide pools in different compartments. It was shown that specific activity of TrxR in the ER is undetectable, whereas higher activities were measured in the cytoplasm and mitochondria. None of the Trx/TrxR isoforms were expressed in the ER by Western blot analysis. Co-localization studies of various isoforms of Trx and TrxR with ER marker Grp94 by immunofluorescent analysis further confirmed their absence from the lumen. The probability of luminal localization of each isoform was also predicted to be very low by several in silico analysis tools. ER-targeted transient transfection of HeLa cells with Trx1 and TrxR1 significantly decreased cell viability and induced apoptotic cell death. In conclusion, the absence of this electron transfer chain may explain the uncoupling of the redox systems in the ER lumen, allowing parallel presence of a reduced pyridine nucleotide and a probably oxidized protein pool necessary for cellular viability.
Assuntos
Retículo Endoplasmático , Oxirredução , Tiorredoxina Dissulfeto Redutase , Tiorredoxinas , Humanos , Tiorredoxinas/metabolismo , Tiorredoxinas/genética , Retículo Endoplasmático/metabolismo , Células HeLa , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxina Dissulfeto Redutase/genética , Mitocôndrias/metabolismo , Apoptose , Sobrevivência CelularRESUMO
The somatic haploidy is unstable in diplontic animals, but cellular processes determining haploid stability remain elusive. Here, we found that inhibition of mevalonate pathway by pitavastatin, a widely used cholesterol-lowering drug, drastically destabilized the haploid state in HAP1 cells. Interestingly, cholesterol supplementation did not restore haploid stability in pitavastatin-treated cells, and cholesterol inhibitor U18666A did not phenocopy haploid destabilization. These results ruled out the involvement of cholesterol in haploid stability. Besides cholesterol perturbation, pitavastatin induced endoplasmic reticulum (ER) stress, the suppression of which by a chemical chaperon significantly restored haploid stability in pitavastatin-treated cells. Our data demonstrate the involvement of the mevalonate pathway in the stability of the haploid state in human somatic cells through managing ER stress, highlighting a novel link between ploidy and ER homeostatic control.Key words: haploid, ER stress, Mevalonate pathway.
Assuntos
Estresse do Retículo Endoplasmático , Homeostase , Linhagem Celular , Colesterol , Haploidia , HumanosRESUMO
Besides the liver, which has always been considered the major source of endogenous glucose production in all post-absorptive situations, kidneys and intestines can also produce glucose in blood, particularly during fasting and under protein feeding. However, observations gained in different experimental animals have given ambiguous results concerning the presence of the glucose-6-phosphatase system in the small intestine. The aim of this study was to better define the species-related differences of this putative gluconeogenic organ in glucose homeostasis. The components of the glucose-6-phosphatase system (i.e., glucose-6-phosphate transporter and glucose-6-phosphatase itself) were analyzed in homogenates or microsomal fractions prepared from the small intestine mucosae and liver of rats, guinea pigs, and humans. Protein and mRNA levels, as well as glucose-6-phosphatase activities, were detected. The results showed that the glucose-6-phosphatase system is poorly represented in the small intestine of rats; on the other hand, significant expressions of glucose-6-phosphate transporter and of the glucose-6-phosphatase were found in the small intestine of guinea pigs and homo sapiens. The activity of the recently described fructose-6-phosphate transporter-intraluminal hexose isomerase pathway was also present in intestinal microsomes from these two species. The results demonstrate that the gluconeogenic role of the small intestine is highly species-specific and presumably dependent on feeding behavior (e.g., fructose consumption) and the actual state of metabolism.
Assuntos
Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Intestino Delgado/enzimologia , Animais , Frutose/metabolismo , Cobaias , Humanos , Microssomos/enzimologia , Ratos , Especificidade da EspécieRESUMO
Glucose is a basic nutrient in most of the creatures; its transport through biological membranes is an absolute requirement of life. This role is fulfilled by glucose transporters, mediating the transport of glucose by facilitated diffusion or by secondary active transport. GLUT (glucose transporter) or SLC2A (Solute carrier 2A) families represent the main glucose transporters in mammalian cells, originally described as plasma membrane transporters. Glucose transport through intracellular membranes has not been elucidated yet; however, glucose is formed in the lumen of various organelles. The glucose-6-phosphatase system catalyzing the last common step of gluconeogenesis and glycogenolysis generates glucose within the lumen of the endoplasmic reticulum. Posttranslational processing of the oligosaccharide moiety of glycoproteins also results in intraluminal glucose formation in the endoplasmic reticulum (ER) and Golgi. Autophagic degradation of polysaccharides, glycoproteins, and glycolipids leads to glucose accumulation in lysosomes. Despite the obvious necessity, the mechanism of glucose transport and the molecular nature of mediating proteins in the endomembranes have been hardly elucidated for the last few years. However, recent studies revealed the intracellular localization and functional features of some glucose transporters; the aim of the present paper was to summarize the collected knowledge.
Assuntos
Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Glucose/metabolismo , Proteínas de Transporte de Sódio-Glucose/metabolismo , Animais , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Glucose-6-Fosfatase/metabolismo , Complexo de Golgi/metabolismo , HumanosRESUMO
Accumulation of misfolded/unfolded proteins in the endoplasmic reticulum (ER) leads to the activation of three branches (Protein kinase (RNA)-like endoplasmic reticulum kinase [PERK], Inositol requiring protein 1 [IRE-1] and Activating trascription factor 6 [ATF6], respectively) of unfolded protein response (UPR). The primary role of UPR is to try to drive back the system to the former or a new homeostatic state by self-eating dependent autophagy, while excessive level of ER stress results in apoptotic cell death. Our study focuses on the role of PERK- and IRE-1-induced arms of UPR in life-or-death decision. Here we confirm that silencing of PERK extends autophagy-dependent survival, whereas the IRE-1-controlled apoptosis inducer is downregulated during ER stress. We also claim that the proper order of surviving and self-killing mechanisms is controlled by a positive feedback loop between PERK and IRE-1 branches. This regulatory network makes possible a smooth, continuous activation of autophagy with respect to ER stress, while the induction of apoptosis is irreversible and switch-like. Using our knowledge of molecular biological techniques and systems biological tools we give a qualitative description about the dynamical behavior of PERK- and IRE-1-controlled life-or-death decision. Our model claims that the two arms of UPR accomplish an altered upregulation of autophagy and apoptosis inducers during ER stress. Since ER stress is tightly connected to aging and age-related degenerative disorders, studying the signaling pathways of UPR and their role in maintaining ER proteostasis have medical importance.
Assuntos
Estresse do Retículo Endoplasmático/genética , Transdução de Sinais/genética , Biologia de Sistemas/métodos , eIF-2 Quinase/genética , Apoptose/genética , Autofagia/genética , Western Blotting , Sobrevivência Celular/genética , Endorribonucleases/genética , Endorribonucleases/metabolismo , Retroalimentação Fisiológica , Expressão Gênica , Células HEK293 , Homeostase/genética , Humanos , Modelos Biológicos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Resposta a Proteínas não Dobradas/genética , eIF-2 Quinase/metabolismoRESUMO
GLUT10 belongs to a family of transporters that catalyze the uptake of sugars/polyols by facilitated diffusion. Loss-of-function mutations in the SLC2A10 gene encoding GLUT10 are responsible for arterial tortuosity syndrome (ATS). Since subcellular distribution of the transporter is dubious, we aimed to clarify the localization of GLUT10. In silico GLUT10 localization prediction suggested its presence in the endoplasmic reticulum (ER). Immunoblotting showed the presence of GLUT10 protein in the microsomal, but not in mitochondrial fractions of human fibroblasts and liver tissue. An even cytosolic distribution with an intense perinuclear decoration of GLUT10 was demonstrated by immunofluorescence in human fibroblasts, whilst mitochondrial markers revealed a fully different decoration pattern. GLUT10 decoration was fully absent in fibroblasts from three ATS patients. Expression of exogenous, tagged GLUT10 in fibroblasts from an ATS patient revealed a strict co-localization with the ER marker protein disulfide isomerase (PDI). The results demonstrate that GLUT10 is present in the ER.
Assuntos
Artérias/anormalidades , Retículo Endoplasmático/metabolismo , Fibroblastos/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Instabilidade Articular/metabolismo , Dermatopatias Genéticas/metabolismo , Malformações Vasculares/metabolismo , Artérias/metabolismo , Imunofluorescência , Humanos , Espaço Intracelular/metabolismo , Instabilidade Articular/genética , Microssomos/metabolismo , Ligação Proteica , Transporte Proteico , Dermatopatias Genéticas/genética , Malformações Vasculares/genéticaRESUMO
Polyunsaturated fatty acids are susceptible to peroxidation and they yield various degradation products, including the main α,ß-unsaturated hydroxyalkenal, 4-hydroxy-2,3-trans-nonenal (HNE) in oxidative stress. Due to its high reactivity, HNE interacts with various macromolecules of the cell, and this general toxicity clearly contributes to a wide variety of pathological conditions. In addition, growing evidence suggests a more specific function of HNE in electrophilic signaling as a second messenger of oxidative/electrophilic stress. It can induce antioxidant defense mechanisms to restrain its own production and to enhance the cellular protection against oxidative stress. Moreover, HNE-mediated signaling can largely influence the fate of the cell through modulating major cellular processes, such as autophagy, proliferation and apoptosis. This review focuses on the molecular mechanisms underlying the signaling and regulatory functions of HNE. The role of HNE in the pathophysiology of cancer, cardiovascular and neurodegenerative diseases is also discussed.
Assuntos
Aldeídos/metabolismo , Fenômenos Fisiológicos Celulares/fisiologia , Doença , Transdução de Sinais/fisiologia , Aldeídos/química , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/fisiopatologia , Humanos , Estrutura Molecular , Neoplasias/metabolismo , Neoplasias/fisiopatologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/fisiopatologiaRESUMO
Beyond its general role as antioxidant, specific functions of ascorbate are compartmentalized within the eukaryotic cell. The list of organelle-specific functions of ascorbate has been recently expanded with the epigenetic role exerted as a cofactor for DNA and histone demethylases in the nucleus. Compartmentation necessitates the transport through intracellular membranes; members of the GLUT family and sodium-vitamin C cotransporters mediate the permeation of dehydroascorbic acid and ascorbate, respectively. Recent observations show that increased consumption and/or hindered entrance of ascorbate in/to a compartment results in pathological alterations partially resembling to scurvy, thus diseases of ascorbate compartmentation can exist. The review focuses on the reactions and transporters that can modulate ascorbate concentration and redox state in three compartments: endoplasmic reticulum, mitochondria and nucleus. By introducing the relevant experimental and clinical findings we make an attempt to coin the term of ascorbate compartmentation disease.
Assuntos
Ácido Ascórbico/metabolismo , Compartimento Celular , Doença , Animais , Regulação da Expressão Gênica , Humanos , Modelos Biológicos , Organelas/metabolismoRESUMO
The endoplasmic reticulum (ER) is central for many essential cellular activities, such as folding, assembly and quality control of secretory and membrane proteins, disulfide bond formation, glycosylation, lipid biosynthesis, Ca(2+) storage and signaling. In addition, this multifunctional organelle integrates many adaptive and/or maladaptive signaling cues reporting on metabolism, proteostasis, Ca(2+) and redox homeostasis. We are beginning to understand how these functions and pathways are integrated with one another to regulate homeostasis at cell, tissue and organism levels. The mechanisms underlying the introduction of the proper set of disulfide bonds into secretory proteins (oxidative folding) are strictly related to redox homeostasis, ER stress sensing and signaling and provide a good example of the integration systems operative in the early secretory compartment.
Assuntos
Oxirredução , Dobramento de Proteína , Transdução de Sinais , Animais , Compartimento Celular , Células/metabolismo , HumanosRESUMO
Significance: Cardiovascular disorders are the most important cause of morbidity and mortality in the Western world. Monogenic developmental disorders of the heart and vessels are highly valuable to study the physiological and pathological processes in cardiovascular system homeostasis. The arterial tortuosity syndrome (ATS) is a rare, autosomal recessive connective tissue disorder showing lengthening, tortuosity, and stenosis of the large arteries, with a propensity for aneurysm formation. In histopathology, it associates with fragmentation and disorganization of elastic fibers in several tissues, including the arterial wall. ATS is caused by pathogenic variants in SLC2A10 encoding the facilitative glucose transporter (GLUT)10. Critical Issues: Although several hypotheses have been forwarded, the molecular mechanisms linking disrupted GLUT10 activity with arterial malformations are largely unknown. Recent Advances: The vascular and systemic manifestations and natural history of ATS patients have been largely delineated. GLUT10 was identified as an intracellular transporter of dehydroascorbic acid, which contributes to collagen and elastin cross-linking in the endoplasmic reticulum, redox homeostasis in the mitochondria, and global and gene-specific methylation/hydroxymethylation affecting epigenetic regulation in the nucleus. We revise here the current knowledge on ATS and the role of GLUT10 within the compartmentalization of ascorbate in physiological and diseased states. Future Directions: Centralization of clinical, treatment, and outcome data will enable better management for ATS patients. Establishment of representative animal disease models could facilitate the study of pathomechanisms underlying ATS. This might be relevant for other forms of vascular dysplasia, such as isolated aneurysm formation, hypertensive vasculopathy, and neovascularization. Antioxid. Redox Signal. 34, 875-889.
Assuntos
Artérias/anormalidades , Ácido Ascórbico/genética , Proteínas Facilitadoras de Transporte de Glucose/genética , Homeostase/genética , Instabilidade Articular/genética , Dermatopatias Genéticas/genética , Malformações Vasculares/genética , Animais , Artérias/metabolismo , Artérias/patologia , Ácido Ascórbico/metabolismo , Ácido Ascórbico/uso terapêutico , Tecido Elástico/metabolismo , Tecido Elástico/patologia , Humanos , Instabilidade Articular/metabolismo , Instabilidade Articular/patologia , Instabilidade Articular/terapia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mutação/genética , Oxirredução , Dermatopatias Genéticas/metabolismo , Dermatopatias Genéticas/patologia , Dermatopatias Genéticas/terapia , Malformações Vasculares/metabolismo , Malformações Vasculares/patologia , Malformações Vasculares/terapiaRESUMO
Oxidative protein folding is mediated by a proteinaceous electron relay system, in which the concerted action of protein disulfide isomerase and Ero1 delivers the electrons from thiol groups to the final acceptor. Oxygen appears to be the final oxidant in aerobic living organisms, although the existence of alternative electron acceptors, e.g. fumarate or nitrate, cannot be excluded. Whilst the protein components of the system are well-known, less attention has been turned to the role of low molecular weight electron carriers in the process. The function of ascorbate, tocopherol and vitamin K has been raised recently. In vitro and in vivo evidence suggests that these redox-active compounds can contribute to the functioning of oxidative folding. This review focuses on the participation of small molecular weight redox compounds in oxidative protein folding.
Assuntos
Proteínas/química , Animais , Ácido Ascórbico/química , Catálise , Glutationa/química , Humanos , Oxirredução , Isomerases de Dissulfetos de Proteínas/química , Isomerases de Dissulfetos de Proteínas/metabolismo , Dobramento de Proteína , Proteínas/metabolismo , Compostos de Sulfidrila/química , Tocoferóis/química , Vitamina K/químicaRESUMO
Preimplantation genetic testing for aneuploidy (PGT-A) is a suitable technique to identify euploid embryos, which have the highest potential to implant, thus increase the chance of a healthy live birth. The main indications of PGT-A are advanced maternal age, repeated implantation failure, repeated miscarriages and severe male infertility. Several studies have already proven that testing embryos for genetic abnormalities in the above cases results in higher implantation rate and reduced number of pregnancy loss. In spite of these - due to a legislative change in Hungary in 2015 - PGT-A was reclassified as an experimental procedure and its use became banned throughout the country. For this reason, after 4 years of successful practice, Hungarian patients were not able to participate in IVF procedure combined with PGT-A anymore. In this retrospective analysis, efficacy of PGT-A-based embryo selection was evaluated and was compared to the conventional morphology-based selection (MBS) in patients with advanced maternal age, between 2013 and 2017â¯at our private fertility clinic. PGT-A was performed with array comparative genomic hybridization. We found that implantation rate was significantly higher (43.62% vs. 27.88%; pâ¯=â¯0.0208) and miscarriage rate was significantly lower (17.07% vs. 37.93%; pâ¯=â¯0.0492) in the PGT-A group compared to the MBS group from 2013 to 2015. These outcomes were achieved with a significantly lower number of transferred embryos in the PGT-A group (1.25 vs. 1.58; pâ¯=â¯0.0003). In 2016-2017, the number of transferred embryos were significantly reduced in the MBS group as well (1.14 vs. 1.58; pâ¯<â¯0.0001). However, outcomes of the IVF treatments did not change significantly compared to the previous two years (2013-2015). Our results imply that PGT-A-based embryo selection is more efficient than morphology-based selection in patients with advanced maternal age. Therefore, prohibition of the use of PGT-A had significant consequences on the efficiency and safety of IVF treatment in the country.
Assuntos
Aborto Espontâneo/diagnóstico , Aneuploidia , Fertilização in vitro/métodos , Diagnóstico Pré-Implantação , Aborto Espontâneo/epidemiologia , Aborto Espontâneo/genética , Aborto Espontâneo/patologia , Adulto , Blastocisto/metabolismo , Blastocisto/patologia , Hibridização Genômica Comparativa , Transferência Embrionária/métodos , Feminino , Humanos , Hungria/epidemiologia , Assistência ao Paciente , GravidezRESUMO
Ascorbate requiring Fe2+/2-oxoglutarate-dependent dioxygenases located in the nucleoplasm have been shown to participate in epigenetic regulation of gene expression via histone and DNA demethylation. Transport of dehydroascorbic acid is impaired in the endomembranes of fibroblasts from arterial tortuosity syndrome (ATS) patients, due to the mutation in the gene coding for glucose transporter GLUT10. We hypothesized that altered nuclear ascorbate concentration might be present in ATS fibroblasts, affecting dioxygenase activity and DNA demethylation. Therefore, our aim was to characterize the subcellular distribution of vitamin C, the global and site-specific changes in 5-methylcytosine and 5-hydroxymethylcytosine levels, and the effect of ascorbate supplementation in control and ATS fibroblast cultures. Diminished nuclear accumulation of ascorbate was found in ATS fibroblasts upon ascorbate or dehydroascorbic acid addition. Analyzing DNA samples of cultured fibroblasts from controls and ATS patients, a lower global 5-hydroxymethylcytosine level was found in ATS fibroblasts, which could not be significantly modified by ascorbate addition. Investigation of the (hydroxy)methylation status of specific regions in six candidate genes related to ascorbate metabolism and function showed that ascorbate addition could stimulate hydroxymethylation and active DNA demethylation at the PPAR-γ gene region in control fibroblasts only. The altered DNA hydroxymethylation patterns in patient cells both at the global level and at specific gene regions accompanied with decreased nuclear accumulation of ascorbate suggests the epigenetic role of vitamin C in the pathomechanism of ATS. The present findings represent the first example for the role of vitamin C transport in epigenetic regulation suggesting that ATS is a compartmentalization disease.
Assuntos
Artérias/anormalidades , Ácido Ascórbico/metabolismo , Núcleo Celular/metabolismo , Metilação de DNA/genética , Fibroblastos/metabolismo , Fibroblastos/patologia , Genoma Humano , Instabilidade Articular/genética , Dermatopatias Genéticas/genética , Malformações Vasculares/genética , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Células Cultivadas , Epigênese Genética , Humanos , Modelos Biológicos , PPAR gama/genética , PPAR gama/metabolismoRESUMO
Enzyme activities localized in the luminal compartment of the endoplasmic reticulum are integrated into the cellular metabolism by transmembrane fluxes of their substrates, products and/or cofactors. Most compounds involved are bulky, polar or even charged; hence, they cannot be expected to diffuse through lipid bilayers. Accordingly, transport processes investigated so far have been found protein-mediated. The selective and often rate-limiting transport processes greatly influence the activity, kinetic features and substrate specificity of the corresponding luminal enzymes. Therefore, the phenomenological characterization of endoplasmic reticulum transport contributes largely to the understanding of the metabolic functions of this organelle. Attempts to identify the transporter proteins have only been successful in a few cases, but recent development in molecular biology promises a better progress in this field.
Assuntos
Proteínas de Transporte/metabolismo , Retículo Endoplasmático/metabolismo , Enzimas/metabolismo , Acetilcoenzima A/metabolismo , Transporte Biológico/fisiologia , Metabolismo dos Carboidratos/fisiologia , Carnitina/metabolismo , Cinética , Nucleotídeos/metabolismo , Oligopeptídeos/metabolismo , Fosfatos/metabolismo , Especificidade por Substrato , Sulfatos/metabolismoRESUMO
The aim of the present study was the investigation of the occurrence of NADPH-generating pathways in the endoplasmic reticulum others then hexose-6-phosphate dehydrogenase. A significant isocitrate and a moderate malate-dependent NADP+ reduction were observed in endoplasmic reticulum-derived rat liver microsomes. The isocitrate-dependent activity was very likely attributable to the appearance of the cytosolic isocitrate dehydrogenase isozyme in the lumen. The isocitrate dehydrogenase activity of microsomes was present in the luminal fraction; it showed a strong preference towards NADP+ versus NAD+, and it was almost completely latent. Antibodies against the cytosolic isoform of isocitrate dehydrogenase immunorevealed a microsomal protein of identical molecular weight; the microsomal enzyme showed similar kinetic parameters and oxalomalate inhibition as the cytosolic one. Measurable luminal isocitrate dehydrogenase activity was also present in microsomes from rat epididymal fat. The results suggest that isocitrate dehydrogenase is an important NADPH-generating enzyme in the endoplasmic reticulum.
Assuntos
Retículo Endoplasmático/enzimologia , Isocitrato Desidrogenase/metabolismo , NADP/metabolismo , Tecido Adiposo/metabolismo , Animais , Citosol/enzimologia , Isoenzimas/metabolismo , Cinética , Masculino , Microssomos/enzimologia , Peso Molecular , NAD/metabolismo , Oxirredução , Ratos , Ratos WistarRESUMO
Toxic endogenous or exogenous compounds can be inactivated by various conjugation reactions. Glucuronidation (i.e. conjugation with glucuronate) is especially important due to the large number of drugs and chemical carcinogens that are detoxified through this pathway. Stable and harmless glucuronides can be reactivated by enzymatic hydrolysis thus inhibitors of glucuronidase activity reduce the risk of chemical carcinogenesis. The aim of this study was to reveal whether this mechanism contributes to the anti-cancer effect of green tea flavanols, which has been shown in various animal models. Therefore, we investigated the effect of these polyphenols on deglucuronidation in rat liver microsomes and in Hepa 1c1c7 mouse hepatoma cells, using 4-methylumbelliferyl glucuronide as model substrate. Tea flavanols inhibited beta-glucuronidase in intact vesicles, where glucuronide transport across the microsomal membrane is rate-limiting, but were almost ineffective in permeabilized vesicles. Epigallocatechin gallate, the major green tea flavanol was shown to have a concentration-dependent inhibitory effect on both beta-glucuronidase activity and glucuronide transport in native vesicles. Epigallocatechin gallate also inhibited beta-glucuronidase activity in native Hepa 1c1c7 mouse hepatoma cells, while failed to affect the enzyme in alamethicin-permeabilized cells, where the endoplasmic membrane barrier was eliminated. Our findings indicate that tea flavanols inhibit deglucuronidation in the endoplasmic reticulum at the glucuronide transport stage. This phenomenon might potentially contribute to the cancer-preventing dietary or pharmacological effect attributed to these catechins.
Assuntos
Catequina/análogos & derivados , Retículo Endoplasmático/metabolismo , Flavonoides/farmacologia , Glucuronídeos/metabolismo , Fenóis/farmacologia , Chá/química , Animais , Anticarcinógenos/farmacologia , Transporte Biológico/efeitos dos fármacos , Catequina/química , Catequina/farmacologia , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Retículo Endoplasmático/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Glucuronidase/metabolismo , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Polifenóis , Ratos , Ratos WistarRESUMO
Effect of 5-100 microM epigallocatechin gallate (EGCG) on hepatic glucose 6-phosphatase (G6Pase) system was investigated. EGCG inhibited G6Pase in intact but not in permeabilized rat liver microsomes, suggesting the interference with the transport. However, EGCG did not hinder microsomal glucose 6-phosphate (G6P) uptake. Instead, it increased the accumulation of radioactivity after the addition of [(14)C]G6P, presumably due to a slower release of [(14)C]glucose, the product of luminal hydrolysis. Indeed, EGCG was found to inhibit microsomal glucose efflux. Since G6Pase activity is depressed by glucose in a concentration-dependent manner, we concluded that EGCG inhibits G6Pase through an elevated luminal glucose level.
Assuntos
Catequina/análogos & derivados , Flavonóis/farmacologia , Glucose-6-Fosfatase/antagonistas & inibidores , Fígado/enzimologia , Chá/química , Animais , Catequina/isolamento & purificação , Catequina/farmacologia , Flavonóis/isolamento & purificação , Glucose/farmacologia , Glucose-6-Fosfatase/análise , Glucose-6-Fosfato/metabolismo , Fígado/efeitos dos fármacos , Fígado/ultraestrutura , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , RatosRESUMO
Stress is the imbalance of homeostasis, which can be sensed even at the subcellular level. The stress-sensing capability of various organelles including the endoplasmic reticulum (ER) has been described. It has become evident that acute or prolonged ER stress plays an important role in many human diseases; especially those involving organs/tissues specialized in protein secretion. This article summarizes the emerging role of ER stress in diverse human pathophysiological conditions such as carcinogenesis and tumor progression, cerebral ischemia, plasma cell maturation and apoptosis, obesity, insulin resistance, and type 2 diabetes. Certain components of the ER stress response machinery are identified as biomarkers of the diseases or as possible targets for therapeutic intervention.
Assuntos
Retículo Endoplasmático/fisiologia , Resposta ao Choque Térmico/fisiologia , Animais , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico/biossíntese , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/fisiologia , Humanos , Chaperonas Moleculares/biossíntese , Chaperonas Moleculares/genética , Chaperonas Moleculares/fisiologiaRESUMO
Loss-of-function mutations in the gene encoding GLUT10 are responsible for arterial tortuosity syndrome (ATS), a rare connective tissue disorder. In this study GLUT10-mediated dehydroascorbic acid (DAA) transport was investigated, supposing its involvement in the pathomechanism. GLUT10 protein produced by in vitro translation and incorporated into liposomes efficiently transported DAA. Silencing of GLUT10 decreased DAA transport in immortalized human fibroblasts whose plasma membrane was selectively permeabilized. Similarly, the transport of DAA through endomembranes was markedly reduced in fibroblasts from ATS patients. Re-expression of GLUT10 in patients' fibroblasts restored DAA transport activity. The present results demonstrate that GLUT10 is a DAA transporter and DAA transport is diminished in the endomembranes of fibroblasts from ATS patients.
Assuntos
Artérias/anormalidades , Ácido Desidroascórbico/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/genética , Instabilidade Articular/genética , Dermatopatias Genéticas/genética , Malformações Vasculares/genética , Ácido Ascórbico/metabolismo , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/genética , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Facilitadoras de Transporte de Glucose/antagonistas & inibidores , Humanos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Interferência de RNA , RNA Interferente Pequeno/farmacologiaRESUMO
Cellular metabolism provides various sources of hydrogen peroxide (H2O2) in different organelles and compartments. The suitability of H2O2 as an intracellular signaling molecule therefore also depends on its ability to pass cellular membranes. The propensity of the membranous boundary of the endoplasmic reticulum (ER) to let pass H2O2 has been discussed controversially. In this essay, we challenge the recent proposal that the ER membrane constitutes a simple barrier for H2O2 diffusion and support earlier data showing that (i) ample H2O2 permeability of the ER membrane is a prerequisite for signal transduction, (ii) aquaporin channels are crucially involved in the facilitation of H2O2 permeation, and (iii) a proper experimental framework not prone to artifacts is necessary to further unravel the role of H2O2 permeation in signal transduction and organelle biology.