Pro- és antioxidáns hatások szerepe az endoplazmás retikulum eredetű stresszben és apoptózisban = Role of pro- and antioxidant effects in the endoplasmic reticulum stress and apoptosis

Az endoplazmás retikulum számos környezeti és metabolikus hatás szenzora. A jelátvitel gyakran a luminális redoxon keresztül valósul meg. Munkánk során azonosítottuk a hexóz-6-foszfát dehidrogenázt, mint a luminális piridin nukleotidok redox státuszának meghatározóját több sejttípusban. A luminális redox fontos a tápláltság érzékelésében, valamint a sejt apotózis/autofágia szabályozásában. A luminális redoxot befolyásoló antioxidánsok, hepatotoxinok, hormonanalógok és más környezeti hatások alapvetően befolyásolhatják a sejt életképességét. | The endoplasmic reticulum is an important sensor and integrator of environmental and metabolic stimuli. The signaling often involves the changes in luminal redox. We have identified the hexose-6-phosphate dehydrogenase as the main determinant of the redox state of luminal pyridine nucleotides in several cell types. Luminal redox is important in nutrient sensing, and in the regulation of programmed cell death. Antioxidants, hepatotoxins, endocrine disruptors and other environmental agents affecting luminal redox can profoundly alter the viability of the cell

Az intraluminális hidrogén peroxid elimináció mechanizmusai az endoplazmás retikulumban = Mechanisms of intraluminal hydrogen peroxide elimination in the endoplasmic reticulum

Az endoplazmás retikulum (ER) oxidatív protein foldingjának luminális H2O2 termelése hozzájárul az organellum oxidatív környezetének kialakulásához. Kimutattuk, hogy az Ero1α, az oxidatív hajtogatás és ER redox homeosztázis egyik legfontosabb szabályozója, feldúsul a MAM frakcióban, és szabályozza a Ca2+ áramokat. Az Ero1α szintjének mind növelésével mind csökkentésével módosítani lehetett az ER Ca2+ fluxusokat, amely feltárja a fehérje kulcsfontosságú szerepét a korai szekréciós kompartimentumban. Azt is megfigyeltük, hogy a máj ER luminális H2O2 szint emelkedése in vivo egerekben a mikroszómális GSH és fehérje tiol tartalom csökkenését, valamint luminális oxidoreduktázok redox állapotának eltolódását eredményezte. Az oxidatív hatás kiváltotta ar ER tágulását, mely redukálószerekkel kivédhető volt. ER-be célzott katalázt overexpresszáló, antitest termelő sejtekben az érett antitest polimerek csökkent szekrécióját, míg az antitest prekurzor monomerek/dimerek intracelluláris felhalmozódását észleltük. Az eredmények szerint a helyi H2O2 termelés elősegíti, míg a H2O2 eltávolítása rontja a diszulfidok kialakulását. Három review-t közöltünk az ER redox viszonyairól. Egy tanulmányban az oxidatív protein foldingra új paradigmát javasoltunk: a több oxidáns hipotézist. Két átfogó review-ban a jelenlegi ismereteket foglaltuk össze az ER legfontosabb redox rendszereiről. Másik két cikkben pedig a kompartimentáció jelentőségét alátámasztó eredményeket tárgyaltuk. | Oxidative protein folding in the endoplasmic reticulum (ER) results in luminal H2O2 production, contributing to the formation of the oxidative environment of the organelle. We showed that Ero1α, a key controller of oxidative folding and ER redox homeostasis, is enriched in mitochondrial-associated ER membranes (MAM) and regulates Ca2+ fluxes. Either increasing or decreasing the levels of Ero1α affected Ca2+ fluxes, which reveals a pivotal role for this oxidase in the early secretory compartment. We also observed that the elevation of hepatic ER luminal H2O2 levels of mice in vivo resulted in a decrease in microsomal GSH and protein-thiol contents and in a redox shift of certain luminal oxidoreductases. The oxidative wave was accompanied by reversible dilation of ER, prevented by concomitant reducing treatment. ER targeted catalase overexpressing antibody producing cells showed diminished secretion of mature antibody polymers, while incomplete antibody monomers/dimers were accumulated and/or secreted. The results indicate that local H2O2 production promotes, while quenching of H2O2 impairs disulfide formation. We published three reviews on the redox conditions in the ER. In a Hypothesis paper we proposed a new paradigm for the oxidative folding: the multiple oxidant hypothesis. In two comprehensive reviews we summarized the present knowledge on the major redox systems in the ER. We summarized the facts showing the importance of compartmentation in two other reviews

Composition of the redox environment of the endoplasmic reticulum and sources of hydrogen peroxide

The endoplasmic reticulum (ER) is a metabolically active organelle, which has a central role in proteostasis by translating, modifying, folding, and occasionally degrading secretory and membrane proteins. The lumen of the ER represents a separate compartment of the eukaryotic cell, with a characteristic proteome and metabolome. Although the redox metabolome and proteome of the compartment have not been holistically explored, it is evident that proper redox conditions are necessary for the functioning of many luminal pathways. These redox conditions are defined by local oxidoreductases and the membrane transport of electron donors and acceptors. The main electron carriers of the compartment are identical with those of the other organelles: glutathione, pyridine and flavin nucleotides, ascorbate, and others. However, their composition, concentration, and redox state in the ER lumen can be different from those observed in other compartments. The terminal oxidases of oxidative protein folding generate and maintain an "oxidative environment" by oxidizing protein thiols and producing hydrogen peroxide. ER-specific mechanisms reutilize hydrogen peroxide as an electron acceptor of oxidative folding. These mechanisms, together with membrane and kinetic barriers, guarantee that redox systems in the reduced or oxidized state can be present simultaneously in the lumen. The present knowledge on the in vivo conditions of ER redox is rather limited; development of new genetically encoded targetable sensors for the measurement of the luminal state of redox systems other than thiol/disulfide will contribute to a better understanding of ER redox homeostasis

A systems biological view of life-and-death decision with respect to endoplasmic reticulum stress—The role of PERK pathway

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. © 2017 by the authors; licensee MDPI, Basel, Switzerland

Glucose Transport and Transporters in the Endomembranes

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

Uncoupled redox systems in the lumen of the endoplasmic reticulum. Pyridine nucleotides stay reduced in an oxidative environment.

The redox state of the intraluminal pyridine nucleotide pool was investigated in rat liver microsomal vesicles. The vesicles showed cortisone reductase activity in the absence of added reductants, which was dependent on the integrity of the membrane. The intraluminal pyridine nucleotide pool could be oxidized by the addition of cortisone or metyrapone but not of glutathione. On the other hand, intraluminal pyridine nucleotides were slightly reduced by cortisol or glucose 6-phosphate, although glutathione was completely ineffective. Redox state of microsomal protein thiols/disulfides was not altered either by manipulations affecting the redox state of pyridine nucleotides or by the addition of NAD(P)+ or NAD(P)H. The uncoupling of the thiol/disulfide and NAD(P)+/NAD(P)H redox couples was not because of their subcompartmentation, because enzymes responsible for the intraluminal oxidoreduction of pyridine nucleotides were distributed equally in smooth and rough microsomal subfractions. Instead, the phenomenon can be explained by the negligible representation of glutathione reductase in the endoplasmic reticulum lumen. The results demonstrated the separate existence of two redox systems in the endoplasmic reticulum lumen, which explains the contemporary functioning of oxidative folding and of powerful reductive reactions

Species-Specific Glucose-6-Phosphatase Activity in the Small Intestine-Studies in Three Different Mammalian Models

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

Participation of Low Molecular Weight Electron Carriers in Oxidative Protein Folding

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

Elektron transzfer rendszerek élettani szerepe = The physiological role of electron transfer systems

Fagocitákban leírtuk a NADPH oxidázt szabályozó két különböző GTPáz aktiváló fehérje szabályozását és a kísérő K+ transzport baktérium ölő hatását. Agyi mitokondriumokban (mito) a légzési lánc I. komplexének szubsztrátjai membránpotenciál (Em) függően reaktív oxigénszármazékot (ROS) képeznek. Az alfa-glicerofoszfát (aGP) ROS-t képez az I. komplexen és az aGP-dehidrogenáz enzimen, utóbbit a Ca2+ aktivája. Idegvégződésekben a mito ROS képzését az Em nem befolyásolja. A mito-k elektromos szincíciumot képeznek, de a Ca2+ diffúziója korlátozott. Alacsony O2.- szint a Ca2+ -mobilizáló agonista Ca2+ jel képző hatását glomerulóza sejtben gátolja. A ROS támadáspontja a belső raktárból történő Ca2+ felszabadulás. UV hatására a mito Ca2+ felvétele is csökkent. Angiotenzin II -vel ingerelt H295R sejtben a mito Ca2+ jel képzés sebessége a mito és az endoplazmás retikulum (ER) közelségével korrelál. A p38 MAPK és az újtípusú PKC izoformák egyidejű gátlása a Ca2+ jelnek a citoszolból a mito-ba történő áttevődését gátolja és a fenti korrelációt megszünteti. Az ER lumenében a tiol/diszulfid rendszertől elkülönülő NAD(P)+/NAD(P)H rendszer működik. Redox állapotát a glukóz-6-foszfát transzporter és az intraluminális oxidoreduktázok határozzák meg. A redukált állapot fenntartása szükséges a glukokortikoidok prereceptoriális aktiválásához, s egyes sejtekben antiapoptotikus hatású. Jellemeztük az ER szulfát transzporterét, valamint a transzlokon peptid csatorna anion permeabilitását. | We described in phagocytes the regulation of two GTPase activating proteins, terminating the activity of plasmalemmal NADPH oxidase and the role of K+ movements in bacterial killing. In brain mitochondria complex I dependent substrates show a membrane potential (Em) dependent reactive oxygen species (ROS) formation. ROS production by alpha-glycerophosphate (aGP) occured at complex I and on the aGP-dehydrogenase enzyme. The latter is activited by Ca2+. Mitochondria form an electric syntitium but the diffusion of Ca2+ is limited. In glomerulosa cells, at low [O2.-] angiotensin-induced Ca2+ signalling is attenuated, the site of ROS action is Ca2+ release from the internal stores. The rate of mitochondrial Ca2+ uptake in angiotensin-stimulated cells correlates with the vicinity of the mitochondrion and the endoplasmic reticulum (ER). Simultaneous activation of p38 MAPK and the novel isoforms of PKC attenuates the transfer of cytosolic Ca2+ signal into the mitochondria and abolishes this correlation. In the ER we observed a novel NAD(P)+/NAD(P)H system different from the thiol/disulphide system. Its reduced state is tuned by the glucose-6-phosphate transporter and the luminal oxidoreductases and is required for the prereceptorial activation of glucocorticoids. We have characterized the sulphate transport in the ER, and the contribution of the translocon peptide channel to the membrane permeation of small anions