Pro- és antioxidáns hatások szerepe az endoplazmás retikulum eredetű stresszben és apoptózisban = Pro- and antioxidant effects in 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. A nemzetközi együttműködés keretében elsősorban a hexóz-6-foszfát dehidrogenázt tanulmányoztuk mint szenzort és mint a luminális redox meghatározóját. | 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. In the frame of the international cooperation hexose-6-phosphate dehydrogenase was studied as a nutrient sensor and as the main regulator of luminal redox

Depletion of luminal pyridine nucleotides in the endoplasmic reticulum activates autophagy with the involvement of mTOR pathway

It has been recently shown that redox imbalance of luminal pyridine nucleotides in the endoplasmic reticulum (ER) together with oxidative stress results in the activation of autophagy. In the present study we demonstrated that decrease of luminal NADPH/NADP+ ratio alone by metyrapone was sufficient to promote the mechanism of "self-eating" detected by the activation of LC3. Depletion of luminal NADPH had also significant effect on the key proteins of mTOR pathway, which got inactivated by dephosphorylation. These findings were also confirmed by silencing the proteins (glucose-6-phosphate transporter and hexose-6-phosphate dehydrogenase) responsible for NADPH generation in the ER lumen. However, silencing the key components and addition of metyrapone had different effects on downstream substrates 4EBP1 and p70S6K of mTOR. The applied treatments did not compromise the viability of the cells. Our data suggest that ER stress caused by luminal NADPH depletion activates a pro-survival autophagic mechanism firmly coupled to the activation of mTOR pathway. © 2013 Orsolya Kapuy and Gábor Bánhegyi

A glukóz-6-foszfatáz rendszer extrahepatikus megjelenésének patofiziológiai szerepe = The pathophysiological role of the extrahepatic manifestations of glucose-6-phosphatase system

A mikroszómális glukóz-6-foszfát transzporter extrahepatikus nem glukoneogenetikus sejt-tipusokban történő manifesztációit vizsgálva kimutattuk a fehérje jelenlétét és funkcióját humán granulocitákban és patkány epididimális zsírszövetben. Eredményeink alapján valószínűsíthető, hogy a nem-glukoneogenetikus szövetekben a transzporter legfontosabb feladata a hexóz-6-foszfát dehidrogenáz szubsztrátellátása, s így az intraluminális pentóz foszfát ciklusban a NADPH generálása. A NADPH termelés célja lehet egyrészt az antioxidáns homeosztázis fenntartása (granulocita), vagy a 11β-hidroxiszteroid dehidrogenáz 1-es típusának kofaktor ellátása (máj, zsírszövet) s ennek révén részvétel a kortizon termelésben. Az eredményekből következően a glukóz-6-foszfát transzporter és a hexóz-6-foszfát dehidrogenáz fontos terápiás célpont lehet a metabolikus szindróma gyógyszeres kezelésében. | The presence and function of the microsomal glucose-6-phosphate transporter was investigated in certain non gluconeogenic cell-types, such as human granulocytes and rat epidydymal adipose tissues. Based on our observations the substrate supply for the hexose-6-phosphate dehydrogenase and this way the generation of NADPH in the intralumenal pentose- phosphate pathway is one of the main tasks of the transporter. Thus NADPH production can participate in the maintenance of the antioxidant homeostasis in granulocytes, or in liver and adipose tissues it can contribute to the cofactor supply for the 11β-hydroxysteroid dehydrogenase type 1 and this way it is involved in cortisone production. It is suggested that glucose-6-phosphate transporter and hexose-6-phosphate dehydrogenase are potential therapeutic targets in treatment of the metabolic syndrome

MAP kináz jelátvitel funkcionális vizsgálat mitokondriumban = Functional studies on mitochondrial MAP kinase signalling

Munkánk során a növényekre specifikus, kevésbé vizsgált, D típusú MAPKok családjának egyik tagját az AtMPK9-t tanulmányoztuk. Élesztő kettős-hibrid rendszerrel a kalmodulint, mint lehetséges AtMPK9 fehérje partnert azonosítottuk, majd a kölcsönhatást in vitro transzlációval előállított fehérjékkel többféle megközelítéssel igazoltuk. Az AtMPK9 poszttranszlációs módosításokon keresztül történő szabályozása korábban ismeretlen volt. A pályázat keretében tömegspektrometriás vizsgálatokkal és in vitro mutagenezissel előállított AtMPK9 variánsokkal bizonyítottuk, hogy az aktiválásért felelős T hurok régióban elhelyezkedő TDY aminosav triplet treoninjának és tirozinjának foszforilálása nélkül a kináz nem rendelkezik aktivitással. A tömegspektrometriás adatok alapján az is nyilvánvaló vált, hogy az AtMPK9 kináz doménjét követő C-terminális doménben további négy aminosav foszforilálódik. Vizsgálataink szerint az összes általunk azonosított foszforiláció autofoszforiláció eredménye. Feltételezésünk szerint a kináz autofoszforilációs aktivitásának szabályozásában a kölcsönható partnerként azonosított kalmodulin kaphat szerepet. Az AtMPK9 in planta funkcióját protoplaszt tranziens expresszióval és null-mutáns növényekkel tanulmányoztuk. Vizsgálataink alapján a fehérje kináz abiotikus stresszel aktiválható, azonban ennek ellenére a null-mutáns növények fenotípusa még stressz körülmények között sem tér el a vadtípusétól, így az AtMPK9 valószínűsíthetően funkcionálisan redudáns kináz. | The project aimed at studying AtMPK9, a member of plant specific, D type mitogen activated protein kinase (MAPK). We identified calmodulin as its putative protein interacting partner by yeast two-hybrid assay. In order to evaluate this result, AtMPK9 and calmodulin were produced by in vitro translation and the interaction was confirmed by pull-down assays and surface plasmone resonance analysis. The kinase activity regulation of AtMPK9 was unknown previously. We demonstrated by mass spectrometry and in vitro mutagenesis studies that phosphorylation of threonine and tyrosine of TDY amino acid triad of T loop is inevitable for kinase activity. Further mass spectrometry analysis revealed another four phosphorylated amino acids in the C-terminal domain of AtMPK9. According to our in vitro translation based data, all the identified phosphorylations are caused by autophosphorylation. We hypothesize that the interacting partner calmodulin regulates the autophosphorylation activity of kinase. The in planta function of the protein kinase was studied by protoplast transient overexpression and application of AtMPK9 knock-out plants. Although the kinase activity of AtMPK9 was inducible by abiotic stress, the knock-out plants did not show any difference in phenotype, not even in stress conditions. These data imply that AtMPK9 is a functionally redundant protein kinase

Mitokondrium, oxidatív stressz és öregedés

A z ötvenes években látott napvilágot az öregedés szabad gyökös elmélete, amely szerint a metabolikus útvonalak által termelt reaktív oxigénvegyületek lényeges szerepet kapnak az öregedés folyamatában. A teória később, a mitokond - rium reaktív oxigénvegyületek fő forrásaként történő azonosításával, módosításra került és az öregedés mitokondriá - lis elmélete néven vált ismertté. Ezt követően felvetették egy öregedési „ördögi kör” meglétét, amely szerint a mito - kondriális respiráció során képződő reaktív oxigénvegyületek károsítják a mitokondriális DNS-t, a mitokondriális funkciókat. A mitokondriális diszfunkció következtében megnő a termelődő reaktív oxigénvegyületek mennyisége. Ez az „ördögi kör” a mitokondriális DNS- mutációk felszaporodását válthatja ki, amely öregedéshez vezethet. A kö - zelmúltban létrehozott mtDNS-mutátor egerek mitokondriális DNS-replikációjáért felelős DNS-polimeráz γ exo - nukleáz aktivitását elrontották. Ennek következtében a mitokondriális DNS-ben a szomatikus mutációk száma megnövekedett és egy öregedő fenotípus alakult ki. Érdekes módon a mutáns egerekben sem emelkedett reaktívoxi - génvegyület- termelést , sem oxidatív károsodást nem tapasztaltak, amelyek erősen megkérdőjelezték az „ördögi kör” meglétét

The inter-relationship of ascorbate transport, metabolism and mitochondrial, plastidic respiration.

Abstract Significance: Ascorbate, this multifaceted small molecular weight carbohydrate derivative, plays important roles in a range of cellular processes in plant cells, from the regulation of cell cycle, through cell expansion and senescence. Beyond these physiological functions, ascorbate has a critical role in responses to abiotic stresses, such as high light, high salinity, or drought. The biosynthesis, recycling, and intracellular transport are important elements of the balancing of ascorbate level to the always-changing conditions and demands. Recent Advances: A bidirectional tight relationship was described between ascorbate biosynthesis and the mitochondrial electron transfer chain (mETC), since L-galactono-1,4-lactone dehydrogenase (GLDH), the enzyme catalyzing the ultimate step of ascorbate biosynthesis, uses oxidized cytochrome c as the only electron acceptor and has a role in the assembly of Complex I. A similar bidirectional relationship was revealed between the photosynthetic apparatus and ascorbate biosynthesis since the electron flux through the photosynthetic ETC affects the biosynthesis of ascorbate and the level of ascorbate could affect photosynthesis. Critical Issues: The details of this regulatory network of photosynthetic electron transfer, respiratory electron transfer, and ascorbate biosynthesis are still not clear, as are the potential regulatory role and the regulation of intracellular ascorbate transport and fluxes. Future Directions: The elucidation of the role of ascorbate as an important element of the network of photosynthetic, respiratory ETC and tricarboxylic acid cycle will contribute to understanding plant cell responses to different stress conditions. Antioxid. Redox Signal. 00, 000-000

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

A cellular stress-directed bistable switch controls the crosstalk between autophagy and apoptosis

Decision-making between life and death is one of the most important tasks of cells to maintain their genetic integrity. While the surviving mechanism is driven by Beclin1-dependent autophagy, the suicide processes are controlled by caspases-mediated apoptosis. Interestingly, both these processes share regulators such as Bcl2 and influence each other through feedback loops. The physiological relevance of the crosstalk between autophagy and apoptosis is still unclear. To gain system level insights, we have developed a mathematical model of the autophagy-apoptosis crosstalk. Our analysis reveals that a combination of Bcl2-dependent regulation and feedback loops between Beclin1 and caspases robustly enforces a sequential activation of cellular responses depending upon the intensity and duration of stress levels. The amplifying loops for caspases activation involving Beclin1-dependent inhibition of caspases and cleavage of Beclin1 by caspases (Beclin1 caspases Beclin1; caspases → cleaved Beclin1 → caspases) not only make the system bistable but also help to switch off autophagy at high stress levels. The presence of an additional positive feedback loop between Bcl2 and caspases helps to maintain the caspases activation by making the switch irreversible. Our results provide a framework for further experiments and modelling. © 2013 The Royal Society of Chemistry

Effect of pulsed electromagnetic fields on endoplasmic reticulum stress

The maintenance of protein homeostasis in the endoplasmic reticulum (ER) is crucial in cell life. Disruption of proteostasis results in ER stress that activates the unfolded protein response (UPR); a signalling network assigned to manage the accumulated misfolded or unfolded proteins. Prolonged or unresolved ER stress leads to apoptotic cell death that can be the basis of many serious diseases. Our aim was to study the effect of pulsed electromagnetic fields (PEMF), an alternative, non-invasive therapeutic method on ER stressed cell lines. First, the effect of PEMF treatment on the expression of ER stress markers was tested in three different cell lines. PEMF had no remarkable effect on ER stress protein levels in human embryonic kidney (HEK293T) and human liver carcinoma (HepG2) cell lines. However, the expression of BiP, Grp94 and CHOP were increased in HeLa cells upon PEMF exposure. Therefore, HepG2 cell line was selected for further experiments. Cells were stressed by tunicamycin and exposed to PEMF. Grp94, PDI, CHOP and PARP expression as markers of stress were monitored by Western blot and cell viability was also investigated. Tunicamycin treatment, as expected, increased the expression of Grp94, PDI, CHOP and inactivated PARP. Analysis of protein expression showed that PEMF was able to decrease the elevated level of ER chaperons Grp94, PDI and the apoptosis marker CHOP. The truncated, inactive form of PARP was also decreased. Accordingly, cell viability was also improved by PEMF exposure. These results indicate that PEMF is able to moderate ER stress induced by tunicamycin in HepG2 cells. However, our results clearly draw attention to that different cell lines may vary in the response to PEMF treatment. © 2016, Polish Physiological Society. All rights reserved

A Systems Biological Perspective of Cellular Stress-Directed Programmed Cell Death

Each eukaryotic cell of multicellular organisms must be able to maintain its integrity by sensing both external and internal stimuli. The primary goal of the generated response mechanism is to drive back the system to the former or to a new homeostatic state. Moreover, the response has to provide an accurate survival-or-death decision to avoid any “misunderstanding” and its unwanted consequences. New data revealed that a systems-level crosstalk of molecular networks has an essential role in achieving the correct characteristic of the response. Although many molecular components of these processes already have been revealed, several elements and regulatory connections of crosstalk are still missing. These “gaps” of the complex control networks make hardly impossible to present comprehensive models. Therefore we approach the questions from a systems biology aspect by combining the experimental results with the special technique of mathematical modelling. In this short report we discuss some novel and preliminary data gained by this approach on the crosstalk between life and death decisions under cellular stress, to get a systems biological view of these networks