Mgr. Daniela Fornuskova PhD thesis Biochemical and molecular studies of cytochrome c oxidase and ATP synthase deficiencies ABSTRACT The mammalian organism fully depends on the oxidative phosphorylation system (OXPHOS) as the major energy (ATP) producer of the cell. Disturbances of OXPHOS may be caused by mutations in either mitochondrial DNA (mtDNA) or nuclear DNA (nDNA). One part of the thesis is focused on the role of early and late assembled nuclear-encoded structural subunits of cytochrome c oxidase (CcO) as well as Oxa1l, the human homologue of the yeast mitochondrial Oxa1 translocase, in the biogenesis and function of the human CcO complex using stable RNA interference of COX4, COX5A, COX6A1 and OXA1L, as well as expression of epitope-tagged Cox6a, Cox7a and Cox7b, in HEK (human embryonic kidney)- 293 cells. Our results indicate that, whereas nuclear- encoded CcO subunits Cox4 and Cox5a are required for the assembly of the functional CcO complex, the Cox6a subunit is required for the overall stability of the holoenzyme. In OXA1L knockdown HEK-293 cells, intriguingly, CcO activity and holoenzyme content were unaffected, although the inactivation of OXA1 in yeast was shown to cause complete absence of CcO activity. In addition, we compared OXPHOS protein deficiency patterns in mitochondria from skeletal...

ATP synthase represents the key enzyme of cellular energy provision and ATP synthase disorders belong to the most deleterious mitochondrial diseases affecting pediatric population. The aim of this thesis was to identify nuclear genetic defects and describe the pathogenic mechanism of altered biosynthesis of ATP synthase that leads to isolated deficiency of this enzyme manifesting as an early onset mitochondrial encephalo-cardiomyopathy. Studies in the group of 25 patients enabled identification of two new disease-causing nuclear genes responsible for ATP synthase deficiency. The first affected gene was TMEM70 that encodes an unknown mitochondrial protein. This protein was identified as a novel assembly factor of ATP synthase, first one specific for higher eukaryotes. TMEM70 protein of 21 kDa is located in mitochondrial inner membrane and it is absent in patient tissues. TMEM70 mutation was found in 23 patients and turned to be the most frequent cause of ATP synthase deficiency. Cell culture studies also revealed that enzyme defect leads to compensatory-adaptive upregulation of respiratory chain complexes III and IV due to posttranscriptional events. The second affected gene was ATP5E that encodes small structural epsilon subunit of ATP synthase. Replacement of conserved Tyr12 with Cys caused...

We have studied the inhibitory effect of five polyphenols namely, resveratrol, piceatannol, quercetin, quercetrin, and quercetin-3-β-d glucoside on Escherichia coli ATP synthase. Recently published X-ray crystal structures of bovine mitochondrial ATP synthase inhibited by resveratrol, piceatannol, and quercetin, suggest that these compounds bind in a hydrophobic pocket between the γ-subunit C-terminal tip and the hydrophobic inside of the surrounding annulus in a region critical for rotation of the γ-subunit. Herein, we show that resveratrol, piceatannol, quercetin, quercetrin, or quercetin-3-β-d glucoside all inhibit E. coli ATP synthase but to different degrees. Whereas piceatannol inhibited ATPase essentially completely (∼0 residual activity), inhibition by other compounds was partial with ∼20% residual activity by quercetin, ∼50% residual activity by quercetin-3-β-d glucoside, and ∼60% residual activity by quercetrin or resveratrol. Piceatannol was the most potent inhibitor (IC50 ∼14 μM) followed by quercetin (IC50 ∼33 μM), quercetin-3-β-d glucoside (IC50 ∼71 μM), resveratrol (IC50 ∼94 μM), quercitrin (IC50 ∼120 μM). Inhibition was identical in both F1Fo membrane preparations as well as in isolated purified F1. In all cases inhibition was reversible. Interestingly, resveratrol and piceatannol inhibited both ATPase and ATP synthesis whereas quercetin, quercetrin or quercetin-3-β-d glucoside inhibited only ATPase activity and not ATP synthesis.

The role of αPhe-291 residue in phosphate binding by Escherichia coli F1F0-ATP synthase was examined. X-ray structures of bovine mitochondrial enzyme suggest that this residue resides in close proximity to the conserved βR246 residue. Herein, we show that mutations αF291D and αF291E in E. coli reduce the ATPase activity of F1F0 membranes by 350-fold. Yet, significant oxidative phosphorylation activity is retained. In contrast to wild-type, ATPase activities of mutants were not inhibited by MgADP-azide, MgADP-fluoroaluminate, or MgADP-fluoroscandium. Whereas, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) inhibited wild-type ATPase essentially completely, ATPase in mutants was inhibited maximally by ∼75%, although reaction still occurred at residue βTyr-297, proximal to αPhe-291 in the phosphate-binding pocket. Inhibition characteristics supported the conclusion that NBD-Cl reacts in βE (empty) catalytic sites, as shown previously by X-ray structure analysis. Phosphate protected against NBD-Cl inhibition in wild-type but not in mutants. In addition, our data suggest that the interaction of αPhe-291 with phosphate during ATP hydrolysis or synthesis may be distinct.

1. Aufgrund der Resultate mit der Mutante MM10, die trotz Disulfidbrücke noch unverminderte Aktivität und Rotation zeigte, wurde der Frage nachgegangen, ob der EF1-Komplex in der Lage ist, die Rotation durch einen Bruch der alpha-helikalen Struktur von gamma oder durch Rotation um eine Einfachbindung der Disulfidbrücke aufrechtzuerhalten. Quervernetzungen vom Hexagon mit gamma in der Mitte und am unteren Ende konnten das Enzym blockieren. Von den vier betrachteten Mutanten KG11, MM26, MM25 und MM24 fiel der MM26 bereits nach der Isolierung raus. Der MM25 wies nicht mehr als 70% Quervernetzung auf. Bei dem KG11 und dem MM24 konnte jedoch eine 99%-ige Quervernetzung nachgewiesen werden. Mit diesen zwei Cystein-Doppelmutanten wurden weitere Quervernetzungen gefunden, die ebenso wie der MM10 aktiv nach Oxidation sind, aber tiefer im Enzym liegen und die ATP-Hydrolyse trotz Blockade durch Quervernetzung aufrecht erhalten. Es konnte gezeigt werden, dass eine Rotation um die Einzelbindungen innerhalb der Disulfidbrücke unwahrscheinlich ist, und daher die Aktivität des quervernetzten Enzyms nur durch eine Aufwindung der gamma-Helix erklärt werden kann. 2. Die Voraussetzung für ein EFOF1-Kostrukt zum optischen Nachweis der Relativrotation unter Synthesebedingungen war der Einbau von zwei verschiedenen Tags zur spezifischen Bindung. Von den vier Mutanten SE3, SE4, SW7 und WH1 zeigten die beiden SE-Mutanten keine Stabilität bei der Isolierung im Bezug auf die Kopplung zwischen FO- und F1-Teil. Mit dem SW7-EFOF1 wurde eine Mutante gefunden, die mit einer guten Aktivitäts- und Rotationsausbeute nach einer Aufreinigung mittels Streptactin-Affinitätchromatographie durch ihre Stabilität als Ausgangspunkt für das Rotationsexperiment unter ATP-Synthese dienen kann. Der WH1, dessen atp-Operon dem des SW7 gleicht, brachte trotz seines veränderten Vektorursprungs keine Verbesserung.

Calcium tolerant pig and rabbit cardiomyocytes were isolated using retrograde aortic perfusion of nominally calcium-free collagenase. Preconditioning protocols used 1 or 3 x l0-min episodes of ischemic pelleting or pre-incubation with 100 μM adenosine, followed by a 15-min post-incubation and 180-240-min ischemic pelleting. Control cells were incubated and washed in parallel with the experimental groups. Injury was assessed by determination of cell morphology, trypan blue permeability following osmotic swelling, lactate and HPLC analysis of adenine nucleotides. Preconditioned pig cardiomyocytes had a reduced rate of ischemic contracture, but protection occurred without conservation of ATP. Preconditioned rabbit cardiomyocytes were protected without significant changes in rates of ischemic contracture or ATP depletion. Incubation of ischemic cells with the protein phosphatase inhibitor, fostriecin, at PP2A-selective concentrations (0.1-10 μM), mimicked preconditioning in both rabbit and pig cardiomyocytes. In rabbits, the K(ATP) channel blocker, 5-hydroxydecanoate (5-HD), did not block preconditioning or fostriecin protection. In the pig, 5-HD blocked both preconditioning and fostriecin protection, with return of the rates of ischemic contracture to control. However, 5-HD was an effective blocker of protection only in early ischemia. Fostriecin mimicked preconditioning in the rabbit and the early responses of the preconditioned pig. Preconditioning appears associated with protein phosphorylation in both the rabbit and the pig, but major pathways leading to protection may differ in the two species.

We have studied the inhibitory effect of natural and structurally modified polyphenols on Escherichia coli ATP synthase to test (I) if the beneficial dietary effects of polyphenols are related to their inhibitory actions on ATP synthase, (II) if inhibitory effects of polyphenolic compound could be augmented through structural modifications, and (III) if they can act as antimicrobial agent through their actions on ATP synthesis. X-ray crystal structures of polyphenol binding sites suggested that polyphenols bind at a distinct polyphenol binding pocket, at the interface of α,β,γ-subunits. We found that both natural and modified polyphenols inhibit E. coli ATP synthase to varying degrees and structural modifications resulted in augmented inhibition. Inhibition was reversible in all cases. Both natural and modulated compounds inhibited E. coli cell growth to varying degrees. We conclude that dietary benefits of polyphenols may be in part due to the inhibition of ATP synthase.

Kriminalvårdares attityd till intagna har betydelse för att den intagne ska kunna genomgå en positiv förändring under sin verkställighetstid i anstalt. Studiens syfte var därför att undersöka kriminalvårdares attityder gentemot intagna och om olika variabler som t.ex. kön och utbildning påverkade den. En självdeklarationsstudie med ATP-skalan gjordes på tre olika anstalter och ett häkte. Totalt svarade 28 kriminalvårdare på enkäten varav två tolkades som extremvärden och inte togs med i resultatet. Resultatet visade att svenska kriminalvårdare har en mer negativ attityd till intagna än vad som mätts upp i tidigare undersökningar i USA och Norge. Studien fann också att kvinnliga kriminalvårdare hade en mer positiv attityd än sina manliga medarbetare och att utbildning inte alltid är bra för attityden. / Correctional officers’ attitudes toward prisoners are of importance for prisoners to positively change during their sentence in prison. The purpose of this study was to review the attitudes of correctional officers toward prisoners and if different variables like gender and education had any effect on the attitude. A self-declaration survey was done with the ATP-scale at three different prisons and one custody facility. A total of 28 correctional officers responded to the survey, two of them was counted as outliers and was not included in the result. The result showed that Swedish correctional officers had more negative attitudes toward prisoners than found in earlier studies from USA and Norway. The study also found that female correctional officers had more positive attitudes than their male co-workers and that education not always led to a more positive attitude toward inmates.

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Plusieurs types cancéreux prolifèrent par leur capacité à exprimer les marqueurs de régulation négative du système immunitaire, tels que les récepteurs PD-L1 et CD80/86 qui inhibent l’activation et la prolifération des lymphocytes T. L’inhibition de ces voies par des anticorps peut ainsi réactiver la réponse immunitaire chez certains patients. D’autres voies de signalisations sont aujourd’hui explorées, incluant la signalisation purinergique (ATP/adénosine) dans la modulation du microenvironnement tumoral. L’adénosine triphosphate extracellulaire (ATPe) est classifiée parmi les molécules de danger extracellulaire et joue un rôle crucial dans l’activation de l’inflammasome NLRP3, un médiateur important de l’activation des réactions pro-inflammatoires. Les ultrasons sont des ondes mécaniques de haute pression capable d’engendrer la cavitation inertielle des microbulles. Il a été démontré que les microbulles (MB) stimulées par ultrasons (US) libèrent de l’ATP dans le muscle squelettique et dans le muscle cardiaque. Nous posons l’hypothèse selon laquelle le traitement US+MB appliqué sur une tumeur de cancer du sein murin (4T1) in vivo peut libérer de l’ATPe localement dans le but d’activer des réactions pro-inflammatoires pour l’immunothérapie du cancer. Dans ce mémoire, nous présentons la quantification du signal d’ATPe d’une culture de cellules 4T1, puis in vivo dans le muscle et dans une tumeur solide sous-cutanée chez la souris à la suite d’une stimulation par US+MB. Nos études démontrent que la thérapie US+MB libère de l’ATP in vitro et in vivo. En comparant le signal découlant de l’injection IM d’ATP avec celui du muscle et des tumeurs post-US+MB, nous pouvons conclure que le traitement US+MB libère une quantité d’ATPe supérieure à 250 µM, ce qui est supérieur à la quantité d’ATPe dans un microenvironnement tumoral et qui persiste pour une durée d’au moins 60 min dans le muscle et 45 min dans la tumeur. La transfection stable de cellules MC38 (carcinome colorectal) à travers le gène PLenti-PmeLUC, codant la synthèse de luciférase sur la face externe de la membrane cellulaire, est explorée afin d’augmenter le rapport signal sur bruit en bioluminescence (annexe A). L’utilisation de POM-1 (inhibiteur pharmacologique de CD39) et l’utilisation de souris knockout du gène CD39 sont discutées pour la suite du projet afin d’inhiber la dégradation de l’ATP extracellulaire (Annexe B). / Several cancer types proliferate due to their ability to express the negative regulatory markers of the immune system (PD-L1 and CD80/86) which inhibit the activation and proliferation of T cells. Inhibition of these pathways by antibodies (anti-PDL-1, anti-PD-1, anti-CTLA-4) can thus reactivate the immune system in some patients. Other signaling pathways are currently being explored, including purinergic signaling (ATP/adenosine) in the modulation of the tumor microenvironment. Extracellular Adenosine triphosphate (eATP) is classified as danger signal plays a critical role in the activation of the NLRP3 inflammasome, an important mediator of the innate immune response. Ultrasound (US) and microbubbles (MB) have been shown to release ATP in skeletal and cardiac muscle. Thus, we hypothesized that US+MB treatment in 4T1 breast cancer cells could locally activate pro-inflammatory responses by releasing an eATP in tumors for cancer immunotherapy. In this thesis, I present the quantification of the eATP signal after US+MB stimulation in vitro (4T1 cell culture), then in muscle and subcutaneous solid tumors in the mouse. Our studies demonstrate that US+MB treatment releases ATP both in vitro and in vivo. In comparison with the IM injection of ATP, we can conclude that US+MB released a large amount of ATP (>250 µM), which is more than the eATP concentration in the untreated tumor microenvironment, and which persisted for at least 60 min in muscle and 45 min in tumor. The stable transfection of MC38 cells (colorectal carcinoma) through the Plenti-PmeLUC gene, encoding the synthesis of luciferase on the external surface of cell membrane is explored to increase the signal to noise ratio in bioluminescence (see appendix A). The use of POM-1 (pharmacological inhibitor of CD39) and CD39 gene knockout mice to inhibit the degradation of eATP signal are discussed for the continuation of the project.