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...

O objetivo deste trabalho foi ampliar o estudo do mecanismo de ação antidepressivo de Hypericum polyanthemum (POL) e de seu composto derivado da classe dos floroglucinóis uliginosina B (ULI). Considerando o potencial desta planta para o desenvolvimento de novos fármacos, realizou-se um estudo pré-clínico de toxicidade aguda e doses repetidas de POL em camundongos. A administração via oral (v.o.) do extrato ciclo-hexano de POL (90 mg/kg) foi capaz de reduzir significativamente o tempo de imobilidade de ratos e camundongos no teste de natação forçada (TNF). A administração de ULI (10 mg/kg, v.o.) igualmente reduziu o tempo de imobilidade no TNF e também no teste de suspensão pela cauda (TSC). A administração simultânea (v.o), das subdoses de POL (45 mg/kg) e ULI (5 mg/kg) foram capazes de potencializar o efeito antidepressivo das subdoses de imipramina (10 mg/kg), bupropiona (3 mg/kg) e fluoxetina (15 mg/kg), no TNF em camundongos. O pré- tratamento de camundongos, pela via intraperitoneal, com SCH 23390 (antagonista de receptor dopaminérgico D1); sulpirida (antagonista de receptor dopaminérgico D2); prazosin (antagonista de receptor α1-adrenérgico); ioimbina (antagonista de receptor α2-adrenérgico) e p-clorofenilalanina metil éster (pCPA- inibidor da síntese de serotonina (5-HT), preveniu o efeito anti-imobilidade de ULI no TNF. In vitro, ULI inibiu a recaptação sinaptossomal de dopamina ([3H]-DA), noradrenalina ([3H]-NA) e serotonina ([3H]-5-HT), sem ligação aos transportadores, e isto foi demonstrado através das diferentes concentrações de ULI que não afetaram a ligação de [3H]- mazindol, [3H]-nisoxetina e [3H]-citalopram aos sítios de recaptação de DA, NA e 5- HT, respectivamente. Estes resultados sugerem que ULI tem ação não-seletiva sobre o sistema monoaminérgico, o que pode representar um novo composto com inibição tripla da recaptação; além disso possui mecanismo de ação diferente dos antidepressivos clássicos, inibindo a recaptação das monoaminas sem se ligar aos respectivos sítios específicos dos transportadores. Também realizou-se estudo bioquímico da atividade da Na+,K+-ATPase em córtex cerebral e hipocampo de camundongos. O tratamento agudo e repetido por via oral (3 dias, 1 x ao dia) aumentou a atividade desta enzima em córtex cerebral de camundongos em diferentes tempos. ULI não alterou a atividade da Na+,K+-ATPase em hipocampo dos camundongos, e esse resultado corrobora com a hipótese de que ULI pode estar contribuindo para a manutenção da excitabilidade neuronal estimulando esta enzima, e agindo seletivamente no córtex cerebral. Além disso, investigou-se o papel de ULI sobre os canais de sódio dependentes de voltagem (Na+), através da pré-administração de veratrina (ativador de canal de Na+). A veratrina bloqueou o efeito antidepressivo de ULI no TNF e este resultado indica a possibilidade de ULI estar bloqueando os canais de Na+ alterando o gradiente iônico através da estimulação da atividade da Na+,K+-ATPase e, prejudicando ou inibindo a recaptação das monoaminas. Nos experimentos de toxicidade, as mudanças fisiológicas, bioquímicas e histopatológicas mais pronunciadas foram observadas nos camundongos que receberam POL nas doses 5 e/ou 10 vezes maiores que a dose efetiva no TNF em camundongos. Entretanto, POL apresenta constituição química diferente de H. perforatum, sugerindo baixo risco desta espécie em relação à fototoxicidade (ausência de hipericina). Através dos dados obtidos neste estudo, acredita-se que POL pode se tornar uma espécie promissora no desenvolvimento de um novo fitoterápico com ação neuroativa. A inibição da recaptação das monoaminas pode estar relacionada a uma alteração do gradiente iônico de Na+, através da estimulação da atividade da Na+,K+-ATPase, o que leva a crer que ULI possui um perfil de ação lamotrigina-like. Dessa forma, ULI torna-se um novo e promissor padrão molecular de substância com atividade no sistema nervoso central. / The aim of this work was to continue studying the antidepressant mechanism of Hypericum polyanthemum (POL) and its compound derived from phloroglucinols class uliginosin B (ULI). Considering the potential for development of new drugs with this plant, a preclinical study of toxicity with POL was accomplished, administering acute and repeated-doses in mice. The cyclo-hexane extract administration by oral route (p.o.) of POL (90 mg/kg) was able to significantly reduce the immobility time of rats and mice in the forced swimming test (FST). ULI (10 mg/kg, p.o.) also produced a reduction in immobility time in two models predictive of antidepressant activity in mice, FST and the tail suspension test (TST). Simultaneously administration (p.o) of subeffective doses of POL (45 mg/kg) and ULI (5 mg/kg) were able to potentiate the antidepressant effect of subeffective dose of imipramine (10 mg/kg), bupropion (3 mg/kg ) and fluoxetine (15 mg/kg), in the mouse FST. The pretreatment of mice, by i.p. route, with SCH 23390 (dopamine D1 receptor antagonist); sulpiride (dopamine D2 receptor antagonist); (α1 adrenoceptor antagonist); yohimbine (α2 adrenoceptor antagonist) and p-chlorophenylalanine methyl ester (pCPA- an inhibitor of serotonin (5-HT) synthesis) prevented anti- immobility effect of ULI in FST. In vitro, ULI inhibited synaptosomal uptake of dopamine ([3H]-DA), noradrenaline ([3H]-NA) and 5-HT ([3H]-5-HT), without binding with monoaminergic transporters, and this was demonstrated by different concentrations of ULI that did not affect the binding of [3H]-mazindol, [3H]-nisoxetine and [3H]-citalopram to DA, NA and 5-HT uptake sites, respectively. These results suggest that ULI has non-selective action on the monoaminergic system, which may represent a new compound with triple reuptake inhibition; furthermore, ULI has a mechanism of action different from the classical antidepressants by inhibiting monoamine reuptake without bind to respective neurotransporters. In addition we have performed a biochemical study for activity of Na+, K+-ATPase in cerebral cortex and hippocampus of mice. Acute and repeated oral treatment (3 days, 1 x per day) increased enzyme activity in cortex at different times. ULI did not alter Na+, K+- ATPase activity in hippocampus, and this result confirms the hypothesis that ULI can be contributing to the maintenance of neuronal excitability by stimulating this enzyme, and acting selectively in cerebral cortex. Moreover, we investigated the role of ULI on voltage-gated sodium channels (Na+), through pre-administration of veratrine ( Na+ channel oppener). Veratrine was able to abolish the antidepressant effect of ULI in TNF and this result indicates the possibility that ULI blocks the Na+ channels by altering the ionic gradient through the stimulation of Na+, K+-ATPase and impairing or inhibiting the reuptake of monoamines. In toxicity experiments, the more pronounced physiological, biochemical and histopathological changes were observed in mice that received POL at doses 5 and/or 10 fold higher than the effective dose in TNF. However, POL has chemically different from H. perforatum, suggesting low risk of this species in relation to phototoxicity (absence of hypericin). Considering all results, we supposed that POL could be a product with potential for the development of new drugs. The monoamine reuptake inhibition can be related to a change on the gradient of Na+ ion, by stimulation of Na+, K+-ATPase, which suggests that ULI has a lamotrigine-like profile. Therefore, we suggest that phloroglucinol derivative ULI represents a promising new molecular pattern with central nervous system activity.

Symmetric cell division in gram-negative bacteria is essential for generating two equal-sized daughter cells, each containing cellular material crucial for growth and future replication. The Min system, comprised of proteins MinC, MinD and MinE, is particularly important for this process since its deletion leads to minicells incapable of further replication. This thesis focuses on the interactions involving MinE that are important for allowing cell division at the mid-cell and for directing the dynamic localization of MinD that is observed in vivo. Previous experiments have shown that the MinE protein contains an N-terminal region that is required to stimulate MinD-catalyzed ATP hydrolysis in the Min protein interaction cycle. However, MinD-binding residues in MinE identified by in vitro MinD ATPase assays were subsequently found to be buried in the hydrophobic dimeric interface in the MinE structure, raising the possibility that these residues are not directly involved in the interaction. To address this issue, the ability of N-terminal MinE peptides to stimulate MinD activity was studied to determine the role of these residues in MinD activation. Our results implied that MinE likely undergoes a change in conformation or oligomerization state before binding MinD. In addition we performed circular dichroism spectroscopy of MinE. The data suggest that direct interactions between MinE and the lipid membrane can lead to conformational changes in MinE. Using NMR spectroscopy in an attempt to observe this structure change, different membrane-mimetic environments were tested. However the results strongly suggest that structural studies on the membrane-bound state of MinE will pose significant challenges. Taken together, the results in this thesis open the door for further exploration of the interactions involving MinE in order to gain a better understanding of the dynamic localization patterns formed by these proteins in vivo.

Drug addiction, opiates respectively, is a social problem which seriousness is currently on the rise. One of key elements causing addiction is tolerance to increasing doses of drug causing abstinence syndrome during withdrawal and craving. Opioid receptors are members of a large group of receptors coupled with heterotrimeric G-proteins (GPCR), whose properties can be investigated using agonist- stimulated binding [35 S] GTPγS. Many extracellular signals are transferred into a cell through GPCR. Opioid receptor agonists inhibit the activity of adenylyl cyclase and are coupled with G-protein group Gi/Go. This work is devoted to the study of changes in isolated plasma membranes of rat forebrain containing opioid receptors of healthy subjects with membranes acquired from morphine addicted subjects. The rats were long-term morphine treated in increasing doses, to develop the dependency. The comparison is done firstly by binding of [3 H]ouabain to Na,K-ATPase, which proves to be a negative standard of changes, secondly by binding [35 S]GTPγS to G-proteins, thereby providing the functional activity of G-protein in stimulating the binding by the agonist of δ-opioid receptors DADLE or agonist of µ-opioid receptors DAMGO. Furthermore, it has been studied the influence of prostaglandin E1 on binding [35...

The hydration and dynamics of the biomolecules appear to be vital for their proper biological functioning. In the presented thesis, various fluorescence techniques were developed and applied to access these properties and their changes upon the mutual interactions of the biomolecules. Initially, the solvent relaxation method based on recording time-dependent fluorescence shift (TDFS) was used to map DNA interactions with proteins and lipids by the newly synthesised fluorene dye covalently bound to the DNA. Secondly, copper-transporting ATPase was probed by Badan attached to the copper-binding cysteine-proline-cysteine motif. The variations in hydration were found to be crucial for the proper ATPase function. Third, a detailed study on quenching of Badan/Prodan fluorescence by tryptophan revealed the limitations of the TDFS method for protein studies, which is essential finding for further applications of TDFS. Fourth application involves investigations of heavy atom effects on the excited state relaxation processes by up-conversion approach in iodinated metallocorroles, which are promising dyes for biological imaging. The obtained findings shall help in further tuning of the optical properties of the corroles desired for the variety of applications. Finally, fluorescence correlation spectroscopy...

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...

La pompe à sodium, la Na+/K+-ATPase, est un échangeur ionique transmembranaire impliqué dans le maintien de l’homéostasie ionique. Elle est principalement constituée de deux sous-unités, la sous-unité alpha, qui correspond au site catalytique de la pompe, et la sous-unité beta qui régule son activité. <p>La sous-unité alpha de la pompe à sodium possède un site de fixation pour les stéroïdes cardiotoniques, molécules bien connues pour leur utilisation dans le traitement des décompensations cardiaques sévères et des arythmies auriculaires. Les stéroïdes cardiotoniques peuvent être subdivisés en deux groupes, les cardénolides et les bufadiénolides.<p>Diverses études épidémiologiques ont mis en évidence l’activité anticancéreuse des stéroïdes cardiotoniques. En effet, lorsque l’on compare le risque de développement d’un cancer, la mortalité liée au cancer ainsi que le taux de récidive d’un cancer du sein ou d’un cancer prostatique chez les patients traités par de la digoxine dans le cadre de leur traitement cardiaque et les patients non traités, on observe un réel bénéfice des stéroïdes cardiotoniques.<p>Il a déjà été démontré que la pompe à sodium, en plus de sa fonction d’échangeur ionique, possède un rôle central dans de nombreux mécanismes de signalisation cellulaire, impliquant notamment les processus de migration, d’invasion et de mort cellulaire. <p>De plus, de nombreux cancers au pronostic sombre présentent des altérations dans l’expression des sous-unités alpha de la pompe à sodium. C’est le cas par exemple des cancers pulmonaires non à petites cellules (NSCLC), des gliomes, des mélanomes et des cancers rénaux qui surexpriment la sous-unité & / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Venom peptides are known to have strong antimicrobial activity and anticancer properties. King cobra cathelicidin or OH-CATH (KF-34), banded krait cathelicidin (BF-30), wolf spider lycotoxin I (IL-25), and wolf spider lycotoxin II (KE-27) venom peptides were found to strongly inhibit Escherichia coli membrane bound F1Fo ATP synthase. The potent inhibition of wild-type E. coli in comparison to the partial inhibition of null E. coli by KF-34, BF-30, Il-25, or KE-27 clearly links the bactericidal properties of these venom peptides to the binding and inhibition of ATP synthase along with the possibility of other inhibitory targets. The four venom peptides KF-34, BF-30, IL-25, and KE-27, caused ≥85% inhibition of wild-type membrane bound E.coli ATP synthase. Venom peptide induced inhibition of ATP synthase and the strong abrogation of wild-type E. coli cell growth in the presence of venom peptides demonstrates that ATP synthase is a potent membrane bound molecular target for venom peptides. Furthermore, the process of inhibition was found to be fully reversible.

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.