Search

The cytochrome bc1 complex (cyt bc1 or complex III) is a promising target of numerous antibiotics and fungicides. With an aim to indentify new lead structures for the bc1 complex, a series of novel inhibitors were discovered from the natural product karrikinolide for the first time. Extensive screening results suggested variable inhibitory activities of these compounds against succinate-cytochrome reductase [SCR, a mixture of respiratory complex II (SQR) and complex III (the bc1 complex)], implying the essential role of a 4-substituted phenyl group for the high potency. Exceptionally, compound 12g showed excellent inhibition potency having an IC50 value in the sub-micromolar range, demonstrating its higher potency than the commercial control amisulbrom by over two orders of magnitude. Further experiments inferred that these newly prepared compounds mainly target the bc1 complex. Seemingly, this work has presented a new lead scaffold for further development of bc1 complex inhibitors.

A marked stimulation of complex II enzymatic activity was detected in cybrids bearing a homoplasmic MTCYB microdeletion causing disruption of both the activity and the assembly of complex III, but not in cybrids harbouring another MTCYB mutation affecting only the complex III activity. Moreover, complex II stimulation was associated with SDHA subunit tyrosine phosphorylation. Despite the lack of detectable hydrogen peroxide production, up-regulation of the levels of mitochondrial antioxidant defenses revealed a significant redox unbalance. This effect was also supported by the finding that treatment with N-acetylcysteine dampened the complex II stimulation, SDHA subunit tyrosine phosphorylation, and levels of antioxidant enzymes. In the absence of complex III, the cellular amount of succinate, but not fumarate, was markedly increased, indicating that enhanced activity of complex II is hampered due to the blockage of respiratory electron flow. Thus, we propose that complex II phosphorylation and stimulation of its activity represent a molecular mechanism triggered by perturbation of mitochondrial redox homeostasis due to severe dysfunction of respiratory complexes. Depending on the site and nature of the damage, complex II stimulation can either bypass the energetic deficit as an efficient compensatory mechanism, or be ineffectual, leaving cells to rely on glycolysis for survival.

Prompted by pronounced structural differences between rat liver and rat hepatocellular carcinoma mitochondria, we suspected these mitochondrial populations to differ massively in their molecular composition. Aiming to reveal these mitochondrial differences, we came across the issue on how to normalize such comparisons and decided to focus on the absolute number of mitochondria. To this end, fluorescently stained mitochondria were quantified by flow cytometry. For rat liver mitochondria, this approach resulted in mitochondrial protein contents comparable to earlier reports using alternative methods. We determined similar protein contents for rat liver, heart and kidney mitochondria. In contrast, however, lower protein contents were determined for rat brain mitochondria and for mitochondria from the rat hepatocellular carcinoma cell line McA 7777. This result challenges mitochondrial comparisons that rely on equal protein amounts as a typical normalization method. Exemplarily, we therefore compared the activity and susceptibility towards inhibition of complex II of rat liver and hepatocellular carcinoma mitochondria and obtained significant discrepancies by either normalizing to protein amount or to absolute mitochondrial number. Importantly, the latter normalization, in contrast to the former, demonstrated a lower complex II activity and higher susceptibility towards inhibition in hepatocellular carcinoma mitochondria compared to liver mitochondria. These findings demonstrate that solely normalizing to protein amount may obscure essential molecular differences between mitochondrial populations.

The effects of α-tocopheryl succinate (α-TOS), α-tocopheryl acetyl ether (α-TEA) and triphenylphosphonium-tagged vitamin E succinate (mitochondrially targeted vitamin E succinate; MitoVES) on energy-related mitochondrial functions were determined in mitochondria isolated from AS-30D hepatoma and rat liver, bovine heart sub-mitochondrial particles (SMPs), and in rodent and human carcinoma cell lines and rat hepatocytes. In isolated mitochondria, MitoVES stimulated basal respiration and ATP hydrolysis, but inhibited net state 3 (ADP-stimulated) respiration and Ca 2 + uptake, by collapsing the membrane potential at low doses (1–10 μM). Uncoupled mitochondrial respiration and basal respiration of SMPs were inhibited by the three drugs at concentrations at least one order of magnitude higher and with different efficacy: MitoVES > α-TEA > α-TOS. At high doses (> 10 μM), the respiratory complex II (CII) was the most sensitive MitoVES target. Acting as an uncoupler at low doses, this agent stimulated total O 2 uptake, collapsed ∆ ψ m , inhibited oxidative phosphorylation and induced ATP depletion in rodent and human cancer cells more potently than in normal rat hepatocytes. These findings revealed that in situ tumor mitochondria are preferred targets of the drug, indicating its clinical relevance.

Mitochondria perform multiple functions critical to the maintenance of cellular homeostasis. Here we report that the downregulation of histone deacetylase 6 (HDAC6) causes a reduction in the net activity of mitochondrial enzymes, including respiratory complex II and citrate synthase. HDAC6 deacetylase and ubiquitin-binding activities were both required for recovery of reduced mitochondrial metabolic activity due to the loss of HDAC6. Hsp90, a substrate of HDAC6, localizes to mitochondria and partly mediates the regulation of mitochondrial metabolic activity by HDAC6. Our finding suggests that HDAC6 regulates mitochondrial metabolism and might serve as a cellular homeostasis surveillance factor.

A procedure is presented for preparation of diffraction-quality crystals of a vertebrate mitochondrial respiratory complex II. The crystals have the potential to diffract to at least 2.0 A with optimization of post-crystal-growth treatment and cryoprotection. This should allow determination of the structure of this important and medically relevant membrane-protein complex at near-atomic resolution and provide great detail of the mode of binding of substrates and inhibitors at the two substrate-binding sites.

This work contributes to our understanding of mitochondrial responses to changing environmental conditions in plants. The first part of this thesis is focused in the study of redox regulation of mitochondrial gene expression. By using inhibitors, the redox state of the components of the mitochondrial respiratory chain was selectively affected. Effects of the altered redox state of these components on mitochondrial translation were studied. This approach allowed the identification of the respiratory complex II as a key component of regulation of mitochondrial translation. Furthermore, results indicating that protein phosphorylation might be part of this regulatory system are also presented. The other aspect investigated in this work is the characterisation of a recently isolated mitochondrial protein, the pea mitochondrial nucleoside diphosphate kinase (pea mtNDPK). Cloning, expression studies, organellar targeting and phylogenetic analysis of this protein are described. Functional characterisation of the pea mtNDPK revealed a role in stress response. It was found, that the pea mtNDPK interacts with a novel 86 kDa protein, of which synthesis is up-regulated upon heat stress in vivo. The pea mtNDPK seems to have various oligomeric states, suggesting its interaction with different types of substrates. The data presented here indicate that the pea mtNDPK most likely is part of the plant mitochondrial response to heat stress, possibly acting as a modulator of the heat up-regulated 86 kDa protein. En del av cellens funktioner utförs i de membranomslutna små "rum" i cellen som kallas organeller. En typ av organeller är mitokondrierna vars främsta funktion är att producera energi – ATP syntes. ATP produceras under en process kallad respiration. Respirationen sker i mitokondriens membran där elektroner transporteras mellan proteinkomplex (komplex I-IV) och elektron bärare (ubiquinone och cytokrom c) (figur 3). Under respirationen konsumeras syre, NAD(P)H och succinat. Mitokondrierna härstammar från frilevande bakterier som inneslutits i en annan organism (Gray, 1993). Detta ledde till uppkomsten av en ny, mer komplex livsform. Under evolutionens gång har de flesta av mitokondriens gener förflyttats till värdorganismens kärna. Ett fåtal gener finns dock fortfarande kvar i mitokondrien. De flesta av mitokondriens proteiner produceras därför utanför mitokondrien i den s.k. cytoplasman och endast ett fåtal är syntetiserade i mitokondrien. För att hela uppsättningen av mitokondriella proteiner skall produceras måste således två genetiska system användas och dessutom koordineras. Varför har då inte alla mitokondriella gener flyttas till kärnan? En förklaring till detta kan vara att uttrycket av generna i mitokondriens genom är, och måste vara, kontrollerade av förändringar av förhållanden, t ex syrekoncentrationen, innuti mitokondrien. Det är kännt att hos vissa bakterier uttrycks en del gener endast vid hög syrekoncentration (Iuchi and Lin, 1988). Komponenter i bakteriernas respiratoriska system kan känna av skillnader i syrekoncentrationen och justera genuttrycket i enlighet med de rådande förhållanderna. På liknanade sätt anses förändringar i mitokondriens respiratoriska aktivitet kunna påverka uttrycket av de mitokondriella generna (Allen, 1993a). För att testa denna hypotes förändrade vi aktiviteten hos respirationens komponenter och studerade vilka effekter dessa förändringar hade på proteinsyntesen i mitokondrierna. Endast då aktiviteten av komplex II var förändrad, kunde vi se påverkan av mitokondriell proteinsyntes (Paper I). Hur kan komplex II-aktiviteten påverka mitokondriellt genuttryck och proteinsyntes? Kan komplex II kommunicera med komponenter av det mitokondriella genuttryckssystemet? Vi kan ännu inte besvara dessa frågor, men tror att translation (ett av de nödvändiga stegen i proteinsyntesen) är kontrollerad av aktiviteten hos komplex II. Dessutom verkar denna reglering involvera fosforylering av ett oidentifierat protein (Paper II). Proteinfosforylering innebär att en fosfatgrupp binds till ett protein. Denna fosfatgrupp förändrar proteinets egenskaper, aktiverar eller inaktiverar proteiner, och kan fungera som medel för kommunikation i cellen. Mitokondriellt nukleosid difosfat kinas (mtNDPK) är ett fosfoprotein vars funktioner ännu inte är fullständigt undersökta. Vi har identifierat den fullständiga genetiska information som kodar för mtNDPK i ärta och visat också att proteinet importeras in i mitokondrien (Paper III). Vi konstaterade vidare att mtNDPK är mer förekommande i blommorna än i andra delar av växten (Paper III & IV). Vi fann också att mtNDPK interagerar med ett protein som nybildas under värmestress. Vi förmodar därför att ärt-mtNDPK har en roll i växtens reaktion på värmestress (Paper IV).