Artemisinin Directly Targets Malarial Mitochondria through Its Specific Mitochondrial Activation

The biological mode of action of artemisinin, a potent antimalarial, has long been controversial. Previously we established a yeast model addressing its mechanism of action and found mitochondria the key in executing artemisinin''s action. Here we present data showing that artemisinin directly acts on mitochondria and it inhibits malaria in a similar way as yeast. Specifically, artemisinin and its homologues exhibit correlated activities against malaria and yeast, with the peroxide bridge playing a key role for their inhibitory action in both organisms. In addition, we showed that artemisinins are distributed to malarial mitochondria and directly impair their functions when isolated mitochondria were tested. In efforts to explore how the action specificity of artemisinin is achieved, we found strikingly rapid and dramatic reactive oxygen species (ROS) production is induced with artemisinin in isolated yeast and malarial but not mammalian mitochondria, and ROS scavengers can ameliorate the effects of artemisinin. Deoxyartemisinin, which lacks an endoperoxide bridge, has no effect on membrane potential or ROS production in malarial mitochondria. OZ209, a distantly related antimalarial endoperoxide, also causes ROS production and depolarization in isolated malarial mitochondria. Finally, interference of mitochondrial electron transport chain (ETC) can alter the sensitivity of the parasite towards artemisinin. Addition of iron chelator desferrioxamine drastically reduces ETC activity as well as mitigates artemisinin-induced ROS production. Taken together, our results indicate that mitochondrion is an important direct target, if not the sole one, in the antimalarial action of artemisinins. We suggest that fundamental differences among mitochondria from different species delineate the action specificity of this class of drugs, and differing from many other drugs, the action specificity of artemisinins originates from their activation mechanism.     

Yeast AMID homologue Ndi1p displays respiration-restricted apoptotic activity and is involved in chronological aging

Apoptosis-inducing factor (AIF) and AIF-homologous mitochondrion-associated inducer of death (AMID) are both mitochondrial flavoproteins that trigger caspase-independent apoptosis. Phylogenetic analysis suggests that these two proteins evolutionarily diverge back from their common prokaryote ancestor. Compared with AIF, the proapoptotic nature of AMID and its mode of action are much less clarified. Here, we show that overexpression of yeast AMID homologue internal NADH dehydrogenase (NDI1), but not external NADH dehydrogenase (NDE1), can cause apoptosis-like cell death, and this effect can be repressed by increased respiration on glucose-limited media. This result indicates that the regulatory network of energy metabolism, in particular the cross-talk between mitochondria and the rest of the cell, is involved in Ndi1p-induced yeast cell apoptosis. The apoptotic effect of NDI1 overexpression is associated with increased production of reactive oxygen species (ROS) in mitochondria. In addition, NDI1 overexpression in sod2 background causes cell lethality in both fermentable and semifermentable media. Interruption of certain components in the electron transport chain can suppress the growth inhibition from Ndi1p overexpression. We finally show that disruption of NDI1 or NDE1 decreases ROS production and elongates the chronological life span of yeast, accompanied by the loss of survival fitness. Implication of these findings for Ndi1p-induced apoptosis is discussed.     

Artemisinin inhibits chloroplast electron transport activity: mode of action

Artemisinin, a secondary metabolite produced in Artemisia plant species, besides having antimalarial properties is also phytotoxic. Although, the phytotoxic activity of the compound has been long recognized, no information is available on the mechanism of action of the compound on photosynthetic activity of the plant. In this report, we have evaluated the effect of artemisinin on photoelectron transport activity of chloroplast thylakoid membrane. The inhibitory effect of the compound, under in vitro condition, was pronounced in loosely and fully coupled thylakoids; being strong in the former. The extent of inhibition was drastically reduced in the presence of uncouplers like ammonium chloride or gramicidin; a characteristic feature described for energy transfer inhibitors. The compound, on the other hand, when applied to plants (in vivo), behaved as a potent inhibitor of photosynthetic electron transport. The major site of its action was identified to be the Q(B); the secondary quinone moiety of photosystemII complex. Analysis of photoreduction kinetics of para-benzoquinone and duroquinone suggest that the inhibition leads to formation of low pool of plastoquinol, which becomes limiting for electron flow through photosystemI. Further it was ascertained that the in vivo inhibitory effect appeared as a consequence of the formation of an unidentified artemisinin-metabolite rather than by the interaction of the compound per se. The putative metabolite of artemisinin is highly reactive in instituting the inhibition of photosynthetic electron flow eventually reducing the plant growth.     

Mitochondrial Respiratory Electron Carriers Are Involved in Oxidative Stress during Heat Stress in Saccharomyces cerevisiae

In the present study we sought to determine the source of heat-induced oxidative stress. We investigated the involvement of mitochondrial respiratory electron transport in post-diauxic-phase cells under conditions of lethal heat shock. Petite cells were thermosensitive, had increased nuclear mutation frequencies, and experienced elevated levels of oxidation of an intracellular probe following exposure to a temperature of 50 degrees C. Cells with a deletion in COQ7 leading to a deficiency in coenzyme Q had a much more severe thermosensitivity phenotype for these oxidative endpoints following heat stress compared to that of petite cells. In contrast, deletion of the external NADH dehydrogenases NDE1 and NDE2, which feed electrons from NADH into the electron transport chain, abrogated the levels of heat-induced intracellular fluorescence and nuclear mutation frequency. Mitochondria isolated from COQ7-deficient cells secreted more than 30 times as much H(2)O(2) at 42 as at 30 degrees C, while mitochondria isolated from cells simultaneously deficient in NDE1 and NDE2 secreted no H(2)O(2). We conclude that heat stress causes nuclear mutations via oxidative stress originating from the respiratory electron transport chains of mitochondria.     

Mitochondrial release of the NADH dehydrogenase Ndi1 induces apoptosis in yeast

Saccharomyces cerevisiae NDI1 codes for the internal mitochondrial ubiquinone oxidoreductase, which transfers electrons from NADH to ubiquinone in the respiratory chain. Previously we found that Ndi1 is a yeast homologue of the protein apoptosis-inducing factor–homologous mitochondrion-associated inducer of death and displays potent proapoptotic activity. Here we show that S. cerevisiae NDI1 is involved in apoptosis induced by various stimuli tested, including H₂O₂, Mn, and acetate acid, independent of Z-VAD-fmk (a caspase inhibitor) inhibition. Although Ndi1 also participates in respiration, its proapoptotic property is separable from the ubiquinone oxidoreductase activity. During apoptosis, the N-terminal of Ndi1 is cleaved off in the mitochondria, and this activated form then escapes out to execute its apoptotic function. The N-terminal cleavage appears to be essential for the manifestation of the full apoptotic activity, as the uncleaved form of Ndi1 exhibits much less growth-inhibitory activity. Our results thus indicate an important role of Ndi1 in the switch of life and death fates in yeast: during normal growth, Ndi1 assimilates electrons to the electron transport chain and initiates the respiration process to make ATP, whereas under stresses, it cleaves the toxicity-sequestering N-terminal cap, is released from the mitochondria, and becomes a cell killer.     

青蒿素类药物的作用机制:一个长久未决的基础研究挑战

青蒿素是中国自主研制的抗疟良药,高效、低毒,许多基于青蒿素研发的衍生物具有良好的抗疟效果,近年来已成为抗疟的一线药物,受到世界医疗卫生界的充分肯定.虽然青蒿素结构奇特,抑疟效果显著,但40年来其生物作用机制之谜一直未被彻底破解.针对青蒿素类药物的作用机制,提出了不同的假说,如血红素参与青蒿素的激活并被烷基化从而起到抑疟作用,线粒体参与青蒿素的激活和作用过程,某些特定的蛋白是青蒿素作用靶点等.除抑疟外,青蒿素类药物在杀灭其他种类寄生虫、抑制某些癌症细胞以及抗病毒、治疗类风湿等方面也有一定作用.本文将对青蒿素类药物作用机制的研究进行综述及展望,包括抗疟疾过程中的药物激活、作用靶点以及简要的青蒿素抑制肿瘤细胞作用机制,以期为今后的研究提供帮助.     

Fatty acids decrease mitochondrial generation of reactive oxygen species at the reverse electron transport but increase it at the forward transport

Long-chain nonesterified (“free”) fatty acids (FFA) can affect the mitochondrial generation of reactive oxygen species (ROS) in two ways: (i) by depolarisation of the inner membrane due to the uncoupling effect and (ii) by partly blocking the respiratory chain. In the present work this dual effect was investigated in rat heart and liver mitochondria under conditions of forward and reverse electron transport. Under conditions of the forward electron transport, i.e. with pyruvate plus malate and with succinate (plus rotenone) as respiratory substrates, polyunsaturated fatty acid, arachidonic, and branched-chain saturated fatty acid, phytanic, increased ROS production in parallel with a partial inhibition of the electron transport in the respiratory chain, most likely at the level of complexes I and III. A linear correlation between stimulation of ROS production and inhibition of complex III was found for rat heart mitochondria. This effect on ROS production was further increased in glutathione-depleted mitochondria. Under conditions of the reverse electron transport, i.e. with succinate (without rotenone), unsaturated fatty acids, arachidonic and oleic, straight-chain saturated palmitic acid and branched-chain saturated phytanic acid strongly inhibited ROS production. This inhibition was partly abolished by the blocker of ATP/ADP transfer, carboxyatractyloside, thus indicating that this effect was related to uncoupling (protonophoric) action of fatty acids. It is concluded that in isolated rat heart and liver mitochondria functioning in the forward electron transport mode, unsaturated fatty acids and phytanic acid increase ROS generation by partly inhibiting the electron transport and, most likely, by changing membrane fluidity. Only under conditions of reverse electron transport, fatty acids decrease ROS generation due to their uncoupling action.     

Oxidation and reduction of exogenous cytochrome c by the activity of the respiratory chain.

Oxidation of exogenous NADH by isolated rat liver mitochondria is generally accepted to be mediated by endogenous cytochrome c which shuttles electrons from the outer to the inner mitochondrial membrane. More recently it has been suggested that, in the presence of added cytochrome c, NADH oxidation is carried out exclusively by the cytochrome oxidase of broken or damaged mitochondria. Here we show that electrons can be transferred in and out of intact mitochondria. It is proposed that at the contact sites between the inner and the outer membrane, a "bi-trans-membrane" electron transport chain is present. The pathway, consisting of Complex III, NADH-b5 reductase, exogenous cytochrome c and cytochrome oxidase, can channel electrons from the external face of the outer membrane to the matrix face of the inner membrane and viceversa. The activity of the pathway is strictly dependent on both the activity of the respiratory chain and mitochondrion integrity.     

Subunit VII, the ubiquinone-binding protein, of the cytochrome b-c1 complex of yeast mitochondria is involved in electron transport at center o and faces the matrix side of the membrane

The functional role and topographical orientation in the inner membrane of subunit VII, the ubiquinone-binding protein, of the cytochrome b-c1 complex of yeast mitochondria has been investigated. The apparent molecular weight of this subunit on sodium dodecyl sulfate-urea gels was calculated to be 15,500, while its amino acid composition was similar to that of the Q-binding proteins present in the cytochrome b-c1 complexes isolated from both beef heart and yeast mitochondria. The specific antibody obtained against subunit VII inhibited 30-47% of the ubiquinol-cytochrome c reductase activity in the isolated cytochrome b-c1 complex and in submitochondrial particles but had no effect on cytochrome c reductase activity in mitoplasts, mitochondria from which the outer membrane has been removed. Furthermore, the antibody against subunit VII strongly inhibited (74%) the reduction of cytochrome b by succinate in the presence of antimycin, an inhibitor of center i, but had no effect on cytochrome b reduction in the presence of myxothiazol, an inhibitor of center o. These results suggest that subunit VII, the Q-binding protein, is involved in electron transport at center o of the cytochrome b-c1 complex of the respiratory chain and that subunit VII is localized facing the matrix side of the inner mitochondrial membrane.     

Artemisinins act through at least two targets in a yeast model

Artemisinin and related compounds are potent and widely used antimalarial drugs but their biochemical mode of action is not clear. There is strong evidence that ATP-dependent calcium transporters are a key target in the malarial parasite. However, work using Saccharomyces cerevisiae suggests that disruption of mitochondrial function is critical in the cell killing activity of these compounds. Here it is shown that, in the absence of reducing agents, artemisinin and artesunate targeted the S. cerevisiae calcium channels Pmr1p and Pmc1p. Both compounds affected the growth of yeast on fermentable and nonfermentable media. This growth inhibition was not seen in a yeast strain in which the genes encoding both calcium channels were deleted. In the presence of reducing agents, which break the endoperoxide bridge in the drugs, growth inhibition was only observed in nonfermentable media. This inhibition could be partially relieved by the addition of a free radical scavenger. These results suggest that the drugs have two biochemical modes of action - one acting by specific binding to calcium channels and one involving free radical production in the mitochondria.     

Fatty acids decrease mitochondrial generation of reactive oxygen species at the reverse electron transport but increase it at the forward transport

Long-chain nonesterified ("free") fatty acids (FFA) can affect the mitochondrial generation of reactive oxygen species (ROS) in two ways: (i) by depolarisation of the inner membrane due to the uncoupling effect and (ii) by partly blocking the respiratory chain. In the present work this dual effect was investigated in rat heart and liver mitochondria under conditions of forward and reverse electron transport. Under conditions of the forward electron transport, i.e. with pyruvate plus malate and with succinate (plus rotenone) as respiratory substrates, polyunsaturated fatty acid, arachidonic, and branched-chain saturated fatty acid, phytanic, increased ROS production in parallel with a partial inhibition of the electron transport in the respiratory chain, most likely at the level of complexes I and III. A linear correlation between stimulation of ROS production and inhibition of complex III was found for rat heart mitochondria. This effect on ROS production was further increased in glutathione-depleted mitochondria. Under conditions of the reverse electron transport, i.e. with succinate (without rotenone), unsaturated fatty acids, arachidonic and oleic, straight-chain saturated palmitic acid and branched-chain saturated phytanic acid strongly inhibited ROS production. This inhibition was partly abolished by the blocker of ATP/ADP transfer, carboxyatractyloside, thus indicating that this effect was related to uncoupling (protonophoric) action of fatty acids. It is concluded that in isolated rat heart and liver mitochondria functioning in the forward electron transport mode, unsaturated fatty acids and phytanic acid increase ROS generation by partly inhibiting the electron transport and, most likely, by changing membrane fluidity. Only under conditions of reverse electron transport, fatty acids decrease ROS generation due to their uncoupling action.     

The absence of energy conservation coupled with electron transfer via the alternative pathway in cyanide-resistant yeast mitochondria.

Electron transfer via the alternative pathway in cyanide-resistant mitochondria of the yeast Candida lipolytica is not coupled with ATP synthesis, generation of membrane potential or energy-dependent reverse electron transport in the main respiratory chain. We conclude that during transfer via the alternative pathway no accumulation of energy in the form of high-energy compounds or membrane potential occurs.     

Tetraphenylborate potentiates the respiratory inhibition by the dopaminergic neurotoxin MPP+ in both electron transport particles and intact mitochondria

The cytotoxicity of 1-methyl-4-phenylpyridinium (MPP+) is believed to arise as a consequence of its time- and energy-dependent accumulation inside mitochondria, followed by inhibition of electron transport at Complex I of the respiratory chain. Consistent with our proposal that the accumulation of MPP+ represents a passive Nernstian transport into mitochondria in response to the transmembrane electrochemical potential gradient, tetraphenylborate (TPB-) was found to accelerate the onset of the respiratory inhibition by MPP+ on intact mitochondria. Moreover, the ultimate level of inhibition reached was unexpectedly also increased. The latter is now explained by our finding that TPB- elicits a 12-fold enhancement of MPP+ inhibition of respiration in electron transport particles. It is suggested that TPB- facilitates access of MPP+ to its intramembrane site of inhibitory action in Complex I.     

The absence of energy conservation coupled with electron transfer via the alternative pathway in cyanide-resistant yeast mitochondria

Electron transfer via the alternative pathway in cyanide-resistant mitochondria of the yeast Candida lipolytica is not coupled with ATP synthesis, generation of membrane potential or energy-dependent reverse electron transport in the main respiratory chain. We conclude that during transfer via the alternative pathway no accumulation of energy in the form of high-energy compounds or membrane potential occurs.     

线粒体通透性转移孔与青蒿素抗疟机制研究

目的:为增进对青蒿素作用机制的了解,探讨参与调节线粒体体积的线粒体通透性转移孔在青蒿素抗疟机制中的作用.方法:分离线粒体,采用分光光度法检测青蒿素能否直接作用于离体线粒体导致线粒体体积变化;利用等效应图分析线粒体通透性转移孔抑制剂是否拮抗青蒿素的抗疟作用.结果:青蒿素可以直接导致离体疟原虫线粒体肿胀,而不会影响鼠肝线粒体体积;两种不同的线粒体通透性转移孔抑制剂均可拮抗青蒿素的抑疟效果.结论:青蒿素可以直接作用于离体疟原虫线粒体导致线粒体肿胀,且青蒿素导致线粒体肿胀的物种选择性与细胞毒性的物种选择性一致.此外,利用抑制剂阻断线粒体通透性转移孔的开放可以拮抗青蒿素的抗疟效果,证明线粒体通透性转移孔在青蒿素抗疟过程中起重要作用.     

Isolation and some properties of mitochondria from yeast Candida lipolytica 695]

Mitochondria is obtained from yeast Candida lipolytica 695 grown in the presence of glucose, lactate or citrate. Yeast mitochondria were shown to be practically indistinguishable from animal tissue mitochondria in [ADP]/[O] values and in their sensitivity to electron transport inhibitors, to inhibitors and uncoupling agents of oxidative phosphorylation. The only exception was more low value of the respiration control under succinate oxidation. Mitochondria from yeast, grown in the presence of lactate or citrate were capable of the reduction of endogenous pyridine nucleotides under succinate oxidation for the expense of the reverse electron transport. No reverse electron transport from succinate to NAD(P) was observed in mitochondria from yeast grown in the presence of glucose, but it was found under oxidation of alpha-glycerophosphate. All three types of yeast mitochondria were not capable of the reverse electron transport coupled with the pyridine nucleotides reduction under lactate oxidation.     

The inhibitors of antioxidant cell enzymes induce permeability transition in yeast mitochondria

In this study we investigated the effects of exogenous and endogenous oxidative stress on mitochondrial membrane permeability transition in yeast cells. E. magnusii yeast was used in the study as it is the only yeast strain possessing a natural high-capacity Са²⁺ transport system. The key reactive oxygen species (ROS) detoxifying enzymes in the yeast cells - catalases (CATs) and superoxide dismutases (SODs) - were fully characterized. At least five isoforms of SODs and only one isoform of CATs were found in the E. magnusii mitochondria. The assessment of the main properties of mitochondrial non-specific permeability under physiological conditions such as dynamics of the membrane potential (?Ψ) and swelling in mitochondria showed that under physiological conditions classical inhibitors of CATs (ATZ - 3-amino-1, 2, 4-triazole) and of SODs (DDC - diethyldithiocarbamate) caused irreversible decline in ?Ψ in the yeast mitochondria. This decline was accelerated in the presence of 500 μM Са²⁺. The combined action of the inhibitors (ATZ + DDC) promoted moderate swelling in the isotonic medium, which was confirmed by transmission electron microscopy. Mitochondrial swelling in the cells exposed to antioxidant system inhibitors was accompanied by typical signs of early apoptosis, namely by chromatin margination and condensation, vacuolization of the cytosol, and damage of the plasma membrane. Here we showed, at both cellular and mitochondrial levels, that the deregulation of oxidant-scavenging enzymes directly leads to the opening of the mPTP, followed by induction of apoptotic markers in the whole yeast cells. Our studies are the first to clarify the highly contradictory data in the literature on mPTP in yeast mitochondria.     

The effect of glucagon on hepatic respiratory capacity

Data from numerous laboratories show that mitochondria isolated from livers treated acutely with glucagon have higher rates of state 3 respiration than control mitochondria. The purpose of the present study was to learn whether this phenomenon is an isolation artifact resulting from a stabilization of the mitochondrial membrane or whether it represents a real increase in the maximal respiratory capacity of liver cells due to glucagon treatment. Electron transport was measured through different spans of the electron transport chain by using ferricyanide as an alternate electron acceptor to O2. With isolated intact liver mitochondria, pretreatment with glucagon was found to cause an increase in electron flow, through both Complex I and Complex III, suggesting that the effect of glucagon was not specific for a single site in the electron transport chain. Using intact isolated hepatocytes, different results are obtained. Respiration was measured in isolated hepatocytes after quantitation of the hepatocyte mitochondrial content by assay of citrate synthase. Hepatocyte respiration could therefore be reported per mg of mitochondrial protein. By providing durohydroquinone to the cells, it was possible to measure electron flow from coenzyme Q to O2 in the absence of the physiological regulation of substrate supply. Likewise, the addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone released the in situ mitochondria from control by the cytosolic ATP/ADP ratio and it was possible to measure maximal electron flow rates through Complex III. In the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone, electron flow was higher in mitochondria in the cell than in isolated mitochondria. Glucagon caused no increase in mitochondrial respiration in situ either in the presence of the physiological substrates or in the presence of durohydroquinone. The data obtained do not support a role for the electron transport chain as a target of glucagon action in hepatocytes.     

Inhibition of Candida parapsilosis Fatty Acid Synthase (Fas2) Induces Mitochondrial Cell Death in Serum

We have recently observed that a fatty acid auxotrophic mutant (fatty acid synthase, Fas2Δ/Δ) of the emerging human pathogenic yeast Candida parapsilosis dies after incubation in various media including serum. In the present study we describe the mechanism for cell death induced by serum and glucose containing media. We show that Fas2Δ/Δ yeast cells are profoundly susceptible to glucose leading us to propose that yeast cells lacking fatty acids exhibit uncontrolled metabolism in response to glucose. We demonstrate that incubation of Fas2Δ/Δ yeast cells with serum leads to cell death, and this process can be prevented with inhibition of protein or DNA synthesis, indicating that newly synthesized cellular components are detrimental to the mutant cells. Furthermore, we have found that cell death is mediated by mitochondria. Suppression of electron transport enzymes using inhibitors such as cyanide or azide prevents ROS overproduction and Fas2Δ/Δ yeast cell death. Additionally, deletion of mitochondrial DNA, which encodes several subunits for enzymes of the electron transport chain, significantly reduces serum-induced Fas2Δ/Δ yeast cell death. Therefore, our results show that serum and glucose media induce Fas2Δ/Δ yeast cell death by triggering unbalanced metabolism, which is regulated by mitochondria. To our knowledge, this is the first study to critically define a link between cytosolic fatty acid synthesis and mitochondrial function in response to serum stress in C. parapsilosis.     

Mitochondrial localization of cytochrome c1 with specific antibodies]

A specific antibody against cytochrome c1 (pig heart mitochondria) has been obtained. It inhibits the electron transport of the respiratory chain in the intact mitochondria at the cytochrome c1 site of inner mitochondrial membrane ; but it has no effect on the isolated submitochondrial particles (inside-out inner mitochondrial membrane vesicles free of any outer membrane or outside-out inner membrane). Thus the topologic position of cytochrome c1 in the inner mitochondrial membrane is asymetrically lcoated on the outer side of the inner mitochondrial membrane. These results agree with our previous researches on ATP-ase and cytochromes b, c and a, indicating the location on the inner side for the first one, transmembranous for the last one, on the outer side for the others respiratory chain components. Thus the electron transport from cytochrome b to a takes place in the outer region of inner mitochondrial membrane and the transmembranous location of cytochrome-oxidase facilitates the transfer of the electrons to oxygen.