Uncoupling protein of Acanthamoeba castellanii mitochondria is activated by lipid peroxidation end product hydroxynonenal

Uncoupling proteins, members of the mitochondrial carrier family, are present in mitochondrial inner membrane and mediate free fatty acid-activated, purine-nucleotide-inhibited H+ re-uptake. Since 1995, it has been shown that the uncoupling protein is present in many higher plants and some microorganisms like non-photosynthetic amoeboid protozoon, Acanthamoeba castellanii and non-fermentative yeast Candida parapsilosis. In mitochondria of these organisms, uncoupling protein activity is revealed not only by stimulation of state 4 respiration by free fatty acids accompanied by decrease in membrane potential (these effects being partially released by ATP and GTP) but mainly by lowering ADP/O ratio during state 3 respiration. Plant and microorganism uncoupling proteins are able to divert very efficiently energy from oxidative phosphorylation, competing for deltamicroH+ with ATP synthase. Functional connection and physiological role of uncoupling protein and alternative oxidase, two main energy-dissipating systems in plant-type mitochondria, are discussed.

Download Full-text

Redox state of quinone affects sensitivity of Acanthamoeba castellanii mitochondrial uncoupling protein to purine nucleotides

Biochemical Journal ◽

10.1042/bj20080333 ◽

2008 ◽

Vol 413 (2) ◽

pp. 359-367 ◽

Cited By ~ 14

Author(s):

Aleksandra Swida ◽

Andrzej Woyda-Ploszczyca ◽

Wieslawa Jarmuszkiewicz

Keyword(s):

Respiratory Rate ◽

Redox State ◽

Uncoupling Protein ◽

Acanthamoeba Castellanii ◽

Inhibitory Effect ◽

Proton Conductance ◽

Purine Nucleotides ◽

Protein Activity ◽

Mitochondrial Uncoupling ◽

Quinone Reduction

We studied FFA (free fatty acid)-induced uncoupling activity in Acanthamoeba castellanii mitochondria in the non-phosphorylating state. Either succinate or external NADH was used as a respiratory substrate to determine the proton conductance curves and the relationships between respiratory rate and the quinone reduction level. Our determinations of the membranous quinone reduction level in non-phosphorylating mitochondria show that activation of UCP (uncoupling protein) activity leads to a PN (purine nucleotide)-sensitive decrease in the quinone redox state. The gradual decrease in the rate of quinone-reducing pathways (using titration of dehydrogenase activities) progressively leads to a full inhibitory effect of GDP on LA (linoleic acid) induced proton conductance. This inhibition cannot be attributed to changes in the membrane potential. Indeed, the lack of GDP inhibitory effect observed when the decrease in respiratory rate is accompanied by an increase in the quinone reduction level (using titration of the quinol-oxidizing pathway) proves that the inhibition by nucleotides can be revealed only for a low quinone redox state. It must be underlined that, in A. castellanii non-phosphorylating mitochondria, the transition of the inhibitory effect of GDP on LA-induced UCP-mediated uncoupling is observed for the same range of quinone reduction levels (between 50% and 40%) as that observed previously for phosphorylating conditions. This observation, drawn from the two different metabolic states of mitochondria, indicates that quinone could affect UCP activity through sensitivity to PNs.

Download Full-text

Mitochondrial superoxide and aging: uncoupling-protein activity and superoxide production

Biochemical Society Symposium ◽

10.1042/bss0710203 ◽

2004 ◽

Vol 71 ◽

pp. 203-213 ◽

Cited By ~ 107

Author(s):

Martin D. Brand ◽

Julie A. Buckingham ◽

Telma C. Esteves ◽

Katherine Green ◽

Adrian J. Lambert ◽

...

Keyword(s):

Lipid Peroxidation ◽

Electron Transport ◽

Complex I ◽

Uncoupling Protein ◽

Proton Motive Force ◽

Motive Force ◽

Superoxide Production ◽

Lipid Peroxidation Product ◽

Mild Uncoupling ◽

Reversed Electron Transport

Mitochondria are a major source of superoxide, formed by the one-electron reduction of oxygen during electron transport. Superoxide initiates oxidative damage to phospholipids, proteins and nucleic acids. This damage may be a major cause of degenerative disease and aging. In isolated mitochondria, superoxide production on the matrix side of the membrane is particularly high during reversed electron transport to complex I driven by oxidation of succinate or glycerol 3-phosphate. Reversed electron transport and superoxide production from complex I are very sensitive to proton motive force, and can be strongly decreased by mild uncoupling of oxidative phosphorylation. Both matrix superoxide and the lipid peroxidation product 4-hydroxy-trans-2-nonenal can activate uncoupling through endogenous UCPs (uncoupling proteins). We suggest that superoxide releases iron from aconitase, leading to a cascade of lipid peroxidation and the release of molecules such as hydroxy-nonenal that covalently modify and activate the proton conductance of UCPs and other proteins. A function of the UCPs may be to cause mild uncoupling in response to matrix superoxide and other oxidants, leading to lowered proton motive force and decreased superoxide production. This simple feedback loop would constitute a self-limiting cycle to protect against excessive superoxide production, leading to protection against aging, but at the cost of a small elevation of respiration and basal metabolic rate.

Download Full-text

Hydroxynonenal-stimulated activity of the uncoupling protein in Acanthamoeba castellanii mitochondria under phosphorylating conditions

Biological Chemistry ◽

10.1515/hsz-2012-0326 ◽

2013 ◽

Vol 394 (5) ◽

pp. 649-658 ◽

Cited By ~ 7

Author(s):

Andrzej Woyda-Ploszczyca ◽

Wieslawa Jarmuszkiewicz

Keyword(s):

Respiratory Rate ◽

Atp Synthase ◽

Redox State ◽

Uncoupling Protein ◽

Acanthamoeba Castellanii ◽

Complex Iii ◽

Dependent Manner ◽

Mitochondrial Uncoupling ◽

Concentration Dependent Manner ◽

Succinate Oxidation

Abstract The influence of 4-hydroxy-2-nonenal (HNE), a lipid peroxidation end product, on the activity of the amoeba Acanthamoeba castellanii uncoupling protein (AcUCP) in isolated phosphorylating mitochondria was studied. Under phosphorylating conditions, exogenously added HNE induced GTP-sensitive AcUCP-mediated mitochondrial uncoupling. The HNE-induced proton leak decreased the yield of oxidative phosphorylation in an HNE concentration-dependent manner. The present study describes how the contributions of ATP synthase and HNE-induced AcUCP in phosphorylating respiration vary when the rate of succinate oxidation is decreased by limiting succinate uptake or inhibiting complex III activity within the range of a constant membrane potential. In phosphorylating mitochondria, at a given HNE concentration (100 μm), the efficiency of AcUCP in mitochondrial uncoupling increased as the respiratory rate decreased because the AcUCP contribution remained constant while the ATP synthase contribution decreased with the respiratory rate. HNE-induced uncoupling can be inhibited by GTP only when ubiquinone is sufficiently oxidized, indicating that in phosphorylating A. castellanii mitochondria, the sensitivity of AcUCP activity to GTP depends on the redox state of the membranous ubiquinone.

Download Full-text

Mitochondrial uncoupling protein-2 deficiency protects steatotic mouse hepatocytes from hypoxia/reoxygenation

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00049.2011 ◽

2012 ◽

Vol 302 (3) ◽

pp. G336-G342 ◽

Cited By ~ 10

Author(s):

Zachary P. Evans ◽

Arun P. Palanisamy ◽

Alton G. Sutter ◽

Justin D. Ellett ◽

Venkat K. Ramshesh ◽

...

Keyword(s):

Lipid Peroxidation ◽

Uncoupling Protein ◽

Thiobarbituric Acid ◽

Atp Depletion ◽

Uncoupling Protein 2 ◽

Mitochondrial Uncoupling ◽

Mitochondrial Uncoupling Protein ◽

Ucp2 Expression ◽

Hypoxia Reoxygenation

Steatotic livers are sensitive to ischemic events and associated ATP depletion. Hepatocellular necrosis following these events may result from mitochondrial uncoupling protein-2 (UCP2) expression. To test this hypothesis, we developed a model of in vitro steatosis using primary hepatocytes from wild-type (WT) and UCP2 knockout (KO) mice and subjected them to hypoxia/reoxygenation (H/R). Using cultured hepatocytes treated with emulsified fatty acids for 24 h, generating a steatotic phenotype (i.e., microvesicular and broad-spectrum fatty acid accumulation), we found that the phenotype of the WT and UCP2 KO were the same; however, cellular viability was increased in the steatotic KO hepatocytes following 4 h of hypoxia and 24 h of reoxygenation; Hepatocellular ATP levels decreased during hypoxia and recovered after reoxygenation in the control and UCP2 KO steatotic hepatocytes but not in the WT steatotic hepatocytes; mitochondrial membrane potential in WT and UCP2 KO steatotic groups was less than control groups but higher than UCP2 KO hepatocytes. Following reoxygenation, lipid peroxidation, as measured by thiobarbituric acid reactive substances, increased in all groups but to a greater extent in the steatotic hepatocytes, regardless of UCP2 expression. These results demonstrate that UCP2 sensitizes steatotic hepatocytes to H/R through mitochondrial depolarization and ATP depletion but not lipid peroxidation.

Download Full-text

Chemiluminescence of Acanthamoeba castellanii

Biochemical Journal ◽

10.1042/bj1840149 ◽

1979 ◽

Vol 184 (1) ◽

pp. 149-156 ◽

Cited By ~ 23

Author(s):

D Lloyd ◽

A Boveris ◽

R Reiter ◽

M Filipkowski ◽

B Chance

Keyword(s):

Lipid Peroxidation ◽

Superoxide Dismutase ◽

Exponential Growth ◽

Light Emission ◽

Acanthamoeba Castellanii ◽

Superoxide Anions ◽

Cyanide Concentration ◽

Oxygen Formation ◽

Singlet Oxygen Formation ◽

Similar Intensity

1. Chemiluminescence of Acanthomoeba castellanii in the presence of O2 was of similar intensity in organisms harvested early or late during exponential growth [when cyanide (1 mM) stimulates or inhibits respiration respectively]. 2. Cyanide (up to 1.5 mM) stimulated photoemission in both types of organism by 250–300 photons/s per 10(7) cells above the value observed under aerobic conditions. 3. ‘Dibromothymoquinone’ (2,5-dibromo-6-isopropyl-3-methyl-p-benzoquinone) (up to 80 microM) further increased chemiluminescence. 4. Similar responses were also demonstrated in whole homogenates and in subcellular fractions; 36% of the chemiluminescence was provided by a fraction sedimenting at 100000g-min, and 20% in that fraction that was non-sedimentable at 200000g-min. 5. Mitochondrial substrates (succinate, 2-oxoglutarate, NADH) in the presence or absence of ADP and Pi or peroxisomal substrates (glycollate, urate or ethanol) gave no increases in light emission by whole homogenates or in any of the fractions. 6. It is suggested that reactions responsible for production of chemiluminescence are those primarily producing superoxide anions and leading to lipid peroxidation and singlet-oxygen formation. Photoemission enhancement and superoxide dismutase inhibition showed similar cyanide concentration-dependencies.

Download Full-text