Molecular regulation of cholesterol metabolism: HDL-based intervention through drugs and diet.

The overloading of cholesterol in the arteries remains the principal cause of cardiovascular diseases. Since available anticholesterolemic drugs are not completely effective and have several severe adverse effects, the aim of this review is to analyze current research focused on the emerging, innovative therapeutic strategies based on both pharmacological and nutritional interventions to control cholesterol metabolism. Pharmacological interventions mainly involve the use of molecules capable of interfering with high-density lipoprotien (HDL) metabolism and the reverse cholesterol transport (RCT) through genetic control of apolipoprotein A-I (ApoA-I), agonism at liver X-receptor alpha (LXRalpha), or inhibition of cholesteryl ester transport protein (CETP), scavenger receptor BI(SR-BI), and ecto F0F1ATPase/synthase.

Ectopic F0F 1 ATP synthase contains both nuclear and mitochondrially-encoded subunits.

Over the past few years, several reports have described the presence of F0F1 ATP synthase subunits at the surface of hepatocytes, where the hydrolytic activity of F1 sector faces outside and triggers HDL endocytosis. An intriguing question is whether the ectopic enzyme has same subunit composition and molecular mass as that of the mitochondrial ATP synthase. Also due to the polar nature of hepatocytes, the enzyme may be localized to a particular cell boundary.

The translocator protein (peripheral benzodiazepine receptor) mediates rat-selective activation of the mitochondrial permeability transition by norbormide.

We have investigated the mechanism of rat-selective induction of the mitochondrial permeability transition (PT) by norbormide (NRB). We show that the inducing effect of NRB on the PT (i) is inhibited by the selective ligands of the 18kDa outer membrane (OMM) translocator protein (TSPO, formerly peripheral benzodiazepine receptor) protoporphyrin IX, N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide and 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one; and (ii) is lost in digitonin mitoplasts, which lack an intact OMM. In mitoplasts the PT can still be induced by the NRB cationic derivative OL14, which contrary to NRB is also effective in intact mitochondria from mouse and guinea pig.

Regulation of the inner membrane mitochondrial permeability transition by the outer membrane translocator protein (peripheral benzodiazepine receptor).

We studied the properties of the permeability transition pore (PTP) in rat liver mitochondria and in mitoplasts retaining inner membrane ultrastructure and energy-linked functions. Like mitochondria, mitoplasts readily underwent a permeability transition following Ca(2+) uptake in a process that maintained sensitivity to cyclosporin A. On the other hand, major differences between mitochondria and mitoplasts emerged in PTP regulation by ligands of the outer membrane translocator protein of 18 kDa, TSPO, formerly known as the peripheral benzodiazepine receptor.

Cyclophilin D modulates mitochondrial F0F1-ATP synthase by interacting with the lateral stalk of the complex.

Blue native gel electrophoresis purification and immunoprecipitation of F(0)F(1)-ATP synthase from bovine heart mitochondria revealed that cyclophilin (CyP) D associates to the complex. Treatment of intact mitochondria with the membrane-permeable bifunctional reagent dimethyl 3,3-dithiobis-propionimidate (DTBP) cross-linked CyPD with the lateral stalk of ATP synthase, whereas no interactions with F(1) sector subunits, the ATP synthase natural inhibitor protein IF1, and the ATP/ADP carrier were observed. The ATP synthase-CyPD interactions have functional consequences on enzyme catalysis and are modulated by phosphate (increased CyPD binding and decreased enzyme activity) and cyclosporin (Cs) A (decreased CyPD binding and increased enzyme activity).

Development and characterization of polyspecific anti-mitochondrion antibodies for proteomics studies on in toto tissue homogenates.

We describe the characterization of polyclonal antibodies directed against the whole mitochondrial subproteome, as obtained by hyperimmunization of rabbits with an organelle fraction purified from human skeletal muscle and lysed by sonication. After 2-DE separations with either blue native electrophoresis or IPG as first dimension and blotting, the polyspecific antibodies detect 113 proteins in human muscle mitochondria, representative of all major biochemical pathways and oxidative phosphorylation (OXPHOS) complexes, and cross-react with 28 proteins in rat heart mitochondria. Using as sample cryosections of human muscle biopsies lysed in urea/thiourea/CHAPS, the mitochondrial subproteome can be detected against the background of contractile proteins.

Switch from inhibition to activation of the mitochondrial permeability transition during hematoporphyrin-mediated photooxidative stress. Unmasking pore-regulating external thiols.

We have studied the mitochondrial permeability transition pore (PTP) under oxidizing conditions with mitochondria-bound hematoporphyrin, which generates reactive oxygen species (mainly singlet oxygen, (1)O(2)) upon UV/visible light-irradiation and promotes the photooxidative modification of vicinal targets. We have characterized the PTP-modulating properties of two major critical sites endowed with different degrees of photosensitivity: (i) the most photovulnerable site comprises critical histidines, whose photomodification by vicinal hematoporphyrin causes a drop in reactivity of matrix-exposed (internal), PTP-regulating cysteines thus stabilizing the pore in a closed conformation; (ii) the most photoresistant site coincides with the binding domains of (external) cysteines sensitive to membrane-impermeant reagents, which are easily unmasked when oxidation of internal cysteines is prevented. Photooxidation of external cysteines promoted by vicinal hematoporphyrin reactivates the PTP after the block caused by histidine photodegradation.

Conformational role of the divalent metal in bovine heart mitochondrial F1-ATPase: an electron spin echo envelope modulation study.

The catalytic sites of beef heart mitochondrial F1-ATPase were studied by electron spin echo envelope modulation (ESEEM) spectroscopy, using Mn(II) as a paramagnetic probe, which replaces the naturally occurring Mg(II), maintaining the enzyme catalytic activity. F1-ATPase was purified from beef heart mitochondria. A protein still containing three endogenous nucleotides, named MF1(1,2), is obtained under milder conditions, whereas a harsher treatment gives a fully depleted F1, named MF1(0,0).

Assessing the molecular basis for rat-selective induction of the mitochondrial permeability transition by norbormide.

It was recently demonstrated that the rat-selective toxicant norbormide also induces rat-selective opening of the permeability transition pore (PTP) in isolated mitochondria. Norbormide is a mixture of endo and exo stereoisomers; however, only the endo forms are lethal to rats. In the present study we tested both endo and exo isomers as well as neutral and cationic derivatives of norbormide to: (i) verify if the PTP-regulatory activity by norbormide is stereospecific; (ii) define the structural features of norbormide responsible for PTP-activation, (iii) elucidate the basis for the drug species-specificity.

Species-specific modulation of the mitochondrial permeability transition by norbormide.

In the present study, we show that norbormide stimulates the opening of the permeability transition pore (PTP) in mitochondria from various organs of the rat but not of guinea pig and mouse. Norbormide does not affect the basic parameters that modulate the PTP activity since the proton electrochemical gradient, respiration, phosphorylation and Ca(2+) influx processes are only partially affected. On the other hand, norbormide induces rat-specific changes in the fluidity of the lipid interior of mitochondrial membranes, as revealed by fluorescence anisotropy of various reporter molecules.

Antioxidant defences in cybrids harboring mtDNA mutations associated with Leber's hereditary optic neuropathy.

Oxidative stress and imbalance between free radical generation and detoxification may play a pivotal role in the pathogenesis of Leber's hereditary optic neuropathy (LHON). Mitochondria, carrying the homoplasmic 11778/ND4, 3460/ND1 and 14484/ND6 mtDNA point mutations associated with LHON, were used to generate osteosarcoma-derived cybrids. Enhanced mitochondrial production of reactive oxygen species has recently been demonstrated in these cybrids [Beretta S, Mattavelli L, Sala G, Tremolizzo L, Schapira AHV, Martinuzzi A, Carelli V & Ferrarese C (2004) Brain 127, 2183-2192].

High-affinity metal-binding site in beef heart mitochondrial F1ATPase: an EPR spectroscopy study.

The high-affinity metal-binding site of isolated F(1)-ATPase from beef heart mitochondria was studied by high-field (HF) continuous wave electron paramagnetic resonance (CW-EPR) and pulsed EPR spectroscopy, using Mn(II) as a paramagnetic probe. The protein F(1) was fully depleted of endogenous Mg(II) and nucleotides [stripped F(1) or MF1(0,0)] and loaded with stoichiometric Mn(II) and stoichiometric or excess amounts of ADP or adenosine 5'-(beta,gamma-imido)-triphosphate (AMPPNP). Mn(II) and nucleotides were added to MF1(0,0) either subsequently or together as preformed complexes.

Antioxidant defences and homeostasis of reactive oxygen species in different human mitochondrial DNA-depleted cell lines.

Three pairs of parental (rho+) and established mitochondrial DNA depleted (rho0) cells, derived from bone, lung and muscle were used to verify the influence of the nuclear background and the lack of efficient mitochondrial respiratory chain on antioxidant defences and homeostasis of intracellular reactive oxygen species (ROS). Mitochondrial DNA depletion significantly lowered glutathione reductase activity, glutathione (GSH) content, and consistently altered the GSH2 : oxidized glutathione ratio in all of the rho0 cell lines, albeit to differing extents, indicating the most oxidized redox state in bone rho0 cells. Activity, as well as gene expression and protein content, of superoxide dismutase showed a decrease in bone and muscle rho0 cell lines but not in lung rho0 cells.