Bruno Miroux

Disruption of the uncoupling protein-2 gene in mice reveals a role in immunity and reactive oxygen species production

The gene Ucp2 is a member of a family of genes found in animals and plants, encoding a protein homologous to the brown fat uncoupling protein Ucp1 (refs 1–3). As Ucp2 is widely expressed in mammalian tissues, uncouples respiration and resides within a region of genetic linkage to obesity, a role in energy dissipation has been proposed. We demonstrate here, however, that mice lacking Ucp2 following targeted gene disruption are not obese and have a normal response to cold exposure or high-fat diet. Expression of Ucp2 is robust in spleen, lung and isolated macrophages, suggesting a role for Ucp2 in immunity or inflammatory responsiveness. We investigated the response to infection with Toxoplasma gondii in Ucp2−/− mice, and found that they are completely resistant to infection, in contrast with the lethality observed in wild-type littermates. Parasitic cysts and inflammation sites in brain were significantly reduced in Ucp2−/− mice (63% decrease, P<0.04). Macrophages from Ucp2 −/− mice generated more reactive oxygen species than wild-type mice (80% increase, P<0.001) in response to T. gondii, and had a fivefold greater toxoplasmacidal activity in vitro compared with wild-type mice (P<0.001 ), which was absent in the presence of a quencher of reactive oxygen species (ROS). Our results indicate a role for Ucp2 in the limitation of ROS and macrophage-mediated immunity.

Uncoupling Protein 2, in Vivo Distribution, Induction upon Oxidative Stress, and Evidence for Translational Regulation

Uncoupling protein 2 (UCP2) belongs to the mitochondrial anion carrier family and partially uncouples respiration from ATP synthesis when expressed in recombinant yeast mitochondria. We generated a highly sensitive polyclonal antibody against human UCP2. Its reactivity toward mitochondrial proteins was compared between wild type and ucp2(−/−) mice, leading to non-ambiguous identification of UCP2. We detected UCP2 in spleen, lung, stomach, and white adipose tissue. No UCP2 was detected in heart, skeletal muscle, liver, and brown adipose tissue. The level of UCP2 in spleen mitochondria is less than 1% of the level of UCP1 in brown adipose tissue mitochondria. Starvation and LPS treatments increase UCP2 level up to 12 times in lung and stomach, which supports the hypothesis that UCP2 responds to oxidative stress situations. Stimulation of the UCP2 expression occurs without any change in UCP2 mRNA levels. This is explained by translational regulation of the UCP2 mRNA. We have shown that an upstream open reading frame located in exon two of theucp2 gene strongly inhibits the expression of the protein. This further level of regulation of the ucp2 gene provides a mechanism by which expression can be strongly and rapidly induced under stress conditions.

Protective Role of Uncoupling Protein 2 in Atherosclerosis

Background—Uncoupling protein 2 (UCP2) regulates the production of reactive oxygen species in macrophages. However, its role in atherosclerosis is unknown. Methods and Results—Irradiated low-density lipoprotein receptor deficient mice (LDLR-/-) were transplanted with bone marrow from either UCP2 deficient mice (Ucp2-/-) or wild type mice (Ucp2 +/+). Mice were fed an atherogenic diet for 7 weeks. Engraftment of bone marrow cells was confirmed by the presence of UCP2 protein expression in spleen cell mitochondria of Ucp2 +/+ transplanted mice and its absence in Ucp2-/- transplanted mice. Leukocyte counts and plasma cholesterol levels were comparable in both groups. We found a marked increase in atherosclerotic lesion size in the thoracic aorta of Ucp2-/- transplanted mice compared with control Ucp2 +/+ transplanted mice (8.3±0.9% versus 4.3±0.4%, respectively;P <0.005), as well as in the aortic sinus (150 066±12 388 &mgr;m2 versus 105 689±9 727 &mgr;m2, respectively;P <0.05). This was associated with increased nitrotyrosine staining, which suggests enhanced oxidative stress. Analysis of plaque composition revealed a significant increase in macrophage accumulation (P <0.05) and apoptosis (P <0.05), along with a decrease in collagen content (P <0.05), suggesting a potentially more vulnerable phenotype. Conclusion—These results suggest a protective role for UCP2 against atherosclerosis.

Uncoupling protein-2 controls proliferation by promoting fatty acid oxidation and limiting glycolysis-derived pyruvate utilization

Uncoupling protein‐2 (UCP2) belongs to the mitochondrial carrier family and has been thought to be involved in suppressing mitochondrial ROS production through uncoupling mitochondrial respiration from ATP synthesis. However, we show here that loss of function of UCP2 does not result in a significant increase in ROS production or an increased propensity for cells to undergo senescence in culture. Instead, Ucp2—/— cells display enhanced proliferation associated with a metabolic switch from fatty acid oxidation to glucose metabolism. This metabolic switch requires the unrestricted availability of glucose, and Ucp2— /— cells more readily activate autophagy than wild‐type cells when deprived of glucose. Altogether, these results suggest that UCP2 promotes mitochondrial fatty acid oxidation while limiting mitochondrial catabolism of pyruvate. The persistence of fatty acid catabolism in Ucp2+/ + cells during a proliferative response correlates with reduced cell proliferation and enhances resistance to glucose starvation‐induced autophagy.— Pecqueur C., Bui, T., Gelly, C., Hauchard, J., Barbot, C., Bouillaud, F., Ricquier, D., Miroux, B., Thompson C. B. Uncoupling protein‐2 controls proliferation by promoting fatty acid oxidation and limiting glycolysis‐derived pyruvate utilization. FASEB J. 22, 9–18 (2008)

Uncoupling Protein-2 Controls Adiponectin Gene Expression in Adipose Tissue Through the Modulation of Reactive Oxygen Species Production

Uncoupling protein-2 (UCP2) is a mitochondrial membrane transporter expressed in white adipose tissue. We observed that circulating adiponectin levels and adiponectin gene expression in adipose tissue are reduced in UCP2-null mice. We studied whether mitochondrial activity and its control by UCP2 may regulate adiponectin gene expression. In 3T3-L1 cells, increasing UCP2 mitochondrial levels by adenoviral-mediated gene transfer induced adiponectin gene expression, whereas oligomycin and antimycin A, inhibitors of ATP synthesis and mitochondrial respiration, led to a downregulation. Reactive oxygen species (ROS) scavengers alleviated the repression of adiponectin gene expression caused by oligomycin or antimycin A. The action of ROS involves the transcription factor CHOP-10, the abundance of which was reduced in response to UCP2 and was induced by oligomycin. CHOP-10 inhibited adiponectin gene expression by interfering with the −117/−73 CCAAT/enhancer binding protein–binding region in the adiponectin gene promoter. Moreover, CHOP-10 levels were increased in adipose tissue from UCP2-null mice. Results indicate that the modulation of ROS levels by mitochondrial activity, and specifically as a consequence of the action of UCP2, controls adiponectin gene expression. This provides a physiological mechanism by which the adipose tissue energetic status may determine the extent of adiponectin release and influence systemic insulin sensitivity.

Dangerous Liaisons between Detergents and Membrane Proteins. The Case of Mitochondrial Uncoupling Protein 2

The extraction of membrane proteins from their native environment by detergents is central to their biophysical characterization. Recent studies have emphasized that detergents may perturb the structure locally and modify the dynamics of membrane proteins. However, it remains challenging to determine whether these perturbations are negligible or could be responsible for misfolded conformations, altering the proteins function. In this work, we propose an original strategy combining functional studies and molecular simulations to address the physiological relevance of membrane protein structures obtained in the presence of detergents. We apply our strategy to a structure of isoform 2 of an uncoupling protein (UCP2) binding an inhibitor recently obtained in dodecylphosphocholine detergent micelles. Although this structure shares common traits with the ADP/ATP carrier, a member of the same protein family, its functional and biological significance remains to be addressed. In the present investigation, we demonstrate how dodecylphosphocholine severely alters the structure as well as the function of UCPs. The proposed original strategy opens new vistas for probing the physiological relevance of three-dimensional structures of membrane proteins obtained in non-native environments.

Mitochondrial DNA polymorphisms specifically modify cerebral β-amyloid proteostasis

Several lines of evidence link mutations and deletions in mitochondrial DNA (mtDNA) and its maternal inheritance to neurodegenerative diseases in the elderly. Age-related mutations of mtDNA modulate the tricarboxylic cycle enzyme activity, mitochondrial oxidative phosphorylation capacity and oxidative stress response. To investigate the functional relevance of specific mtDNA polymorphisms of inbred mouse strains in the proteostasis regulation of the brain, we established novel mitochondrial congenic mouse lines of Alzheimer’s disease (AD). We crossed females from inbred strains (FVB/N, AKR/J, NOD/LtJ) with C57BL/6 males for at least ten generations to gain specific mitochondrial conplastic strains with pure C57BL/6 nuclear backgrounds. We show that specific mtDNA polymorphisms originating from the inbred strains differentially influence mitochondrial energy metabolism, ATP production and ATP-driven microglial activity, resulting in alterations of cerebral β-amyloid (Aβ) accumulation. Our findings demonstrate that mtDNA-related increases in ATP levels and subsequently in microglial activity are directly linked to decreased Aβ accumulation in vivo, implicating reduced mitochondrial function in microglia as a causative factor in the development of age-related cerebral proteopathies such as AD.

Highly efficient over-production in E. coli of YvcC, a multidrug-like ATP-binding cassette transporter from Bacillus subtilis

ATP-binding cassette (ABC) transporters have often been refractory to over-expression. Using the C41(DE3) E. coli as a host strain, membrane vesicles highly enriched (>50%) in YvcC, a previously uncharacterized ABC transporter from Bacillus subtilis homologous to P-glycoprotein multidrug transporters, were obtained. The functionality of YvcC was assessed by its high vanadate-sensitive ATPase activity and its ability to transport a fluorescent drug, the Hoechst 33342.

Alteration of Plant Mitochondrial Proton Conductance by Free Fatty Acids UNCOUPLING PROTEIN INVOLVEMENT

We characterized the uncoupling activity of the plant uncoupling protein from Solanum tuberosum (StUCP) using mitochondria from intact potato tubers or from yeast (Saccharomyces cerevisiae) expressing the StUCPgene. Compared with mitochondria from transfected yeast, StUCP is present at very low levels in intact potato mitochondrial membranes (at least thirty times lower) as shown by immunodetection with anti-UCP1 antibodies. Under conditions that ruled out undesirable effects of nucleotides and free fatty acids on uncoupling activity measurement in plant mitochondria, the linoleic acid-induced depolarization in potato mitochondria was insensitive to the nucleotides ATP, GTP, or GDP. In addition, sensitivity to linoleic acid was similar in potato and in control yeast mitochondria, suggesting that uncoupling occurring in potato mitochondria was because of a UCP-independent proton diffusion process. By contrast, yeast mitochondria expressing StUCP exhibited a higher sensitivity to free fatty acids than those from the control yeast and especially a marked proton conductance in the presence of low amounts of linoleic acid. However, this fatty acid-induced uncoupling was also insensitive to nucleotides. Altogether, these results suggest that uncoupling of oxidative phosphorylation and heat production cannot be the dominant feature of StUCP expressed in native potato tissues. However, it could play a role in preventing reactive oxygen species production as proposed for mammalian UCP2 and UCP3.

Production of UCP1 a membrane protein from the inner mitochondrial membrane using the cell free expression system in the presence of a fluorinated surfactant

Structural studies of membrane protein are still challenging due to several severe bottlenecks, the first being the overproduction of well-folded proteins. Several expression systems are often explored in parallel to fulfil this task, or alternately prokaryotic analogues are considered. Although, mitochondrial carriers play key roles in several metabolic pathways, only the structure of the ADP/ATP carrier purified from bovine heart mitochondria was determined so far. More generally, characterisations at the molecular level are restricted to ADP/ATP carrier or the uncoupling protein UCP1, another member of the mitochondrial carrier family, which is abundant in brown adipose tissues. Indeed, mitochondrial carriers have no prokaryotic homologues and very few efficient expression systems were described so far for these proteins. We succeeded in producing UCP1 using a cell free expression system based on E. coli extracts, in quantities that are compatible with structural approaches. The protein was synthesised in the presence of a fluorinated surfactant, which maintains the protein in a soluble form. Further biochemical and biophysical analysis such as size exclusion chromatography, circular dichroism and thermal stability, of the purified protein showed that the protein is non-aggregated, monodisperse and well-folded.