Corinne Levi-Meyrueis

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.

A limited cytoplasmic region of the prolactin receptor critical for signal transduction

Prolactin receptors (PRL-R) are members of the cytokine receptor superfamily, which have in common, an absence of any known consensus sequence for signal transduction in their cytoplasmic domains. Four areas of high sequence homology have been identified in the cytoplasmic domains of PRL and growth hormone (GH) receptors, which may be important for signal transduction. The aim of this study was to investigate the role of these cytoplasmic regions in the functional activity of the PRL-R. Several mutant forms of PRL-R were constructed either by truncation or by deletion of the cDNA. Biological activities of these mutant receptors were assayed in CHO cells using a functional assay consisting in the co-transfection of PRL-R cDNA, along with a PRL responsive promoter fused to the coding sequence of the chloramphenicol acetyl transferase (CAT) gene. Progressive truncation of the cytoplasmic domain led to a progressive loss of ability to transactivate the CAT gene. Fully active PRL-R could be obtained when 217 of 358 aa of the cytoplasmic domain were present. Deletion of the first region of homology with the GH-R (residues 245-267) abolished the functional activity of PRL-R, whereas deletion of the second region of homology (residues 322-333) was without effect. These results indicate that a critical cytoplasmic region of 23 residues proximal to the transmembrane domain is essential for PRL signal transduction. There is strong homology within an 8-residue segment of this region with other members of the cytokine receptor superfamily, suggesting it contains a sequence necessary for signal transduction.

Contributions of studies on uncoupling proteins to research on metabolic diseases.

Abstract. Ricquier D, Fleury C, Larose M, Sanchis D, Pecqueur C, Raimbault S, Gelly C, Vacher D, Cassard‐Doulcier A‐M, Lévi‐Meyrueis C, Champigny O, Miroux B, Bouillaud F (Centre National de la Recherche Scientifique, Centre de Recherche sur l’Endocrinologie Moléculaire et le Développement, Meudon, France). Contributions of studies on uncoupling proteins to research on metabolic diseases. (Minisymposium: Genes & Obesity). J Intern Med 1999; 245: 637–642.

Contribution to the Identification and Analysis of the Mitochondrial Uncoupling Proteins

This review is primarily focused on the contribution of our laboratory to study of themitochondrial uncoupling UCPs. The initial stage was the description of a 32-kDamembranous protein specifically induced in brown adipose tissue mitochondria of cold-adaptedrats. This protein was then shown by others to be responsible for brown fat thermogenesisand was referred to as the uncoupling protein-UCP (recently renamed UCP1). cDNA andgenomic clones of UCP1 were isolated and used to investigate the topology and functionalorganization of the protein in the membrane and the mechanisms of control of UCP1 genetranscription. Orientation of the transmembrane fragments was proposed and specificamino acid residues involved in the inhibition of UCP1 by purine nucleotides wereidentified in recombinant yeast. A potent enhancer mediating the response of the UCP1gene to retinoids and controlling the specific transcription in brown adipocytes wasidentified using transgenic mice. More recently, we identified UCP2, an UCP homologwidely expressed in human and rodent tissues we also collaborated to characterize theplant UCP. Although the biochemical activities and physiological roles of the novel UCPsare not well understood, these recent data stimulate research on mitochondrial carriers,mitochondrial bioenergetics, and energy expenditure.

The mitochondrial uncoupling protein UCP / genetic and structural studies

Heat production by brown adipocytes is due to uncoupling of the mitochondrial respiratory chain by the uncoupling protein UCP, a nucleotide-inhibitable and free fatty acid-activable proton carrier in the inner membrane, unique to brown adipocyte mitochondria. Our laboratory is studying the mechanisms that restrict UCP gene transcription to brown adipocytes, and the functional organization of the UCP which belongs to the family of mitochondrial transporters. Using cell transfection and transgenic mice evidence was obtained that a region encompassing 3 kb of DNA upstream the transcription start site contains positive and negative elements controlling UCP gene transcription. Transfection experiments based on DNA-CAT constructs identified a strong enhancer at −2.4 kb. This enhancer as well as the proximal region of the promoter were analyzed in detail using DNAse I footprint analysis and band-shift experiments. To study UCP topological organization, antibodies directed against certain subsequences were selected and used. The orientation of 5 out of 6 predicted α-helices was determined and allowed to propose a membranous folding. In collaboration with E. Rial (Madrid), wild and mutated UCP was expressed in yeasts. This strategy was used to demonstrate that none cysteine is essential for UCP and that lysine 268 and glycine 269 are involved in its inhibition by nucleotides.

Molecular Studies of the Mitochondrial Uncoupling Protein

The brown adipocytes play an important role in the regulation of body temperature in hibernating as well as in small and newborn mammals. These thermogenic adipocytes express an Uncoupling Protein — UCP. This mitochondrial carrier, unique to these cells, uncouples mitochondrial ATP synthesis from the respiratory chain activity and is responsible for heat production by brown adipocytes. Besides its important physiological role, UCP is a proton membranous transporter proving the validity of Mitchell’s chemiosmotic theory (Nicholls and Locke 1984, Klingenberg 1990). UCP is also able to transport Cl- ions electrophoretically (Garlid 1990). Both H+ and Cl- transports are inhibited by purine nucleotides and free fatty acids activate H+ translocation (Rial and Nicholls 1989). Another characteristic property of UCP, is that it is a member of an expanding family of mitochondrial ion transporters sharing sequence and structural homologies. In addition to UCP, the main members forming this family are the Adenine Nucleotide Translocator, the Phosphate Carrier and the Oxoglutarate Carrier (Walker 1992).