Glutamine stimulates translation of uncoupling protein 2mRNA.

Uncoupling protein 2 (UCP2) belongs to a family of transporters/exchangers of the mitochondrial inner membrane. Using cell lines representing natural sites of UCP2 expression (macrophages, colonocytes, pancreatic beta cells), we show that UCP2 expression is stimulated by glutamine at physiological concentrations. This control is exerted at the translational level. We demonstrate that the upstream open reading frame (ORF1) in the 5' untranslated region (5'UTR) of the UCP2 mRNA is required for this stimulation to take place. Cloning of the 5' UTR of the UCP2 mRNA in front of a GFP cDNA resulted in a reporter gene with which GFP expression could be induced by glutamine. An effect of glutamine on translation of a given mRNA has not been identified before, and this is the first evidence for a link between UCP2 and glutamine, an amino acid oxidized by immune cells or intestinal epithelium and playing a role in the control of insulin secretion.

Translation control of UCP2 synthesis by the upstream open reading frame.

Uncoupling protein 2 (UCP2) belongs to a family of transporters of the mitochondrial inner membrane. In vivo low expression of UCP2 contrasts with a high UCP2 mRNA level, and induction of UCP2 expression occurs without change in mRNA level, demonstrating a translational control. The UCP2 mRNA is characterized by a long 5' untranslated region (5'UTR), in which an upstream open reading frame (uORF) codes for a 36-amino-acid sequence. The 5'UTR and uORF have an inhibitory role in the translation of UCP2. The present study demonstrates that the 3' region of the uORF is a major determinant for this inhibitory role. In this 3' region, a single-base substitution that kept the codon sense unchanged significantly modified UCP2 translation, whereas some important amino acid changes had no effect. We discuss our results within the framework of the existing models explaining initiation of translation downstream of a uORF.

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 the ucp2 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.

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

The coupling of O2 consumption to ADP phosphorylation in mitochondria is partial. This is particularly obvious in brown adipocyte mitochondria which use a regulated uncoupling mechanism generating heat production from substrate oxidation, and catalysing thermogenesis in rodents or infants in response to cold, and arousing hibernators. In the case of brown adipose tissue, the uncoupling mechanism is related to a specific protein in the inner mitochondrial membrane referred to as UCP1. Although the biological importance of UCP1 in human adults is not demonstrated, genetic analysis of various human cohorts suggested a participation of UCP1 to control of fat content and body weight. Very recently, the cloning of UCP2 and UCP3, two homologues of UCP1, has renewed the field of research on the importance of respiration control in metabolic processes and metabolic diseases. UCP2 is widely expressed in organs, whereas UCP3 is mainly present in muscles. These proteins may explain why the coupling of respiration to ADP phosphorylation is less than perfect. Their biological importance should be studied. They also represent new putative targets for drugs against metabolic diseases such as obesity.

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 the mitochondrial uncoupling UCPs. The initial stage was the description of a 32-kDa membranous protein specifically induced in brown adipose tissue mitochondria of cold-adapted rats. This protein was then shown by others to be responsible for brown fat thermogenesis and was referred to as the uncoupling protein-UCP (recently renamed UCP1). cDNA and genomic clones of UCP1 were isolated and used to investigate the topology and functional organization of the protein in the membrane and the mechanisms of control of UCP1 gene transcription. Orientation of the transmembrane fragments was proposed and specific amino acid residues involved in the inhibition of UCP1 by purine nucleotides were identified in recombinant yeast. A potent enhancer mediating the response of the UCP1 gene to retinoids and controlling the specific transcription in brown adipocytes was identified using transgenic mice. More recently, we identified UCP2, an UCP homolog widely expressed in human and rodent tissues we also collaborated to characterize the plant UCP. Although the biochemical activities and physiological roles of the novel UCPs are not well understood, these recent data stimulate research on mitochondrial carriers, mitochondrial bioenergetics, and energy expenditure.

BMCP1, a novel mitochondrial carrier with high expression in the central nervous system of humans and rodents, and respiration uncoupling activity in recombinant yeast.

We report here the cloning and functional analysis of a novel homologue of the mitochondrial carriers predominantly expressed in the central nervous system and referred to as BMCP1 (brain mitochondrial carrier protein-1). The predicted amino acid sequence of this novel mitochondrial carrier indicates a level of identity of 39, 31, or 30%, toward the mitochondrial oxoglutarate carrier, phosphate carrier, or adenine nucleotide translocator, respectively, and a level of identity of 34, 38, or 39% with the mitochondrial uncoupling proteins UCP1, UCP2, or UCP3, respectively. Northern analysis of mouse, rat, or human tissues demonstrated that mRNA of this novel gene is mainly expressed in brain, although it is 10-30-fold less expressed in other tissues. In situ hybridization analysis of brain showed it is particularly abundant in cortex, hippocampus, thalamus, amygdala, and hypothalamus. Chromosomal mapping indicates that BMCP1 is located on chromosome X of mice and at Xq24 in man. Expression of the protein in yeast strongly impaired growth rate. Analysis of respiration of total recombinant yeast or yeast spheroplasts and in particular of the relationship between respiratory rate and membrane potential of yeast spheroplasts revealed a marked uncoupling activity of respiration, suggesting that although BMCP1 sequence is more distant from the uncoupling proteins (UCPs), this protein could be a fourth member of the UCP family.

Deletion of amino acids 261-269 in the brown fat uncoupling protein converts the carrier into a pore.

The uncoupling protein (UCP) from brown adipose tissue mitochondria is a carrier that catalyzes proton re-entry into the matrix and thus dissipates the proton electrochemical potential gradient as heat. UCP activity is regulated: purine nucleotides inhibit while fatty acids activate transport. We have previously reported that sequence 261-269 of the UCP has a closely related counterpart in the adenine nucleotide translocator, as well as in the DNA binding domain of the estrogen receptor. Site-directed mutagenesis of the UCP showed that deletion of amino acids 267-269 in the UCP abolished nucleotide inhibition [Bouillaud, F., et al. (1994) EMBO J. 13, 1990-1997]. Complete deletion of the homologous domain (UCPDelta9) produced a highly deleterious mutant that collapsed the mitochondrial membrane potential and halted yeast growth. Since under our growth conditions revertants appeared rapidly, it was not possible to characterize this mutant. In this article, we have designed conditions to isolate mitochondria containing significant amounts of the UCPDelta9 mutant protein. These mitochondria show no respiratory control and are insensitive to nucleotides. Investigation of the permeability properties revealed that UCPDelta9 mitochondria swell rapidly in potassium salts in the absence of valinomycin, thus indicating a loss of specificity. The size exclusion properties of this mutant were determined with polyethylene glycols of various molecular masses (400-20000 Da), and it was found that UCPDelta9 can catalyze permeation of molecules of up to 1000 Da. We conclude that the deletion of amino acids 261-269 converts the UCP into an unspecific pore.

Kupffer cells are a dominant site of uncoupling protein 2 expression in rat liver.

The mechanisms underlying thermogenesis in liver are not well understood. They may involve proteins related to the mitochondrial uncoupling protein (UCP1) of brown adipocytes. In this paper, it is demonstrated that UCP1 is not expressed in any liver cell type of rat while UCP2, a recently cloned homologue of UCP1, is expressed at a very high level in Kupffer cells but not in hepatocytes. This high level of expression of UCP2 in Kupffer cells allowed cross immunoreactivity with antibodies directed against UCP1. This cross reactivity was confirmed by the detection of UCP2 with anti-UCP1 antibody, in western blotting analysis of transfected yeasts expressing rat UCP2. The high level expression of UCP2 in Kupffer cells suggests a particular function of UCP2 in macrophages.

Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia.

A mitochondrial protein called uncoupling protein (UCP1) plays an important role in generating heat and burning calories by creating a pathway that allows dissipation of the proton electrochemical gradient across the inner mitochondrial membrane in brown adipose tissue, without coupling to any other energy-consuming process. This pathway has been implicated in the regulation of body temperature, body composition and glucose metabolism. However, UCP1-containing brown adipose tissue is unlikely to be involved in weight regulation in adult large-size animals and humans living in a thermoneutral environment (one where an animal does not have to increase oxygen consumption or energy expenditure to lose or gain heat to maintain body temperature), as there is little brown adipose tissue present. We now report the discovery of a gene that codes for a novel uncoupling protein, designated UCP2, which has 59% amino-acid identity to UCP1, and describe properties consistent with a role in diabetes and obesity. In comparison with UCP1, UCP2 has a greater effect on mitochondrial membrane potential when expressed in yeast. Compared to UCP1, the gene is widely expressed in adult human tissues, including tissues rich in macrophages, and it is upregulated in white fat in response to fat feeding. Finally, UCP2 maps to regions of human chromosome 11 and mouse chromosome 7 that have been linked to hyperinsulinaemia and obesity. Our findings suggest that UCP2 has a unique role in energy balance, body weight regulation and thermoregulation and their responses to inflammatory stimuli.

Activation of the uncoupling protein by fatty acids is modulated by mutations in the C-terminal region of the protein.

The transport properties of the uncoupling protein (UCP) from brown adipose tissue have been studied in mutants where Cys304 has been replaced by either Gly, Ala, Ser, Thr, Ile or Trp. This position is only two residues away from the C-terminus of the protein, a region that faces the cytosolic side of the mitochondrial inner membrane. Mutant proteins have been expressed in Saccharomyces cerevisiae and their activity determined in situ by comparing yeast growth rates in the presence and absence of 2-bromopalmitate. Their bioenergetic properties have been studied in isolated mitochondria by determining the effects of fatty acids and nucleotides on the proton permeability and NADH oxidation rate. It is revealed that substitution of Cys304 by non-charged residues alters the response of UCP to fatty acids. The most effective substitution is Cys for Gly since it greatly enhances the sensitivity to palmitate, decreasing threefold the concentration required for half-maximal stimulation of respiration. The opposite extreme is the substitution by Ala which increases twofold the half-maximal concentration. We conclude that the C-terminal region participates in the fatty acid regulation of UCP activity. The observed correlation between yeast growth rates in the presence of bromopalmitate and the calculated activation constants for respiration in isolated mitochondria validates growth analysis as a method to screen the in situ activity of UCP mutants.

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.

A sequence related to a DNA recognition element is essential for the inhibition by nucleotides of proton transport through the mitochondrial uncoupling protein.

The uncoupling protein (UCP) is uniquely expressed in brown adipose tissue, which is a thermogenic organ of mammals. The UCP uncouples mitochondrial respiration from ATP production by introducing a proton conducting pathway through the mitochondrial inner membrane. The activity of the UCP is regulated: nucleotide binding to the UCP inhibits proton conductance whereas free fatty acids increase it. The similarities between the UCP, the ADP/ATP carrier and the DNA recognition element found in the DNA binding domain of the estrogen receptor suggested that these proteins could share common features in their respective interactions with free nucleotides or DNA, and thus defined a putative 'nucleotide recognition element' in the UCP. This article provides demonstration of the validity of this hypothesis. The putative nucleotide recognition element corresponding to the amino acids 261-269 of the UCP was gradually destroyed, and these mutant proteins were expressed in yeast. Flow cytometry, measuring the mitochondrial membrane potential in vivo, showed increased uncoupling activities of these mutant proteins, and was corroborated with studies with isolated mitochondria. The deletion of the three amino acids Phe267, Lys268 and Gly269, resulted in a mutant where proton leak could be activated by fatty acids but not inhibited by nucleotides.

Cysteine residues are not essential for uncoupling protein function.

The uncoupling protein (UCP) of brown adipose tissue is a regulated proton carrier which allows uncoupling of mitochondrial respiration from ATP synthesis and, therefore, dissipation of metabolic energy as heat. In this article we demonstrate that, when UCP is expressed in Saccharomyces cerevisiae, it retains all its functional properties: proton and chloride transport, high-affinity binding of nucleotides and regulation of proton conductance by nucleotides and fatty acids. Site-directed mutagenesis demonstrates that sequential replacement by serine of cysteine residues in the UCP does not affect either its uncoupling activity or its regulation by nucleotides and fatty acids, and therefore establishes that none of the seven cysteine residues present in the wild-type UCP is critical for its activity. These data indicate that transport models involving essential thiol groups can be discounted and that chemical modification data require critical re-evaluation.

Lack of hormone binding in COS-7 cells expressing a mutated growth hormone receptor found in Laron dwarfism.

A single point mutation in the growth hormone (GH) receptor gene generating a Phe-->Ser substitution in the extracellular binding domain of the receptor has been identified in one family with Laron type dwarfism. The mutation was introduced by site-directed mutagenesis into cDNAs encoding the full-length rabbit GH receptor and the extracellular domain or binding protein (BP) of the human and rabbit GH receptor, and also in cDNAs encoding the full length and the extracellular domain of the related rabbit prolactin (PRL) receptor. All constructs were transiently expressed in COS-7 cells. Both wild type and mutant full-length rabbit GH and PRL receptors, as well as GH and prolactin BPs (wild type and mutant), were detected by Western blot in cell membranes and concentrated culture media, respectively. Immunofluorescence studies showed that wild type and mutant full-length GH receptors had the same cell surface and intracellular distribution and were expressed with comparable intensities. In contrast, all mutant forms (full-length receptors or BPs), completely lost their modify the synthesis ligand. These results clearly demonstrate that this point mutation (patients with Laron syndrome) does not modify the synthesis or the intracellular pathway of receptor proteins, but rather abolishes ability of the receptor or BP to bind GH and is thus responsible for the extreme GH resistance in these patients.

Identification and sequence analysis of a second form of prolactin receptor by molecular cloning of complementary DNA from rabbit mammary gland.

Two lambda gt11 clones containing fragments of cDNA encoding the prolactin receptor from rabbit mammary gland were isolated using a rat liver prolactin receptor cDNA probe. An 1848-base-pair open reading frame encodes a mature prolactin-binding protein of 592 amino acids that contains three domains: (i) the extracellular, amino-terminal, prolactin-binding region of 210 residues; (ii) the transmembrane region of 24 residues; and (iii) the intracellular, carboxyl-terminal domain of 358 residues. This latter domain is much longer than the cytoplasmic domain (57 residues) previously described for the rat liver prolactin receptor. In addition, the sequence identity of this form of prolactin receptor with the growth hormone receptor is extended in the cytoplasmic domain.

Further studies on recombination in diploid clones from Bacillus subtilis protoplast fusion.

Diploid prototrophs were obtained from protoplast fusion of Bacillus subtilis strains. They are unstable but upon further cultivation they stabilize retaining diploidy but are genetically inactive. It has been suggested that recombination between the parental chromosomes is involved in the production of stable prototrophs and recombinants. In this work the occurrence of this recombination was searched for by determining genetic linkages in transformation experiments. In prototrophs two alleles: hisH2 and trpE8 carried originally on each parental chromosome, were shown to be 48% co-transformable in a stable clone whereas they were only cotransformed in 10% of the unstable colonies. For Trp- recombinants (the most frequent type of a Leu- Met- Thr- x Ade- Ura- Trp- fusion pair) lysed protoplasts were used as donor DNA for the transformations. High values of co-transfer for Ura+ Met+ were obtained. These results confirm the occurrence of recombination in stable diploid clones, prototrophs or recombinants.

Complementation and genetic inactivation: two alternative mechanisms leading to prototrophy in diploid bacterial clones.

Evidence for diploidy at loci located all around the Bacillus subtilis chromosome previously led us to refer to the prototrophic bacterial clones produced by fusion of polyauxotrophic protoplasts as complementing diploid clones (Lévi-Meyrueis et al. 1980; Sanchez-Rivas 1982). In this paper, evidence is presented that gene inactivation may occur in such clones, as judged from the unequal expression of three unselected markers and their low transforming activity in cell lysates, an established property of inactivated genes (Bohin et al. 1982). The insensitivity to protease treatment of the lysates and also the low transforming activity observed with purified DNA may indicate that chromosome inactivation does not necessarily result from the mere attachment of proteins to DNA. Cotransfer by transformation of similarly expressed genes, initially located on separate chromosomes, suggests that genetic recombination has taken place, resulting in the reassortment of active and inactive genes on separate chromosomes. Several genetic structures compatible with the observations are presented which illustrate that prototrophy may result from such reassortment as well as from functional complementation.

Diploid state of phenotypically recombinant progeny arising after protoplast fusion in Bacillus subtilis.

After fusion of Bacillus subtilis protoplasts the phenotypically recombinant clones isolated, whether immediately or as segregants of complementing diploid clones, have in common the following properties. They appear independently of the recN+ gene, most often as the result of apparently non-reciprocal recombination occurring in genetic intervals encompassing the origin and the terminus of replication. First indicated by reciprocal fusion crosses between ø105-lysogenic and ø105-sensitive strains, the diploidy of the recombinants was confirmed by studying the transforming activities of their DNA. These experiments establish heterozygosity at eight loci scattered on the chromosome map. By revealing the presence of the trpF+ allele in trpF7 recombinants, the results also strongly suggest that stable phenotypic recombinants may arise by genetic inactivation. Two possible genetic structures for these recombinants are discussed, one implying total inactivation of one recombinant chromosome, the other a segmentary inactivation of one unrecombined chromosome. Whatever the structure, genetic stability is not a reliable sign of haploidy in bacterial clones produced after protoplast fusion.