PubMed Health⌕ Search

Biomedical subjects

F Bouillaud

Publications and source records attributed to F Bouillaud.

At least 55 records · Page 3Linked to original sources
PubMed1994

In vitro interactions between nuclear proteins and uncoupling protein gene promoter reveal several putative transactivating factors including Ets1, retinoid X receptor, thyroid hormone receptor, and a CACCC box-binding protein.

Previous studies of rat ucp (uncoupling protein) gene organization carried out in this laboratory identified regulatory sequences located in the 5'-flanking region. In this work, DNase I footprint analysis of the enhancer revealed two domains at base pairs (bp) -2444 to -2423 and bp -2352 to -2319. The former domain can bind in vitro, in a cooperative manner, factors related to nuclear factor 1 and Ets1; the latter domain contains a type 3 directly repeated sequence that was shown to be able to bind the retinoid X and triiodothyronine receptors. Moreover, a positive effect of retinoic acid on ucp mRNA levels in immortalized brown adipocytes was observed. DNase I footprint analysis identified two hypersensitive regions, A and B, at bp -509 to -472 and bp -403 to -350, respectively; region A contains a repeated CACCC box, and region B can bind protein related to Ets1. The A box differentially binds liver and brown adipose tissue nuclear proteins and could be involved in uncoupling protein induction. Further analysis showed three foot-printed boxes, C-E, at bp -182 to -159, -147 to -120, and -111 to -85, able to bind in vitro proteins related to nuclear factor 1, cAMP response element-binding protein, and Sp1, respectively.

Adipose Tissue, Brown↗
PubMed1994

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.

Amino Acid Sequence↗
PubMed1993

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.

Base Sequence↗
PubMed1993

The topology of the brown adipose tissue mitochondrial uncoupling protein determined with antibodies against its antigenic sites revealed by a library of fusion proteins.

The uncoupling protein (UCP) of brown adipose tissue mitochondria is a specialized member of the family of evolutionarily related mitochondrial membrane transporters, which also includes the ADP/ATP translocator and the phosphate carrier. We have generated a library of bacterial clones randomly expressing short subsequences of the UCP fused to the MalE periplasmic protein of Escherichia coli. Anti-UCP sera were used to select clones expressing antigenic sequences of the UCP. Ten different fusion proteins representing eight non-overlapping subsequences of the UCP were obtained. The ability of fusion proteins to select antibodies directed against a short segment of the UCP was used to study the topological organization of the UCP in the inner mitochondrial membrane. Four different fusion proteins were used to determine the orientation of the N-terminal extremities of the first, second, third and fourth predicted alpha-helices of the UCP. This topological study together with previous data on the UCP provides an experimental basis for the predicted structure of the UCP and for other homologous carrier proteins.

ATP-Binding Cassette Transporters↗
PubMed1993

Tissue-specific and beta-adrenergic regulation of the mitochondrial uncoupling protein gene: control by cis-acting elements in the 5'-flanking region.

Uncoupling protein (UCP) gene expression is tightly restricted to thermogenic brown adipocytes and is rapidly activated by norepinephrine released after cold exposure. To identify cis-acting regulatory elements controlling this gene, a region encompassing 4.5 kilobases of DNA upstream of the transcription start site was analyzed using hybrid UCP-chloramphenicol acetyltransferase reporter gene constructs. Evidence for the presence of both tissue-specific and beta-adrenergic response elements in this 4.5-kilobase region was obtained by comparing the expression of these reporter genes in transfected brown adipocytes (in vitro differentiated), brown preadipocytes, white adipocytes, and Chinese hamster ovary (CHO) cells and from experiments in transgenic animals. Deletion analyses in transfected cells indicated that the minimal region exhibiting promoter activity and tissue specificity is located between -157 and -57 base pairs (bp). A 211-bp activator element located between -2494 and -2283 bp was necessary for full expression in brown adipocytes. This element also activated expression of the homologous -157-bp promoter and expression of a heterologous promoter in both brown adipocytes and CHO cells. A second region, downstream of the activator and possibly located between positions -400 and -157 bp, inhibited the UCP promoter in CHO cells. In mice transgenic for a chloramphenicol acetyltransferase reporter gene containing these elements, expression was both tissue specific and regulatable by environmental temperature changes. These results indicate that both positive and negative cis-acting elements participate in the regulation of UCP gene expression.

Adipose Tissue, Brown↗
PubMed1992

Activation by ATP of a proton-conducting pathway in yeast mitochondria.

The growth of Saccharomyces cerevisiae cells under aerobic conditions, in the presence of an energy-rich source, leads to production of an excess of NAD(P)H. Since the redox balance must be maintained, it has been postulated that NAD(P)H reoxidation is accelerated by the activation of energy-dissipating reactions, which would, in turn, explain the low growth efficiencies observed. It has been demonstrated already in S. cerevisiae cultures that these putative energy-dissipating reactions are stimulated both by oxygen and high cytosolic ATP levels. In this paper, we show that ATP induces a proton-permeability pathway in mitochondria at concentrations which are within the physiological range, as revealed both from the ATP stimulation of respiration and from the induction of H(+)-dependent swelling. We also demonstrate that phosphate acts as a competitive inhibitor of the nucleotide, and since activation is observed even in the presence of atractylate, we postulate that the ATP-binding site is located in the outer face of the mitochondrial inner membrane.

Adenosine Triphosphate↗
PubMed1992

Antibodies selected from whole antiserum by fusion proteins as tools for the study of the topology of mitochondrial membrane proteins. Evidence that the N-terminal extremity of the sixth alpha-helix of the uncoupling protein is facing the matrix.

The reactivity to freeze-thawed mitochondria or submitochondrial particles of a whole antiserum raised against the uncoupling protein has been investigated. Incubation with freeze-thawed brown adipose tissue mitochondria trapped antibodies reactive toward accessible parts of the uncoupling protein. One-third to one-half of antibodies against uncoupling protein which were present in the serum remained free. These antibodies were highly reactive with the vesicles obtained by sonication of mitochondria, in which the matricial side of the inner membrane was made accessible. To define epitopes recognized by the antiserum, different fusion proteins made up of MalE protein and uncoupling protein fragments were used. Immunoaffinity chromatography, using an immobilized purified fusion protein containing amino acids 253 to 290 of uncoupling protein, selected antibodies specifically directed against this part of the protein. A more precise localization of the main epitope recognized by these antibodies is proposed. These purified antibodies reacted with the protein only in submitochondrial particles, indicating a matricial orientation of this epitope. This result, associated with other data concerning uncoupling protein or related mitochondrial carriers such as the ADP/ATP translocator and the phosphate carrier, allowed us to determine the orientation of the sixth alpha-helix of the uncoupling protein.

Amino Acid Sequence↗
PubMed1992

Comment to Shinohara et al. (1991) FEBS Letters 293, 173-174. The uncoupling protein is not expressed in rat liver.

Using Northern blot analysis, immunoblotting with purified antibodies and Polymerase Chain Reaction analysis, we were unable to detect the Uncoupling Protein-UCP or its mRNA in liver of control, cold-exposed or newborn rats. The unique expression of this protein in brown adipocytes was confirmed. These data refute the surprising recent report on UCP expression in rat liver (Shinohara (1991) FEBS Lett. 293, 173-174). Moreover we report that the hybridization signal obtained by these authors is probably non-specific and due to the 3' non-coding domain of the UCP cDNA probe.

Animals↗
PubMed1991

Molecular studies of the uncoupling protein.

The uncoupling protein (UCP) is a proton/anion transporter found in the inner mitochondrial membrane of brown adipocyte. Although UCP has not been detected in mitochondria from any other tissue, it shares structural and catalytic properties with several other mitochondrial carrier proteins. Although UCP was discovered only recently it is one of the most extensively studied mitochondrial carrier proteins. Many tools useful in research on UCP have been developed such as antibodies and cDNAs corresponding to UCP of several animal species. More recently, the mouse, rat, and human genes encoding for UCP have been isolated and sequenced. The availability of these various tools has led to several significant observations. UCP gene expression is strongly controlled at the level of transcription by signals that are activated after the stimulation of brown adipocytes by norepinephrine. The comparison of UCP gene with the genes encoding the adenine nucleotide translocator revealed the existence of structural and evolutionary homologies. Moreover, in humans the UCP gene and one form of adenine nucleotide translocator gene are located on the same chromosome. Recently, the expression of functional UCP in various heterologous systems was achieved (Xenopus oocytes, CHO cells, yeasts). These data will facilitate studies of the structure/function relationship in UCP (identification of residues involved in H+ transport, Cl- transport, nucleotide binding, mitochondrial targeting...). Another aspect of the present research on UCP is the understanding of mechanisms that control the UCP gene and the differentiated commitment of adipose precursor cells to thermogenic brown adipocytes. The multifaceted aspects of research on UCP make this protein interesting in areas of research as different as studies of ion translocating mechanisms, cellular specificity of gene transcription, control of gene expression by neuromediators, adipocyte differentiation, and the pharmacological treatment of obesity.

Amino Acid Sequence↗
PubMed1990

Expression of the brown fat mitochondria uncoupling protein in Xenopus oocytes and important into mitochondrial membrane.

Non shivering thermogenesis of brown adipose tissue is due to the uncoupling protein (UCP), located in the inner mitochondrial membrane, which functions as a proton translocator and can thus uncouple mitochondrial respiration. We describe here the expression of UCP in Xenopus laevis oocytes after injection of UCP mRNA, which was transcribed in vitro. UCP seems to be correctly transported into mitochondria and integrated into the membrane, but we were not able to establish definitely the functionality of this UCP. We conclude that this expression system could be suitable for the study of the mitochondrial import mechanism but not for the examination of physiological properties of UCP.

Adipose Tissue, Brown↗
PubMed1990

Human uncoupling protein gene: structure, comparison with rat gene, and assignment to the long arm of chromosome 4.

The uncoupling protein (UCP) gene encodes a unique mammalian mitochondrial proton carrier that induces heat production in brown adipocytes. Human UCP gene was isolated and its organization analyzed. A comparison was made with rat UCP gene. Human UCP gene spans 13 Kb and contains a transcribed region that covers 9 Kb of the human genome. All of the exons were also sequenced except the extreme end of the 3' untranslated region. Two Kb DNA upstream the TATA box were also sequenced. This region contains several fragments that are highly homologous to the gene of rat UCP. Neither CCAAT sequence nor Sp 1 binding motif were detected. Human UCP gene is split into six exons. The complete amino acid sequence of the protein was determined. Human UCP has 305 amino acids and a molecular weight of 32,786. It has no N-terminal targeting sequence. It is 79% homologous to rat UCP both at nucleotidic and amino acid levels. The primary structure of UCP is significantly homologous to the primary structure of the human T1 ADP/ATP carrier, particularly in the C-terminal extremity, which is supposed to contain a nucleotide-binding site in both proteins. Human UCP gene is single type, as it is in rodents. Two genomic fragments were used to detect a 1.9 Kb mRNA in human perirenal brown adipose tissue. Using in situ hybridization, UCP gene was assigned in humans to chromosome 4 in q31. Interestingly, the T1 gene encoding the heart-skeletal muscle ADP/ATP carrier has recently been shown to be on the same chromosome (Li et al. Biol Chem 264:13998, 1989).

Amino Acid Sequence↗
PubMed1990

Stable expression of functional mitochondrial uncoupling protein in Chinese hamster ovary cells.

The mitochondrial uncoupling protein (UCP) is a membranous proton carrier exclusively synthesized in brown adipocytes. The cDNA for the rat UCP was placed in an expression vector and transfected into mammalian cells. Its expression was tested in transiently transfected CHO cells. In these cells the UCP was detected in mitochondria by using antibodies. Permanent expression of the UCP was achieved in stable transformed CHO cell lines. In these cells the UCP was characterized in mitochondrial membranes, by using antibodies and hydroxyapatite purification. The protein expressed in CHO cells displayed the functional characteristics of brown adipocyte UCP. It induced the uncoupling of respiration in isolated CHO mitochondria. The membrane potential of transformed mitochondria was also significantly lowered, as a result of the proton translocating activity of the UCP. GDP is known to inhibit the proton pathway in brown fat mitochondria. Addition of GDP to CHO mitochondria containing UCP resulted in a recoupling of respiration and an increase in membrane potential. Thus we conclude that functional UCP is expressed in CHO cells and that the insertion of the UCP alone in any mitochondria is sufficient to induce the uncoupling of respiration. This approach should allow studies on the structure-function relationship of the UCP and of several other related mitochondrial carriers.

Adipose Tissue, Brown↗
PubMed1989

Sequential changes in the expression of mitochondrial protein mRNA during the development of brown adipose tissue in bovine and ovine species. Sudden occurrence of uncoupling protein mRNA during embryogenesis and its disappearance after birth.

Samples of adipose tissue were obtained from different sites in bovine and ovine foetuses and newborns. RNA was isolated and analysed using bovine cDNA and ovine genomic probe for uncoupling protein (UCP), cDNA for subunits III and IV of cytochrome c oxidase and cDNA for ADP/ATP carrier. UCP mRNA was characterized for the first time in foetal bovine and ovine adipose tissue. It appeared later than mRNA of cytochrome c oxidase subunit III, and increased dramatically at birth (10-fold). ADP/ATP carrier mRNA was expressed at a lower level but also increased 10-fold at birth. It was demonstrated that UCP mRNA reached its highest level at birth in all bovine adipose tissues studied, except subcutaneous tissue. It disappeared quickly afterwards, being no longer detectable two days after birth. Similar variations were observed in newborn lambs. ADP/ATP carrier mRNA showed the same pattern of expression as UCP mRNA; although it was still lightly expressed two days after birth, it disappeared soon afterwards. Only mRNAs for cytochrome c oxidase subunits III and IV remained at the same level during the first postnatal week. On the basis of these data and of observations reported in the literature a sequence of events for the development of brown adipose cells in vivo is proposed. Soon after birth the perirenal adipose tissue of ruminants, which still contains mitochondria of typical brown adipose tissue morphology and high levels of cytochrome c oxidase mRNA, lacks UCP mRNA. Can it still be considered as brown fat? Ruminant species appear to be attractive models to study both the differentiation of brown adipose tissue and its possible conversion to white fat in large animals.

Adenosine Diphosphate↗
PubMed1988

The gene for rat uncoupling protein: complete sequence, structure of primary transcript and evolutionary relationship between exons.

The complete nucleotide sequence of rat Uncoupling Protein gene has been determined. 4.5 Kb of the 5'-flanking region have also been sequenced. The site of transcription start as well as 3'-end extremities were identified. Transcription unit spans 8.4 Kb and contains 6 exons and 5 introns. Uncoupling protein as well as related mitochondrial carriers such as ADP/ATP carrier and phosphate carrier has a triplicated structure and each repeat of Uncoupling Protein corresponds to 2 exons. Two gene duplications could explain the triplicated structure of UCP and the more recent event of duplication concerned exons III and V. Evidence for homology between Uncoupling Protein and Neurospora crassa ADP/ATP carrier at the gene level was also noticed.

Animals↗