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Haemophilus influenzae protein F (PF) is an important virulence factor interacting with laminin, an extracellular matrix protein ubiquitously expressed in the respiratory tract. Here we defined PF orthologs in Pseudomonas aeruginosa, Moraxella catarrhalis, and Staphylococcus aureus, bacteria that occasionally colonize and infect the human airways. Despite low sequence homology (48.2%-77.3% similarity), all orthologs (Paf, AfeA, and MntC) interacted with laminin. Interestingly, all proteins bound at the heparin-binding sites of laminin, including the globular domains, and also attached to laminin expressed on respiratory epithelial cells. Laminin is thus a highly important target for PF orthologs of the bacterial species examined.

Haemophilus influenzae is a Gram-negative human pathogen that resides in the upper respiratory tract. Encapsulated H. influenzae type b (Hib) and type f (Hif) are the most common serotypes associated with invasive disease. H. influenzae displays various strategies to circumvent the host innate immune response, including the bactericidal effect of the complement system. In this study, we identified an H. influenzae lipoprotein having the ability to bind factor H (FH), the major regulator of the alternative pathway of complement activation. This protein, named protein H (PH), was surface exposed and was found in all clinical Hib and Hif isolates tested. Deletion of the gene encoding for PH (lph) in Hib and Hif significantly reduced the interaction between bacteria and FH. When Hib and Hif PH variants were separately expressed in nontypeable (unencapsulated) H. influenzae, which did not bind FH, an increased FH affinity was observed. We recombinantly expressed the two PH variants in Escherichia coli, and despite sharing only 56% identical amino acids, both FH-binding Haemophilus proteins similarly interacted with the complement regulator FH short consensus repeats 7 and 18–20. Importantly, Hib and Hif resistance against the bactericidal effect of human serum was significantly reduced when bacterial mutants devoid of PH were tested. In conclusion, we have characterized a hitherto unknown bacterial protein that is crucial for mediating an interaction between the human pathogen H. influenzae and FH. This novel interaction is important for H. influenzae resistance against complement activation and will consequently promote bacterial pathogenesis.

The β-lactamase genes of Haemophilus influenzae are commonly positioned on large integrative and conjugative elements, but a group of blaTEM-carrying small plasmids (4000-6000 bp) with a common structural backbone have recently been characterized. In this study we investigated the epidemiological significance and potential for transfer of this group of small plasmids.We developed a two-step PCR assay to screen for and type this group of resistance plasmids in H. influenzae. A large collection of respiratory isolates (n = 2845) from south Sweden, obtained from 2009 to 2011, as well as a collection of invasive Swedish H. influenzae from 1997 to 2010 (n = 310) was screened. The distribution of plasmid types among clinical isolates was investigated using multilocus sequence typing (MLST).In the collection, 15.8% of β-lactamase-producing isolates and 1.4% of total isolates possessed a small plasmid with the signature structure. The plasmids were genetically conserved and widely spread geographically. MLST revealed that the spread of small plasmids occurred by both clonal expansion and horizontal transfer. In vitro experiments suggested that one plasmid type, pN223, can transfer ampicillin resistance to susceptible Escherichia coli.Small β-lactamase-encoding plasmids constitute a significant mechanism for β-lactam resistance in H. influenzae and can spread through clonal expansion of resistant clones as well as through horizontal plasmid transfer.

The incidence of invasive Haemophilus influenzae type b (Hib) disease has significantly decreased since the introduction of an efficient vaccine against Hib. However, in contrast to Hib, infections caused by H. influenzae serotype f (Hif) are emerging. We recently did a whole genome sequencing of an invasive Hif isolate, and reported that Hif interacts with factor H by expressing protein H (PH). In this study, upon screening with various human complement regulators, we revealed that PH is also a receptor for vitronectin (Vn), an abundant plasma protein that regulates the terminal pathway of the human complement system in addition to being a component of the extracellular matrix. Bacterial Vn binding was significantly reduced when the lph gene encoding PH was deleted in an invasive Hif isolate. The dissociation constant (KD) of the interaction between recombinant PH and Vn was 2.2 μM, as revealed by Biolayer interferometry. We found that PH has different regions for simultaneous interaction with both Vn and factor H, and that it recognized the C-terminal part of Vn (aa 352–362). Importantly, PH-dependent Vn binding resulted in better survival of the wild-type Hif or PH-expressing Escherichia coli when exposed to human serum. Finally, we observed that PH mediated an increased bacterial adherence to alveolar epithelial cells in the presence of Vn. In conclusion, our study reveals that PH most likely plays an important role in Hif pathogenesis by increasing serum resistance and adhesion to the airways.

Factor H is an important complement regulator of the alternative pathway commonly recruited by pathogens to achieve increased rates of survival in the human host. The respiratory pathogen Moraxella catarrhalis, which resides in the mucosa, is highly resistant to the bactericidal activity of serum and causes otitis media in children and respiratory tract infections in individuals with underlying diseases. In this study, we show that M. catarrhalis binds factor H via the outer membrane protein OlpA. M. catarrhalis serum resistance was dramatically decreased in the absence of either OlpA or factor H, demonstrating that this inhibition of the alternative pathway significantly contributes to the virulence of M. catarrhalis.

The human pathogen Haemophilus influenzae causes mainly respiratory tract infections such as acute otitis media in children and exacerbations in patients with chronic obstructive pulmonary disease. We recently revealed the crystal structure of H. influenzeae protein E (PE), a multifunctional adhesin that is involved in direct interactions with lung epithelial cells and host proteins. Based upon the PE structure we here suggest a hypothetical binding pocket that is compatible in size with a hemin molecule. An H. influenzae mutant devoid of PE bound significantly less hemin in comparison to the PE-expressing wild type counterpart. In addition, E. coli expressing PE at the surface resulted in a hemin-binding phenotype. An interaction between hemin and recombinant soluble PE was also demonstrated by native-PAGE and UV-visible spectrophotometry. Surface plasmon resonance revealed an affinity (Kd) of 1.6 × 10(-6)M for the hemin-PE interaction. Importantly, hemin that was bound to PE at the H. influenzae surface, was donated to co-cultured luciferase-expressing H. influenzae that were starved of hemin. When hemin is bound to PE it thus may serve as a storage pool for H. influenzae. To our knowledge this is the first report showing that H. influenzae can share hemin via a surface-located outer membrane protein.

13 páginas, 6 figuras, 1 tabla -- PAGS nros. 137-149, It has been reported that the region 261-269 of the uncoupling protein from brown adipose tissue mitochondria, UCP1, has an important role in the control of its proton translocating activity. Thus the deletion of residues Phe267-Lys268-Gly269 leads to the loss of the nucleotide regulation of the protein, while the complete deletion of the segment leads to the formation of a pore. The region displays sequence homology with the DNA-binding domain of the estrogen receptor. The present report analyzes the structure, by NMR and circular dichroism, of a 20 amino acid residue peptide containing the residues of interest. We demonstrate that residues 263-268 adopt an α-helical structure. The helix is at the N-terminal end of the sixth transmembrane domain. The functional significance of this helix has been examined by site-directed mutagenesis of the protein expressed recombinantly in yeasts. Alterations in the structure or orientation of the region leads to an impairment of the regulation, by nucleotides and fatty acids, of the transport activity. UCP1 is one member of the family formed by the carriers of the mitochondrial inner membrane. The family is characterized by a tripartite structure with three repeated segments of about 100 amino acid residues. Two of the mutations have also been performed in the first and second matrix loops and the effect on UCP1 function is very similar. We conclude that the three matrix loops contribute to the formation of the gating domain in UCP1 and propose that they form a hydrophobic pocket that accommodates the purine moiety of the bound nucleotide, E.R. and M.A.J. are supported by research grants from the Spanish Ministry of Education and Culture (PB95-0118 and PB93-0189). F.B. and C.F. are supported by the CNRS and INSERM with grants from the Association pour la Recherche sur le Cancer and Danone (C.F.). The collaboration (CSIC-CNRS) project is funded by the Human Frontier Science Program Organization (RG0307/1998-M). M.G.B. was supported by a fellowship of the Spanish Ministry of Education and Culture

Human and mouse UCP2 genes were cloned and sequenced. Transcriptional start sites were identified using primer extension analysis. The transcription unit of UCP2 gene is made of 2 untranslated exons followed by 6 exons encoding UCP2. In vitro translation analysis demonstrated that an open-reading-frame for a putative peptide of 36 residues present in exon 2 did not prevent UCP2 translation and confirmed that the initiation site of translation was in exon 3 as predicted from sequencing data. Short (bp -125 to +93) and long (bp -1383 and +93) CAT-constructs containing DNA upstream of the transcriptional start site of the human gene were made and transfected in adipocytes or HeLa cells allowing characterization of a potent promoter. Analysis of several genomic clones encompassing UCP2 and/or UCP3 genes demonstrated that the 2 genes are adjacent, the human UCP2 gene being located 7 kb downstream of the UCP3 gene.

Uncoupling protein 2 (UCP2), a member of the family of mitochondrial carrier proteins, has been implicated in the control of whole-body energy balance. The coding region of the human UCP2 gene has now been shown to comprise six exons, and the sequences of the exon-intron boundaries were determined. With the use of this sequence information, 25 Japanese patients with obesity and noninsulin-dependent diabetes mellitus (NIDDM) and 25 subjects with simple obesity were screened for mutations in the entire coding region of UCP2 by PCR and single-strand conformation polymorphism analysis. Two nucleotide polymorphisms resulting in Ala55 → Val and Ala232 → Thr substitutions were detected. With the use of PCR and restriction fragment length polymorphism analysis, the allele frequencies for each of these polymorphisms were determined in 210 Japanese patients with NIDDM, 42 obese individuals, and 218 normal control subjects. The frequency of the Val55 allele did not differ significantly among the NIDDM group (46.0%), the obesity group (48.8%), and the normal control group (48.4%). The Thr232 allele was detected in only three subjects, who were heterozygotes and in the NIDDM group (allele frequency, 0.7%). However, expression in yeast of the human wild-type UCP2 protein and UCP2 containing Thr232 revealed no difference in functional activity. These results indicate that the Ala55 → Val and Ala232 → Thr variants of UCP2 do not play an important role in the pathogenesis of NIDDM or obesity in the Japanese population.

The uncoupling protein of brown adipose tissue (UCP1) is a transporter that allows the dissipation as heat of the proton gradient generated by the respiratory chain. The discovery of new UCPs in other mammalian tissues and even in plants suggests that the proton permeability of the mitochondrial inner membrane can be regulated and its control is exerted by specialised proteins. The UCP1 is regulated both at the gene and the mitochondrial level to ensure a high thermogenic capacity to the tissue. The members of the mitochondrial transporter family, which includes the UCPs, present two behaviours with carrier and channel transport modes. It has been proposed that this property reflects a functional organization in two domains: a channel and a gating domain. Mounting evidence suggest that the matrix loops contribute to the formation of the gating domain and thus they are determinants to the control of transport activity.

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.

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.

Transgenic mice were generated with a transgene containing the 211-base pair (bp) enhancer and 0.4 kilobase pairs of 5'-flanking DNA of the uncoupling protein (ucp) gene. Expression of this transgene was restricted to brown adipose tissue and was inducible by cold exposure or treatment of transgenic mice by norepinephrine, retinoic acid (RA), or CL-316,243 beta3-adrenoreceptor agonist. A search for retinoic acid response elements in the ucp gene enhancer was undertaken using mutagenesis and transfection of cultured cells with chloramphenicol acetyltransferase constructs. Deletion or mutations of several putative retinoic acid response elements were ineffective. Mutations of a TGAATCA region dramatically decreased the transcriptional activity in the presence of RA. In vitro this region was able to bind a complex containing proteins recognized by antibodies against Jun or Fos. Mutations of an adjacent region related to an inverted repeat of type 2 also markedly decreased RA effect. This region was able to bind in vitro retinoid X receptor alpha and retinoic acid receptor beta. The two regions form an activating region between bp -2421 and -2402 (referred to as the ucp gene-activating region), which has an enhancer activity but cannot confer RA response to a promoter. This response was obtained with a larger DNA fragment (bp -2489 to -2398) constituting a complex RA response domain.

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.

In invertebrates, members of the collagen family have been found in various phyla. Surprisingly, in mollusc, little is known about such molecules. In this study, we characterize the full-length abalone type IV collagen and we analysed its biological effects on human fibroblast in order to gain insights about this molecule in molluscs and particularly clues about its roles. We screened a cDNA library of Haliotis tuberculata hemocytes. The expression pattern of the transcript is determined using real-time polymerase chain reaction and in situ hybridization. The close identity between α1(IV) C-terminal domain and the vertebrate homologue led us to produce, purify and test in vitro a recombinant protein corresponding to this region using human dermal fibroblasts cell culture. The biological effects were evaluated on proliferation and on differentiation. We found that the 5,334-bp open reading frame transcript encodes a protein of 1,777 amino acids, including an interrupted 1,502-residue collagenous domain and a 232-residue C-terminal non-collagenous domain. The expression pattern of this transcript is mainly found in the mantle and hemocytes. The recombinant protein corresponding α1(IV) C-terminal domain increased fibroblast proliferation by 69% and doubled collagen synthesis produced in primary cultures. This work provides the first complete primary structure of a mollusc non-fibrillar collagen chain and the biological effects of its C-terminal domain on human cells. In this study, we prove that the NC1 domain from a molluscan collagen can improve human fibroblast proliferation as well as differentiation.

Laminin (Ln) and collagen are multifunctional glycoproteins that play an important role in cellular morphogenesis, cell signalling, tissue repair and cell migration. These proteins are ubiquitously present in tissues as a part of the basement membrane (BM), constitute a protective layer around blood capillaries and are included in the extracellular matrix (ECM). As a component of BMs, both Lns and collagen(s), thus function as major mechanical containment molecules that protect tissues from pathogens. Invasive pathogens breach the basal lamina and degrade ECM proteins of interstitial spaces and connective tissues using various ECM-degrading proteases or surface-bound plasminogen and matrix metalloproteinases recruited from the host. Most pathogens associated with the respiratory, gastrointestinal, or urogenital tracts, as well as with the central nervous system or the skin, have the capacity to bind and degrade Lns and collagen(s) in order to adhere to and invade host tissues. In this review, we focus on the adaptability of various pathogens to utilize these ECM proteins as enhancers for adhesion to host tissues or as a targets for degradation in order to breach the cellular barriers. The major pathogens discussed are Streptococcus, Staphylococcus, Pseudomonas, Salmonella, Yersinia, Treponema, Mycobacterium, Clostridium, Listeria, Porphyromonas and Haemophilus; Candida, Aspergillus, Pneumocystis, Cryptococcus and Coccidioides; Acanthamoeba, Trypanosoma and Trichomonas; retrovirus and papilloma virus.

Manduca sexta allatotropin (Manse-AT) is a multifunctional neuropeptide whose actions include the stimulation of juvenile hormone biosynthesis, myotropic stimulation, cardioacceleratory functions, and inhibition of active ion transport. Manse-AT is a member of a structurally related peptide family that is widely found in insects and also in other invertebrates. Its precise role depends on the insect species and developmental stage. In some lepidopteran insects including M. sexta, structurally-related AT-like (ATL) peptides can be derived from alternatively spliced mRNAs transcribed from the AT gene. We have isolated a cDNA for an AT receptor (ATR) from M. sexta by a PCR-based approach using the sequence of the ATR from Bombyx mori. The sequence of the M. sexta ATR is similar to several G protein-coupled receptors from other insect species and to the mammalian orexin receptor. We demonstrate that the M. sexta ATR expressed in vertebrate cell lines is activated in a dose-responsive manner by Manse-AT and each Manse-ATL peptide in the rank order ATL-I > ATL-II > ATL-III > AT, and functional analysis in multiple cell lines suggest that the receptor is coupled through elevated levels of Ca(2+) and cAMP. In feeding larvae, Manse-ATR mRNA is present at highest levels in the Malpighian tubules, followed by the midgut, hindgut, testes, and corpora allata, consistent with its action on multiple target tissues. In the adult corpora cardiaca--corpora allata complex, Manse-ATR mRNA is present at relatively low levels in both sexes. ispartof: Insect Biochemistry and Molecular Biology vol:41 issue:10 pages:804-814 ispartof: location:England status: published

The mitochondrial uncoupling protein of brown adipose tissue (UCP1) was expressed in skeletal muscle and heart of transgenic mice at levels comparable with the amount found in brown adipose tissue mitochondria. These transgenic mice have a lower body weight, and when related to body weight, food intake and energy expenditure are increased. A specific reduction of muscle mass was observed but varied according to the contractile activity of muscles. Heart and soleus muscle are unaffected, indicating that muscles undergoing regular contractions, and therefore with a continuous mitochondrial ATP production, are protected. In contrast, the gastrocnemius and plantaris muscles showed a severely reduced mass and a fast to slow shift in fiber types promoting mainly IIa and IIx fibers at the expense of fastest and glycolytic type IIb fibers. These observations are interpreted as a consequence of the strong potential dependence of the UCP1 protonophoric activity, which ensures a negligible proton leak at the membrane potential observed when mitochondrial ATP production is intense. Therefore UCP1 is not deleterious for an intense mitochondrial ATP production and this explains the tolerance of the heart to a high expression level of UCP1. In muscles at rest, where ATP production is low, the rise in membrane potential enhances UCP1 activity. The proton return through UCP1 mimics the effect of a sustained ATP production, permanently lowering mitochondrial membrane potential. This very likely constitutes the origin of the signal leading to the transition in fiber types at rest.

Programmed cell death (PCD) serves as a major mechanism for the precise regulation of cell numbers, and as a defense mechanism to remove unwanted and potentially dangerous cells. Despite the striking heterogeneity of cell death induction pathways, the execution of the death program is often associated with characteristic morphological and biochemical changes termed apoptosis. Although for a long time the absence of mitochondrial changes was considered as a hallmark of apoptosis, mitochondria appear today as the central executioner of programmed cell death. This crucial position of mitochondria in programmed cell death control is not due to a simple loss of function (deficit in energy supplying), but rather to an active process in the regulation of effector mechanisms. The large diversity of regulators of apoptosis in mammals and their numerous interactions complicate the analysis of their individual functions. Yeast, eukaryotic but unicellular organism, lack the main regulators of apoptosis (caspases, Bcl-2 family members, …) found in mammals. This absence render them a powerful tool for heterologous expression, functional studies, and even cloning of new regulators of apoptosis. Great advances have thus been made in our understanding of the molecular mechanisms of Bcl-2 family members interactions with themselves and other cellular proteins, specially thanks to the two hybrid system and the easy manipulation of yeast (molecular biology and genetics). This review will focus on the use of yeast as a tool to identify new regulators and study function of mammalian apoptosis regulators.

There is considerable interest in determining the conditions leading to enhanced inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) biosynthesis. Using in vivo footprinting, we demonstrate that heat shock of murine macrophages concurrent with lipopolysaccharide (LPS) treatment stimulated changes in guanine methylation sensitivity at ?898/9, at a putative partial heat shock element (HSE) and at -893/4, a site bordering an E-box, within the iNOS gene enhancer, suggesting inducible occupation by transcription factors at these regions. LPS treatment accompanied by heat shock provoked increased iNOS gene transcription, increased levels of iNOS protein, and increased production of NO compared with LPS treatment alone. Electrophoretic mobility shift analysis revealed low constitutive levels of specific binding to an E-box and a partial HSE within the iNOS enhancer. Binding to the E-box was increased by LPS treatment or by heat shock, achieving a greater increase by a combination of both treatments. The proteins occupying this site were identified as belonging to the USF family of transcription factors. Heat shock or LPS increased binding to the HSE, and the factor responsible for this interaction was identified as heeat shock factor-1 (HSF-1). Mutations at the HSE revealed the importance of HSF-1 in the induction of iNOS by LPS. Thus, our data reveal two novel regulatory sites in the murine iNOS gene, one of which is implicated in enhancing iNOS expression via LPS stimulation, and provide the first evidence that heat shock enhances transcription of the iNOS gene. These results could have implications in the host response mechanism to fever-associated gram-negative infection.

7 páginas, 5 figuras, 1 tabla -- PAGS nros. 5827-5833, In mammalian brown adipose tissue, thermogenesis is explained by uncoupling mitochondrial respiration from ATP synthesis. Uncoupling protein-1 (UCP1) is responsible for this uncoupled state, because it allows proton re-entry into the matrix and thus dissipates the proton gradient generated by the respiratory chain. Proton transport by UCP1 is regulated negatively by nucleotides and positively by fatty acids. Adrenergic stimulation of brown adipocytes stimulates lipolysis and therefore enhances uncoupling and thermogenesis. Adrenergic stimulation also boosts ucp1 gene transcription. Since retinoic acid also promotes ucp1 gene transcription and its structure makes it a possible activator of UCP1, we hypothesized that retinoic acid, like noradrenaline, could have a dual action and trigger the activity of the protein UCP1 itself. Here we show that retinoic acid strongly increases proton transport by UCP1 in brown adipose tissue mitochondria and that it is much more potent than fatty acids. These data are corroborated with yeast mitochondria where UCP1 was introduced by genetic manipulation. The yeast expression system allows the comparison of the UCP1 with the newly described homologues UCP2 and UCP3. The search for regulators of UCP2 has demonstrated that it is positively regulated by retinoids in a pH-dependent manner, This work is supported by grants of the Spanish Ministry of Education and Culture (PB 95-0118 and Petri 95-0262-OP) to E.R., by the French Centre National de la Recherche Scientifique, the Association pour la Recherche contre le Cancer, and the Institut National de la Santé et Recherche Medicale (F.B. and D.R.), the European Commission, Grant TMR No. ERBFMBICT972845 (D.S.), and the Human Frontier Science Program Organization (D.R. and E.R.)

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 mitochondrialcarrier 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.

5 páginas, 2 figuras -- PAGS nros. 15528-15532, The activity of the brown fat uncoupling protein (UCP1) is regulated by purine nucleotides and fatty acids. Although the inhibition by nucleotides is well established, the activation by fatty acids is still controversial. It has been reported that the ADP/ATP carrier, and possibly other members of the mitochondrial carrier family, mediate fatty acid uncoupling of mitochondria from a variety of sources by facilitating the transbilayer movement of the fatty acid anion. Brown fat mitochondria are known to be more sensitive to fatty acid uncoupling, a property that has been assigned to the presence of UCP1. We have analyzed the transport properties of UCP1 and conclude that fatty acids are not essential for UCP1 function, although they increase its uncoupling activity. In order to establish the difference between the proposed carrier-mediated uncoupling and that exerted through UCP1, we have studied the facility with which fatty acids uncouple respiration in mitochondria from control yeast and strains expressing UCP1 or the mutant Cys-304 → Gly. The concentration of free palmitate required for half-maximal activation of respiration in UCP1-expressing mitochondria is 80 or 40 nm for the mutant protein. These concentrations have virtually no effect on the respiration of mitochondria from control yeast and are nearly 3 orders of magnitude lower than those reported for carrier-mediated uncoupling. We propose that there exist two modes of fatty acid-mediated uncoupling; nanomolar concentrations activate proton transport through UCP1, but only if their concentrations rise to the micromolar range do they become substrates for nonspecific carrier-mediated uncoupling. The ability of long-chain fatty acids to uncouple mitochondrial respiration from ATP synthesis (see Ref. 1; reviewed in Ref. 2) has been generally assumed to resemble that of other weak acids, which can permeate through the lipid bilayer in both protonated and unprotonated forms. However, recent data have established that fatty acid anion flip-flop across the phospholipid bilayer occurs far too slowly (t > 1 min) (3) to allow the anion to complete a classic protonated/deprotonated cycle as for classical uncouplers. The protonophoric activity observed when extremely high fatty acid concentrations (above 0.1 mm) are employed is probably caused by the disruption of the lipid bilayer in a detergent-like mode (1, 4, 5), inasmuch as, for example, effects are not reversed by albumin. The situation is less clear when lower fatty acid concentrations are employed and detergent effects are negligible. It has been shown recently that, under these conditions, fatty acid uncoupling becomes sensitive to inhibitors such as carboxyatractylate, and consequently it has been proposed that permeation of the fatty acid anion is mediated by the ADP/ATP carrier (6-9) and possibly by other anion carriers of the mitochondrial inner membrane (10, 11). Brown fat is a specialized tissue, the function of which is to produce heat. The thermogenic mechanism is centered around the brown fat uncoupling protein (UCP1),which allows dissipation of the proton electrochemical potential gradient and therefore uncouples respiration from ATP synthesis (12, 13). Recently, three new uncoupling proteins have been identified with homology to UCP1 and termed UCP2 (14), UCP3 (15), and StUCP (16). Free fatty acids, liberated by the noradrenergic stimulation of lipolysis, are the substrate for brown fat thermogenesis and also act as the cytosolic second messengers by which noradrenaline activates UCP1 (17, 18)., The mechanism by which fatty acids activate UCP1 is a matter of debate. Two main hypothesis are current. The first is an extension of the observation of carboxyatractylate-sensitive mitochondrial fatty acid uncoupling, and proposes that UCP1 and other mitochondrial anion carriers (19, 20) can catalyze the transport of the fatty acid anion (21, 22). The second model proposes that fatty acids act as a prosthetic group in UCP1 delivering protons to a site from which they are translocated to the other side of the membrane (23). The anion transporting activity would have little physiological importance and thus would be a vestige of its evolutionary relationship with the rest of mitochondrial metabolite carriers. The present report will examine the characteristics of fatty acid-mediated uncoupling of yeast mitochondria expressing recombinant UCP1. Two questions are addressed. First, does UCP1 function in the absence of both nucleotides and fatty acids? Second, what is the difference between UCP1-mediated uncoupling and that mediated by other carriers? We conclude that UCP1 retains the capacity to transport H+(OH−) in the absence of fatty acids and that fatty acids activate UCP1 at much lower concentrations than are required for other mitochondrial carriers, This work was supported by Spanish Ministry of Education and Culture Grant PB95-0118 and by grants from the French Direction des Recherches, Etudes et Techniques and the Association de Recherches sur le Cancer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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.

The possible regulation of the expression of uncoupling protein-2 (UCP2) mRNA by thyroid hormones in different tissues was examined in rats, Triiodothyronine (T-3) was found to produce an organ-specific enhancement of UCP2 expression in rat tissues. The effect of T-3 was markedly observed in heart, whereas a moderate effect was seen in skeletal muscle and no effect in kidney or liver, These results suggest that UCP2 is a protein that may be involved in the nuclear-mediated effect of T-3 on resting metabolic rate in the rat. (C) 1997 Federation of European Biochemical Societies.

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.

In mammals, body temperature can be raised by the action of uncoupling proteins (UCPs), which dissipate the proton electrochemical gradient across the inner mitochondrial membrane to produce heat rather than synthesize ATP1. Any similar mechanism of thermogenesis in plants is not so well understood. We have now identifiedcomplementary DNA from potatoes (Solanum tuberosum) that encodes a plant uncoupling protein, named StUCP, which is induced by cold treatment and so may be involved in thermoregulation in plants.

Although on-line calorimetry has been widely used to detect transitions in global metabolic activity during the growth of microorganisms, the relationships between oxygen consumption flux and heat production are poorly documented. In this work, we developed a respirometric and calorimetric approach to determine the enthalpy efficiency of respiration-linked energy transformation of isolated yeast mitochondria and yeast cells under growing and resting conditions. On isolated mitochondria, the analysis of different phosphorylating and non-phosphorylating steady states clearly showed that the simultaneous measurements of heat production and oxygen consumption rates can lead to the determination of both the enthalpy efficiency and the ATP/O yield of oxidative phosphorylation. However, these determinations were made possible only when the net enthalpy change associated with the phosphorylating system was different from zero. On whole yeast cells, it is shown that the simultaneous steady state measurements of the heat production and oxygen consumption rates allow the enthalpy growth efficiency (i.e. the amount of energy conserved as biomass compared to the energy utilised for complete catabolism plus anabolism) to be assessed. This method is based on the comparison between the calorimetric-respirometric ratio (CR ratio) determined under growth versus resting conditions during a purely aerobic metabolism. Therefore, in contrast to the enthalpy balance approach, this method does not rely on the exhaustive and tedious determinations of the metabolites and elemental composition of biomass. Thus, experiments can be performed in the presence of non-limiting amounts of carbon substrate, an approach which has been successfully applied to slow growing cells such as yeast cells expressing wild-type or a mutant rat uncoupling protein-1.

In eukaryotic cells ATP is generated by oxidative phosphorylation, an energetic coupling at the mitochondrial level. The oxidative reactions occurring in the respiratory chain generate an electrochemical proton gradient on both sides of the inner membrane. This gradient is used by the ATPsynthase to phosphorylate ADP into ATP. The coupling between respiration and ADP phosphorylation is only partial in brown adipose tissue (BAT) mitochondria, where the uncoupling protein UCP1 causes a reentry of protons into the matrix and abolishes the electrochemical proton gradient. The liberated energy is then dissipated as heat and ATP synthesis is reduced. This property was for a long time considered as an exception and specific to the non-shivering thermogenesis found in BAT. The recent cloning of new UCPs expressed in other tissues revealed the importance of this kind of regulation of respiratory control in metabolism and energy expenditure. The newly characterised UCPs are potential targets for obesity treatment drugs which could favour energy expenditure and diminish the metabolic efficiency. In 1997, we cloned UCP2 and proposed a role for this new uncoupling protein in diet-induced thermogenesis, obesity, hyperinsulinemia, fever and resting metabolic rate. Currently, an abundant literature deals with UCP2, but its biochemical and physiological functions and regulation remain unclear. The present review reports the status of our knowledge of this mitochondrial carrier in terms of sequence, activity, tissue distribution and regulation of expression. The putative physiological roles of UCP2 will be introduced and discussed.

International audience; During apoptosis, mitochondrial membrane permeability (MMP) increases and the release into the cytosol of pro-apoptotic factors (procaspases, caspase activators and caspase-independent factors such as apoptosis-inducing factor (AIF)) leads to the apoptotic phenotype. Apart from this pivotal role of mitochondria during the execution phase of apoptosis (documented in other reviews of this issue), it appears that reactive oxygen species (ROS) produced by the mitochondria can be involved in cell death. These toxic compounds are normally detoxified by the cells, failing which oxidative stress occurs. However, ROS are not only dangerous molecules for the cell, but they also display a physiological role, as mediators in signal transduction pathways. ROS participate in early and late steps of the regulation of apoptosis, according to different possible molecular mechanisms. In agreement with this role of ROS in apoptosis signaling, inhibition of apoptosis by anti-apoptotic Bcl-2 and Bcl-x L is associated with a protection against ROS and/or a shift of the cellular redox potential to a more reduced state. Furthermore, the fact that active forms of cell death in yeast and plants also involve ROS suggests the existence of an ancestral redox-sensitive death signaling pathway that has been independent of caspases and Bcl-2.