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.
Rat uncoupling protein 1 (UCP1) was successfully translated in transfected
Leishmania major promastigotes. Immune electron microscopy revealed that the protein was exclusively in the mitochondria. ...UCP1 expression was about 350 000 copies per promastigote, accounting for 4.7% of the total mitochondrial protein. In intact parasites, expression of UCP1 induced a slight increase in respiratory rate and a modest decrease in mitochondrial membrane potential (ΔΨ
m). In contrast, in digitonin-permeabilized parasites, a significantly lower value both in ΔΨ
m (57±10 vs 153±12 mV) and respiratory control ratio (0.99 vs 1.54) were observed for UCP1 versus control parasites, although when UCP1 activity was inhibited by bovine serum albumin (BSA) and GDP, control values were restored. Therefore, a fully functional UCP1 was present and only partially inhibited in vivo by endogenous purine nucleotides. However, neither ATP levels, growth rate nor mitochondrial protein import differed significantly between both types of parasites. Expression of the pore-like mutant UCP1Δ9 was deleterious to the organism. Consequently,
Leishmania was capable of expressing and importing into mitochondria proteins from higher eukaryotes lacking an N-terminal targeting pre-sequence as UCP1. As described previously, parasite metabolism had only a limited tolerance to mitochondrial disfunction. Transfection of
Leishmania with foreign proteins which play an important regulatory role in metabolism is a useful tool to study both parasite metabolism in general, and alternative pathways involved in maintaining internal homeostasis.
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.
Couplage respiratoire, UCP et ion superoxyde Ricquier, Daniel; Miroux, Bruno; del Mar Gonzalez-Barroso, Maria ...
M.S. Médecine sciences,
05/2002, Letnik:
18, Številka:
5
Journal Article
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.
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.
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.
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.
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 bromoplamitate 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.
Uncoupling proteins (UCPs) are mitochondrial transporters present in the inner membrane of mitochondria. They are found in all mammals and in plants. They belong to the family of anion mitochondrial ...carriers including adenine nucleotide transporters. The term "uncoupling protein" was originally used for UCP1, which is uniquely present in mitochondria of brown adipocytes, the thermogenic cells that maintain body temperature in small rodents. In these cells, UCP1 acts as a proton carrier activated by free fatty acids and creates a shunt between complexes of the respiratory chain and ATP synthase. Activation of UCP1 enhances respiration, and the uncoupling process results in a futile cycle and dissipation of oxidation energy as heat. UCP2 is ubiquitous and highly expressed in the lymphoid system, macrophages, and pancreatic islets. UCP3 is mainly expressed in skeletal muscles. In comparison to the established uncoupling and thermogenic activities of UCP1, UCP2 and UCP3 appear to be involved in the limitation of free radical levels in cells rather than in physiological uncoupling and thermogenesis. Moreover, UCP2 is a regulator of insulin secretion and UCP3 is involved in fatty acid metabolism. Diabetes 53 (Suppl. 1):S130-S135, 2004
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