WO1998045438A1 - Ucp3: un homologue de proteine decouplante - Google Patents

Ucp3: un homologue de proteine decouplante Download PDF

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Publication number
WO1998045438A1
WO1998045438A1 PCT/US1998/006959 US9806959W WO9845438A1 WO 1998045438 A1 WO1998045438 A1 WO 1998045438A1 US 9806959 W US9806959 W US 9806959W WO 9845438 A1 WO9845438 A1 WO 9845438A1
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gly
leu
ala
pro
ucp3
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PCT/US1998/006959
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Bradford B. Lowell
Jeffrey S. Flier
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Beth Israel Deaconess Medical Center
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Priority to AU74661/98A priority Critical patent/AU7466198A/en
Publication of WO1998045438A1 publication Critical patent/WO1998045438A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • UCP1 the first uncoupling protein to be identified (Lin, C.S., et al . , FEES Lett . , 113:299-303 (1980); Jacobsson, A., et al . , J. Biol . Chem . , 260 : 16250 - 16254 (1985); Bouillaud, F., et al., J. Biol . Chem . , 261 : 1487-1490 (1986)), is expressed exclusively in brown adipose tissue, an important site of energy expenditure in rodents (Himms-Hagen, J., Prog. Lipid Res . , 28:67-115 (1989) ) .
  • UCP1 may be of lesser importahce in humans, in whom the amount of brown adipose tissue is limited.
  • a second uncoupling protein referred to UCP2 , was recently identified (Fleury, C., et al . , Nature Genetics, 15:269-272 (1997)) or UCPH (Gimeno, R.E., et al . , Diabetes, 46:900-906 (1997)).
  • UCP2 is expressed in many tissues, including sites not thought to mediate energy expenditure which occurs in response to environmental temperature or diet (adaptive thermogenesis) .
  • the present invention relates to an uncoupling protein (UCP3) gene which is selectively expressed in skeletal muscle and brown fat, two tissues involved in energy expenditure in mammals.
  • UCP3 uncoupling protein
  • the invention relates to an alternative form of UCP3 designated UCP3-sl ⁇ .ort form
  • UCP3sh which is also expressed in skeletal muscle. Skeletal muscle particularly has a capacity for energy expenditure, or adaptive thermogenesis, in humans.
  • UCP3 refers to UCP3 and UCP3sh.
  • the present invention relates to isolated (e.g., purified, essentially pure) nucleic acids (oligonucleotides, nucleotide sequences) which encode a mammalian (e.g., human) UCP3 protein, and include for example, nucleic acids (DNA, RNA) obtained from natural sources, recombinantly produced or chemically synthesized.
  • the nucleic acids of the present invention include nucleic acids encoding human UCP3 (SEQ ID NO : 1) , human UCP3sh (SEQ ID NO: 2) , mouse UCP3 (SEQ ID NO: 7) and characteristic portions thereof (e.g., probes, primers).
  • the invention also includes complementary sequences (i.e., a complement) of SEQ ID NO: 1, SEQ ID NO : 2, SEQ ID NO : 7 and . characteristic portions thereof.
  • the nucleic acids of the present invention encompass nucleic acids encoding a human UCP3 amino acid sequence (SEQ ID NO: 3) , a human UCP3sh form amino acid sequence (SEQ ID NO: 4) , a mouse UCP3 amino acid sequence (SEQ ID NO: 8) and characteristic portions thereof .
  • the present invention further relates to isolated, recombinantly produced or synthetic nucleic acids which hybridize to the nucleic acids described herein (e.g., SEQ ID NO: 1, SEQ ID NO : 2, SEQ ID NO : 7 or characteristic portions thereof) and encode UCP3 protein (a protein having the same amino acid sequence as the amino acid sequences included herein and/or a protein which exhibits the same characteristics as the UCP3 protein described herein) .
  • the invention relates to nucleic acids which hybridize, under moderate or high stringency, conditions, to SEQ ID NO: 1, SEQ ID NO : 2, SEQ ID NO : 7, characteristic portions thereof or other sequences which encode, UCP3.
  • nucleic acid construct comprising nucleic acid which encodes a UCP3 protein (e.g., SEQ ID NO : 1, SEQ ID NO: 2, SEQ ID NO : 7 and characteristic portions thereof) , wherein the nucleic acid of the construct is expressed when the construct is present in an appropriate host cell.
  • the nucleic acid construct of the present invention is operably linked to exogenous regulatory sequence (s) such as a promoter and/or enhancer, whereby mammalian UCP3 is expressed when the host cell is maintained under conditions suitable for expression.
  • the present invention also relates to a host cell comprising nucleic acid encoding mammalian UCP3 protein.
  • Also encompassed by the present invention is a method for producing a mammalian UCP3 protein (human) .
  • a nucleic acid construct comprising a nucleotide sequence (DNA, RNA) which encodes a mammalian UCP3 protein is introduced into a host cell, resulting in production of a recombinant host cell which contains a UCP3 coding sequence operably linked to an (i.e., at least one) expression control sequence.
  • the host cells produced are maintained in a suitable medium under conditions appropriate for the nucleotide sequence to be expressed, whereby the encoded UCP3 is produced.
  • the present invention also relates to isolated (e.g., purified, essentially pure) UCP3 protein and includes, for example, UCP3 protein obtained from natural sources, recombinantly produced or chemically synthesized.
  • the UCP3 protein can be human UCP3 protein (SEQ ID NO: 3), human UCP3sh (SEQ ID N0:4), mouse UCP3 protein (SEQ ID NO: 8) or functional portions thereof.
  • the present invention also pertains to a method of identifying agents which modulate or alter (e.g., inhibit or enhance) UCP3 activity.
  • An inhibitor of UCP3 interferes (partially or completely) with the function or bioactivity of UCP3 , directly or indirectly.
  • An enhancer (activator) of UCP3 increases or enhances the function or bioactivity of UCP3 , directly or indirectly.
  • the present invention relates to a method of identifying an agent which alters UCP3 activity, wherein a nucleic acid construct comprising nucleic acid which encodes a mammalian UCP3 is introduced into a host cell(s) .
  • the host cells produced are maintained under conditions appropriate for expression of the encoded mammalian UCP3 , whereby the nucleic acid is expressed.
  • the host cells are then contacted with a compound to be assessed (an agent) and the mitochondrial electrical potential of the cells is detected in the presence of the compound to be assessed. Detection of a change in mitochondrial electrical potential in the presence of the agent indicates that the agent alters UCP3 activity.
  • the invention relates to a method of identifying an agent which is an activator of UCP3 activity wherein a nucleic acid construct comprising nucleic acid which encodes a mammalian UCP3 is introduced into a host cell (s) .
  • the host cells produced are maintained under conditions appropriate for expression of the encoded mammalian UCP3 , whereby the nucleic acid is expressed.
  • the host cells are then contacted with a compound to be assessed (an agent) and the mitochondrial electrical potential of the cells is detected in the presence of the compound to be assessed. Detection of a decrease or reduction of mitochondrial electrical potential in the presence of the agent indicates that the agent activates UCP3 activity.
  • the invention relates to a method of identifying an agent which is an inhibitor of UCP3 activity, wherein a nucleic acid construct comprising nucleic acid which encodes a mammalian UCP3 is introduced into a host cell (s) .
  • the host cells produced are maintained under conditions appropriate for expression of the encoded mammalian UCP3 , whereby the nucleic acid is expressed.
  • the host cells are then contacted with a compound to be assessed (an agent) and the mitochondrial electrical potential of the cells is detected in the presence of the compound to be assessed. Detection of an increase of mitochondrial electrical potential in the presence of the agent indicates that the agent inhibits UCP3 activity.
  • Methods of identifying agents which alter UCP3 activity can also be performed, as described herein, using a mixture of a membrane fraction, mitochondria and UCP3 (Jezek, et al . , J " . Biol . Chem . 271:6199-6205 (1996)).
  • an agent which interacts with UCP3 directly or indirectly, and inhibits or enhances UCP3 function is an agent which interacts with UCP3 directly or indirectly, and inhibits or enhances UCP3 function.
  • the agent is an inhibitor which interferes with UCP3 directly (e.g., by binding UCP3) or indirectly (e.g., by blocking the ability of UCP3 to regulate thermogenesis in skeletal muscle and/or brown adipose tissue) .
  • an inhibitor of the UCP3 protein is an antibody specific for UCP3 protein or a portion of a UCP3 protein; that is, the antibody binds the UCP3 protein.
  • the antibody can be specific for the human UCP3 protein (SEQ ID NO: 3, SEQ ID NO : 4), the mouse UCP3 protein (SEQ ID NO: 8) or functional portions thereof.
  • the inhibitor can be an agent other than an antibody (e.g., small organic molecule, protein, peptide) which binds UCP3 and blocks its activity.
  • the inhibitor can be an agent which mimics UCP3 structurally but lacks its function.
  • the inhibitor of UCP3 can be an agent which binds to or interacts with a molecule which UCP3 normally binds with or interacts with, thus blocking UCP3 from doing so and preventing it from exerting the effects it would normally exert.
  • the agent is an enhancer of UCP3 which increases the activity of UCP3 (increases thermogenesis in skeletal muscle and/or brown adipose tissue) , increases the length of time it is effective (by preventing its degradation or otherwise prolonging the time during which it is active) or both, either directly or indirectly.
  • the present invention also relates to antibodies (monoclonal or polyclonal) or functional portions thereof (e.g., an antigen binding portion such as an Fv, Fab, Fab', or F(ab') 2 fragment) which bind mammalian UCP3.
  • antibodies monoclonal or polyclonal
  • functional portions thereof e.g., an antigen binding portion such as an Fv, Fab, Fab', or F(ab') 2 fragment
  • the present invention also relates to a method of detecting mammalian UCP3 in a sample (e.g., skeletal muscle, brown adipose tissue) obtained from an individual, such as a human.
  • a sample e.g., skeletal muscle, brown adipose tissue
  • the sample is treated to render nucleic acids in the sample available for hybridization to a nucleic acid probe (e.g., SEQ ID NO: 1, SEQ ID NO : 2, SEQ ID NO : 7 and/or characteristic portions thereof which bind to characteristic regions of UCP3 -encoding nucleic acids).
  • the treated sample is combined with a nucleic acid probe (labeled or unlabeled) comprising or complementary to all or a characteristic portion of the nucleotide sequence encoding UCP3 protein, under conditions appropriate for hybridization of complementary nucleic acids to occur. Hybridization of nucleic acids in the treated sample with the nucleic acid probe is detected; the occurrence of hybridization indicates the presence of UCP3 protein in the sample.
  • the sample is contacted with an antibody which binds to UCP3 protein (e.g., SEQ ID NO: 3, SEQ ID NO : 4, SEQ ID NO : 8 or functional portions thereof) under conditions suitable for binding of the antibody to the mammalian UCP3. Binding of the antibody to a component of the sample is detected; binding of the antibody to a component of the sample indicates the presence of UCP3 protein in the sample.
  • Isolation of UCP3 also makes it possible to identify a promoter (s) and/or enhancer (s) of the UCP3 gene. Identification of promoters and/or enhancers of the UCP3 gene allow for identification of regulators of UCP3 transcription .
  • the present invention relates to transgenic non human animals (e.g., mice) which lack the UCP3 gene or contain a nonfunctional UCP3 gene such that UCP3 activity is lacking (e.g., UCP3 knockout mouse).
  • the invention also relates to methods of producing UCP3 gene knockout animals, such as mice.
  • UCP3 knockout mice can be used to further study the UCP3 gene and to assay for inhibitors and enhancers of UCP3.
  • the present invention also relates to a method of inhibiting (partially, completely) protein catabolism in a mammal (e.g., human) comprising administering to the mammal an effective amount of an inhibitor of UCP3.
  • the invention also relates to a method of enhancing protein catabolism in a mammal comprising administering to the mammal an effective amount of an enhancer of UCP3.
  • Also encompassed by the present invention is a method of inhibiting muscle wasting in a mammal comprising administering an effective amount of an inhibitor of UCP3 to the mammal .
  • UCP3 gene provides for selective modulation (enhancement, inhibition) of the expression and/or function of the UCP3 gene in skeletal muscle and brown fat, two tissues involved in adaptive thermogenesis.
  • Figures 1A-1C are the nucleotide sequence of human UCP3 (SEQ ID NO: 1) and three different amino acid sequences (SEQ ID NO : 27, SEQ ID NO: 28 and SEQ ID NO : 29) translated from SEQ ID NO: 1.
  • Figures 2A-2B are the nucleotide sequence of the UCP3- short form (UCP3sh) gene (SEQ ID NO : 2) and three different amino acid sequences (SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32) translated from SEQ ID NO: 2.
  • Figure 3 is a comparison of the human UCP3 amino acid sequence (SEQ ID NO: 3) , the human UCP3sh amino acid sequence (SEQ ID NO: 4) , the human UCP1 amino acid sequence (SEQ ID NO: 5) and the human UCP2 amino acid sequence (SEQ ID NO: 6) ; sequence alignments were performed using the
  • Figure 4 is a graph of the hydrophilicity plots of human UCP2 and human UCP3 showing the hydrophobicity of protein across linear sequence; hydrophilicity plots for hUCP2 and hUCP3 were generated using the methods .of Kyte and Doolittle (Kyte, J. and Doolittle, R.F., J. Mol . Biol . 157:105-132 (1982) ) .
  • Figures 5A-5C are the nucleotide sequence of mouse UCP3 (SEQ ID NO: 7) and three different amino acid sequences (SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35) translated from SEQ ID NO: 7.
  • Figure 6 is the amino acid sequence of mouse UCP3 (SEQ ID NO: 7)
  • Figure 7 is a comparison of the mouse UCP3 amino acid sequence (SEQ ID NO: 8) with the mouse UCP1 amino acid sequence (SEQ ID NO: 9) , the mouse UCP2 amino acid sequence (SEQ ID NO: 10) and the human UCP3 amino acid sequence (SEQ ID NO: 3) ; the attached sequence and amino acid alignments, mUCP3 is 46% identical to mUCPl, 62% identical to mUCP2 but is 82% identical to hUCP3.
  • Figure 8 is a graphic representation of the genomic organization of the human UCP3 gene, and shows the splice donor sequence (SEQ ID NO: 11) and splice acceptor sequence (SEQ ID NO: 12) between exons 1 and 2, the splice donor sequence (SEQ ID NO: 13) and splice acceptor sequence (SEQ ID NO: 14) between exons 2 and 3, the splice donor sequence (SEQ ID NO: 15) and splice acceptor sequence (SEQ ID NO: 16) between exons 3 and 4, the splice donor sequence (SEQ ID NO: 17) and splice acceptor sequence (SEQ ID NO: 18) between exons 4 and 5, the splice donor sequence (SEQ ID NO: 19) and splice acceptor sequence (SEQ ID NO: 20) between exons 5 and 6, and the splice donor sequence (SEQ ID NO: 21) and splice acceptor sequence (SEQ ID NO: 22) between exons 6 and 7 of the UCP
  • the present invention relates to an uncoupling protein (UCP3) gene which is selectively expressed in skeletal muscle and brown fat, two tissues involved in energy expenditure in mammals.
  • UCP3 uncoupling protein
  • the invention relates to an alternative form of UCP3 designated UCP3-short form (UCP3sh) , which is also expressed in skeletal muscle.
  • UCP3 refers to UCP3 and UCP3sh.
  • the present invention relates to isolated (e.g., purified, essentially pure) UCP3 gene which is involved in regulation of thermogenesis (energy expenditure) in mammals.
  • the present invention relates to nucleic acids (e.g., DNA, RNA, oligonucleotides, polynucleotides) or characteristic portions thereof as described herein, obtained from natural sources, recombinantly produced or chemically synthesized which encode a mammalian UCP3 or functional portion thereof.
  • Nucleic acids referred to herein as “isolated” are nucleic acids substantially free of (separated away from) the nucleic acids of the genomic DNA or cellular RNA of their biological source of origin (e.g., as it exists in cells or in a mixture of nucleic acids such as a library) , and may have undergone further processing.
  • isolated nucleic acids include nucleic acids obtained by methods described herein, similar methods or other suitable methods, including essentially pure nucleic acids, nucleic acids produced by chemical synthesis or by combinations of biological and chemical methods, and recombinantly produced nucleic acids which are isolated (see e.g., Daugherty, B.L. et al . , Nucleic Acids Res .
  • Nucleic acids referred to herein as "recombinant” are nucleic acids which have been produced by recombinant DNA methodologies (recombinantly produced) .
  • Recombinant DNA methodologies include, for example, expression of UCP3 in a host cell containing or modified to contain DNA or RNA encoding UCP3 or expression of UCP3 using polymerase chain reaction (PCR) techniques .
  • PCR polymerase chain reaction
  • a "characteristic portion" of nucleic acids described herein refers to portions of a nucleotide sequence which encode a protein or polypeptide having at least one property, function or activity characteristic of UCP3 protein (e.g., predominantly expressed in brown adipose tissue and skeletal muscle; activity in regulating thermogenesis in skeletal muscle and brown adipose tissue; selectively uncoupling mitochondrial respiration in brown adipocytes and skeletal muscle) .
  • the term includes a nucleotide sequence which, through the degeneracy of the genetic code, encodes the same peptide as a peptide whose sequence is presented herein (e.g., SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO : 7).
  • the nucleic acids described herein may also contain a modification of the molecule such that the resulting gene product is sufficiently similar to that encoded by the unmodified sequence that it has essentially the same activity as the unmodified sequence.
  • Such a modification would be a "silent" codon substitution or an amino acid substitution, for instance, substitution of one codon encoding a hydrophobic amino acid to another codon encoding the same hydrophobic amino acid or substitution of one acidic amino acid for another acidic amino acid. See Ausubel, F.M., et al . , Current Protocols in Molecular Biology, Greene Publ . Assoc. and Wiley- Interscience 1989.
  • the nucleic acid or characteristic portion thereof encodes a protein or polypeptide having at least one property, activity or function characteristic of a mammalian UCP3 (as defined herein) , such as activity or function characteristic of a mammalian UCP3 (as defined herein) , such as activity in regulation of thermogenesis in skeletal muscle and brown adipose tissue.
  • the present invention also relates more specifically to isolated nucleic acids or a characteristic portion thereof, which encode mammalian UCP3 or variants thereof.
  • the invention relates to isolated nucleic acids that: (1) hybridize to (a) a nucleic acid encoding a mammalian UCP3 (e.g., human) , such as a nucleic acid having a nucleotide sequence as set forth or substantially as set forth in Figures 1A-1C (SEQ ID NO:l), Figures 2A-2B (SEQ ID NO: 2) or Figures 5A-5C (SEQ ID NO: 7) ; (b) the complement of the sequences of (a) ; or (c) characteristic portions of either of the foregoing (e.g., a portion comprising the open reading frame) ;
  • a mammalian UCP3 e.g., human
  • a protein or polypeptide having at least one property, activity of function characteristic of a UCP3 protein e.g., predominantly expressed in brown adipose tissue and skeletal muscle; activity in regulating thermogenesis in skeletal muscle and brown adipose tissue; selectively uncoupling mitochondrial respiration in brown adipocytes and skeletal muscle
  • a polypeptide having the amino acid sequence of a mammalian UCP3 e.g., SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7 ; or
  • the nucleic acid shares at least about 75% nucleotide sequence similarity, and more preferably, at least about 90% nucleotide sequence similarity, to the sequence shown in Figures 1A-1C (SEQ ID NO:l), Figures 2A-2B (SEQ ID NO: 2) or Figures 5A-5C (SEQ ID NO: 7) .
  • Isolated nucleic acids meeting these criteria include nucleic acids having sequences identical to sequences of naturally occurring mammalian UCP3 or variants of the naturally occurring sequences which encode mammalian (human) UCP3. Such variants include mutants differing by the addition, deletion or substitution of one or more residues, modified nucleic acids in which one or more residues are modified (e.g., DNA or RNA analogs), and mutants comprising one or more modified residues.
  • Nucleic acids of the present invention may be RNA or DNA (e.g., cDNA, genomic DNA, and synthetic DNA).
  • the DNA may be double-stranded or single-stranded and, if single stranded, may be the coding strand or non-coding (anti- sense) strand.
  • the coding sequence which encodes the polypeptide may be identical to the coding sequence shown in Figures 1A-1C (SEQ ID NO:l), Figures 2A-2C (SEQ ID NO:
  • Figures 5A-5C (SEQ ID NO: 7) or may be a different coding sequence which, as a result of the redundancy or degeneracy of the genetic code, encodes the same polypeptide as the polypeptide encoded by the DNA of Figures 1A-1C (SEQ ID N0:1), Figures 2A-2B (SEQ ID NO : 2 ) or Figures 5A-5C (SEQ ID NO: 7) .
  • the nucleic acid (polynucleotide) which encodes a UCP3 polypeptide encoded by the UCP3 cDNA may include: only the coding sequence of a polypeptide; the coding sequence for a polypeptide and additional coding sequence such as a leader or secretory sequence; the coding sequence for a polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5' and/or 3' of the coding sequence.
  • Nucleic acids of the present invention including those which hybridize to a selected nucleic acid as described above, can be detected or isolated under high stringency conditions or moderate stringency conditions, for example.
  • High stringency conditions and “moderate stringency conditions” for nucleic acid hybridizations are explained at pages 2.10.1-2.10.16 (see particularly 2.10.8- 11) and pages 6.3.1-6 in Current Protocols in Molecular Biology (Ausubel, F.M. et al., eds., Vol. 1, Suppl . 26, 1991) , the teachings of which are hereby incorporated by reference.
  • Factors such as probe length, base composition, percent mismatch between the hybridizing sequences, temperature and ionic strength influence the stability of nucleic acid hybrids.
  • high or moderate stringency conditions can be determined empirically, and depend in part upon the characteristics of the known nucleic acid (e.g., DNA) and the other nucleic acids to be assessed for hybridization thereto.
  • Nucleic acids of the present invention that are characterized by their ability to hybridize (e.g., under high or moderate stringency conditions) to (a) a nucleic acid encoding a mammalian UCP3 (for example, the nucleic acid depicted in Figures 1A-1C (SEQ ID NO:l), Figures 2A-2B (SEQ ID NO:2), Figures 5A-5B (SEQ ID NO : 7) or characteristic portions thereof) ; (b) the complement of the nucleic acids of (a); or (c) a portion thereof, can also encode a protein or polypeptide having at least one property, activity or function characteristic of a mammalian UCP3 as defined herein, such as activity in regulation of thermogenesis in skeletal muscle and brown adipose tissue.
  • a nucleic acid encoding a mammalian UCP3 for example, the nucleic acid depicted in Figures 1A-1C (SEQ ID NO:l), Figures 2A-2B (SEQ ID
  • nucleic acid encodes a polypeptide which retains substantially the same biological function or activity as the polypeptide encoded by the DNA of Figures 1A-1C (SEQ ID NO:l), or Figures 2A-2B (SEQ ID NO:2) or Figures 5A-5C (SEQ ID NO: 7) .
  • Nucleic acids of the present invention can be used in the production of proteins or polypeptides.
  • a nucleic acid e.g., DNA
  • encoding a mammalian UCP3 can be incorporated into various constructs and vectors created for further manipulation of sequences or for production of the encoded polypeptide in suitable host cells as described above .
  • a further embodiment of the invention is antisense nucleic acid, which is complementary, in whole or in part, to a UCP3 sense strand, and can hybridize with it.
  • the antisense strand hybridizes to DNA, or its RNA counterpart (i.e., wherein T residues of the DNA are U residues in the RNA counterpart) .
  • antisense nucleic acid hybridizes to and inhibits the expression of the sense strand.
  • Antisense nucleic acids can be produced by standard techniques .
  • the antisense nucleic acid is wholly or partially complementary to and can hybridize with a target nucleic acid which encodes a mammalian UCP3.
  • antisense nucleic acid can be complementary to a target nucleic acid having the sequence shown as the open reading frame in Figures 1A-1C (SEQ ID N0:1), Figures 2A-2B (SEQ ID NO:2) , Figures 5A-5C (SEQ ID NO: 7) or to a portion thereof sufficient to allow hybridization.
  • the nucleic acids can also be used as probes (e.g., for in si tu hybridization) to assess regulation of thermogenesis in skeletal muscle and/or brown adipose tissue.
  • the nucleic acids can also be used as probes to detect and/or isolate (e.g., by hybridization with RNA or DNA) polymorphic or allelic variants, for example, in a sample (e.g., skeletal muscle, brown adipocytes, ,white blood cells) obtained from a host (e.g., a human) .
  • a sample e.g., skeletal muscle, brown adipocytes, ,white blood cells
  • a host e.g., a human
  • the presence or level of a particular variant in a sample (s) obtained from an individual as compared with the presence or level in a sample (s) from normal individuals, can be indicative of an association between abnormal regulation of thermogenesis (e.g., obesity) and a particular variant, which in turn can be used in the diagnosis of the condition.
  • thermogenesis e.g., obesity
  • the present invention also relates to isolated (e.g., pure, essentially pure) proteins or polypeptides designated mammalian UCP3 and variants of mammalian UCP3.
  • the isolated proteins of the present invention have at least one property, activity or function characteristic of a mammalian UCP3 (as defined herein) , such as activity in regulating (mediating) thermogenesis in skeletal muscle and brown adipose tissue or selectively uncoupling mitochondrial respiration in brown adipocytes and in skeletal muscle.
  • isolated proteins are proteins or polypeptides purified to a state beyond that in which they exist in mammalian cells.
  • isolated proteins or polypeptides include proteins or polypeptides obtained by methods described herein, similar methods or other suitable methods. They include essentially pure proteins or polypeptides, proteins or polypeptides produced by chemical synthesis (e.g., synthetic peptides) , or by combinations of biological and chemical methods, and recombinant proteins or polypeptides which are isolated.
  • the proteins can be obtained in an isolated state of at least about 50 % by weight, preferably at least about 75 % by weight, and more preferably, in essentially pure form.
  • Proteins or polypeptides referred to herein as “recombinant” are proteins or polypeptides produced by the expression of recombinant nucleic acids.
  • "mammalian UCP3" protein ref,ers to naturally occurring or endogenous mammalian UCP3s, proteins having an amino acid sequence which is the same as that of a naturally occurring or endogenous corresponding mammalian UCP3 (e.g., recombinant proteins), and functional variants of each of the foregoing (e.g., functional fragments and/or mutants produced via mutagenesis and/or recombinant techniques) .
  • the term includes mammalian UCP3 , glycosylated or unglycosylated UCP3 , polymorphic or allelic variants, and other isoforms of mammalian UCP3 (e.g., produced by alternative splicing or other cellular processes), and functional fragments.
  • Naturally occurring or endogenous mammalian UCP3s include wild type proteins such as mammalian UCP3 , polymorphic or allelic variants and other isoforms which occur naturally in mammals (e.g., primate, preferably human, murine, bovine) . Such proteins can be recovered from a source in which UCP3 is naturally produced, for example.
  • These mammalian proteins have the same amino acid sequence as naturally occurring or endogenous corresponding mammalian UCP3.
  • “Functional variants" of mammalian UCP3 include functional fragments, functional mutant proteins, and/or functional fusion proteins.
  • fragments or portions of mammalian UCP3 encompassed by the present invention include those having one or more amino acid deletions relative to the naturally occurring mammalian UCP3 protein (such as N-terminal, C-terminal or internal deletions) . Fragments or portions in which only contiguous amino acids have been deleted or in which non-contiguous amino acids have been deleted relative to naturally occurring mammalian UCP3 are also encompassed by the invention.
  • mutants or derivatives of mammalian UCP3 encompassed by the present invention include natural or artificial variants differing by the addition, deletion and/or substitution of one or more contiguous or non-contiguous amino acid residues, or modified polypeptides in which one or more residues is modified, and mutants comprising one or more modified residues.
  • mutants can be natural or artificial variants of mammalian UCP3 which differ from naturally occurring UCP3 by the addition, deletion and/or substitution of one or more contiguous or non-contiguous amino acid residues.
  • a “functional fragment or portion”, “functional mutant” and/or “functional fusion protein” of a mammalian UCP3 refers to an isolated protein or oligopeptide which has at least one property, activity or function characteristic of a mammalian UCP3 , such as activity in regulating (mediating) thermogenesis in skeletal muscle and brown adipose tissue or activity in selectively uncoupling mitochondrial respiration in brown adipocytes and in skeletal muscle.
  • Suitable fragments or mutants can be identified by screening.
  • the N-terminal, C-terminal, or internal regions of the protein can be deleted in a step- wise fashion and the resulting protein or polypeptide can be screened using a suitable assay, for example, by measuring mitochondrial membrane potential in a host cell expressing UCP3. Where the resulting protein displays activity in the assay, the resulting protein ("fragment") is functional.
  • the invention also encompasses fusion proteins, comprising a mammalian UCP3 as a first moiety, linked to a second moiety not occurring in the mammalian UCP3 found in nature.
  • the second moiety can be, for example, an amino acid, oligopeptide or polypeptide.
  • the first moiety can be in an N-terminal location, C-terminal location or internal location of the fusion protein.
  • the fusion protein comprises a mammalian UCP3 or portion thereof as the first moiety, and a sec.ond moiety comprising an affinity ligand (e.g., an enzyme, an antigen, epitope tag) joined to the first moiety.
  • the two components can be joined by a linker.
  • human UCP3 examples include proteins having an amino acid sequence as set forth or substantially as set forth in Figure 3 (SEQ ID NO: 3, SEQ ID NO: 4) and functional portions thereof.
  • An example of “mouse UCP3” includes a protein having an amino acid sequence as set forth or substantially set forth in Figure 6 (SEQ ID NO: 8) .
  • a human UCP3 protein, a mouse UCP3 protein or a variant thereof has an amino acid sequence which has at least about 75% identity, and more preferably at least about 90% identity, to the protein shown in Figure 3 (SEQ ID NO: 3, SEQ ID NO: 4) or Figure 6 (SEQ ID NO: 8) .
  • Another aspect of the invention relates to a method of producing a human UCP3 or variant (e.g., portion) thereof.
  • Recombinant protein can be obtained, for example, by the expression of a recombinant DNA molecule encoding a mammalian UCP3 or variant thereof in a suitable host cell.
  • Constructs suitable for the expression of a mammalian UCP3 or variant thereof are also provided. The constructs can be introduced into a suitable host cell, and cells which express a recombinant mammalian UCP3 or variant thereof, can be produced and maintained in culture.
  • Suitable host cells can be procaryotic, including bacterial cells such as E. coli , B . subtilis and or other suitable bacteria (e.g., Streptococci ) or eucaryotic, such as fungal or yeast cells (e.g., Pichia pastoris , Aspergillus species,
  • insects e.g., Sf9 insect cells
  • mammals e.g., Chinese hamster ovary cells (CHO) , COS cells, HuT 78 cells, 293 cells
  • CHO Chinese hamster ovary cells
  • COS cells HuT 78 cells
  • 293 cells See, e.g., Ausubel, F.M. et al., eds. Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons Inc., (1993)).
  • Host cells which produce a recombinant mammalian UCP3 or variants thereof can be produced as follows.
  • nucleic acid encoding all or part of the UCP3 protein or a functional portion thereof can be inserted into a nucleic acid vector, e.g., a DNA vector, such as a plasmid, virus or other suitable replicon for expression.
  • a nucleic acid vector e.g., a DNA vector, such as a plasmid, virus or other suitable replicon for expression.
  • a variety of vectors is available, including vectors which are maintained in single copy or multiple copy, or which become integrated into the host cell chromosome.
  • the transcriptional and/or translational signals of a mammalian UCP3 gene can be used to direct expression.
  • suitable expression vectors for the expression of a nucleic acid encoding all or part of the desired protein are available.
  • Suitable expression vectors can contain a number of components, including, but not limited to, one or more of the following: an origin of replication; a selectable marker gene; one or more expression control elements, such as a transcriptional control element (e.g., a promoter, an enhancer, terminator), and/or one or more translation signals; a signal sequence or leader sequence for membrane targeting or secretion (of mammalian origin or from a heterologous mammal or non-mammalian species) .
  • a signal sequence can be provided by the vector, the mammalian UCP3 coding sequence, or other source.
  • a promoter can be provided for expression in a suitable host cell. Promoters can be constitutive or inducible. The promoter is operably linked to nucleic acid encoding the mammalian UCP3 or variant thereof, and is capable of directing expression of the encoded polypeptide in the host cell .
  • suitable promoters for procaryotic e.g., lac, tac, T3 , T7 promoters for E. coli
  • eucaryotic e.g., yeast alcohol dehydrogenase (ADH1) , SV40, CMV
  • the expression vectors typically comprise a selectable marker for selection of host cells carrying the vector, and in the case of a replicable expression vector, also comprise an origin of replication.
  • Genes encoding products which confer antibiotic or drug resistance are common selectable markers and may be used in procaryotic (e.g., /3-lactamase gene (ampicillin resistance) , Tet gene for tetracycline resistance) and eucaryotic cells (e.g., neomycin (G418 or geneticin) , gpt (mycophenolic acid) , ampicillin, or hygromycin resistance genes) .
  • Dihydrofolate reductase marker genes permit selection with methotrexate in a variety of hosts.
  • auxotrophic markers of the host e.g., LEU2 , URA3 , HIS3
  • vectors which are capable of integrating into the genome of the host cell such as retroviral vectors
  • the present invention also relates to cells carrying these expression vectors.
  • a nucleic acid encoding a mammalian UCP3 or variant thereof is incorporated into a vector, operably linked to one or more expression control elements, and the construct is introduced into host cells which are maintained under conditions suitable for expression, whereby the encoded polypeptide is produced.
  • the construct can be introduced into cells by a method appropriate to the host cell selected (e.g., transformation, transfection, electroporation, infection) .
  • host cells comprising the construct are maintained under conditions appropriate for expression, (e.g., in the presence of inducer, suitable media supplemented with appropriate salts, growth factors, antibiotic, nutritional supplements, etc.) .
  • the encoded protein e.g., human UCP3 can be isolated from the host cells or medium.
  • Fusion proteins can also be produced in this manner.
  • some embodiments can be produced by the insertion of a mammalian UCP3 cDNA or portion thereof into a suitable expression vector, such as Bluescript ® II SK +/- (Stratagene) , pGEX-4T-2 (Pharmacia) , pcDNA-3 (Invitrogen) and pET-15b (Novagen) .
  • the resulting construct can then be introduced into a suitable host cell for expression.
  • fusion protein can be isolated or purified from a cell lysate by means of a suitable affinity matrix (see e.g., Current Protocols in Molecular Biology (Ausubel, F.M. et al . , eds., Vol.
  • affinity labels provide a means of detecting a fusion protein.
  • the cell surface expression or presence in a particular cell fraction of a fusion protein comprising an antigen or epitope affinity label can be detected by means of an appropriate antibody.
  • UCP3 nucleic acids DNA, RNA
  • protein can be used in a variety of ways.
  • UCP3 nucleic acids and proteins can be used to identify agents (e.g., molecules) that alter or modulate (enhance, inhibit) UCP3 expression and/or function.
  • agents e.g., molecules
  • UCP3 can be expressed in a host cell and effects of test compounds on mitochondrial membrane potential in the host cell could be assessed.
  • evaluation of mitochondrial respiration could also be performed in the host cell.
  • the present invention relates to a method of identifying an agent which alters UCP3 activity, wherein a nucleic acid construct comprising nucleic acid which encodes a mammalian UCP3 is introduced into , a host cell(s) .
  • the host cells produced are maintained under conditions appropriate for expression of the encoded mammalian UCP3 , whereby the nucleic acid is expressed.
  • the host cells are then contacted with a compound to be assessed (an agent) and the mitochondrial electrical potential (mitochondrial membrane potential) of the cells is detected in the presence of the compound to be assessed. Detection of a change in mitochondrial electrical potential in the presence of the agent indicates that the agent alters UCP3 activity.
  • the invention relates to a method of identifying an agent which is an activator of UCP3 activity wherein a nucleic acid construct comprising nucleic acid which encodes a mammalian UCP3 is introduced into a host cell(s) .
  • the host cells produced are maintained under conditions appropriate for expression of the encoded mammalian UCP3 , whereby the nucleic acid is expressed.
  • the host cells are then contacted with a compound to be assessed (an agent) and the mitochondrial electrical potential of the cells is detected in the presence of the compound to be assessed. Detection of a decrease or reduction of mitochondrial electrical potential in the presence of the agent indicates that the agent activates UCP3 activity.
  • the invention in another embodiment, relates to a method of identifying an agent which is an inhibitor of UCP3 activity, wherein a nucleic acid construct comprising nucleic acid which encodes a mammalian UCP3 is introduced into a host cell (s) .
  • the host cells produced are maintained under conditions appropriate for expression of the encoded mammalian UCP3 , whereby the nucleic acid is expressed.
  • the host cells are then contacted with a compound to be assessed (an agent) and the mitochondrial electrical potential of the cells is detected in the presence of the compound to be assessed. Detection of an increase of mitochondrial electrical potential in the presence of the agent indicates that the agent inhibits UCP3 activity.
  • Detection of a change in mitochondrial electrical potential can be performed using a variety of techniques. For example, a change in mitochondrial electrical potential can be detected by measuring fluorescence of recombinant cells expressing UCP3. Decrease of fluorescence in the presence of the test compound, indicates a decrease of mitochondrial membrane potential (mitochondrial ⁇ ) , and vice versa for cases where fluorescence is increased. That is, increase of fluorescence in the presence of the test compound indicates an increase of mitochondrial ⁇ . If decrease in fluorescence is observed in UCP3 expressing cells, but not in control cells, then the test compound is an activator of UCP3. If an increase in fluorescence is observed in UCP3 expressing cells, but not in control cells, then the test compound is an inhibitor of UCP3.
  • a high throughput screen can be used to identify agents that activate (enhance) or inhibit UCP3 activity.
  • the method of identifying an agent which alters UCP3 activity can be performed as follows.
  • a nucleic acid construct comprising nucleic acid which encodes a mammalian UCP3 is introduced into a host cell(s) to produce recombinant host cells.
  • the recombinant host cells produced are maintained under conditions appropriate for expression of the encoded mammalian UCP3 , whereby the nucleic acid is expressed.
  • a fluorescent dye and the compound to be assessed are added to the recombinant host cells; the resulting combination is referred to as a test sample. Fluorescence is detected.
  • a decrease of fluorescence in the presence of the test compound occurs with a decrease in the mitochondrial electrical potential of the cells, which indicates that the agent is an activator of UCP3. Conversely, an increase of fluorescence in the presence of the test compound occurs with an increase in the mitochondrial electrical potential of the cells, which indicates that the agent is an inhibitor of UCP3.
  • Suitable dyes for use in this embodiment of the invention include, for example, JC-1, rhodamine 123, DiOCc[3], or tetramethylhydrosamine .
  • control can be used in the methods of detecting agents which alter UCP3 activity.
  • the control sample includes the same reagents but lacks the compound or agent being assessed; it is treated in the same manner as the test sample.
  • an agent which interacts with UCP3 directly or indirectly, and inhibits or enhances UCP3 expression and/or function is an agent which interacts with UCP3 directly or indirectly, and inhibits or enhances UCP3 expression and/or function.
  • the agent is an inhibitor which interferes with UCP3 directly (e.g., by binding UCP3) or indirectly (e.g., by blocking the ability of UCP3 to function in thermogenesis) .
  • an inhibitor of UCP3 protein is an antibody specific for UCP3 protein or a functional portion of UCP3 ; that is, the antibody binds the UCP3 protein.
  • the antibody can be specific for the protein encoded by the amino acid sequence of human UCP3 (SEQ ID NO: 3) , human UCP3sh (SEQ ID NO: 4) , mouse UCP3 (SEQ ID NO : 8) or portions thereof.
  • the inhibitor can be an agent other than an antibody (e.g., small organic molecule, protein or peptide) which binds UCP3 and blocks its activity.
  • the inhibitor can be an agent which mimics UCP3 structurally, but lacks its function.
  • it can be an agent which binds to or interacts with a molecule which UCP3 normally binds with or interacts with, thus blocking UCP3 from doing so and preventing it from exerting the effects it would normally exert.
  • the agent is an enhancer
  • UCP3 activator of UCP3 which increases the activity.of UCP3 (increases the effect of a given amount or level of UCP3) , increases the length of time it is effective (by preventing its degradation or otherwise prolonging the time during which it is active) or both either directly or indirectly.
  • UCP3 nucleic acids and proteins can be used to identify anti-obesity drugs which enhance UCP3 to induce uncoupling in brown fat and/or skeletal muscle, with the result that stored energy is released as heat.
  • sequences described herein can be used to detect UCP3 or DNA encoding UCP3 in a sample.
  • a labeled nucleic acid probe having all or a functional portion of the nucleotide sequence of UCP3 can be used in a method to detect UCP3 in a sample.
  • the sample is treated to render the nucleic acids in the sample available for hybridization to a nucleic acid probe, which can be DNA or RNA.
  • the resulting treated sample is combined with a labeled nucleic acid probe having all or a portion of the nucleotide sequence of UCP3 , under conditions appropriate for hybridization of complementary sequences to occur.
  • Detection of hybridization of nucleic acids from the sample with the labeled nucleic probe indicates the presence of UCP3 in a sample.
  • the presence of UCP3 mRNA is indicative of UCP3 expression.
  • Such a method can be used, for example, as a screen for normal or abnormal thermogenesis in skeletal muscle or brown adipose tissue.
  • a method of detecting UCP3 in a sample can be accomplished using an antibody directed against UCP3 or a portion of UCP3. Detection of specific binding to the antibody indicates the presence of UCP3 in the sample (e.g., ELISA) . This could reflect a pathological state associated with UCP3 and, thus, can be used diagnostically .
  • the sample for use in the methods of the present invention includes a suitable sample from, for example, a mammal, particularly a human.
  • the sample can be blood, skeletal muscle or brown adipose tissue.
  • the UCP3 sequences of the present invention can also be used to generate nonhuman gene knockout animals, such as mice, which lack UCP3 and transgenically overexpress UCP3.
  • nonhuman gene knockout animals such as mice, which lack UCP3 and transgenically overexpress UCP3.
  • UCP3 gene knockout mice can be generated and used to obtain further insight into the function of UCP3 as well as assess the specificity of UCP3 activators and inhibitors.
  • overexpression of UCP3 (e.g., human UCP3) in transgenic mice can be used as a means of creating a test system for UCP3 activators and inhibitors (e.g., against human UCP3) .
  • the UCP3 gene can be used to clone the UCP3 promoter/enhancer in order to identify regulators of UCP3 transcription.
  • UCP3 gene knockout animals include animals which completely or partially lack the UCP3 gene and/or UCP3 activity or function.
  • UCP3 plays a role in controlling protein wasting and production of gluconeogenic precursors by skeletal muscle via transport of one or more metabolites, which indicates that inhibitors of UCP3 can be used as a means of curtailing muscle wasting due to, for example, infection, (e.g., human immunodeficiency virus) cancer, tumor cachexia, muscle diseases (e.g., muscular dystrophy) or as a possible treatment for non- insulin dependent diabetes mellitus (NIDDM) .
  • infection e.g., human immunodeficiency virus
  • tumor cachexia e.g., muscle diseases (e.g., muscular dystrophy) or as a possible treatment for non- insulin dependent diabetes mellitus (NIDDM) .
  • NIDDM non- insulin dependent diabetes mellitus
  • the present invention relates to a method of inhibiting (partially, completely) protein catabolism in a mammal (e.g., human) comprising administering to the mammal an effective amount of an inhibitor of UCP3.
  • the invention also relates to a method of enhancing protein catabolism in a mammal comprising administering to the mammal an effective amount of an enhancer UCP3.
  • Also encompassed by the present invention is a method of inhibiting muscle wasting in a mammal comprising administering an effective amount of an enhancer of UCP3 to the mammal .
  • brown adipose tissue plays an important role in regulating energy balance in rodents (Himms-Hagen, J., Prog. Lipid Res . , 28:67-115 (1989)) .
  • the tissue is highly specialized for stimulated energy expenditure with a rich vascular supply, dense sympathetic innervation, and numerous mitochondria.
  • brown adipocytes are further distinguished from other cell types by their expression of all three uncoupling proteins: UCP1, which is expressed exclusively in brown adipocytes, UCP2 , which is expressed widely (Fleury, C, et al . f Nature Genetics , 15:269-272 (1997); Gimeno, R.E., et al . , Diabetes , in press (1997)) and, as demonstrated herein, UCP3 which is expressed selectively and abundantly in brown adipocytes and skeletal muscle. These features make brown fat ideally suited to regulated thermogenesis. 1
  • brown adipose tissue in large mammals is relatively limited and therefore brown fat may not be a significant regulator of human energy expenditure.
  • a number of studies in humans have implicated skeletal muscle as an important mediator of adaptive thermogenesis in humans (Astrup, A., et al . , Am. J. Physiol . , 248:E507- 515 (1985); Astrup, A., et al . , Am. J “ . Physiol . , 257:E340- 345 (1989); Zurlo, F., et al., J “ . Clin . Invest . , 86:1423- 1427. (1990) ; Simonsen, L., et al., Am.
  • brown fat and skeletal muscle have many features in common: a rich blood supply, a dense sympathetic innervation, and abundant mitochondria. In addition, both tissues express high levels of UCP3 mRNA.
  • UCP3 is minimally expressed in cardiac tissue. This is especially true given the general tendency for non-contractile muscle-specific genes to be expressed in both striated muscle types (skeletal and cardiac) . Abundant expression of UCP3 in two thermogenic tissues, skeletal muscle and brown fat, and relative lack of expression in other sites such as the heart, demonstrates that UCP3 is an important molecular mediator of adaptive thermogenesis.
  • the present invention provides for anti-obesity drug development wherein the UCP3 nucleic acids and protein can be used to identify, for example, enhancers (activators) of UCP3 which can be used to induce uncoupling.
  • enhancers activators
  • UCP3 which increase UCP1 expression and activity in brown fat are presently under development, but may have limited effects given the paucity of brown fat in humans.
  • UCP2 is another potential target. However, it is expressed in a number of critical organs and tissues and its activation could produce unwanted side effects.
  • Specific activators of UCP3 expression and/or function selectively increase energy expenditure in skeletal muscle and brown fat, two tissues that have the capacity for adaptive energy expenditure .
  • the present invention is further illustrated by the following examples, which are not intended to be limiting in any way.
  • Human Multiple Tissue Northern Blots (#7760-1, #7759-1 and ⁇ 7767-1) containing approximately 2 ⁇ g of polyA RNA per lane were purchased from Clontech Laboratories (Palo Alto, CA) . All hybridizations, membranes washes and membrane strippings were performed according to manufacturer' s specifications. The blots were first hybridized to a hUCP3 probe, washed and exposed to film for 1-18 hours, then stripped, rehybridized to a hUCP2 probe and exposed to film for 18 hours.
  • the hUCP3 probe was a 293 bp fragment corresponding to residues #211-308.
  • the hUCP2 probe was a 1125 bp fragment spanning the entire open reading frame.
  • Mouse Northern blots were generated using total RNA isolated from a number of tissues and equal loading of lanes was established using ethidium bromide florescence. The mouse Northern blots were hybridized using the hUCP3 probe described above .
  • Skeletal muscle and heart RNA was obtained from Clontech. Aliquots of 1, 3, 5 and 10 ⁇ g of adipose tissue and skeletal muscle I?NA and 10 ⁇ g aliquot of heart RNA were used for determination of UCP3 and mRNA levels.
  • the Rnase protection assay was performed as previously described (Vidal-Puig, A., et al., J " . Clin . Invest . , 97:2553-2561 (1997)).
  • a UCP-3 cDNA fragment was generated by reverse transcriptase-PCR using total RNA from human muscle as follows: two primers (5' GGA CTA CCA CCT GCT CAC TG 3' (SEQ ID NO : 23) and 5' CCC GTA ACA TAT GGA CTT T3 ' (SEQ ID NO: 24)) were designed to amplify 302 bp of the hUCP-3 sequence corresponding to residues #209-308.
  • the PCR product was subcloned into PGMT easy TA cloning vector (Promega Corp., Madison, WI) and linearized for riboprobe synthesis using Spe I . Identity and orientation of the UCP3 probe was confirmed by sequencing.
  • the antisense [ 32 P] -labeled UCP3 template was synthesized using T& RNA polymerase.
  • a human cyclin riboprobe was used as an internal control (Ambion, Inc., Austin, TX) .
  • UCP3 a third uncoupling homologue designated UCP3 has been cloned. It is distinguished from UCP1 and UCP2 by its selective expression in skeletal muscle and brown adipose tissue, two important sites for regulated energy expenditure in humans (Astrup, A., et al., Am. J. Physiol . , 248 -. E501-515 (1985); Astrup, A., et al., Am. J. Physiol . , 257:E340-345 (1989); Zurlo, F., et al . , J. Clin . Invest . , 86:1423-1427 (1990); Simonsen, L., et al., Am . J.
  • hUCP3 is 71% identical to hUCP2 and 57% identical to hUCPl . Because UCP3 is abundantly and selectively expressed in skeletal muscle and brown adipose tissue, UCP3 is likely to be an important mediator of regulated thermogenesis in humans.
  • UCP3 is minimally expressed in heart and other critical organs, it ,is a promising target for anti-obesity drug development aimed at increasing thermogenesis.
  • the expressed sequence tag (EST) database http://www.ncbi.nlm.gov) was screened for sequences homologous to UCP1.
  • One human EST deposited by the Washington University, St. Louis - Merck & Co. EST project, was identified which was similar but not identical to hUCPl and hUCP2 (accession no. AA192136, IMAGE clone no. 628529) .
  • This clone originated from a human skeletal muscle cDNA library (#937209, Stratagene, La Jolla, CA) .
  • the bacterial stock for clone 628529 was obtained from Genome Systems (St. Louis, MI) and was found to contain an insert of approximately 1.3kb, which included the C- terminal third of the open reading frame.
  • the coding region within clone 628529 was fully resequenced.
  • Full-length cDNA sequences were generated using the Marathon cDNA Amplification Kit, human skeletal muscle Marathon-Ready cDNA (both from Clontech Laboratories, Palo Alto, CA) and an antisense primer (5' -TTC ACC ACG TCC ACC CGG GGG GAT GCC ACC-3') (SEQ ID NO: 25) corresponding to the coding sequence presumed to represent hUCP3.
  • UCP3 cDNA sequence contains a 5' untranslated region of at least 183 bases, an open reading from of 936 bases, a 3' untranslated region of approximately 1.1 kb, a polyadenylation signal and a polyA tail ( Figures 1A-1C) .
  • the UCP3 mRNA transcript is predicted to be equal to or greater than 2.2 kb .
  • UCP3 protein, as deduced from the open reading frame, is composed of 312 amino acids and is estimated to have a molecular weight of approximately 34 kD ( Figure 3) .
  • hUCP3 is 71% identical to hUCP2 and 57% identical to hUCPl; and hUCP2 is 59% identical to hUCPl .
  • Many of the nonidentical residues in hUCP3 are conservative substitutions which in most cases correspond to residues found in either mUCP2 (Fleury, C, et al., Nature Genetics, 15:269-272 (1997); Gimeno, R.E., et al . , Diabetes, 46:900- 906 (1997)) or in UCP1 from various species (Klaus, S., et al . , Int . J. Biochem . , 23:791-801 (1991)).
  • UCP3 uncouples mitochondrial respiration.
  • Northern blot analyses were performed. UCP3 was abundantly expressed in skeletal muscle, generating a dominant mRNA transcript of approximately 2.4 kb. With longer exposure (18 hours), a much weaker UCP3 signal (2.4 kb) was detected in a large number of other tissues and organs. The longer exposures (18 hours) of the human UCP3 Northern blots also revealed the presence of a smaller mRNA transcript which had a similar size (approximately 1.6 kb) .
  • the 294 bp hUCP3 probe employed was 75% identical to hUCP2. Rehybridization of the same blots with hUCP2 confirmed that this smaller 1.6 kb signal was UCP2.
  • the UCP2 signal as previously reported (Fleury, C, et al . , Nature Genetics, 15:269-272 (1997); Gimeno, R.E., et al., Diabetes, 446:900-906 (1997)) was widely expressed. It was being most abundant in spleen, thymus, bone marrow, trachea, and lymph node, and somewhat less abundant in skeletal muscle as well as a number of other tissues.
  • UCP2 was also abundantly expressed in white adipose tissue as reported Gimeno, R.E., et al . , Diabetes, 446:900-906 (1997)) .
  • Gimeno, R.E., et al . , Diabetes, 446:900-906 (1997)) A comparison of hybridization signals for UCP2 and UCP3 suggests that UCP3 may be the dominant uncoupling protein transcript in human skeletal muscle.
  • a sensitive RNase protection assay was used to assess UCP3 mRNA expression in heart, skeletal muscle and white adipose tissue. No UCP3 signal could be detected in white adipose tissue. In heart, a very weak UCP3 signal was detected. The signal in heart was less than 1% of that detected in skeletal muscle.
  • mice In mice, abundant UCP3 expression was detected in skeletal muscle and brown fat. As with humans, little or o UCP3 expression was detected in other mouse tissues such as white adipose tissue, brain, kidney, liver and colon. s was observed in the human mRNA studies, a smaller transcript was detected in mouse samples as well. This smaller transcript most likely represents mUCP2 given that it was most abundant in white adipose tissue, a site of high-level UCP2 expression (Fleury, C, et al . , Nature Genetics, 15:269-272 (1997); Gimeno, R.E., et al . , Diabetes, in press (1997) ) . Of note, the hUCP3 probe is 73% identical to mUCP2.
  • Figure 4 is a hydrophilicity plot of human UCP2 and human UCP3 showing the hydrophobicity of protein across linear sequence.
  • UCP3 UCP3-short form
  • the UCP3sh transcript encodes a shortened version of the UCP3 protein. As shown in Figure 8, the UCP3sh transcript results when a polyadenylation/transcription termination signal (AATAAA) (SEQ ID NO: 26) located within intron 6 terminates transcription (see Figure 3) . However, this AATAAA (SEQ ID NO: 26) seems to be only partially effective in terminating transcription. When it does succeed in terminating transcription, the UCP3sh transcript is generated. When it fails to terminate transcription, transcription continues on through exon 7 and terminates at the exon 7 7AATAAA (SEQ ID NO: 26) signal. Splicing between exon 6 and exon 7 then occurs to generate the UCP3 transcript .
  • AATAAA polyadenylation/transcription termination signal
  • UCP3sh differs from UCP3 only by the absence of the last 37 amino acids. It is reasonable to expect that this is significant, since the region missing in UCP3sh is highly homologous to a region in UCP1 which has been implicated in mediating inhibition of uncoupling activity by purine nucleotides (Murdza-Inglis, D.L., et al . , J Biol Chem . 269:7435-7438 (1994)), As a result, it is reasonable to expect that UCP3sh is more active as an uncoupler than UCP3.
  • UCP3sh mRNA like UCP3 mRNA is extremely abundant in human skeletal muscle. In normal individuals, the leve.1 of UCP3sh mRNA is skeletal muscle is equal to or greater that the level of UCP3 mRNA. Preliminary studies have indicated that UCP3sh mRNA levels are reduced in obese individuals compared to lean individual. In contrast, UCP3 mRNA levels seem to be unchanged in obese individuals. These preliminary findings raise the possibility that UCP3sh is the more important UCP3 protein for body weight regulation.
  • mouse UCP3 gene was isolated using methods similar to those described in Example 1.
  • the mouse UCP3 nucleotide sequence (SEQ ID NO: 7) is shown in Figures 5A-5C, and the mouse UPC3 amino acid sequence is shown in Figure 6. Comparisions of mUCP3 versus mUCPl and mUP2 and human UCP3 are shown in Figure 7.
  • Recombinant cells expressing hUCP3 and cells not expressing UCP3 are grown in 96 well plates. On the day of analysis, the plates are rinsed and JC-1 dye is added to all wells plus or minus test compounds. Later, plates are washed and then, in the presence of the test compound, fluorescence is determined in a fluorometer. Decrease of fluorescence in the presence of the test compound, indicates a decrease of mitochondrial ⁇ (and vice versa for cases where fluorescence is increased) . That is, increase of fluorescence in the presence of the test compound indicates an increase of mitochondrial ⁇ . If decrease in fluorescence is observed in UCP3 expressing cells but not in control cells, then the test compound is an activator of UCP3. If an increase in fluorescence is observed in UCP3 expressing cells, but not in control cells, then the test compound is an inhibitor of JCP3.
  • JC-1 dye a delocalized lipophilic cation (DLC)
  • DLC delocalized lipophilic cation
  • the distinguishing feature of DLCs is that they are positively charged, yet lipophilic.
  • the lipophilic feature allows then to traverse membranes and the positive charge causes then to accumulate within mitochondria (negatively charged on the inside) .
  • This accumulation is proportional to ⁇ , the membrane electrical potential across the inner mitochondrial membrane, and follows the Nernst Equation shown below.
  • the mitochondrial ⁇ results from the protein electrochemical gradient across the inner mitochondrial membrane and represents the electrical portion of this gradient ( ⁇ pH represents the chemical portion of the gradient) .
  • a ⁇ of -60 mV corresponds to a DLC in/out ratio of 10 to 1
  • a ⁇ of -120 mV corresponds to a DLC in/out ratio of 100 to 1.
  • a change in ⁇ is amplified by a change in F jn /F ou[ .
  • ⁇ for most mitochondrial range between -50 mV and -160 mV.
  • Protonophore uncouplers such as DNP (dinitrophenol) , CCCP (carbonyl cyanide m-chlorophenyllhydrazone) , decrease ⁇ and, as a result, markedly decrease the accumulation of JC-1. Any drug which increases UCP activity is expected to have the same effect as DNP, CCCP or FCCP .
  • JC-1 has fluorescent features which makes it extremely useful as a monitor of mitochondrial ⁇ . Many dyes aggregate at high concentrations and this reduces fluorescence greatly (for example, rhodamine 123). Aggregates of JC-1 fluoresce intensely, and at higher wavelength than JC-1 monomers. Specifically, monomers emit at 527 nM (green) while J-aggregates emit at 590 nM (red) . Thus, high concentrations of JC-1 accumulate in mitochondria permitting the formation of aggregates. The accumulation of JC-1 and therefore the formation of aggregates is proportional to mitochondrial ⁇ . Aggregates do not form in other cellular locations due to insufficient accumulation of JC-1. Thus, detection of aggregates (as measured by fluorescence at 590 nM) is a sensitive indicator of mitochondrial ⁇ .
  • CX-1 cells were incubated with JC-1 (lOug/ml) with or without the uncoupler, FCCP, for 10 minutes, washed 3 times, trypsinized and then transferred as a cell suspension to a 1 cm quartz cuvette, in which fluorescence was monitored using a Kontron SFM25 fluorescent spectrophotometer .
  • JC-1 aggregate fluorescence can be monitored in living cells and that an uncoupler (FCCP) which is expected to have the same effect as a UCP activator markedly lowers "red” fluorescence. Fluorescence can also be monitored using a FACScan flow cytometer or in a single cell using fluorescence microscopy.
  • FCCP uncoupler
  • UCP3 is expressed abundantly and preferentially in skeletal muscle
  • UCP3 is expressed abundantly in skeletal muscle and brown fat.
  • Starvation - UCP3 was dramatically increased by starvation in mice and rats (-5-10 fold) .
  • 5 days of food restriction causes a 2.5-fold increase in UCP3 mRNA expression.
  • human UCP3 mRNA is significantly upregulated when transgenic mice bearing a human UCP3 PI clone are starved.
  • humans like rodents, increase UCP3 gene expression with starvation.
  • Starvation increases lipolysis in adipose tissue, causing a marked increase in blood levels of FFAs.
  • the increase in FFAs is thought to promote conservation of protein in skeletal muscle (when lipid fuels are abundant, the requirement for gluconeogenesis from muscle protein is reduced). Nicotinic acid inhibits lipolysis, restores FFA levels to fed values, and stimulates protein catabolism in skeletal muscle (Lowell and Goodman, Diabetics, 36:14-19 (1987) ) .
  • the experiment described herein shows that nicotinic acid treatment of fasted animals returned FFA levels to fed values, but increased UCP3 mRNA to .levels 2- fold higher than those observed in saline treated fasted controls.
  • Endotoxin Endotoxin - Endotoxin treatment of rats and mice resulted in a very large increase in UCP3 mRNA levels in skeletal muscle, but not in other tissues.
  • Endotoxin is a well known stimulator of protein catabolism in skeletal muscle .
  • Dexamethasone High dose dexamethasone treatment markedly stimulated UCP3 mRNA levels in skeletal muscle, but not in other tissues.
  • Dexamethasone is also a well known stimulator of protein catabolism in skeletal muscle.
  • Thyroid Hormone High dose thyroid treatment in rats stimulated UCP3 mRNA levels. Thyroid hormones seemed to have little or no effect in mice.
  • Thyroid hormone is also a well known stimulator of protein catabolism in skeletal muscle .
  • ob/ob and db/db mice fa/fa rats - These genetically obese rodents were generated and shown to have markedly increased UCP3 mRNA levels in skeletal muscle. It is likely that increased UCP3 mRNA levels in ob/ob mice contributed to elevated production of gluconeogenic precursors by muscle, thereby promoting non-insulin dependent diabetes mellitus (NIDDM) in these animals.
  • NIDDM non-insulin dependent diabetes mellitus
  • UCP3 plays an important role in regulating skeletal muscle protein catabolism (conversion of muscle protein to gluconeogenic precursors) .
  • Possible mechanisms by which UCP3 plays a role are the following:
  • UCP3 is a mitochondrial carrier which transports biosynthetic metabolites in and out of mitochondria during skeletal muscle protein catabolism (i.e., conversion of aspartate, glutamate, valine, isoleucine and leucine to gluconeogenic precursors alanine and glutamine) .
  • UCP3 is the aspartate/glutamate carrier and is rate limiting for operation of the aspartate/malate shuttle (transfers cytosolic NADH into the mitochondria) . Increased operation of this shuttle would reduce the cytosolic NADH/NAD ratio. It has been suggested that the cytosolic NADH/NAD ratio regulates muscle protein catabolism.
  • UCP3 is indeed a genuine uncoupling protein and increased UCP3 activity in catabolic states oxidizes the whole cell redox state (NADH/NAD ratio) , thereby stimulating protein catabolism and amino acid metabolism.
  • amino acids released from muscle protein are significantly metabolized inside the myocytes prior to their release into the bloodstream.
  • Alanine and glutamine represent approximately 12% amino acids in muscle protein but together represent > 50% of amino acids released by muscle during starvation. Thus, much of the alanine and glutamine released must be synthesized.
  • aspartate, asparginine, glutamate, leucine, isoleucine and valine represent > 30% of amino acids in muscle protein but are released in only small amounts during starvation. These amino acids are interconverted to alanine and glutamine by muscle.
  • amino acids such as glycine, cysteine, serine, threonine, methionine, proline, lysine, arginine, histidine, phenylalanine, tyrosine and tryptophan represents approximately 50% of muscle protein and are released either unchanged or as deaminated ⁇ -ketoacids .
  • Alanine is generated by the transamination of pyruvate.
  • the pyruvate (i.e., carbon) for alanine synthesis come from glycolysis while the nitrogen originates from aspartate, asparginine, glutamate, leucine, isoleucine and valine.
  • the released alanine is taken up by the liver and used to synthesize glucose.
  • the glucose is then returned to the muscle and is metabolized into pyruvate, thus completing the glucose-alanine cycle. It is important to note that no new glucose is synthesized by this process, the carbons are simply recycled.
  • the glucose-alanine cycle functions to conserve carbohydrate, but does not generate new carbohydrate.
  • the cycle also functions to transfer NH2 from amino acids with are metabolized (aspartate, asparginine, glutamate, leucine, isoleucine and valine) to the liver where it can be detoxified via the urea cycle.
  • Glutamine synthetase is the enzyme which converts glutamate to glutamine, the final step in glutamine synthesis.
  • glutamine synthetase gene expression in muscle is induced by starvation, streptozotocin diabetes, endotoxin treatment and dexamethasone. It is also interesting to note, as was seen with UCP3 , that these effects on glutamine synthetase gene expression are observed in skeletal muscle, but not in other tissues.
  • ADDRESSEE HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • GGCCCATTCC CCGGGACCAT GGTTGGACTT
  • CAGCCCTCCG AAGTGCCTCC CACAACGGTT 240
  • MOLECULE TYPE protein
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • DESCRIPTION: /desc "DNA”
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid

Abstract

Cette invention a trait à des acides nucléiques isolés et/ou de recombinaison codant une protéine 3 découplante (UCP3) mammalienne (humaine, murine) ainsi qu'une autre forme de la protéine UCP3, nommée UCP3-forme courte (UCP3sh). Elle concerne également des acides nucléiques s'hybridant aux acides nucléiques de la protéine UCP3 ainsi qu'à leurs éléments fonctionnels. L'invention porte également sur un produit de recombinaison d'acide nucléique comprenant un acide nucléique codant la protéine UCP3 et une cellule hôte, cette dernière comportant le produit de recombinaison d'acide nucléique codant la protéine UCP3. Elle concerne, en outre, une technique de production de protéine UCP3 mammalienne consistant à introduire dans une cellule hôte le produit de recombinaison d'acide nucléique codant la protéine UCP3, ce qui détermine une expression de l'acide nucléique. Cette invention a trait, de surcroît, à une protéine UCP3 isolée, ou produite par recombinaison, ainsi qu'à des éléments fonctionnels de celle-ci. Elle décrit aussi une technique d'identification d'un inhibiteur et/ou d'un activateur de l'expression et/ou de la fonction de la protéine UCP3 (un anticorps, par exemple) et l'utilisation qui est faite de ces inhibiteurs et activateurs. Elle porte encore sur une technique permettant de détecter la présence de la protéine UCP3 dans un prélèvement pris sur un sujet.
PCT/US1998/006959 1997-04-09 1998-04-08 Ucp3: un homologue de proteine decouplante WO1998045438A1 (fr)

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US60/043,447 1997-04-09
US4625497P 1997-05-12 1997-05-12
US60/046,254 1997-05-12
US89274597A 1997-07-15 1997-07-15
US08/892,745 1997-07-15

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WO2000002577A1 (fr) * 1998-07-09 2000-01-20 Smithkline Beecham Plc Utilisation therapeutique de la proteine decouplante hhfcw60
EP1027424A1 (fr) * 1997-10-09 2000-08-16 Tularik, Inc. Regulateurs de l'expression du gene ucp3
WO2000047617A1 (fr) * 1999-02-09 2000-08-17 Lexicon Genetics Incorporated Proteines humaines bruleuses de graisses excedentaires et polynucleotides les codant
WO2000032624A3 (fr) * 1998-11-30 2000-11-09 Genentech Inc Ucp5
WO2000068686A1 (fr) * 1999-05-10 2000-11-16 Tularik Inc. Bioanalyses haute capacite de criblage de modulateurs de potentiel de membrane mitochondriale
WO2000078941A3 (fr) * 1999-06-23 2001-02-22 Univ Vermont Procedes et produits permettant la manipulation de l'expression de proteines ucp
WO2001024625A1 (fr) * 1999-10-01 2001-04-12 Smithkline Beecham P.L.C. Rongeur transgenique comprenant un polynucleotide codant pour un polypeptide humain ucp3
WO2002007754A2 (fr) * 2000-07-25 2002-01-31 Smithkline Beecham Plc Nouvel usage
US6620594B1 (en) 1997-05-07 2003-09-16 Novartis Ag Uncoupling protein homologue: UCP 3
US7105718B2 (en) 2000-03-31 2006-09-12 The Regents Of The University Of Colorado Compositions and methods for regulating metabolism in plants
US7342102B2 (en) 1998-11-30 2008-03-11 Genentech, Inc. Uncoupling protein 5 (UCP5)
US7381413B1 (en) 1998-04-17 2008-06-03 University Of Vermont And State Agricultural College Methods and products related to metabolic interactions in disease
US7510710B2 (en) 2004-01-08 2009-03-31 The Regents Of The University Of Colorado Compositions of UCP inhibitors, Fas antibody, a fatty acid metabolism inhibitor and/or a glucose metabolism inhibitor

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DA-WEI GONG ET AL: "Uncoupling protein-3 is a mediator of thermogenesis regulated by thyroid hormone, beta3-adrenergic agonists, and leptin", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 272, no. 39, 26 September 1997 (1997-09-26), MD US, pages 24129 - 24132, XP002075965 *
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Cited By (24)

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Publication number Priority date Publication date Assignee Title
US6620594B1 (en) 1997-05-07 2003-09-16 Novartis Ag Uncoupling protein homologue: UCP 3
EP1027424A1 (fr) * 1997-10-09 2000-08-16 Tularik, Inc. Regulateurs de l'expression du gene ucp3
EP1027424A4 (fr) * 1997-10-09 2004-04-07 Tularik Inc Regulateurs de l'expression du gene ucp3
US7390782B2 (en) 1998-04-17 2008-06-24 University Of Vermont And State Agricultural College Methods and products related to metabolic interactions in disease
US7381413B1 (en) 1998-04-17 2008-06-03 University Of Vermont And State Agricultural College Methods and products related to metabolic interactions in disease
WO2000002577A1 (fr) * 1998-07-09 2000-01-20 Smithkline Beecham Plc Utilisation therapeutique de la proteine decouplante hhfcw60
US7342102B2 (en) 1998-11-30 2008-03-11 Genentech, Inc. Uncoupling protein 5 (UCP5)
WO2000032624A3 (fr) * 1998-11-30 2000-11-09 Genentech Inc Ucp5
US6967245B2 (en) 1998-11-30 2005-11-22 Genentech, Inc. Ucp5
JP2002533062A (ja) * 1998-11-30 2002-10-08 ジェネンテック・インコーポレーテッド Ｕｃｐ５
US6403784B1 (en) 1999-02-09 2002-06-11 Lexicon Genetics Incorporated Human uncoupling proteins and polynucleotides encoding the same
WO2000047617A1 (fr) * 1999-02-09 2000-08-17 Lexicon Genetics Incorporated Proteines humaines bruleuses de graisses excedentaires et polynucleotides les codant
US6987178B2 (en) 1999-02-09 2006-01-17 Lexicon Genetics Incorporated Human uncoupling proteins and polynucleotides encoding the same
WO2000068686A1 (fr) * 1999-05-10 2000-11-16 Tularik Inc. Bioanalyses haute capacite de criblage de modulateurs de potentiel de membrane mitochondriale
WO2000078941A3 (fr) * 1999-06-23 2001-02-22 Univ Vermont Procedes et produits permettant la manipulation de l'expression de proteines ucp
AU780815B2 (en) * 1999-06-23 2005-04-21 University Of Vermont And State Agricultural College, The Methods and products for manipulating uncoupling protein expression
JP2003503319A (ja) * 1999-06-23 2003-01-28 ザ ユニバーシティ オブ バーモント アンド ステイト アグリカルチュラル カレッジ アンカップリングタンパク質発現を操作する方法および産物
US7816319B2 (en) 1999-06-23 2010-10-19 University Of Vermont And State Agricultural College Methods and products for manipulating uncoupling protein expression
WO2001024625A1 (fr) * 1999-10-01 2001-04-12 Smithkline Beecham P.L.C. Rongeur transgenique comprenant un polynucleotide codant pour un polypeptide humain ucp3
US7105718B2 (en) 2000-03-31 2006-09-12 The Regents Of The University Of Colorado Compositions and methods for regulating metabolism in plants
WO2002007754A2 (fr) * 2000-07-25 2002-01-31 Smithkline Beecham Plc Nouvel usage
WO2002007754A3 (fr) * 2000-07-25 2003-06-05 Smithkline Beecham Plc Nouvel usage
US7510710B2 (en) 2004-01-08 2009-03-31 The Regents Of The University Of Colorado Compositions of UCP inhibitors, Fas antibody, a fatty acid metabolism inhibitor and/or a glucose metabolism inhibitor
US8293240B2 (en) 2004-01-08 2012-10-23 The Regents Of The University Of Colorado Method of treating drug-resistant cancer

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