WO1997045439A1 - Adn encodant une acylcoenzyme a (acyltransferase de cholesterol) et emplois dudit produit - Google Patents

Adn encodant une acylcoenzyme a (acyltransferase de cholesterol) et emplois dudit produit Download PDF

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WO1997045439A1
WO1997045439A1 PCT/US1997/009460 US9709460W WO9745439A1 WO 1997045439 A1 WO1997045439 A1 WO 1997045439A1 US 9709460 W US9709460 W US 9709460W WO 9745439 A1 WO9745439 A1 WO 9745439A1
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nucleic acid
acylcoenzyme
cholesterol acyltransferase
leu
phe
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PCT/US1997/009460
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English (en)
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Stephen L. Sturley
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The Trustees Of Columbia University In The City Of New York
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Priority to AU32259/97A priority Critical patent/AU3225997A/en
Publication of WO1997045439A1 publication Critical patent/WO1997045439A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Cholesterol or related sterols required for the viability of eukaryotic cells, exist in the free form or as esters conjugated to fatty acids.
  • the concentration of free sterol determines the fluidity of eukaryotic cell membranes, whereas esterified sterols cannot participate in membrane assembly.
  • cholesterol depletion of the rough endoplasmic reticulum (ER) relative to the smooth ER (3) may modulate protein translocation or membrane-associated transcriptional activators such as the Sterol Response Element Binding proteins (SREBP, 4 ) .
  • SREBP Sterol Response Element Binding proteins
  • production of cholesterol ester (CE) by acylcoenzyme A cholesterol acyltransferase m the rough ER may influence the transport of sterol between intracellular pools. Similar este ⁇ fication activities have been observed m other eukaryotes such as plants and yeasts (5) .
  • Elevations in acylcoenzyme A cholesterol acyltransferase activity perturb several pathways that contribute to hyperlipidemia and atherosclerosis.
  • Sterol esterification modifies the activity of the low density lipoprotein (LDL) receptor and alters serum lipoprotein composition to be pro-atherogenic (6, 7) . It may also be a rate limiting step m intestinal sterol absorption (8) .
  • CE deposition m the arterial wall is an important initial step in atherogenesis (9) .
  • the understanding of the acylcoenzyme A cholesterol acyltransferase reaction has been hampered by the difficulty of biochemical purification and by a poor grasp of the relevant genetic determinants.
  • a human acylcoenzyme A cholesterol acyltransferase I gene from macrophages was identified by complementation of Chinese Hamster Ovary cell lines deficient in acylcoenzyme A: cholesterol acyltransferase activity (10) and was functionally expressed m insect cells devoid of endogenous activity (11) .
  • This invention provides an isolated nucleic acid which encodes an acylcoenzyme A: cholesterol acyltransferase II or an acylcoenzyme A: cholesterol acyltransferase III.
  • This invention also provides a vector which includes the isolated nucleic acid which encodes an acylcoenzyme A: cholesterol acyltransferase II or an acylcoenzyme A: cholesterol acyltransferase III and a host vector system which includes a vector.
  • This invention also provides a method of producing a polypeptide which comprises growing such host vector system of claim 14 under suitable conditions permitting production of the polypeptide and recovering the polypeptide so produced.
  • This invention also provides a purified wildtype acylcoenzyme A: cholesterol acyltransferase II or an acylcoenzyme A: cholesterol acyltransferase III.
  • This invention also provides an oligonucleotide of at least 15 nucleotides capable of specifically hybridizing with a unique sequence of nucleotides present withm a nucleic acid which encodes a wildtype acylcoenzyme A: cholesterol acyltransferase II or an acylcoenzyme A: cholesterol acyltransferase III without hybridizing to a nucleic acid which encodes a mutant acylcoenzyme A: cholesterol acyltransferase II or an acylcoenzyme A: cholesterol acyltransferase III.
  • This invention also provides an oligonucleotide of at least 15 nucleotides capable of specifically hybridizing with a unique sequence of nucleotides present withm the nucleic acid which encodes a mutant acylcoenzyme A: cholesterol acyltransferase II or an acylcoenzyme A: cholesterol acyltransferase III without hybridizing to a nucleic acid which encodes a wildtype acylcoenzyme A: cholesterol acyltransferase II or an acylcoenzyme A: cholesterol acyltransferase III.
  • This invention also provides a method for determining whether a subject known to have an imbalance in sterol levels has the imbalance due to a defect m esterification of sterol and for treating a subject who has an imbalance m sterol levels due to a defect in esterification of sterol.
  • This invention also provides methods for inhibiting wildtype acylcoenzyme A: cholesterol acyltransferase II or an acylcoenzyme A: cholesterol acyltransferase III m a subject.
  • This invention also provides a method for identifying a chemical compound which is capable of inhibiting acylcoenzyme A: cholesterol acyltransferase II or an acylcoenzyme A: cholesterol acyltransferase III in a subject and a pharmaceutical composition comprising of the chemical compound so identified.
  • This invention also provides a transgenic, nonhuman mammal comprising the isolated nucleic acid which encodes acylcoenzyme A: cholesterol acyltransferase II or an acylcoenzyme A: cholesterol acyltransferase III.
  • amino acid residues are abbreviated as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gin; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
  • A Ala
  • C Cys
  • D Asp
  • E Glu
  • F Phe
  • G Gly
  • H His
  • I Ile
  • K Lys
  • L Leu
  • M Met
  • N Asn
  • P Pro
  • Q Gin
  • R Arg
  • S Ser
  • T Thr
  • V Val
  • W Trp
  • Y Tyr
  • Figures IA and IB Protein sequence alignments predicted from candidate genes for the human acylcoenzyme A: cholesterol acyltransferase gene I, the yeast homologs, acylcoenzyme A: cholesterol acyltransf erase-related enzyme I and acylcoenzyme A: cholesterol acyltransf erase- related enzyme II, and a consensus sequence of all three sequences.
  • acylcoenzyme A cholesterol acyltransferase (Sequence I.D. No.: 2)
  • acylcoenzyme A cholesterol acyl transf erase- rela ted enzyme I
  • acylcoenzyme A cholesterol acyl transf erase- related enzyme II (Sequence I.D. No. : 6) , respectively.
  • R07932 denotes the partial sequence of another human acylcoenzyme A: cholesterol acyltransferase candidate cDNA (residues 500 to 600) (Sequence I.D. No.: 14) .
  • the asterisks indicate the residues in R07932 identical to those of the other sequences. IA.
  • FIGS. 2A, 2B, 2C, 2D and 2E Construction and analysis of acylcoenzyme A: cholesterol acyltransferase genes and deletion mutants.
  • the schematic depicts a fragment from yeast chromosome III in plasmid pH3(34) . Strategic restriction endonucleases are indicated (H, Hind III; B, Bam HI) .
  • 2B The autoradiogram depicts Bam HI digested DNA from wild-type or disrupted diploid strains probed with the 2993-bp Bam-HI fragment. This produced a fragment corresponding to the wild-type acylcoenzyme A: cholesterol acyltransferase-related enzyme I locus and a 1984-bp fragment characterizing the arel ⁇ NA allele.
  • the diploid is heterozygous for the acylcoenzyme A: cholesterol acyltransferase- related enzyme I deletion.
  • C Reduced stringency hybridization of yeast genomic DNA with acylcoenzyme A: cholesterol acyltransferase-related enzyme I coding sequences. Genomic DNA from wild-type or ⁇ RE1/ arel ⁇ NA diploids were reprobed with an Nhe
  • I-Avr II fragment corresponding to the acylcoenzyme A cholesterol acyltransferase- related enzyme I open reading frame ("ORF") . Hybridizations and washes were performed at 60°C m the absence of formamide. D. The are2 ⁇ deletion.
  • step 1 PCR amplifying oligonucleotides, KO-5' and KO-3' and a LEU2 template were used to produce the selectable yeast gene flanked at the 5' and 3' ends by acylcoenzyme A: cholesterol acyltransferase- related enzyme II.
  • step 2 this was used to direct homologous recombination at acylcoenzyme A: cholesterol acyltransferase-related enzyme II by transformation of a diploid strain and selection for leucine protrophy.
  • step 3 integrants to acylcoenzyme A: cholesterol acyltransferase-related enzyme II were identified by a PCR reaction using oligonucleotides flanking ARE2 (are2-5' and are2-3') and a 3' amplimer within LEU2 (L2-3' ) .
  • a 999-bp fragment identifies are2 ⁇ , as shown in the ethidium bromide stained agarose gel.
  • the wild-type fragment (2206-bp) is also produced in the same reaction.
  • Leucine prototrophic transformants with deletions of acylcoenzyme A: cholesterol acyltransferase-related enzyme II were obtained at a frequency of -2° .
  • M indicates the 50-2,000-bp ladder markers
  • FIGS 3A and 3B Fluorescent staining of triglyceride and sterol ester.
  • the cells were grown in YEPD to stationary phase, washed with deionized H,0, and incubated with 1 ⁇ g/ml Nile Red (1 mg/ml m acetone) .
  • Fluorescent images were obtained with a BioRad MRC600 laser scanning confocal microscope (BioRad Microscience, Hercules, CA) on an inverted Zeiss Atiovert microscope (Zeiss, OberKochem, Germany) using 63X (NA1.4) Zeiss Plan-apo infinity corrected objective.
  • Figures 4A, 4B, 4C and 4D Neutral lipid and sterol biosynthesis in ARE deletion mutants.
  • 4C Sterol ester biosynthesis in wild-type and mutant cells transformed with vector control (black box) or acylcoenzyme A: cholesterol acyltransferase-related enzyme I over-expression plasmids, YEp3-16 (increased copy number, shaded box) and pADH5-36 (transcription from the ADH promoter, open boxes) .
  • Cells were grown in selective media to maintain the acylcoenzyme A: cholesterol acyltransferase-related enzyme I expression plasmids. Lipids were labeled, extracted and analyzed as above. 4D.
  • Sterol biosynthesis in acylcoenzyme A cholesterol acyltransferase-related enzyme deletion mutants. Lipids were labeled in synthetic complete media containing [l- ⁇ C] acetate, saponified and extracted with hexane and subjected to thin layer chromatography analysis. The data is representative of three separate experiments and expressed as the ratio of incorporation into sterols to incorporation into fatty acids.
  • Figures 5A, 5B, 5C, 5D, 5E and 5F The nucleic acid and amino acid or predicted amino acid sequences. 5A-1 - 5A-3.
  • the nucleic acid sequence of human acylcoenzyme A cholesterol acyltransferase I designated Sequence ID No.: 1.
  • the amino acid sequence of human acylcoenzyme A cholesterol acyltransferase I designated Sequence ID No.: 2. 5A-1.
  • Nucleic acid sequence of human acylcoenzyme A cholesterol acyltransferase I from nucleic acid bases 1-1624.
  • Amino acid sequence of human acylcoenzyme A cholesterol acyltransferase I from ammo acid residues 1-76.
  • Nucleic acid sequence of human acylcoenzyme A cholesterol acyltransferase I from nucleic acid bases 1625-2524.
  • Amino acid sequence of human acylcoenzyme A cholesterol acyltransferase I from amino acid residues 77-376.
  • Nucleic acid sequence of human acylcoenzyme A cholesterol acyltransferase I from nucleic acid bases 2525-3649.
  • Amino acid sequence of human acylcoenzyme A cholesterol acyltransferase I from ammo acid residues 377-551.
  • A cholesterol acyltransferase-related enzyme
  • A cholesterol acyltransferase-related enzyme I from nucleic acid bases 1-1289.
  • Ammo acid sequence of acylcoenzyme A cholesterol acyltransferase-related enzyme I from ammo acid residues 1-209. 5 5B-2.
  • A cholesterol acyltransferase- related enzyme I from nucleic acid bases 1290-2114.
  • Ammo acid sequence of acylcoenzyme A cholesterol 10 acyltransferase-related enzyme I from ammo acid residues 210-484. 5B-3.
  • Nucleic acid sequence of acylcoenzyme A cholesterol acyltransferase- related enzyme I from nucleic acid 15 bases 2115-2601.
  • Ammo acid sequence of acylcoenzyme A cholesterol acyltransferase-related enzyme I from ammo acid residues 485-611. 5C-1 - 5C-3.
  • 20 The nucleic acid sequence of yeast acylcoenzyme
  • A cholesterol acyltransferase-related enzyme II designated Sequence ID No.: 5.
  • the ammo acid sequence of yeast acylcoenzyme A cholesterol acyltransferase-related enzyme II 25 designated Sequence ID No.: 6.
  • Nucleic acid sequence of acylcoenzyme A cholesterol acyltransferase- related enzyme II from nucleic acid bases 1-1061.
  • Ammo acid sequence of 30 acylcoenzyme A cholesterol acyltransferase-related enzyme II from amino acid residues 1-238.
  • acylcoenzyme A cholesterol acyltransferase-related enzyme II from ammo acid residues 239-538. 5C-3. Nucleic acid sequence of acylcoenzyme
  • A cholesterol acyltransferase-related enzyme II from nucleic acid bases 1962-2421.
  • Ammo acid sequence of acylcoenzyme A cholesterol acyltransferase-related enzyme II from ammo acid residues 539-643.
  • A cholesterol acyltransferase II designated Sequence ID No.: 11.
  • the ammo acid sequence of mouse acylcoenzyme A cholesterol acyltransferase II designated Sequence ID No.: 12.
  • Figure 6A A restriction map of the expression vector YepAB-ACAT2.
  • FIG. 6B and 6C Expression of human macrophage ACAT in pRS426GP. 6B.
  • the ACAT open reading frame was inserted at the Notl and Sacl sites, downstream of the promoter of the GAL1 /1 0 gene
  • hACAT hACAT
  • pRS426GP vector
  • M Molecular weight reference markers
  • the arrow indicates the position of the DM10 immunoreactive product in extracts from murine adrenals.
  • the expressed form of hACAT in yeast is of coincident mobility.
  • Figures 7A and 7B Multiple human tissue Northern analysis of poly (A)+ RNAs probed with 32 P-labeled cDNA
  • 7A Tissue specific expression of wildtype human acylcoenzyme A: cholesterol acyltransferase II using a wildtype acylcoenzyme A: cholesterol acyltransferase II specific probe.
  • 7B Tissue specific expression of wildtype human acylcoenzyme A: cholesterol acyltransferase I using a wildtype acylcoenzyme A: cholesterol acyltransferase I specific probe.
  • FIG. 8A, 8B, 8C and 8D Tissue specific expression of ARGP1 and hACAT.
  • ARGP1 specific probe as described in the text. 8C and 8D.
  • the same blots were also analyzed using a hACAT specific probe.
  • the first panel is ldentical to that published by Chang et al (8) .
  • the second panel is the same blot as in A and B, probed with the ACAT cDNA 1600 bp probe.
  • FIG. 10 Cultured cell expression of AGRP1.
  • RNA samples from HepG2 and CVI were reverse transcribed and PCR amplified as described m the text.
  • P indicate a plasmid template control.
  • the blank lanes represent water or no RT controls.
  • sequences shown were identified in genome databases and aligned based on protem sequence using GCG Ine software (pileup) . They were subsequently arranged to their sequence conservation to determine approximate evolutionary relatedness.
  • Figure 15A and 15B Nucleotide and predicted protein sequence of ARGP1.
  • Figure 16. Nucleotide and predicted protein sequence of ARGP2.
  • references to specific nucleotides are to nucleotides present on the coding strand of the nucleic acid.
  • the following standard abbreviations are used throughout the specification to indicate specific nucleotides:
  • a “gene” means a nucleic acid molecule, the sequence of which includes all the information required for the normal regulated production of a particular protein, including the structural coding sequence, promoters and enhancers .
  • nucleic acids or oligonucleotides of the subject invention also include nucleic acids or oligonucleotides coding for polypeptide analogs, fragments or derivatives which differ from naturally-occurring forms in terms of the identity or location of one or more amino acid residues (deletion analogs containing less than all of the residues specified for the protein, substitution analogs wherein one or more residues specified are replaced by other residues and addition analogs where in one or more amino acid residues is added to a terminal or medial portion of the polypeptides) and which share some or all properties of naturally-occurring forms.
  • nucleic acids or oligonucleotides include: the incorporation of codons "preferred" for expression by selected non-mammalian hosts; the provision of sites for cleavage by restriction endonuclease enzymes; and the provision of additional initial, terminal or intermediate DNA sequences that facilitate construction of readily expressed vectors.
  • nucleic acids and oligonucleotides described and claimed herein are useful for the information which they provide concerning the amino acid sequence of the polypeptide and as products for the large scale synthesis of the polypeptide by a variety of recombinant techniques.
  • the molecule is useful for generating new cloning and expression vectors, transformed and transfected prokaryotic and eukaryotic host cells, and new and useful methods for cultured growth of such host cells capable of expression of the polypeptide and related products.
  • An isolated nucleic acid which encodes an acylcoenzyme A: cholesterol acyltransferase II may be DNA or RNA, specifically cDNA or genomic DNA. Specifically, the isolated nucleic acid has the sequence designated Seq. I.D. No.: 7. The isolated nucleic acid encodes a human wildtype acylcoenzyme A: cholesterol acyltransferase II having substantially the same amino acid sequence as the sequence designated Seq. I.D. No. : 8. Specifically the isolated nucleic acid has the sequence designated Seq. I.D. No.: 11.
  • the isolated nucleic acid encodes a mouse wildtype acylcoenzyme A: cholesterol acyltransferase II having substantially the same amino acid sequence as the sequence designated Seq. I.D. No.: 12. Further, the isolated nucleic acid of encodes a mutant acylcoenzyme A: cholesterol acyltransferase II.
  • An isolated nucleic acid which encodes an acylcoenzyme A: cholesterol acyltransferase III may be DNA or RNA, specifically cDNA or genomic DNA. Specifically, the isolated nucleic acid has the sequence as set forth in Fig. 16. The isolated nucleic acid encodes a human wildtype acylcoenzyme A: cholesterol acyltransferase III having substantially the same amino acid sequence as set forth in Fig. 16. Further, the isolated nucleic acid of encodes a mutant acylcoenzyme A: cholesterol acyltransferase III.
  • acylcoenzyme A cholesterol acyltransferase III
  • acylcoenzyme A cholesterol acyltransferase III
  • this term includes any such polypeptide whether naturally occurring and obtained by purification from natural sources or non-naturally occurring and obtained synthetically, e.g. by recombinant DNA procedures.
  • the term includes any such polypeptide whether its sequence is substantially the same as, or identical to the sequence of any mammalian homolog of the human polypeptide, e.g. murine, bovine, porcine, etc. homologs. Additionally, the term includes mutants or other variants of any of the foregoing which retain at least some of the enzymatic activity of nonmutants or nonvariants.
  • acylcoenzyme A cholesterol acyltransferase II
  • this term includes any such polypeptide whether naturally occurring and obtained by purification from natural sources or non-naturally occurring and obtained synthetically, e.g. by recombinant DNA procedures.
  • the term includes any such polypeptide whether its sequence is substantially the same as, or identical to the sequence of any mammalian homolog of the human polypeptide, e.g. murine, bovine, porcine, etc. homologs. Additionally, the term includes mutants or other variants of any of the foregomg which retain at least some of the enzymatic activity of nonmutants or nonvariants.
  • the invention also encompasses DNAs and cDNAs which encode ammo acid sequences which differ from those of acylcoenzyme A: cholesterol acyltransferase II, but which do not produce phenotypic changes.
  • the mvention also encompasses DNAs and cDNAs which encode ammo acid sequences which differ from those of acylcoenzyme A: cholesterol acyltransferase III, but which do not produce phenotypic changes.
  • the nucleic acid of the subject invention also include nucleic acids that encode for polypeptide analogs, fragments or derivatives which differ from naturally- occurring forms m terms of the identity or location of one or more amino acid residues (including deletion analogs containing less than all of the residues specified for the protein, substitution analogs wherein one or more residues specified are replaced by other residues and addition analogs wherem one or more ammo acid residues is added to a terminal or medial portion of the polypeptides) and which share some or all properties of the naturally-occurring forms.
  • polypeptide of the subject invention also includes analogs, fragments or derivatives which differ from naturally-occumng forms, but having acylcoenzyme A: cholesterol acyltransferase activity.
  • This invention also provides a vector comprising an isolated nucleic acid encoding acylcoenzyme A: cholesterol acyltransferase II or III.
  • the isolated nucleic acid of the vectors is operatively linked to a promoter of RNA transcription which maybe, or is identical to, a bacterial, yeast, insect or mammalian promoter.
  • the vector may be a plasmid, cosmid, yeast artificial chromosome (YAC) , bacteriophage or eukaryotic viral DNA.
  • YAC yeast artificial chromosome
  • this invention provides a vector designated YepAB-ACAT2 ( Figure 6) .
  • vector backbones known in the art as useful for expressing proteins may be employed.
  • Such vectors include but are not limited to: adenovirus, simian virus 40 (SV40) , cytomegalovirus (CMV) , mouse mammary tumor virus (MMTV) , Moloney murine leukemia virus, murine sarcoma virus, and Rous sarcoma virus, DNA delivery systems, i.e liposomes, and expression plasmid delivery systems.
  • This invention also provides a vector system for the production of a polypeptide which comprises the vector m a suitable host.
  • Suitable host includes a cell which includes, but is not limited, prokaryotic or eukaryotic cells, e.g. bacterial cells (including gram positive cells) , yeast cells, fungal cells, insect cells and animal cells.
  • Suitable animal cells include, but are not limited to, HeLa cells, Cos cells, CVI cells and various primary mammalian cells. Numerous mammalian cells may be used as hosts, including, but not limited to, the mouse fibroblast cell NIH 3T3, CHO cells, Ltk " cells, etc. Expression plasmids such as that described supra may be used to transfect mammalian cells by methods well known in the art such as calcium phosphate precipitation, electroporation.
  • This invention also provides a method for producmg a polypeptide (e.g. acylcoenzyme A: cholesterol acyltransferase) which comprises growing a host vector system under suitable conditions permitting production of the polypeptide and recovering the polypeptide so produced.
  • Methods of recovering polypeptides produced m such host vector systems are well-known m the art and typically include steps involving cell lysis, solubilization and chromatography.
  • This invention also provides a method of obtaining a polypeptide in purified form which comprises : (a) introducing a vector, as described above, into a suitable host cell; (b) culturing the resulting cell so as to produce the polypeptide; (c) recovering the polypeptide produced in step (b) ; and (d) purifying the polypeptide so recovered.
  • the vector may include a plasmid, cosmid, yeast artificial chromosome, bacteriophage or eukaryotic viral DNA.
  • the host cell may be a bacterial cell (including gram positive cells) , yeast cell, fungal cell, insect cell or animal cell.
  • Suitable animals cells include, but are not limited to HeLa cells, Cos Cells, CVI cells and various primary mammalian cells. Culturing methods useful for permitting transformed or transfected host cells to produce polypeptides are well known in the art as are the methods for recovering polypeptides from such cells and for purifying them. Usmg the aforementioned method, this mvention also provides a purified wildtype acylcoenzyme A: cholesterol acyltransferase II or III and a purified mutant acylcoenzyme A: cholesterol acyltransferase II or III.
  • This invention also provides an oligonucleotide of at least 15 nucleotides capable of specifically hybridizing with a unique sequence of nucleotides present withm a nucleic acid which encodes a wildtype acylcoenzyme A: cholesterol acyltransferase II or III without hybridizing to a nucleic acid which encodes a mutant acylcoenzyme A: cholesterol acyltransferase II or III.
  • this invention also provides an oligonucleotide of at least 15 nucleotides capable of specifically hybridizing with a unique sequence of nucleotides present withm the nucleic acid which encodes a mutant acylcoenzyme A: cholesterol acyltransferase II or III without hybridizing to a nucleic acid which encodes a wildtype acylcoenzyme A: cholesterol acyltransferase II or III.
  • These oligonucleotide DNA or RNA.
  • Such oligonucleotides may be used m accordance with well known standard methods for known purposes, for example, to detect the presence m a sample of DNA which will hybridize thereto.
  • oligonucleotides include, but are not limited to, oligonucleotides that hybridize to mRNA encoding acylcoenzyme A: cholesterol acyltransferase II or III so as to prevent translation of the protem.
  • This invention also provides a nucleic acid having a sequence complementary to the sequence of the isolated nucleic acid which encodes acylcoenzyme A: cholesterol acyltransferase II or III.
  • This invention also provides a method for determinmg whether a subject known to have an imbalance in sterol levels has the imbalance due to a defect m esterification of sterol which comprises (a) obtaining from the subject an appropriate sample containing a mixture of all of the subject's nucleic acids; and (b) determining whether any nucleic acid in the sample from step (a) is, or is derived from, a nucleic acid which encodes a mutant acylcoenzyme A: cholesterol acyltransferase so as to thereby determine whether the subject's imbalance m sterol levels is due to a defect m esterification of sterol.
  • the determination step (b) may comprises: (I) contacting the sample of step (a) with the isolated nucleic acid which encodes acylcoenzyme A: cholesterol acyltransferase II or III or the oligonucleotide of at least 15 nucleotides capable of specifically hybridizing with a unique sequence of nucleotides present withm a nucleic acid which encodes a wildtype acylcoenzyme A: cholesterol acyltransferase II or III without hybridizing to a nucleic acid which encodes a mutant acylcoenzyme A: cholesterol acyltransferase II or III under conditions permitting binding of any nucleic acid in the sample which is, or is derived from, a nucleic acid which encodes a mutant acylcoenzyme A: cholesterol acyltransferase to the nucleic acid or oligonucleotide so as to form a complex; (ii) isolating the complex so formed; and (m)
  • the isolated nucleic acid or the oligonucleotide is labeled with a detectable marker.
  • the detectable marker may be a radioactive isotope, a fluorophore or an enzyme.
  • the nucleic acid sample may be bound to a solid matrix before performing step (I) .
  • This invention also provides a method for treating a subject who has an imbalance m sterol levels due to a defect m esterification of sterol which comprises introducing an isolated nucleic acid which encodes a wildtype acylcoenzyme A: cholesterol acyltransferase II or III mto the subject under conditions such that the nucleic acid expresses a wildtype acylcoenzyme A: cholesterol acyltransferase II or III, so as to thereby treat the subject.
  • This invention also provides a method for inhibiting wildtype acylcoenzyme A: cholesterol acyltransferase II or III in a subject which comprises transforming appropriate cells from the subject with a vector which expresses the nucleic acid complementary to the isolated nucleic acid which encodes a wildtype acylcoenzyme A: cholesterol acyltransferase II or III, and introducing the transformed cells mto the subject so as to thereby inhibit wildtype acylcoenzyme A: cholesterol acyltransferase II or III.
  • the nucleic acid is capable of specifically hybridizing to a mRNA molecule encoding acylcoenzyme A: cholesterol acyltransferase II or III so as to prevent translation of the mRNA molecule.
  • This invention also provides a method for inhibiting the wildtype acylcoenzyme A: cholesterol acyltransferase II or III in a subject which comprises introducing an oligonucleotide of at least 15 nucleotides capable of specifically hybridizing with a unique sequence of nucleotides present within a nucleic acid which encodes a wildtype acylcoenzyme A: cholesterol acyltransferase II or III without hybridizing to a nucleic acid which encodes a mutant acylcoenzyme A: cholesterol acyltransferase II or III into the subject so as to thereby inhibit the wildtype acylcoenzyme A: cholesterol acyltransferase II or III.
  • the oligonucleotide is capable of specifically hybridizing to a mRNA molecule encoding acylcoenzyme A: cholesterol acyltransferase II or III so as to prevent translation of the mRNA molecule.
  • This invention also provides for a method for identifying a chemical compound which is capable of inhibiting acylcoenzyme A: cholesterol acyltransferase II or III in a subject which comprises (a) contacting a wildtype acylcoenzyme A: cholesterol acyltransferase II or III with the chemical compound under conditions permitting binding between the acylcoenzyme and the chemical compound (b) detecting specific binding of the chemical compound to the acylcoenzyme; and ⁇ determining whether the chemical compound inhibits the activity of the coenzyme so as to identify a chemical compound which is capable of inhibiting acylcoenzyme A: cholesterol acyltransferase II or III in a subject.
  • This invention also provides method for differentially inhibiting one acylcoenzyme A: cholesterol acyltransferase but not others using the above methods.
  • only acylcoenzyme A: cholesterol acyltransferase I is inhibited.
  • only acylcoenzyme A: cholesterol acyltransferase II is inhibited.
  • only acylcoenzyme A: cholesterol acyltransferase III (ARGP2) IS inhibited.
  • two of the acylcoenzyme A: cholesterol acyltransferases may be inhibited.
  • This invention further provides pharmaceutical compositions which will differentially inhibit one or more acylcoenzyme A: cholesterol acyltransferases .
  • This invention also provides for a pharmaceutical composition
  • a pharmaceutical composition comprismg the chemical compound identified by the above-described method in an amount effective to inhibit acylcoenzyme A: cholesterol acyltransferase II or III m a subject and a pharmaceutically effective carrier.
  • This invention also provides a method of treating a subject who has atherosclerosis comprising the above- described pharmaceutical composition.
  • a method of treating a subject who has hyperlipidemia comprising the above-described pharmaceutical composition.
  • This invention also provides a transgenic, nonhuman mammal comprising the isolated nucleic acid which encodes acylcoenzyme A: cholesterol acyltransferase II or III.
  • the mammal includes, but is not limited to, a mouse, bovine, cat or dog.
  • Transformation of yeast was performed with lithium acetate (15) by amino-acid prototrophy selection.
  • a diploid strain (5051) was constructed between two isogenic derivatives of W303 (16); W1346-3C (MATa , ade2-l , canl -100, hi s3-l l , 15, leu2-3 , 112 , trpl -1 , ura3-l ) and W1134-2C (MATa, canl -1 00, hi s3-l l , 15, leu2-3, 112, trpl -1 , ura3-l , metl 4DHpaI-Sal I) . Growth on complete (YEPD) or synthetic medium, sporulation and dissection was performed as described (17) .
  • Competent cells of Escherichia coli strain DH5a (Gibco-BRL) and DNA modifying enzymes (Promega) were used according to the manufacturers instructions. pH3(34), from L.A. Grivell, was digested with Nhe I, blunt-ended with Klenow sequences, and digested with Avr II to liberate a 1614-bp fragment. An Xba I, Sma I fragment of pJH-Hl encoding the HIS3 gene was then inserted at these sites in the vector backbone to produce the arel ⁇ NA allele. This construct was digested with Bsa I to liberate a 3821-bp fragment which was then transformed into strain 5051. Disruption of ARE1 was confirmed by Southern blot analysis.
  • Radioactive probes of acylcoenzyme A cholesterol acyltransferase-related enzyme I were prepared by random priming (Pharmacia) with 32 P-dCTP. Genomic DNA (18) was transferred to Hybond membranes (Amersham) and hybridized in the absence of formamide at 65° or 60°C (19) .
  • acyl coenzyme A chol esterol acyl transferase-rela ted enzyme I and acyl coenzyme A : chol esterol acyl transferase-rela ted enzyme II genes are deposited at GenBank (P25628 and U51790, respectively) .
  • acylcoenzyme A cholesterol acyltransferase-related enzyme I gene by copy number under the control of its own promoter in YEp3-16, a 2354 bp Cia I fragment from pH3(34), encompassing the entire acylcoenzyme A: cholesterol acyltransferase-related enzyme I gene, was made blunt-ended with Klenow DNA polymerase I and introduced into the Sma I site of YEp352.
  • acylcoenzyme A cholesterol acyltransferase-related enzyme I from the ADH promoter in pADH5-36, a 2290 bp Nar I fragment of pH3(34), starting 70 bp 5' to the ORF was blunt-ended with Klenow and ligated to Klenow-treated, Eco RI digested, pDC-ADH (a derivative of pS5) (26) .
  • Increased expression of the acylcoenzyme A cholesterol acyltransferase-related enzyme I transcripts, relative to a wild-type cell, was confirmed by northern blot analysis .
  • acylcoenzyme A cholesterol acyltransferase sequence was used to search for homologous yeast genes and subsequently to identify an additional human isoform ( Figures IA and IB) .
  • Acylcoenzyme A cholesterol acyltransferase related enzyme I, an 1830-bp open reading frame (ORF) on yeast chromosome III, encodes a 610- residue protem with 23% identity and 49% similarity to human acylcoenzyme A: cholesterol acyltransferasel
  • Figures IA and IB The yeast and human proteins possess leucine zipper motifs that could mediate protem-protem mteractic .s (esterification is probably performed by a multimeric complex) (12), and possess at least two predicted transmembrane domains that may mediate the membrane association of the acylcoenzyme A: cholesterol acyltransferase reaction (13, 14) .
  • acylcoenzyme A cholesterol acyltransferase-related enzyme I in sterol esterification
  • the deletion mutant arel ⁇ NA
  • Fig. 2A homologous recombination (15, 16, 17)
  • Fig. 2B Southern hybridization
  • acylcoenzyme A cholesterol acyltransferase-related enzyme I
  • this gene designated acylcoenzyme A: cholesterol acyltransferase-related enzyme II
  • acylcoenzyme A cholesterol acyltransferase-related enzyme II
  • Figures IA and IB The genomic sequence (20, 21, 22) encompassing acylcoenzyme A: cholesterol acyltransferase-related enzyme II on chromosome XIV predicts a 5997-bp Bam HI fragment and a 1929-bp ORF, which translates into a 643-res ⁇ due polypeptide.
  • the yeast acylcoenzyme A cholesterol acyltransferase related enzymes genes are 61% and 49 ⁇ > identical at the DNA and predicted protem levels, respectively.
  • Arelp, Are2p and the human acylcoenzyme A cholesterol acyltransferasel protem are most related at the COOH-terminal region (42% identity over a 90-res ⁇ due sequence) ( Figures IA and IB) .
  • acylcoenzyme A cholesterol acyltransferase-related enzyme II coding sequence was deleted from the genome of an AREl /arel ⁇ NA heterozygous diploid by a polymerase chain reaction approach (23) (Fig. 2D) .
  • Haploid progeny representing the smgle arel ⁇ NA and are2 ⁇ deletions and the arel ⁇ NAare2 ⁇ double mutant were obtained.
  • acylcoenzyme A cholesterol acytransferase-related enzymes genes upon cytoplasmic lipid storage
  • the neutral lipid components (triglyceride and sterol ester) of the yeast cells were detected by fluorescence microscopy after staining with Nile Red (24) .
  • cytoplasmic fluorescent droplets accumulated m stationary phase cultures (Fig. 3A) .
  • No differences m are single mutants were detected.
  • the number of droplets observed in arel ⁇ NAare2 ⁇ double mutants was one-third to that m wild-type strains (Fig. 3B; over multiple fields, 5.57 ⁇ 2.73 vs. 16.73 + 4.6 droplets/cell, P ⁇ 0.05) .
  • Sterol ester levels of are2 ⁇ smgle mutants were reduced to less than 26% of wild-type suggesting the acylcoenzyme A: cholesterol acyltransferase-related enzyme II isoform to confer the majority of acyltransferase activity.
  • the arel ⁇ NAare2 ⁇ double mutant was almost totally deficient m sterol esterification (less than 1% of wild-type levels) .
  • microsomes from double mutant yeast cells lacked acylcoenzyme A: cholesterol acyltransferase activity when assayed m vi tro .
  • acylcoenzyme A cholesterol acyltransferase-related enzymes ORF was sufficient for sterol esterification
  • the acylcoenzyme A: cholesterol acyltransferase-related enzyme I coding sequence was over-expressed in vectors with increased copy number (YEp3-16) or elevated transcription (the alcohol dehydrogenase promoter m pADH5-36) (26) . There were no detectable changes in triglyceride or phospholipid biosynthesis resulting from acylcoenzyme A: cholesterol acyltransferase-related enzyme I over-expression.
  • acylcoenzyme A cholesterol acyltransferase-related enzyme I over-expression complemented the sterol esterification defect (Fig. 4C) .
  • the high level expression of acylcoenzyme A: cholesterol acyltransferase-related enzyme I did not elevate sterol ester synthesis above untransformed controls. This suggests that either substrates are limiting in acylcoenzyme A: cholesterol acyltransferase-related enzymes strains or that the enzyme is post-translationally regulated as m mammalian cells (27) .
  • the arel ⁇ Naare2 ⁇ double mutants had a two to three-fold lower level of sterol biosynthesis than wild-type cells, although no changes were observed m the single mutants (Fig. 4D) . In fact, free sterol concentrations were roughly equivalent in all cells.
  • Feedback regulation of sterol biosynthesis by acylcoenzyme A cholesterol acyltransferase activity has been observed m mammalian cells (31) and may be a common mechanism that maintains intracellular sterol at non-toxic concentrations.
  • yeast acylcoenzyme A cholesterol acyltransferase-related enzymes
  • human acylcoenzyme A cholesterol acyltransferasel sequences was used to identify an additional cDNA with significant identity (47%) to human acylcoenzyme A: cholesterol acyltransferasel and the yeast proteins ( Figure IB, Genbank accession # R07932) .
  • Sterol homeostasis is a complex event under subtle regulatory controls, one component of which is sterol esterification.
  • the analysis of esterification reactions in yeast is likely to impact the understanding of sterol homeostasis and atherosclerosis in humans.
  • acylcoenyme A cholesterol acyltransferase II was analyzed by Northern blot RNA hybridization of RNA obtained from the described tissues. Using the same materials and procedures of Chang, et al .
  • acylcoenyme A cholesterol acyltransferase II in liver and muscle is documents, in contrast to similar experiments using the previously known acylcoenyme A: cholesterol acyltransferase I (10) ( Figures 7A and 7B) .
  • Acylcoenyme A cholesterol acyltransferase II was also detected and specifically expressed in adrenal, thyroid and testicular tissues .
  • Example 3
  • the fragment was cut out with restrictions enzymes Bgl II and Not I.
  • the resulting fragment was introduced into the yeast expression vector pRS426 at Bgl II and Not I sites downstream of the yeast promoter (GAL1/GA110) which is regulated by carbon sources.
  • the resultant vector was designated YepAB-ACAT2 ( Figure 6) .
  • Antisense RNA technology can be used to create mice, or mouse or human cell lines incapable of translating acylcoenzyme A: cholesterol acyltransferase II RNA into protein. Standard methods may be used to create an antisense oligonucleotide to the human homolog of acylcoenzyme A: cholesterol acyltransferase II These methods are well known in the art (36) .
  • acylcoenzyme A cholesterol acyltransferase II is ligated adjacent to a mammalian promoter in the opposite orientation.
  • the promoter and other replicatory mechanisms mside the cell will transcribe a human homolog of acylcoenzyme A: cholesterol acyltransferase II encoding, nonsense strand.
  • This strand will bind with the coding mRNA which is normally synthesized to form a complex. Due to the formation of this complex, the antisense strand prevents the translation of the coding mRNA into protem.
  • oligonucleotide m vi tro which is capable of binding the mRNA that encodes a human homolog of acylcoenzyme A: cholesterol acyltransferase II so as to inhibit the translation of the mRNA mto protem.
  • the oligonucleotides can then be introduced mto the subject using a pharmaceutically acceptable carrier.
  • Methods of synthesizing naturally and non-naturally occurring oligonucleotides which are capable of inhibiting the translation of the mRNA mto protein are well known in the art.
  • means of transfecting an organism with such oligonucleotides are well known m the field.
  • Mice can be made with an alteration in their genome, specifically at the acylcoenyme A: cholesterol acyltransferase II gene site. Standard methods may be used to alter the genome. These methods are well known in the art (37, 38) .
  • One such process to achieve this goal involves disrupting the wildtype mouse homolog of acylcoenyme A: cholesterol acyltransferase II in vi tro, then introducing the altered gene into mouse embryonal stem cells m such a way as to taret integration mto the corresponding genomic region.
  • This process can be performed such that both copies of the wildtype acylcoenyme A: cholesterol acyltransferase II are replaced by the altered, knock-out version.
  • These modified cells can be introduced into blastocysts which will be allowed to develop into chimeric adults.
  • Mice bearing the altered acylcoenyme A: cholesterol acyltransferase II gene will be mated to each other to generate homozygous mutant acylcoenyme A: cholesterol acyltransferase II animals.
  • mice who are heterozygous for mutant acylcoenzyme A: cholesterol acyltransferase II. From their progeny, one skilled in the art could select the progeny who are homozygous for mutant acylcoenzyme A: cholesterol acyltransferase II. Breeding and selecting such progeny are well known in the art.
  • ACAT acyl coenzyme A-cholesterol acyltransferase
  • ARGP ACAT related gene products
  • acylcoenzyme A cholesterol acyltransferase II
  • acylcoenzyme A cholesterol acyltransferase III respectively.
  • the ARGPs exhibit marked sequence conservation to the human ACAT sequence (hACAT) originally identified by Chang and colleagues.
  • ARGP1 is expressed at high levels in intestine and liver in contrast to the expression of hACAT which is of low abundance in these tissues.
  • knock-out mutant mice deficient in the murine homolog if hACAT retain sterol esterification activity in liver and intestine (Meiner et al .
  • ARGP1 is a candidate for sterol esterification in these tissues.
  • ARGP2 seems to be restricted to the fetal liver, suggesting it to have a role in lipid metabolism during development.
  • ACAT-like gene families are a common occurrence in multiple organisms. It is hypothesize that multiple enzymes for sterol esterification will provide flexibility in response to differing sterol and fatty acid substrates encountered by different tissues. This further suggests specific roles for these enzymes in lipoprotein production, lipid homeostasis, and disease progression.
  • a critical component of this homeostasis is the intracellular neutralization of sterol by an esterification reaction between the C 3 -0H group of cholesterol and fatty acyl-coenzyme A. This reaction is performed in mammalian cells by the enzyme acyl coenzyme A-cholesterol acyltransferase (ACAT) . Since the process of sterol esterification converts sterol into a cytoplasmic storage form, it is critical to all eukaryote, including the microorganism Saccharomyces cerevisiae (budding yeast) .
  • a critical test of the role of the ACAT gene product in cholesterol homeostasis and atherosclerosis was initiated by Farese and colleagues, by the production of "knock ⁇ outs" at the Acact locus corresponding to the mouse homolog of hACAT (10) .
  • the fidelity of the mutation was confirmed by sequencing of cDNA from the disrupted allele and by the failure to detect immunoreactive protein in Acact ⁇ cell extracts.
  • the animals were healthy and fertile and had residual, but significant, sterol esterification activity in fibroblasts and macrophages. Cholesterol ester levels and ACAT activity in the adrenals were also severely reduced. Conversely, Acact ⁇ livers contained significant levels of cholesterol ester, and esterification activity was not altered.
  • ARGP1 ACAT Related Gene Products
  • ACAT related sequence A sequence corresponding to the strongest region of protein conservation between the human macrophage ACAT and yeast ARE sequences was used to identify protein sequences predicted to be encoded by entries in the best using the tblastn software (NCBLI) .
  • NCBLI tblastn software
  • Escherichia coli clones with the largest inserts corresponding to these sequences were obtained from the I.M.A.G.E. consortium and resequenced from both ends using commercial primers, T3 and T7, or internal primers derived from a consensus. Nucleotide sequencing was performed at the Columbia University Combined Center core facility using an Applied Biosystems fluorescent sequencing machine.
  • Table 1 Entries of human ACAT related gene products in the products in the data base of expressed sequence tags .
  • a nested PCT reaction was performed using internal gene specific primers and library adaptors.
  • primer extension cDNA products were identified from mRNA extracted from human intestine (a kind gift of P. Dawson) . Amplification products of the predicted size were confirmed as gene specific, using southern hybridization to sequences predicted to be at the 3'-end of these products. The products were isolated from agarose gels using Geneclean and subcloned into TA variants of pBluescript (Stratagene ® ) vectors of klenow/kinase treated and blunt end ligated to pGEM2 (Promega ® ) . Positive clones were identified by colony hybridization or by PCR amplifications using an internal ARGP specific primer. Clones with the largest inserts were sequenced to obtain novel sequence and where necessary, this process was reiterated with ARGP 5' specific primers derived from the new sequence.
  • RT-PCR reverse- transcriptase PCR
  • primers were designed to be conserved between rodents and humans (as described below, the mouse sequence homolog to ARGPl) has been identified. Alternatively, PCR conditions were optimized to permit moderate mismatches. The ARGP amplification primers were designed to be gene specific
  • LN E FGDR-FY GDWWN single letter amino-acid code
  • ACAT Related Gene Products ARGP 1 (acylcoenzyme A: cholesterol acyltransferase II) and 2 (acylcoenzyme A: cholesterol acyltransferase III) .
  • the sequence identified by Chang et al (8) will be referred to as hACAT, hereon.
  • a limited protein sequence to a founder clone (R07932) to ARGPl has been presented previously (11) .
  • the entries in the best that define these two genes, including their insert sizes are described in table 1.
  • the majority of inserts (with the exception of a chimeric clone ZA3867) are less than lKbp.
  • the northern and sequence analysis presented indicated them to be incomplete clones.
  • ARGPl a ubiquitously expressed member of the ACAT gene family.
  • probed multiple tissue northerns of human mRNA was probed, using a fragment close to the 3' end of the gene. Although this region displays the maximum conservation at the protein level in this gene family, the genes are sufficiently divergent at the DNA level to be able to design gene specific hybridization probes.
  • the ARGPl sequence is expressed at abundant levels in may tissues with the exception of lung and kidney. The majority of tissues express a 2. Okb message but, some tissues (e.g. adrenal, small intestine, thymus) also express a 2.4kb mRNA at varying levels. The same northerns were hybridized with a probe to the human macrophage ACAT sequence.
  • the hACAT sequence detects 4 messages of approximately 3.0, 4.0, 4.7 and 7.4Kb. Upon comparison of the two hybridization results, an overlapping but occasionally differential expression pattern was observed.
  • Adrenal tissues express the highest levels of both hACAT and ARGPl message.
  • hACAT messages are rare in liver and intestine in contrast to ARGPl which is highly expressed in these tissues.
  • ARGPl was poorly expressed in kidney, lung and placenta although hACAT mRNA was easily detected. This tissue specific expression suggests that ARGPl is an ideal candidate for sterol esterification in tissues such as liver and intestine, which retain sterol esterification activity in ACAT k/o mice (10) .
  • ARGP2 an embryonic isoform of the ACAT gene family. Efforts to identify a transcript from ARGP2 in adult tissues were unsuccessful. Therefore embryonic tissue samples were chosen to investigate since the original founder clone was derived from a fetal liver library. A multiple tissue northern of mRNA from human embryonic brain, liver, kidney, and lung, were probed with and ARGP2 specific, COOH-terminal probe. As shown in figure 9, a single message of ⁇ 2.2kb was identified only in embryonic liver tissues, suggesting a high degree of tissue and developmental specificity to the expression of this gene product.
  • ARGPl expression of ARGPl in cell culture models. To develop a system in which to test the effect of reaction substrates on the esterification reaction performed by the ARGP enzymes. The expression of these genes in several tissue specific were examined, cell culture models. As shown in figure 10, ARGPl is clearly expressed in liver (HepG2) and Kidney (CV-1) cell lines. The latter result is somewhat in contrast to the northern blot on human tissue samples. This most likely reflects the sensitivity of the RT-PCR approach compared to filter hybridization and suggests that ARGPl is probably expressed in most tissues. Alternatively it may represent species difference (simian vs. human) or more interestingly the differentiation status of the cells under study. In data not shown here, ARGPl was also clearly expressed in human and mouse macrophage models (THP-1 and J774 cells) .
  • ARGP2 displays a significantly higher level of amino acid conservation with hACAT than does ARGPl. Over the sequence shown (Fig. 12) , the protein is 59% identical and 79% similar to human ACAT. Over the same region ARGPl is only conserved at the level of 32% identity. This striking identity is maintained at the DNA level (62% identity) and may suggest that ARGP2 is more closely analogous to hACAT in both its mechanism of action and its origin, than is ARGPl. As for ARGPl , certain hallmark sequences are retained in ARGP2 (see below) . The ARGP2 predicted protein also possesses several predicted transmembrane domains. One entry to the best for ARGP2 has also been allocated an STS (sequence tagged site) at the Whitehead Institute, (entry # WI-11660) and has thus been mapped to human chromosome 12.
  • STS sequence tagged site
  • Sterol esterification enzymes evolve as gene families in multiple organisms. Using the hACAT and AGRP nucleotide sequences as probes of multiple databases, we sought to establish whether the observation of gene families of ACAT related enzymes in yeast and humans was a common occurrence in other organisms. In general this is the case (Fig. 13) . Sequences from the genome of C. elegans, P. melanogastor and S.pombe, have been identified that are distinct from each other, within an organism, and exhibit approximately 25% identity at the predicted protein level. As for all the ACAT-like proteins, the maximum conservation is observed at the COOH-terminal region, with many of the apparently critical motifs described below, being maintained. As would be anticipated the mouse cDNA for ARGPl exhibits approximately 85% identity with its human homolog.
  • TCCCACTGTA AGATGGGGTT ATGTCGCTAT GAAGTTTGCA CAGGTCTTTG GTTGCTTTTT 2400
  • CTATGTGTAC TACATCTTTG AAAGGCTTTG TGCCCCCTTG TTTCGGAATA TCAAACAGGA 2460
  • ATCCTCGCCC CACGCTCCTG GACAGCGCCA TCAACGTGCC CTTCCAGACG ACTTTCAAAG 1080
  • GGGATGCCAT TTTGAACTGT GTGGCTGAAT TGACAAGATT TGGCGACAGA TATTTCTACG 1920
  • CTCCTGAAGC TGGCGGTCCC CAATCACCTC ATCTGGCTCA TCTTCTTCTA CTGGCTCTTC 240
  • CTGCCTGCAA AACCTGGGGA CCAGGACTTC CTGTCTTGCA TTCCCAAATT TGGGTTCTTG 300

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Abstract

Cette invention décrit un acide nucléique isolé qui encode une acylcoenzyme A (acyltransférase de cholestérol II ou III). L'invention décrit en particulier un acide nucléique isolé qui encode une acylcoenzyme A humaine de type sauvage (acyltransférase de cholestérol II ou III). L'invention décrit aussi divers procédés permettant d'inhiber l'acylcoenzyme A de type sauvage (acyltransférase de cholestérol II ou III) chez un sujet. La présente invention concerne également un procédé permettant d'identifier un composé chimique capable d'inhiber l'acylcoenzyme A (acyltransférase de cholestérol II ou III) et une composition pharmaceutique comprenant le composé chimique inventé par le procédé susmentionné. L'invention concerne en outre un procédé permettant de traiter un sujet atteint d'athérosclérose ou d'hyperlipidémie.
PCT/US1997/009460 1996-05-30 1997-05-30 Adn encodant une acylcoenzyme a (acyltransferase de cholesterol) et emplois dudit produit WO1997045439A1 (fr)

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999063096A2 (fr) * 1998-06-05 1999-12-09 Calgene Llc Acyl coa : sequences d'acide nucleique en rapport avec la cholesterol acyltransferase
WO1999067368A1 (fr) * 1998-06-23 1999-12-29 The Regents Of The University Of California Nouvelle acyl coa cholesterol aclytransferase (acat-2)
WO2000032756A2 (fr) * 1998-12-02 2000-06-08 E.I. Du Pont De Nemours And Company Des diacylglycerol acyltransfease vegetale
EP1099761A1 (fr) * 1999-11-12 2001-05-16 Scandinavian Biotechnology Research AB (ScanBi AB) Utilisation d'une classe d'enzymes pour augmenter la teneur en huile dans des organismes transgéniques
WO2001034814A1 (fr) * 1999-11-12 2001-05-17 Scandinavian Biotechnology Research Ab (Scanbi Ab) Utilisation d'une classe d'enzymes et de leurs genes codants pour augmenter la teneur en huile d'organismes transgeniques
WO2001090329A2 (fr) * 2000-05-23 2001-11-29 Millennium Pharmaceuticals, Inc. 46745, un nouveau membre humain de la famille acyltransferase et utilisations de ce dernier
US6344548B1 (en) 1998-06-24 2002-02-05 The Regents Of The University Of California Diacylglycerol o-acyltransferase
WO2004052311A2 (fr) * 2002-12-10 2004-06-24 Isis Pharmaceuticals, Inc. Modulation de l'expression de l'acetyl-coa acetyltransferase 2
US6791008B1 (en) 1999-11-12 2004-09-14 Scandinavian Biotechnology Research (Scanbi) Ab Use of a class of enzymes and their encoding genes to increase the oil content in transgenic organisms
EP1330552A4 (fr) * 2000-10-31 2005-06-22 Michel Alphonse Julien Georges Selection assistee par marqueurs de bovins a production laitiere amelioree faisant appel au gene diacylglycerol acyltransferase dgat1
US7202357B2 (en) 2001-07-30 2007-04-10 Isis Pharmaceuticals, Inc. Antisense modulation of acyl CoA cholesterol acyltransferase-2 expression
WO2011123401A1 (fr) 2010-03-30 2011-10-06 Novartis Ag Utilisations d'inhibiteurs de dgat1
WO2023085931A1 (fr) 2021-11-11 2023-05-19 Koninklijke Nederlandse Akademie Van Wetenschappen Organoïdes hépatiques

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US5484727A (en) * 1992-10-14 1996-01-16 Trustees Of Dartmouth College Cloned gene encoding acylcoenzyme A: cholesterol acyltransferase (ACAT)

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US5484727A (en) * 1992-10-14 1996-01-16 Trustees Of Dartmouth College Cloned gene encoding acylcoenzyme A: cholesterol acyltransferase (ACAT)

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EXPRESSED SEQUENCE TAG (EST), Database Accession Numbers, R99213 (13 September 1995), R99214 (13 September 1995), H27149 (12 July 1995), N76754 (02 April 1996), H45923 (31 July 1995), H45924 (31 July 1995), H24791 (07 July 1995), Z39933 (21 September 1995), T79408 (15 March 1995), R48474 (18 May 1995), T79494 (15 March *

Cited By (27)

* Cited by examiner, † Cited by third party
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US6444876B1 (en) 1998-06-05 2002-09-03 Calgene Llc Acyl CoA: cholesterol acyltransferase related nucleic acid sequences
US7041872B2 (en) 1998-06-05 2006-05-09 Calgene Llc Acyl CoA:cholesterol acyltransferase related nucleic acid sequences
WO1999063096A3 (fr) * 1998-06-05 2000-01-27 Calgene Llc Acyl coa : sequences d'acide nucleique en rapport avec la cholesterol acyltransferase
WO1999063096A2 (fr) * 1998-06-05 1999-12-09 Calgene Llc Acyl coa : sequences d'acide nucleique en rapport avec la cholesterol acyltransferase
US6579974B1 (en) 1998-06-23 2003-06-17 The Regents Of The University Of California Acyl CoA:cholesterol acyltransferase (ACAT-2)
AU759181B2 (en) * 1998-06-23 2003-04-10 Regents Of The University Of California, The Novel acyl coa:cholesterol acyltransferase (ACAT-2)
US7238779B2 (en) 1998-06-23 2007-07-03 The Regents Of The University Of California Acyl CoA:cholesterol acyltransferase (ACAT-2)
WO1999067368A1 (fr) * 1998-06-23 1999-12-29 The Regents Of The University Of California Nouvelle acyl coa cholesterol aclytransferase (acat-2)
US6869937B1 (en) 1998-06-23 2005-03-22 The Regents Of The Universtiy Of California Acyl CoA:cholesterol acyltransferase (ACAT-2)
US6344548B1 (en) 1998-06-24 2002-02-05 The Regents Of The University Of California Diacylglycerol o-acyltransferase
WO2000032756A3 (fr) * 1998-12-02 2000-10-12 Du Pont Des diacylglycerol acyltransfease vegetale
US7524945B2 (en) 1998-12-02 2009-04-28 E.I. Du Pont De Nemours And Company Plant diacyglycerol acyltransferases
WO2000032756A2 (fr) * 1998-12-02 2000-06-08 E.I. Du Pont De Nemours And Company Des diacylglycerol acyltransfease vegetale
US8497362B2 (en) 1998-12-02 2013-07-30 E I Du Pont De Nemours And Company Plant diacylglycerol acyltransferases
CN1294265C (zh) * 1999-11-12 2007-01-10 斯堪的纳维亚生物技术研究公司 应用一类酶及其编码基因增加转基因生物的含油量
EP1099761A1 (fr) * 1999-11-12 2001-05-16 Scandinavian Biotechnology Research AB (ScanBi AB) Utilisation d'une classe d'enzymes pour augmenter la teneur en huile dans des organismes transgéniques
WO2001034814A1 (fr) * 1999-11-12 2001-05-17 Scandinavian Biotechnology Research Ab (Scanbi Ab) Utilisation d'une classe d'enzymes et de leurs genes codants pour augmenter la teneur en huile d'organismes transgeniques
US6791008B1 (en) 1999-11-12 2004-09-14 Scandinavian Biotechnology Research (Scanbi) Ab Use of a class of enzymes and their encoding genes to increase the oil content in transgenic organisms
WO2001090329A3 (fr) * 2000-05-23 2002-06-06 Millennium Pharm Inc 46745, un nouveau membre humain de la famille acyltransferase et utilisations de ce dernier
WO2001090329A2 (fr) * 2000-05-23 2001-11-29 Millennium Pharmaceuticals, Inc. 46745, un nouveau membre humain de la famille acyltransferase et utilisations de ce dernier
EP1330552A4 (fr) * 2000-10-31 2005-06-22 Michel Alphonse Julien Georges Selection assistee par marqueurs de bovins a production laitiere amelioree faisant appel au gene diacylglycerol acyltransferase dgat1
US7202357B2 (en) 2001-07-30 2007-04-10 Isis Pharmaceuticals, Inc. Antisense modulation of acyl CoA cholesterol acyltransferase-2 expression
US7335764B2 (en) 2001-07-30 2008-02-26 Isis Pharmaceuticals, Inc. Antisense modulation of acyl CoA cholesterol acyltransferase-2 expression
WO2004052311A3 (fr) * 2002-12-10 2005-04-14 Isis Pharmaceuticals Inc Modulation de l'expression de l'acetyl-coa acetyltransferase 2
WO2004052311A2 (fr) * 2002-12-10 2004-06-24 Isis Pharmaceuticals, Inc. Modulation de l'expression de l'acetyl-coa acetyltransferase 2
WO2011123401A1 (fr) 2010-03-30 2011-10-06 Novartis Ag Utilisations d'inhibiteurs de dgat1
WO2023085931A1 (fr) 2021-11-11 2023-05-19 Koninklijke Nederlandse Akademie Van Wetenschappen Organoïdes hépatiques

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