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WO1997045536A1 - Enzymes apparentees a l'acylcoenzyme a:cholesterol acyltransferase dans la levure et leurs utilisations - Google Patents

Enzymes apparentees a l'acylcoenzyme a:cholesterol acyltransferase dans la levure et leurs utilisations Download PDF

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Publication number
WO1997045536A1
WO1997045536A1 PCT/US1997/009160 US9709160W WO9745536A1 WO 1997045536 A1 WO1997045536 A1 WO 1997045536A1 US 9709160 W US9709160 W US 9709160W WO 9745536 A1 WO9745536 A1 WO 9745536A1
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Prior art keywords
acylcoenzyme
cholesterol acyltransferase
related enzyme
wildtype
chemical compound
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PCT/US1997/009160
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English (en)
Inventor
Stephen L. Sturley
Martin Bard
Hongyuan Yang
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The Trustees Of Columbia University In The City Of New York
Indiana University Foundation
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Application filed by The Trustees Of Columbia University In The City Of New York, Indiana University Foundation filed Critical The Trustees Of Columbia University In The City Of New York
Priority to AU32894/97A priority Critical patent/AU3289497A/en
Publication of WO1997045536A1 publication Critical patent/WO1997045536A1/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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • ACYLCOENZYME A CHOLESTEROL ACYLTRANSFERASE-RELATED ENZYMES IN YEAST AND USES THEREOF
  • 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 en ⁇ oplasmic reticulum (ER) relative to the smooth ER (3) may modulate protein translocation or membrane-associated transc ⁇ ptional 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 in the rough ER may influence the transport of sterol between mtracellular 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 lipoprotem (LDL) receptor and alters serum lipoprotein composition to be pro-atherogenic (6, 7) . It may also be a rate limiting step in intestinal sterol absorption (8) . Furthermore, CE deposition in 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 1 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 expression vector comprising an isolated nucleic acid which encodes wildtype acylcoenzyme A: cholesterol acyltransferase-related enzyme I. This invention also provides an expression vector comprising an isolated nucleic acid which encodes wildtype acylcoenzyme A: cholesterol acyltransferase- related enzyme II.
  • This invention provides a method of identifying a chemical compound which is capable of inhibiting wildtype acylcoenzyme A: cholesterol acyltransferase II.
  • This invention also provides a pharmaceutical composition which is capable of inhibiting acylcoenzyme A: cholesterol acyltransferase II.
  • This invention also provides a method of treating a subject who has atherosclerosis or hyperlipidemia
  • This invention also provides a method of identifying a chemical compound which is capable of inhibiting wildtype acylcoenzyme A: cholesterol acyltransferase-related enzyme I, wildtype acylcoenzyme A: cholesterol acyltransferase-related enzyme II or both m a subject.
  • This invention also provides a method of inhibiting the growth of a fungus. Further, this invention provides a method of treating a fungal invasion in a subject. 97/45536 PC17US97/09160
  • amino acid residues are abbreviated as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, He; 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. CON: consensus sequence.
  • Figures 1A and IB Protein sequence alignments predicted from candidate genes for the human acylcoenzyme A: cholesterol acyltransferase I gene, the yeast homologs, acylcoenzyme A: cholesterol acyltransferase-related enzyme I and acylcoenzyme A: cholesterol acyltransferase- related enzyme II, and a consensus sequence of all three sequences.
  • acylcoenzyme A cholesterol acyltransferase I (Sequence I.D. No. : 2)
  • acylcoenzyme A cholesterol acyltransferase- related enzyme I (Sequence I.D. No. : 4)
  • acylcoenzyme A cholesterol acyltransferase- 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.: 8) .
  • the asterisks indicate the residues in R07932 identical to those of the other sequences. 1A.
  • FIGS. 2A, 2B, 2C, 2D and 2E Construction and analysis of Acylcoenzyme A : cholesterol acyltransferase-related enzymes 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 arelzl 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 Acylcoenzyme A: cholesterol acyltransferase-related enzyme 1 / 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 in the absence of formamide . D.
  • 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 acylcoenzyme A: cholesterol acyltransferase-related enzyme II (are2-5' and are2-3' ) and a 3' amplimer within LEU2 (L2-3' ) .
  • E. A 999-bp fragment identifies are2 ⁇ , as shown in the ethidium bromide stained agarose gel. The wild-type fragment (2206-bp) is also produced m the same reaction.
  • Leucme 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 (Bio-Rad Laboratories) .
  • Figures 3A and 3B Fluorescent staining of triglyceride and sterol ester.
  • the cells were grown m YEPD to stationary phase, washed with deionized H ? 0, and incubated with 1 ⁇ g/ml Nile Red (1 mg/ml in acetone) . Fluorescent images were obtained with a BioRad
  • FIGS 4A, 4B, 4C and 4D Neutral lipid and sterol biosynthesis in Acylcoenzyme A : cholesterol acyltransferase-rela ted enzymes deletion mutants.
  • Strain genotypes are as described m the text; dpm/mg dry weight: disintegrations per minute per milligram of dry weight of cells.
  • C Sterol ester biosynthesis m wild-type and mutant cells transformed with vector control
  • acylcoenzyme A cholesterol acyltransferase-related enzyme I over-expression plasmids, YEp3-16 (increased copy number, shaded box) and pADH5-36
  • 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 enzymes deletion mutants. Lipids were labeled in synthetic complete media containing [ 1—'"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 ammo acid or predicted amino acid sequences.
  • 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 1 from nucleic acid bases 1-1624. Amino acid sequence of human acylcoenzyme A: cholesterol acyltransferase 1 from ammo acid residues 1-76. 5A-2. Nucleic acid sequence of human acylcoenzyme A: cholesterol acyltransferase 1 from nucleic acid bases 1625-2524. Amino acid sequence of human acylcoenzyme A: cholesterol acyltransferase 1 f rom amino acid residues 77-376.
  • A cholesterol acyltransferase-related enzym.e I designated Sequence ID No. : 3.
  • the amino acid sequence of yeast acylcoenzyme A cholesterol acyltransferase-related enzyme I designated Sequence ID No. : 4. 5B-1.
  • Nucleic acid sequence of acylcoenzyme 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.
  • A cholesterol acyltransferase-related enzyme I from nucleic acid bases 1290-2114.
  • Ammo acid sequence of acylcoenzyme A cnolesterol 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 bases 2115-2601.
  • Ammo acid sequence of acylcoenzyme A cholesterol acyltransferase-related enzyme I from ammo acid residues 485-611. 3.
  • A cholesterol acyltransferase-related enzyme II designated Sequence ID No. : 5.
  • the ammo acid sequence of yeast acylcoenzyme A cholesterol acyltransferase-related enzyme II designated Sequence ID No. : 6.
  • 5C-1 Nucleic acid sequence of acylcoenzyme A: cholesterol acyltransferase- related enzyme II from nucleic acid bases 1-1061. Ammo acid sequence of acylcoenzyme A: cholesterol acyltransferase-related enzyme II from ammo acid residues 1-238. 5C-2. Nucleic acid sequence of acylcoenzyme A: cholesterol acyltransferase- related enzyme II from nucleic acid bases 1062-1961. Ammo acid sequence of acylcoenzyme A: cholesterol acyltransferase-related enzyme II from ammo acid residues 239-538.
  • 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.
  • Figure 6A and 6B Expression of human macrophage ACAT in pRS426GP.
  • the ACAT open reading frame was inserted at the MotI and Sa cl sites, downstream of the promoter of the GAL1 /1 0 gene
  • hACAT hACAT
  • pRS426GP vector
  • Figure 7A In vivo esterification analysis of yeast cells expressing human ACAT.
  • 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 .
  • This invention provides an expression vector comprising an isolated nucleic acid which encodes wildtype acylcoenzyme A: cholesterol acyltransferase-related enzyme I operatively linked to a promoter of RNA transcription.
  • This invention also provides an expression vector comprising a isolated nucleic acid which encodes wildtype acylcoenzyme A: cholesterol acyltransferase-related enzyme II operatively linked to a promoter of RNA transcription.
  • the promoter may be a bacterial, yeast, insect or mammalian promoter.
  • vector backbones are known in the art and are useful for expressing proteins.
  • Such vectors include plasmid vectors, cosmid vectors, yeast artificial chromosome (YAC) , bacteriophage or eukaryotic viral DNA.
  • YAC yeast artificial chromosome
  • one such class of vectors comprises DNA elements derived from viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MoMLV) , Semliki Forest virus or SV40 virus.
  • viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MoMLV) , Semliki Forest virus or SV40 virus.
  • Such vectors may be obtained commercially or assembled from the sequences described by methods well-known in the art.
  • a wildtype acylcoenzyme A cholesterol acyltransferase-related enzyme I means a polypeptide having the activity of the naturally occurring form of acylcoenzyme A: cholesterol acyltransferase-related enzyme I.
  • the polypeptide has an ammo acid identical to the sequence which is set forth m Sequence I.D. No. : 4.
  • a wildtype acylcoenzyme A: cholesterol acyltransferase-related enzyme II means a polypeptide having the activity of the naturally occurring form of acylcoenzyme A: cholesterol acyltransferase-related enzyme II.
  • the polypeptide has an ammo acid identical to the sequence which is set form m Sequence I.D. No. : 6.
  • polypetides described above include any such polypeptide whether naturally occurring and obtained by purification from natural sources or non-naturally occurring and obtained synthetically, e.g. by recombmant DNA procedures, by methods well-known m the art.
  • nucleic acids and oligonucleotides of the subject invention also include nucleic acids coding for polypeptide analogs, fragments or derivatives of the subject invention which differ from naturally-occurring forms in terms of the identity or location of one or more ammo 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.
  • These molecules 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 ammo acid sequence of the polypeptide and as products for the large scale synthesis of the polypeptide by a variety of recombmant techniques. Further the nucleic acids and oligonucleotides are 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.
  • a host vector system for the production of a polypeptide which comprises the expression vector in a suitable host is provided.
  • Suitable host cell include, but are not limited to, prokaryotic or eukaryotic cells, e.g. bacterial cells
  • mammalian cells including gram positive cells
  • yeast cells including gram positive cells
  • fungal cells including gram positive cells
  • insect cells including gram positive cells
  • animals cells including gram positive cells
  • mammalian cells including, but not limited to, the mouse fibroblast cell NIH 3T3, CHO cells,
  • HeLa cells Ltk cells, Cos cells, etc.
  • Mammalian cells may be used to transfect cells by methods well known m the art such as calcium phosphate precipitation or electroporation may be otherwise introduced into cells, e.g., by micromjection, to obtain transfected cells.
  • This invention also provides a method of producing a polypeptide (e.g. acylcoenzyme A: cholesterol acyltransferase-related enzyme I or acylcoenzyme A: cholesterol acyltransferase-related enzyme II) which comprises growing the host vector system, as described above, under suitable conditions permitting production of the polypeptide and recovering the polypeptide so produced.
  • a polypeptide e.g. acylcoenzyme A: cholesterol acyltransferase-related enzyme I or acylcoenzyme A: cholesterol acyltransferase-related enzyme II
  • 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 metnod of obtaining a polypeptide m purified form which includes
  • the vector may include a plasmid vector, cosmid vector, yeast artificial chromosome (YAC), bacteriophage or eukaryotic viral DNA.
  • the host cells may be a bacterial cell (including gram positive cells) , yeast cell, fungal cell, insect cell or animal cell. Suitable animal cells include, but are not limited to HeLa cells, Cos cells, CV1 cells and various primary mammalian cells.
  • Culturmg methods useful for permitting transformed or transfected host cells to produce polypeptides are well known m the art as are methods for recovering polypeptides from such cells and for purifying the polypeptides.
  • this invention also provides purified wildtype acylcoenzyme A: cholesterol acyltransferase-related enzyme I and purified wildtype acylcoenzyme A: cholesterol acyltransferase-related enzyme II.
  • polypeptide of the subject invention also includes analogs, fragments or derivatives which differ from naturally-occurring forms, but retain the activity of wildtype acylcoenzyme A: cholesterol acyltransferase-related enzyme I or wildtype acylcoenzyme A: cholesterol acyltransferase-related enzyme II..
  • This invention also provides oligonucleotides of at least 15 nucleotides capable of specifically hybridizing with a unique sequence of nucleotides withm a nucleic acid which encodes a wildtype acylcoenzyme A: cholesterol acyltransferase-related enzyme I without hybridizing to any sequence of nucleotides within a nucleic acid which encodes a mutant acylcoenzyme A: cholesterol acyltransferase-related enzyme I.
  • This invention also provides an oligonucleotide of at least 15 nucleotides capable of specifically hybridizing with a unique sequence of nucleotides within a nucleic acid which encodes a wildtype acylcoenzyme A: cholesterol acyltransferase-related enzyme II without hybridizing to any sequence of nucleotides withm a nucleic acid which encodes a mutant acylcoenzyme A: cholesterol acyltransferase-related enzyme II.
  • These oligonucleotide may comprise DNA or RNA or modified nucleotides; all are well known m the art and may be made using standard methods such as automated synthesis.
  • This invention also provides a method for identifying a chemical compound which is capable of inhibiting wildtype acylcoenzyme A: cholesterol acyltransferase II (i.e. preventmg the activity of the enzyme) which comprises (a) obtaining a yeast cell which lacks or expresses deficient levels of wildtype acylcoenzyme A: cholesterol acyltransferase-related enzyme I and wildtype acylcoenzyme A: cholesterol acyltransferase-related enzyme II; (b) transfectmg the obtained cell with a nucleic acid encoding a wildtype human acylcoenzyme A: cholesterol acyltransferase II to render the cell capable of expressing the wildtype acylcoenzyme A: cholesterol acyltransferase; (c) contacting the transfected cell of step (b) with the chemical compound under conditions permitting binding between the chemical compound and the expressed wildtype acylcoenzyme A: cholesterol acyltransferase II to form
  • the wildtype acylcoenzyme A: cholesterol acyltransferase II is human acylcoenzyme A: cholesterol acyltransferase II and the chemical compound identified specifically inhibits wildtype human acylcoenzyme A: cholesterol acyltransferase II.
  • Methods for identifying chemical compounds that inhibit particular polypeptides are well- known m the art.
  • the chemical compound includes agents previously known to inhibit the esterification of sterols, but whose toxicity was unknown.
  • acylcoenzyme A cholesterol acyltransferase II
  • the chemical compound identified may be an inorganic compound, a nucleic acid molecule, an oligonucleotide, an organic compound, a peptide, a peptidomimetic compound, or a protein.
  • This invention also provides a method of inhibiting wildtype acylcoenzyme A: cholesterol acyltransferase II m a subject which comprises administration of the chemical compound identified by the above-described method m an amount effective to inhibit wildtype acylcoenzyme A: cholesterol acyltransferase II m a subject.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the chemical compound identified by the above- described method m an amount effective to inhibit acylcoenzyme A: cholesterol acyltransferase II and a pharmaceutically acceptable carrier.
  • the chemical compound identified by the above-described method may be administered aerosol delivery; anal, nasal, ocular, oral, otic or topical delivery; infusion; mtralesional, intramuscular, mtraperItoneal or intravenous injection; liposome-mediated delivery.
  • effective amount means an amount of the chemical compound effective to inhibit wildtype acylcoenzyme A: cholesterol acyltransferase II.
  • This invention further provides a method of treating a subject who has atherosclerosis or hyperlipidemia with the pharmaceutical composition described above.
  • This invention also provides a method of identifying a chemical compound which is capable of inhibiting wildtype acylcoenzyme A: cholesterol acyltransferase-related enzyme I m a subject which comprises (a) contacting a wildtype acylcoenzyme A: cholesterol acyltransferase-related enzyme I with the chemical compound under conditions permitting binding between the wildtype acylcoenzyme A: cholesterol acyltransferase-related enzyme I and the chemical compound; (b) detecting specific binding of the chemical compound to the wildtype acylcoenzyme A: cholesterol acyltransferase-related enzyme I; and (c) determining whether the chemical compound inhibits the activity of the coenzyme so as to identify a chemical compound which is capable of inhibiting wildtype acylcoenzyme A: cholesterol acyltransferase-related enzyme 1.
  • This invention also provides a metnod c f identifying a chemical compound which is capable of inhibiting wildtype acylcoenzyme A: cholesterol acyl t ransierase-related enzyme II in a subject which comprises (a) contacting a wildtype acylcoenzyme A: cholesterol acyltransferase- related enzyme II with the chemcal compound under conditions permitting binding between the wildtype acylcoenzyme A: cholesterol acyltransferase-related enzyme II and the chemical compound; (b) detecting specific binding of the chemical compound to the wildtype acylcoenzyme A: cholesterol acyltransferase-related enzyme II; and (c) determining whether the chemical compound inhibits the activity of the coenzyme so as to identify a chemical compound which is capable of inhibiting wildtype acylcoenzyme A: cholesterol acyltransferase-related enzyme II.
  • This invention also provides a method of identifying a chemical compound which is capable of inhibiting acylcoenzyme A: cholesterol acyltransferase-related enzyme I and acylcoenzyme A: cholesterol acyltransferase- related enzyme II in a subject which comprises (a) contacting both acylcoenzyme A: cholesterol acyltransferase-related enzyme I and acylcoenzyme A: cholesterol acyltransferase-related enzyme II with the chemical compound under conditions permitting binding between the acylcoenzyme A: cholesterol acyltransferase- related enzyme I and acylcoenzyme A: cholesterol acyltransferase-related enzyme II, and the chemical compound; (b) detecting specific binding of the chemical compound to the acylcoenzyme A: cholesterol acyltransferase-related enzyme I and acylcoenzyme A: cholesterol acyltransferase-related enzyme II; and (c) determining whether the chemical compound inhibits
  • This invention also provides a method of inhibiting the growth of a fungus which comprises contacting the organism with the chemical compound identified by the above-described method in an amount effective to inhibit acylcoenzyme A: cholesterol acyltransferase-related enzyme I and thereby inhibit the growth of the yeast organism.
  • This invention also provides a method of inhibiting the growth of a fungus which comprises contacting the organism with the chemical compound identified by the above-descnbed method in an amount effective to inhibit acylcoenzyme A: cholesterol acyltransferase-related enzyme II and thereby inhibit the growth of the yeast organism.
  • This invention also provides a method of inhibiting the growth of fungus which comprises contacting the organisms with the chemical compound identified by the above- described method in an amount effective to inhibit both acylcoenzyme A: cholesterol acyltransferase-related enzyme I and acylcoenzyme A: cholesterol acyltransferase- related enzyme II, and thereby inhibit the growth of the yeast organism.
  • fungus means an organism including, but not limited to, yeasts and fungi.
  • the fungus is a pathogenic yeast.
  • the fungus is an organism that is normally not pathogenic, but becomes pathogenic in a subject with a lowered immune response.
  • the fungus is an organism that causes such conditions as vaginitis, athlete's foot and other infections to mucosal membranes that are not necessarily pathogenic.
  • the yeast organism may be Candida albicans, Candida tropicalia, Candida parapsilosis, Cryptococcus neoformans, Histoplasma capsulatum, Aspergillus nidulans, Pneumocystis carmii or Coccidoides immitis.
  • This invention also provides a method of treating a fungal invasion in a subject which comprises administering to the subject the chemical compound identified by the above-described method in an amount effective to specifically inhibit acylcoenzyme A: cholesterol acyltransferase-related enzyme I in the subj ect .
  • This invention also provides a method of treating a fungal invasion in a subject which comprises administering to the subject the chemical compound identified by the above-described method m an amount effective to specifically inhibit acylcoenzyme A: cholesterol acyltransferase-related enzyme II m the subject.
  • fungal invasion means an infection caused by either a yeast or fungal organisms that causes either pathogenic reactions m a subject, or becomes pathogenic m a subject with a lower immune response. Further, the “fungal invasion” includes, but is not limited to, infections that cause conditions such as vagmitis, athelete' s foot and other superficial infections. In a preferred embodiment, the fungal invasion is associated with Candida albmcans, Candida tropicalia, Candida parapsilosis, Crytococcus neoformans, Histoplasma capsulatum, Aspergillus nidulans, Pneumocystis carmii or Coccidoides immitis.
  • This invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the chemical compound identified by the above- described method m an amount effective to inhibit acylcoenzyme A: cholesterol acyltransferase-related enzyme I m a subject and a pharmaceutically effective carrier.
  • This invention also provides pharmaceutical composition
  • pharmaceutical composition comprising the chemical compound identified by the above- described method m an amount effective to inhibit acylcoenzyme A: cholesterol acyltransferase-related enzyme II in a subject and a pharmaceutically effective carrier.
  • This invention also provides pharmaceutical composition
  • pharmaceutical composition comprising the chemical compound identified by the above- described method in an amount effective to inhibit both acylcoenzyme A: cholesterol acyltransferase-related enzyme I and acylcoenzyme A: cholesterol acyltransferase- related enzyme II in a subject and a pharmaceutically effective carrier.
  • acylcoenzyme A cholesterol acyltransferase-related enzyme I
  • acylcoenzyme A cholesterol acyltransferase-related enzyme II or both.
  • Transformation of yeast was performed with lithium acetate (15) by ammo-acid prototrophy selection.
  • a diploid strain (5051) was constructed between two isogenic derivatives of W303 (16) ; W1346-3C (MATa, ade2-l , canl -1 00, h ⁇ s3-l l , 1 5 , l eu2-3, 112 , trpl -1 , ura3-l ) and W1134-2C (MATa , canl -1 00, h ⁇ s3-l l r 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 (1/) .
  • Competent cells of Escheri chia col i 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 1, Sma I fragment of pJH-Hl encoding the HIS3 gene was then inserted at these sites m 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 acylcoenzyme A cholesterol acyltransferase-related enzyme I was confirmed by Southern blot analysis.
  • Radioactive probes of acylcoenzyme A cholesterol acyltransferase-related enzyme I were prepared by random priming (Pharmacia) with "P-dCTP. Genomic DNA (18) was transferred to Hybond membranes (Amersham) and hybridized in the absence of formamide at 65° or 60°C (19) .
  • a shotgun library of cosmid 14-21 from chromosome XIV was constructed using the nebulizing technique (20) .
  • the DNA was nebulized (90 seconds, 2 bars), size fractionated, treated with DNA polymerase I (Klenow fragment) and T4 DNA polymerase and blunt-end ligated into pTZl ⁇ R (Pharmazia, Germany) .
  • Nucleotide sequencing was performed by dideoxy-chain-termination with digoxigenin-labeled reverse primer and Sequenase (United States Biochemical) .
  • the reactions were analyzed on the GATC 1500 direct blotting electrophoresis system (GATC GmbH, Germany) using the Boehringer-Mannheim Dig-development protocol. Sequences were aligned by SeqMan (DNA Star Inc.) . Database searching was performed with BLAST (21) and GCG Inc. software (22) .
  • the DNA sequence of the acylcoenzyme A: cholesterol acyltransferase-related enzyme I and acylcoenzyme A: cholesterol acyltransferase-related enzyme II genes are deposited at GenBank (P25628 and U51790, respectively) .
  • acylcoenzyme A cholesterol acyltransferase-related enzyme II locus
  • a PCR was performed on genomic DNA from these strains using are2-5' (CATTGCAGTTACACGTGAATGC) (Sequence ID No.: 11), are2-3' : (TAGCTCCACAGAACAGTTGCAGG) (Sequence ID No . : 12) and a 3' amplimer corresponding to the LEU2 gene (L2-3' CTCTGACAACAACGAAGTCAG) (Sequence ID No. : 13) .
  • acylcoenzyme A cholesterol acyltransferase-related enzyme I gene by copy number under the control of its own promoter in YEp3-16, a 2354 bp Cla 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 .
  • the human acylcoenzyme A cholesterol acyltransferase sequence were used to search for homologous yeast genes and subsequently to identify an additional human isoform ( Figures 1A and IB) .
  • Acylcoenzyme A cholesterol acyltransferase-related enzyme 1, an 1830-bp open reading frame (ORF) on yeast chromosome III, encodes a 610- residue protein with 23V identity and 49 similarity to human acylcoenzyme A: cholesterol acy transferase I ( Figures 1A and IB) .
  • yeast and human proteins possess leucme zipper motifs that could mediate protein-protein interactions (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 1A 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 OPF, which translates into a 643-res ⁇ due polypeptide.
  • the yeast acylcoenzyme A cholesterol acyltransferase-related enzyme genes are 61° and 49% identical at the DNA and predicted protein levels, respectively.
  • Arelp, Are2p and the human acylcoenzyme A cholesterol acyltransferase I protein are most related at the COOH-termmal region (42 identity over a 90-res ⁇ due sequence) ( Figures 1A and IB) .
  • acylcoenzyme A cholesterol acyltransferase-related enzyme II coding sequence was deleted from the genome of an Acyl coenzyme A : chol estero l acyl transferase-rel a t ed enzymel /arel ⁇ NA heterozygous diploid by a polymerase chain reaction approach (23) (Fig. 2D) .
  • Haploid progeny representing the single arel ⁇ NA and are2 ⁇ deletions and the arel ⁇ NAare2 ⁇ double mutant were obtained.
  • acylcoenzyme A cnolesterol acyltransferase-related enzyme genes upon cytoplasmic lipid storage
  • the neutral lipid components (tnglyceride and sterol ester) of the yeast cells were detected by fluorescence microscopy after staining with Nile Red
  • acylcoenzyme A cholesterol acyltransferase-related enzyme 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 in 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 aylcoenzyme A: cholesterol acyltransferase-related enzyme I did not elevate sterol ester synthesis above untransformed controls. This suggests that either substrates are limiting in wildtype strains or that the enzyme is post-translationally regulated as in 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 in the single mutants (Fig. 4D) . In fact, free sterol concentrations were roughly equivalent m all cells.
  • Feedback regulation of sterol biosynthesis by acylcoenzyme A cholesterol acyltransferase activity has been observed in mammalian cells (31) and may be a common mechanism that maintains mtracellular sterol at non-toxic concentrations.
  • yeast Acyl coenzyme A cnol esterol acyl transferase-rel a ted enzyme and human acylcoenzyme A: cholesterol acyltransferase sequences was used to identify an additional cDNA with significant identity (47 o) to human acylcoenzyme A: cholesterol acyltransferase I and the yeast proteins ( Figure IB, Genbank accession # R07932) .
  • yeast genes with similarity to a human cDNA encoding acyl-coenzyme A cholesterol Acyltransferase (ACAT) were identified. Deletion of both yeast genes results in a viable cell with undetectable esterified sterol (1) .
  • the human cDNA in the yeast double mutant was expressed, resulting in high level production of ACAT protein, but low in vi vo esterification of ergosterol, the predominant yeast sterol.
  • the activity of the human enzyme was increased by incubation of these cells with 25- hydroxy-cholesterol, an established positive regulator of mammalian sterol esterification. In contrast, the yeast enzymes were unaffected by this reagent.
  • Free sterols are essential components of all eukaryotic membranes and exert a major influence on membrane fluidity and permeability and the activity of membrane-bound protems (2) .
  • the esterification of sterol i.e. the conjugation of fatty-acids with sterols, plays an important role m sterol homeostasis, since it converts excess free sterol into a cytoplasmic storage form. In human cells, this esterification reaction is mediated by the enzyme, Acyl-Coenzyme A : Cholesterol Acyltransferase [ACAT, (3) ] .
  • ACAT activity has been localized to the rough endoplasmic reticulum (RER) and is present m all tissues tested (4) .
  • ACAT has several physiological functions with possible pathological consequences. In addition to maintaining mtracellular cholesterol homeostasis, the ACAT reaction has been suggested to be involved m absorption of cholesterol from the intestinal lumen (5) . Furthermore, the cholesterol ester synthesis rate in the liver may affect the secretion of very low density lipoprotem (VLDL) and thus plasma cholesterol and triglyceride levels (6) . In adrenal cells, cholesterol ester (CE) is a sterol source for acute steroid hormone production. Most profoundly, the up-regulation of ACAT in macrophages and smooth muscle cells, accompanied by the accumulation of cholesterol ester in the arterial wall (foam cells), is an early event m the formation of an atherosclerotic plaque (7,8) .
  • VLDL very low density lipoprotem
  • CE cholesterol ester
  • Sandoz 58035 one of the first specific ACAT inhibitors, is a competitive fatty acid homologue (14) while DuP 128 is a potent, non-competitive inhibitor of the reaction [IC of 10 nm, (15,16) ] .
  • the sterol esterification reaction is conserved from yeast to humans (17) .
  • the predominant sterol is structurally distinct.
  • the major sterol source is cholesterol; m yeast, it is ergosterol.
  • Microsomal preparations from rat liver exhibit marked specificity of the ACAT reaction for cholesterol. Notably, the reaction was very sensitive to changes in the sterol side chain; the addition of a 24- ⁇ -methyl group, as in ergosterol, resulted in esterification at about 5" the rate of cholesterol.
  • yeast microsomal preparations este ⁇ fied both ergosterol and cholesterol to similar extents (18) .
  • the predominant fatty acyl-CoA in each species has not been as well studied.
  • Yeast strains, transformations, and media Congenic yeast strains with deletions m the yeast homologs of human
  • Yeast extract, Yeast Nitrogen Base, Bacto-peptone and Bacto-agar were from Difco laboratories; D-dextrose, D-galactose, and D- raffmose were from Sigma. Complete (YEPD) , synthetic complete (SC), or SC lacking uracil (SC-ura) media were prepared as described (23,24) .
  • PCR amplification of a Human ACAT cDNA and construction of the yeast expression plasmid Competent cells of Escherichia coli strain DH5 ⁇ (Lift Technologies, Inc.) and DNA modifying enzymes (Promega) were used according to the manufacturers instructions.
  • the human ACAT cDNA sequence was obtained from a human leukocyte cDNA library by PCR amplification as described (25) .
  • the pCMV vector containing ACAT was cut with Apal and made blunt ended by T4 DNA polymerase treatment and then digested with MotI.
  • the ensuing fragment contains a full length human ACAT open reading frame (ORF) with the translation initiation codon four bases away from the MotI site.
  • ORF human ACAT open reading frame
  • the yeast shuttle vector pRS-426-GAL was cut by SacI and blunt ended with the Klenow fragment of DNA Polymerase I and then digested by MotI.
  • the MotI, Apal fragment containing ACAT and the linearized vector were ligated by T4 DNA ligase to construct the ACAT expression vector pRS426-ACAT. This placed the ACAT ORF directly downstream of the yeast GAL1 /1 0 promoter (Fig. 1A) .
  • Yeast cells were grown overnight in SC-ura dextrose (2S) liquid medium. Cells were harvested at mid-log phase and washed with sterile water twice. The cells were resuspended in 10ml of SC-ura medium containing 2V> galactose and 1 raffmose and grown overnight. Cells were lysed by vortexmg with glass beads m 0.01 M borate, 0.15 M NaCl and resuspension of the low speed pellet m 1" SDS and 5M urea.
  • Denaturing gel electrophoresis (5 ⁇ g total protein per lane) was performed using 120 polyacrylamide for the resolving gel m the presence of 0.1% sodium dodecyl sulfate (26) . After SDS-PAGE separation, the proteins were electro- blotted to nitrocellulose. The membrane was blocked in 5 ⁇ . non-fat milk in 20mM T ⁇ s-HCl, 137mM NaCl and 0.1% Tween- 20 (TBST) and probed with 2.8 ⁇ g/ml of the DM10 ⁇ ACAT antibody (27) in TBST-lo, non-fat milk for 1 hour. Detection of the immune complexes was attained using horse radish peroxidase-conjugated secondary anti-rabbit IgG antibody and the ECL Western blotting detection reagent (Amersham) .
  • Yeast strains containing pRS426-ACAT or vector control were grown m 5ml SC-ura, 2V glucose, medium to a density of 10 cells/ml. The cells were harvested, washed twice with sterile water and then resuspended m 10ml SC-ura media containing 2V galactose and 1° raffmose. For experiments to test the effect of 25-hydroxy cholesterol on sterol esterification, 10 or 25 ⁇ g/ml 25-hydroxy cholesterol m ethanol was added to the growth medium at this point.
  • In vitro (microsomal) sterol esterification assay Yeast cells containing pRS426-ACAT or the vector control were grown m 5ml of SC-ura media containing 2 ⁇ glucose to a density of about 10 J cells/ml. The cultures were diluted to 500 ml SC-ura media containing 2 % glucose and grown overnight. The cells were harvested, washed twice with sterile water, and then resuspended m 500ml ura-CM media containing 2o galactose and 1" raffmose and allowed to grow for six hours before freezing as a cell pellet.
  • Frozen yeast pellets were quick-thawed at 37°C, washed in 2x pellet volumes of homogenization buffer (HB; 0.1M potassium phosphate, 0.5mM EDTA, ImM glutathione, 20 ⁇ M leupeptm, lO ⁇ g/ml benzamidme, and 2mM PMSF) and spun at 2,000g x g to re-pellet cells. The supernatant was removed and another 2 volumes of HB added to resuspend the cells. The suspension was shaken with intermittent cooling in the presence of 1.0 g of 0.5mm diameter glass beads in a mmi-beadbeater (Biospec Products) at 5,000rpm for 3 x 1 minute intervals.
  • HB homogenization buffer
  • the resulting homogenate was spun at 1,000 x g for 5 mm and 15,000 x g for 15 mm. The supernatant was removed and spun at 105,000 x g for 1 hour to pellet the microsomes. This pellet was resuspended m ACAT buffer (0.1M potassium phosphate, ImM GSH, pH 7.4) and protein concentration determined (28) . Microsomes were aliquoted and frozen at -70°C. Rat liver microsomes were prepared as described previously (29) .
  • Enzyme activity was determined by the rate of incorporation of [ M C] oleoyl-CoA or f ' C] cholesterol into steryl ester (29) .
  • the standard assay m duplicate or triplicate, contained 200 ⁇ g of microsomal protein, 1 mg BSA, 20 nmol oleoyl-CoA and 20 ⁇ g cholesterol m a final volume of 200 ⁇ l of 0.1M potassium phosphate butter, pH 7.4, containing ImM glutathione.
  • labeled oleoyl-CoA or [ A C ] cholesterol was added after the reaction had been stopped.
  • the inhibitors were added in 5 ⁇ l of Me SO and compared to a solvent control of 5 ⁇ l Me SO.
  • S58035 and DuP 128 were used at a concentrations of 50 and 0.5 ⁇ M, respectively.
  • cold sterols were included whereas those utilizing [ 14 C] cholesterol (40,000 dpm/nmol) used unlabelled acyl-CoAs.
  • sterols were suspended m reaction buffer with the aid of Triton WR-1339 at a ratio of 30:1 (Triton/sterol, w/w) (29) . After premcubation for lb minutes at 37°C, the reaction was initiated by the addition of oleoyl-CoA.
  • the assay was stopped after 2.5 mm by the addition of 4 ml chloroform/methanol (2:1) .
  • Phase separation was induced by the addition of water (800ul) and [ ⁇ ] -cholesteryl oleate (30,000 dpm) and 15 ⁇ g cholcsteryl oleate were added as the internal standard and carrier, respectively.
  • the chloroform layer containing the lipi ⁇ s was dried under nitrogen and resuspended m lOO ⁇ l chloroform for spotting on ITLC-SA thm layer plates (Gelman Sciences) . Lipids were separated and quantitated as described above.
  • the specific activity of ACAT was determined as picomoles of either cholesterol or oleoyl-CoA ccnverted to steryl ester/mm/per mg of protein.
  • yeast eukaryotic micro-organism Saccnaromyces cerevi siae
  • yeast gene products tha ⁇ exhibit significant structural and functional homology to the putative catalytic component of cholesterol esterification in human macrophages were identified.
  • l r was snown that deletion of these genes produces a yeast cell lacking sterol ester
  • AREl are2 " 224+49 arel”
  • ARE2 6371133 arel
  • m vi tro measurements of sterol esterification essentially mirrored the assays performed m vi vo (table 1) .
  • Deletion of both AREl and ARE 2 produces a strain devoid of microsomal ACAT activity.
  • AREl encodes the minor isoform m terms of its contribution to esterification, whereas Are2p is the major esterification enzyme (-80 ⁇ of wild-type), m vi t ro and i n vi vo .
  • the activity of the yeast isoforms in wild-type cells was elevated 2-3 -fold after equilibration of exogenous cholesterol into microsomes prior to the assay (not shown) . This increase in activity is similar to that observed m rat liver microsomes (29) .
  • a human ACAT cDNA originally isolated from macrophages was expressed.
  • the cDNA was fused to the divergent inducible promoter from the yeast GAL1 / GAL1 0 genes in pRS426-ACAT as described above (Fig. 1A) .
  • a conditional promoter (m this case induced by galactose), was used to circumvent possible toxicity of high level expression of human ACAT m yeast. In fact, no toxicity was observed.
  • the plasmid was used to transform AREl are2 , arel ARE2 , arel are2 " , and wild type yeast strains.
  • Transformants were incubated overnight in SC-ura, galactose/raffinose to induce expression of hACAT. Proteins were prepared and analyzed by SDS-PAGE and immunoblotting with DM10 anti- ACAT antibody. A protein of approximately 48 kDa was detected under inducing conditions m the ACAT expressing strain and not in the non-expressing strain (vector control, Fig. IB) . The size of the protein is smaller than the predicted size of the ACAT primary translation product (63.8 kDa) but co-migrates with proteins isolated from mouse adrenal microsomes, consistent with previous reports (27,30) . The lower molecular weight species is most likely a degradation product of the hACAT protein; it is detectable m the mouse adrenal extracts on darker exposure (not shown) , and is also observed m preparations described elsewhere (27) .
  • ACAT expressing strains were assayed m vi vo for their ability to esterify endogenous yeast sterols (primarily ergosterol) .
  • hACAT protein there were no alterations in incorporation of [''el oleate into sterol ester in wild type or single ARE mutant yeast cells.
  • m the are! are2 ' double disruption strain expressing pRS426-ACAT, a low but significant esterification activity was detected at about 4% of a wild type yeast cell (Fig. 2A) .
  • the activity endowed by the hACAT construct was further confirmed as genuine when it was observed to increase in response to incubation of the cells with 25-OH cholesterol (Fig. 2B) .
  • the level of activity was not commensurate with the mass of protein observed in the immunoblot. It is possible that the human ACAT expressed m yeast lost most of its activity due to mis-foldmg or aberrant secondary modification of the protein.
  • yeast the predominant sterol is ergosterol which differs from cholesterol by the presence of unsaturation at the C-7 position and at C-22, with an extra methyl group at C-24. It has been shown that ergosterol is not a good substrate for rat liver microsomal ACAT (18) .
  • yeast an in vitro assay system to measure ACAT activity (Table 2) was utilized.
  • Wild type 317+8.48 762+52.33 0 71 arel " are2 24.514.95 35+25.46 arel " are2 " + hACAT 60.5 ⁇ 27.58 355.5+98.29 94 92
  • microsomes from single arel and are2 mutants were prepared and assayed. ilsmg a range of concentrations of the inhibitor, the IC of Arelp and Are2p were estimated to be 31 and 34 uM respectively, whereas the IO 0 of a preparation from wild type cells was 20 uM. The IC , of rat ACAT in the same experiment was estimated as 40 ⁇ M.
  • Wild-type 66 ⁇ 33 1081+33 386158 353+112 arel ⁇ are2 + hACAT 98+67 305122 35+35 6412
  • acyl-CoA substrate specificity was assessed by the esterification of radiolabelled cholesterol m the presence of various unlabeled acyl-CoAs .
  • Human ACAT expressed m yeast utilized various acyl-CoAs as substrates with palmitoyl, oleoyl, linoleoyl, and arachidonyl CoAs showing similar activity and stearoyl-CoA somewhat less.
  • yeast esterification was specific for C : unsaturated fatty acids .
  • Rat liver enzymes showed a broader specificity than either of the yeast enzymes, but m no case was activity against arachidonyl-CoA observed.
  • microsomes from yeast strains deficient m AREl , ARE2, or both genes display identical relative levels of in vi tro activity to those observed m vivo, rules out a direct role for these proteins in sterol translocation.
  • substrate specificities of both the yeast and human enzymes expressed m yeast are most consistent with the ARE and hACAT proteins acting as catalytic components of the enzymatic complex as opposed to regulatory factors.
  • the human enzyme demonstrates the predicted response to these reagents, namely increased activity and inhibition, respectively. Since the transcription of the hACAT cDNA is driven by a yeast promoter regulated only by carbon source, these responses, particularly the induction by 25- hydroxy cholesterol, clearly act at a post-trancriptional point. This is in confirmation of previous studies (30) .
  • the 71o inhibition observed with S58035 in wild type yeast could be due to preferential inhibition of Are2p or to partial inhibition of both isoforms.
  • An analysis of microsomal preparations from single are mutants refutes this latter hypothesis; Arelp and Are2p were equally sensitive to the fatty-acyl analogue. The observations contradict those of Yu et al .
  • this disclosure also confirms the fidelity of the yeast expression system with which to study the human enzyme. It is clear that the expression of hACAT m yeast occurs at high levels in terms of mass and that the reaction proceeds m a similar manner to that performed in mammalian cells. Thus, it seems likely that the sterol substrate specificities and pnarmacological inhibitions described here for recombmant hACAT, essentially reproduce those observed with the enzyme m its normal cellular context.
  • the acyl-CoA specificity profile is more complex.
  • the hACAl expressed in yeast utilized a much broader range of iatty acids for esterification than either the yeast or the rat liver enzyme (Table 4) .
  • the ratio of lmoleate/oleate m sterol ester was low (0.15), however the ratio m phospholipid was high (3.4) suggesting a preference for Imoleate incorporation into phospholipid under these assay conditions.
  • radiolabelled cholesterol was added as a tracer m acetone, while m these experiments, the cholesterol was equilibrated with microsomes using the detergent Triton WR-1339.
  • the non-ionic detergent may facilitate the interaction of lmoleoyl-CoA with the microsomal ACAT.
  • the predominant mammalian hepatic sterol ester which reflects ACAT activity is cholesteryl oleate, but again this may be due to normally low concentrations of the essential 18:2 fatty acid or to alternate metabolism.
  • cholesteryl oleate when a diet rich in Imoleate was fed to African Green monkeys, the amount of hepatic cholesteryl Imoleate increased by 3.3 - fold (35) .
  • TCCCACTGTA AGATGGGGTT ATGTCGCTAT GAAGTTTGCA CAGGTCTTTG GTTGCTTTTT 2400
  • CTATGTGTAC TACATCTTTG AAAGGCTTTG TGCCCCCTTG TTTCGGAATA TCAAACAGGA 2460
  • Ala Lys Lys lie Lys Leu Thr Ala Glu Ala Glu Glu Leu Lys Pro Phe 50 55 60
  • CTTCTTCTGC TTTTTTCCTC TTTATCACAC TATGTATGTG CTGCTCATCT CTTCTTTTTA 42 0
  • ATCCTCGCCC CACGCTCCTG GACAGCGCCA TCAACGTGCC CTTCCAGACG ACTTTCAAAG 1 08 0
  • GGGATGCCAT TTTGAACTGT GTGGCTGAAT TGACAAGATT TGGCGACAGA TATTTCTACG 1920
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO: 12: TAGCTCCACA GAACAGTTGC AGG 23

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Abstract

L'invention concerne un vecteur d'expression comprenant un acide nucléique isolé qui code le phénotype sauvage de l'enzyme I apparentée à l'acylcoenzyme A:cholestérol acyltransférase dans une liaison opérationnelle avec un promoteur de transcription d'ARN. On décrit également un vecteur d'expression renfermant un acide nucléique isolé qui code le phénotype sauvage de l'enzyme II apparentée à l'acylcoenzyme A:cholestérol acyltransférase dans une liaison opérationnelle avec un promoteur de transcription d'ARN. On décrit enfin un procédé pour identifier un composé chimique capable d'inhiber le phénotype sauvage de l'enzyme II sus-mentionnée, et aussi un procédé pour inhiber la croissance d'un champignon.
PCT/US1997/009160 1996-05-30 1997-05-30 Enzymes apparentees a l'acylcoenzyme a:cholesterol acyltransferase dans la levure et leurs utilisations WO1997045536A1 (fr)

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Citations (1)

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

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5484727A (en) * 1992-10-14 1996-01-16 Trustees Of Dartmouth College Cloned gene encoding acylcoenzyme A: cholesterol acyltransferase (ACAT)

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF BIOLOGICAL CHEMISTRY, 05 October 1993, Volume 268, No. 28, CHANG et al., "Molecular Cloning and Functional Expression of Human Acyl-Coenzyme A: Cholesterol Acyltransferase in Mutant Chinese Hamster Ovary Cells", pages 20747-20755. *
SCIENCE, 31 May 1996, Vol. 272, YANG et al., "Sterol Esterification in Yeast: A Two-Gene Process", pages 1353-1356. *

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