ARV1, a protein involved in sterol uptake and sterol hemeostasis in the budding yeast, S. cerevisiae, and a functional human ARV1

Abstract

This invention provides an isolated nucleic acid which encodes mammalian ACAT-Related Enzyme 2 Required for Viability protein (ARV1p), wherein the encoded ARV1p protein has the amino acid sequence set forth in FIG. 7. This invention provides an isolated nucleic acid which encodes yeast ACAT-Related Enzyme 2 Required for Viability protein (ARV1), wherein the encoded ARV1 protein has the amino acid sequence set forth in FIG. 5. This invention provides a purified mammalian ACAT-Related Enzyme 2 Required for Vialbility protein (ARV1p) having the amino acid sequence set forth in FIG. 7. This invention provides a purified yeast ACAT-Related Enzyme 2 Required for Vialbility protein (ARV1p) having the amino acid sequence set forth in FIG. 5. This invention provides a method for identifying a chemical compound which is capable of inhibiting or stimulating ACAT-Related Enzyme 2 Required for Viability protein (ARV1p) which is capable of inhibiting ARV1p. Methods of treatment using pharmaceutical compositions of the invention asre also provided.

What is claimed is:  
     
         1 . An isolated nucleic acid which encodes mammalian ACAT-Related Enzyme 2 Required for Viability protein (ARV1p), wherein the encoded ARV1p protein has the amino acid sequence set forth in FIG. 7.  
     
     
         2 . An isolated nucleic acid which encodes yeast ACAT-Related Enzyme 2 Required for Viability protein (ARV1p), wherein the encoded ARV1p protein has the amino acid sequence set forth in FIG. 5.  
     
     
         3 . The isolated nucleic acid molecule of  claim 1 , wherein the nucleic acid is DNA or RNA.  
     
     
         4 . The isolated nucleic acid molecule of  claim 2 , wherein the nucleic acid is DNA or RNA.  
     
     
         5 . The isolated nucleic acid of  claim 1 , wherein the nucleic acid is cDNA or genomic DNA.  
     
     
         6 . The isolated nucleic acid of  claim 2 , wherein the nucleic acid is cDNA or genomic DNA.  
     
     
         7 . The isolated nucleic acid of  claim 1 , wherein the mammalian ARV1 is a human ARV1 (hARV1).  
     
     
         8 . The isolated nucleic acid of  claim 7  comprising a nucleic acid sequence as set forth in FIG. 6.  
     
     
         9 . The isolated nucleic acid of  claim 2  comprising a nucleic acid sequence as set forth in FIG. 4.  
     
     
         10 . A vector comprising the isolated nucleic acid of  claim 1 .  
     
     
         11 . The vector of  claim 10  further comprising a promoter of DNA transcription operatively linked to the nucleic acid.  
     
     
         12 . The vector of  claim 11 , wherein the promoter comprises a bacterial, yeast, insect, plant or mammalian promoter.  
     
     
         13 . The vector of  claim 12 , further comprising a plasmid, cosmid, yeast artificial chromosome (YAC), bacteriophage or eukaryotic viral DNA.  
     
     
         14 . A host vector system for the production of a polypeptide which comprises the vector of  claim 11  in a suitable host.  
     
     
         15 . The host vector system of  claim 14 , wherein the suitable host is a prokaryotic or eukaryotic cell.  
     
     
         16 . The host vector system of  claim 15 , wherein the prokaryotic cell is a bacterial cell.  
     
     
         17 . The host vector system of  claim 16 , wherein the eukaryotic cell is a yeast, insect, plant or mammalian cell.  
     
     
         18 . A vector comprising the isolated nucleic acid of  claim 2 .  
     
     
         19 . The vector of  claim 18  further comprising a promoter of DNA transcription operatively linked to the nucleic acid.  
     
     
         20 . The vector of  claim 19 , wherein the promoter comprises a bacterial, yeast, insect, plant or mammalian promoter.  
     
     
         21 . The vector of  claim 20 , further comprising a plasmid, cosmid, yeast artificial chromosome (YAC), bacteriophage or'eukaryotic viral DNA.  
     
     
         22 . A host vector system for the production of a polypeptide which comprises the vector of  claim 19  in a suitable host.  
     
     
         23 . The host vector system of  claim 14 , wherein the suitable host is a prokaryotic or eukaryotic cell.  
     
     
         24 . The host vector system of  claim 23 , wherein the prokaryotic cell is a bacterial cell.  
     
     
         25 . The host vector system of  claim 23 , wherein the eukaryotic cell is a yeast, insect, plant or mammalian cell.  
     
     
         26 . A method for producing a mammalian ACAT-Related Enzyme 2 Required for Vialbility protein (ARV1p) which comprises growing the host vector system of  claim 14  under suitable conditions permitting production of the ARV1p and recovering the ARV1p so produced.  
     
     
         27 . A method of obtaining a mammalian ACAT-Related Enzyme 2 Required for Viability protein (ARV1p) in purified form which comprises: 
 (a) introducing the vector of  claim 14  into a suitable host cell;    (b) culturing the resulting cell so as to produce the ARV1p;    (c) recovering the ARV1p produced in step (b); and    (d) purifying the ARV1p so recovered.    
     
     
         28 . A method for producing a yeast ACAT-Related Enzyme 2 Required for Vialbility protein (ARV1p) which comprises growing the host vector system of  claim 22  under suitable conditions permitting production of the ARV1p and recovering the ARV1p so produced.  
     
     
         29 . A method of obtaining a yeast ACAT-Related Enzyme 2 Required for Viability protein (ARV1p) in purified form which comprises: 
 (a) introducing the vector of  claim 22  into a suitable host cell;    (b) culturing the resulting cell so as to produce the ARV1p;    (c) recovering the ARV1p produced in step (b); and    (d) purifying the ARV1p so recovered.    
     
     
         30 . A purified mammalian ACAT-Related Enzyme 2 Required for Vialbility protein (ARV1p) having the amino acid sequence set forth in FIG. 7.  
     
     
         31 . The purified mammalian ACAT-Related Enzyme 2 Required for Vialbility protein (ARV1p) of  claim 30  which is a human ARV1p.  
     
     
         33 . A purified yeast ACAT-Related Enzyme 2 Required for Vialbility protein (ARV1p) having the amino acid sequence set forth in FIG. 5.  
     
     
         34 . An oligonucleotide of at least 15 nucleotides capable of specifically hybridizing with a unique sequence of complementary nucleotides present within a nucleic acid which encodes a mammalian ACAT-Reated Enzyme 2 Required for Vialbility protein (ARV1) having the amino acid sequence set forth in FIG. 7.  
     
     
         35 . An oligonucleotide of at least 15 nucleotides capable of specifically hybridizing with a unique sequence of complementary nucleotides present within a nucleic acid which encodes a mammalian ACAT-Reated Enzyme 2 Required for Vialbility protein (ARV1) having the amino acid sequence set forth in FIG.  5 .  
     
     
         36 . The oligonucleotide of  claim 34  or  35 , wherein the nucleic acid is genomic DNA or cDNA.  
     
     
         37 . A method for identifying a chemical compound which is capable of inhibiting ACAT-Related Enzyme 2 Required for Viability protein (ARV1p) which comprises: 
 (a) contacting an isolated ARV1p with the chemical compound under conditions permitting an effect to occur upon contact between the ARV1p and the chemical compound; and    (b) determining whether the chemical compound inhibits the activity of the ARV1p so as to identify a chemical compound which is capable of inhibiting ARV1p.    
     
     
         38 . A pharmaceutical composition comprising the chemical compound identified by the method of  claim 37  in an amount effective to inhibit ARV1p in a subject and a pharmaceutically effective carrier.  
     
     
         39 . A method for identifying a chemical compound which is capable of stimulating ACAT-Related Enzyme 2 Required for Viability protein (ARV1p) which comprises: 
 (a) contacting an isolated ARV1p with the chemical compound under conditions permitting an effect to occur upon contact between the ARV1p and the chemical compound; and    (b) determining whether the chemical compound stimulates the activity of the ARV1p so as to identify a chemical compound which is capable of stimulating ARV1p.    
     
     
         40 . A pharmaceutical composition comprising the chemical compound identified by the method of  claim 38  in an amount effective to stimulate ARV1p in a subject and a pharmaceutically effective carrier.  
     
     
         41 . A method of treating a subject who has a disease wherein cellular sterol transport or sterol uptake are defective comprising administering the pharmaceutical composition of  claim 38  or  40 .  
     
     
         42 . The method of  claim 41 , wherein the disease is selected from the group consisting of hypercholesterolemia, obesity, neurodegeneration, atherosclerosis, hyperlipidemia.  
     
     
         43 . The method of  claim 41 , wherein the administration of the pharmaceutical composition stops dietary cholesterol absorption by the intestine.  
     
     
         44 . A method of redistributing intracellular sterol between plasma membrane and endoplasmic reticulum in a cell of a subject whose sterol transport into the plasma membrane or sterol uptake by the plasma membrane is defective which comprises administering to the cell the vector of  claim 11 , thereby increasing ARV1 production in the cell so as to redistribute sterol between the plasma mambrane and the endoplasmic reticulum.  
     
     
         45 . The method of  claim 44 , wherein the defect in sterol transport into the plasma membrane or sterol uptake by the plasma membrane is present in disease selected from the group consisting of hypercholesterolemia, obesity, neuro-degeneration, atherosclerosis, hyperlipidemia.  
     
     
         46 . The method of  claim 44 , wherein the administration of the the vector stops intestinal absorption of dietary cholesterol in the subject.  
     
     
         7 . A pharmaceutical composition comprising ARV1 protein having the amino acid sequence set forth in FIG. 7 and a pharmaceutically acceptable carrier.  
     
     
         48 . A method of treating a subject who has a disease wherein cellular sterol transport or sterol uptake are defective comprising administering the pharmaceutical composition of claim  47  to the subject so as to restore the sterol transport, sterol uptake or sterol distribution.  
     
     
         49 . The method of  claim 48 , wherein the defect in sterol transport into the plasma membrane or sterol uptake by the plasma membrane is present in disease selected from the group consisting of hypercholesterolemia, obesity, neuro-degeneration, atherosclerosis, hyperlipidemia.  
     
     
         50 . The method of  claim 48 , wherein the administration of the the pharmaceutical composition stops intestinal absorption of dietary cholesterol in the subject.  
     
     
         51 . A method of treating a subject who has a disease wherein cellular sterol transport or sterol uptake are defective comprising administering to cells of the subject the pharmaceutical composition of claim  47 , so as to redistribute intracellular sterol between plasma membrane and endoplasmic reticulum in the cells of the subject and thereby treat the disease.  
     
     
         52 . The method of  claim 51 , wherein the defect in sterol transport into the plasma membrane or sterol uptake by the plasma membrane is present in disease selected from the group consisting of hypercholesterolemia, obesity, neuro-degeneration, atherosclerosis, hyperlipidemia.  
     
     
         53 . The method of  claim 51 , wherein the administration of the the pharmaceutical composition stops intestinal absorption of dietary cholesterol in the subject.  
     
     
         54 . A method for determining whether elevated levels of intracellular sterol in absence of sterol esterification in a subject are due to ARV1 mutation which comprises: 
 (a) obtaining a nucleic acid sample from the subject; and    (b) comparing the nucleic acid from the sample with the isolated nucleic acid of  claim 1  encoding ARV1p;    (c) determining whether the nucleic acid from the sample of step (a) differs from the isolated nucleic acid of  claim 1 , a difference in the nucleic acid from the isolated nucleic acid of  claim 1  indicating presence of a mutation in the nucleic acid encoding ARV1 from the sample.    
     
     
         55 . A method for determining whether elevated levels of intracellular sterol in absence of sterol esterification in a subject are due to ARV1 mutation which comprises: 
 (a) obtaining nucleic acid from a sample from the subject; and    (b) contacting the nucleic acid of step (a) with an isolated nucleic acid sequence encoding a mutant ARV1 under conditions permitting binding of any nucleic acid from the sample to the nucleic acid sequence encoding a mutant ARV1 so as to form a complex, formation of the complex indicating presence of a mutation in the nucleic acid encoding ARV1 from the sample.    
     
     
         56 . The method of  claim 55 , wherein the isolated nucleic acid encoding the mutant is labeled with a detectable marker.  
     
     
         57 . The method of  claim 56 , wherein the detectable marker is a radioactive isotope, a fluorophor or an enzyme.  
     
     
         58 . The method of  claim 55 , wherein the nucleic acid from the sample is first bound to a solid matrix before performing step (a).  
     
     
         59 . The method of  claim 55 , wherein the sample comprises plasma or blood cells.  
     
     
         60 . A method for treating a subject who has an imbalance in cell sterol levels due to a defect in sterol esterification which comprises introducing the isolated nucleic acid of  claim 1  into cells of the subject under conditions such that the nucleic acid expresses ARV1p, such that the cells obtain sterol from an extracellular source, so as to thereby treat the subject.  
     
     
         61 . A method for treating a subject who has an imbalance in sterol levels in cells due to a defect in sterol esterification which comprises administering ARV1p to the subject, such that the ARV1p transports to the cells extracellular sterol, so as to thereby treat the subject.  
     
     
         62 . A transgenic nonhuman mammal comprising the isolated nucleic acid of  claim 1 .  
     
     
         63 . A transgenic nonhuman mammal comprising the isolated nucleic acid of  claim 1  which is mutated.  
     
     
         64 . An antibody directed to an epitope of a purified mammalian ARV1p having the amino acid sequence set forth in FIG. 7.  
     
     
         65 . The antibody of  claim 64 , wherein the mammalian ARV1p is a human ARV1.  
     
     
         66 . An antibody of  claim 64  or  65  which is a polyclonal antibody or a monoclonal antibody.  
     
     
         67 . A pharmaceutical composition comprising an amount of the oligonucleotide of either of  claim 34  or  35 , effective to prevent overexpression of a human ARV1 and a pharmaceutically acceptable carrier capable of passing through a cell membrane.  
     
     
         68 . A plant producing seeds whose lipid oils content is altered from the wild-type lipid oils content by a change in the expression of nucleic acid which encodes human ARV1 or plant ARV1.  
     
     
         69 . A plant seed whose lipid oils content is altered from the wild-type lipid oils content by a change in the expression of nucleic acid which encode human ARV1 or plant ARV1.  
     
     
         70 . A method of altering lipid oils content of a plant seed which comprises introducing into the nucleic acid of the plant seed an isolated nucleic acid encoding plant ARV1p, wherein the encoded plant ARV1p differs from the wild-type plant ARV1, such that the lipid oils content of the plant seed is altered.  
     
     
         71 . A pharmaceutical composition comprising lipid oils of a plant seed whose lipid oils content is altered from the wild-type lipid oils content of the plant seed by a change in the expression of nucleic acid which encode ARV1 and a pharmaceutically acceptable carrier.  
     
     
         72 . A method for identifying a chemical compound which has antifungal activity which comprises: 
 (a) contacting a yeast cell comprising an isolated nucleic acid which encodes yeast ACAT-Related Enzyme 2 Required for Viability protein (ARV1p), wherein the encoded ARV1p protein is a deletion mutant with the chemical compound under conditions permitting an effect to occur upon contact between the yeast cell and the chemical compound; and    (b) determining whether the chemical compound inhibits yeast ARV1p so as to identify a chemical compound which has antifungal activity.    
     
     
         73 . The method of  claim 72 , wherein the deletion mutant is arvΔ or arvI-1.  
     
     
         74 . A pharmaceutical composition comprising the compound identified by the method of  claim 72  and a pharmaceutically acceptable carrier.  
     
     
         75 . A method of treating a fungal infection in a subject 15 which comprises administering to the subject the pharmaceutical composition of  claim 74 .  
     
     
         76 . A method of treating a fungal infection in a subject which comprises administering to the subject the pharmaceutical composition of  claim 74  and an antifungal agent.  
     
     
         77 . The method of  claim 76 , wherein the antifungal agent is polyene.  
     
     
         78 . The method of  claim 77 , wherein the polyene is nystatin, amphotericin B or filipin.