WO2001031002A1 - Nouveau polypeptide, proteine humaine active 22 (scap) impliquee dans la coupure des proteines srebp, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, proteine humaine active 22 (scap) impliquee dans la coupure des proteines srebp, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001031002A1
WO2001031002A1 PCT/CN2000/000389 CN0000389W WO0131002A1 WO 2001031002 A1 WO2001031002 A1 WO 2001031002A1 CN 0000389 W CN0000389 W CN 0000389W WO 0131002 A1 WO0131002 A1 WO 0131002A1
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WO
WIPO (PCT)
Prior art keywords
polypeptide
polynucleotide
binding protein
sterol regulatory
active protein
Prior art date
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PCT/CN2000/000389
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
Original Assignee
Shanghai Bio Road Gene Development Ltd.
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Publication date
Application filed by Shanghai Bio Road Gene Development Ltd. filed Critical Shanghai Bio Road Gene Development Ltd.
Priority to AU12652/01A priority Critical patent/AU1265201A/en
Publication of WO2001031002A1 publication Critical patent/WO2001031002A1/fr

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

Definitions

  • LDL receptors reduce the uptake of LDL by hepatocytes, which indirectly leads to an increase in plasma LDL, which ultimately leads to atherosclerosis and heart attacks.
  • Drugs that inhibit HMG CoA reductase open the feedback system by consuming cholesterol from liver cells and trigger an increase in LDL receptors and a decrease in plasma LDL (Goldstein et al., 1993). This drug is expected to reduce heart attacks and prolong the life of atherosclerotic patients.
  • the key molecules of the cholesterol feedback system are a pair of membrane-bound transcription factors SREBP-1 and SREBP-2. SCAP activity can regulate the splicing of SREBP (Nohturff t A et al., 1999).
  • the gene encoded on the third chromosome has 55% homology at the protein level (homologous protein number AF106583), and the Chinese rat SCAP gene is on the newly discovered human promyelocytic lineage KG-1.
  • the gene KIAA0199 is highly homologous.
  • the polypeptide domain of the present invention is similar to the characteristic domain of the SCAP gene family, the reaction region of the hydrophobic region of SCAP (on the structure of amino acid residues 280-444). It has five transmembrane components. Specific to the transmembrane component of the HMG CoA reductase is a sterol-sensitive domain.
  • the C-terminal hydrophobic region contains several WD repeats.
  • a polynucleotide encoding these isolated polypeptides, the polynucleotide comprising a nucleotide sequence having at least 70 nucleotides with a nucleotide sequence selected from the group consisting of % Identity: (a) a polynucleotide encoding the human sterol regulatory component-binding protein cleavage active protein 22; (b) a polynucleotide complementary to the polynucleotide (a).
  • the polynucleotide encodes a polypeptide having the amino acid sequence shown in SEQ ID NO: 2.
  • isolated human sterol regulatory component-binding protein cleavage active protein 22 means that human sterol regulatory component-binding protein cleavage active protein 22 is substantially free of other proteins, lipids, Sugars or other substances. Those skilled in the art can purify human sterol regulatory component binding protein cleavage active protein 22 using standard protein purification techniques. Substantially pure peptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the human sterol regulatory component binding protein cleavage protein 22 can be analyzed by amino acid sequence analysis.
  • polypeptides of the invention may be glycosylated or may be non-glycosylated.
  • the polypeptides of the invention may also or may not include the initial methionine residue.
  • genomic DM is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating the cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Labora tory Manua 1, Cold Spooning Harbor Labora tory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) the determination of human sterol regulatory component binding protein cleavage active protein 22 The level of transcripts; (4) Detecting protein products expressed by genes by immunological techniques or by measuring biological activity. The above methods can be used alone or in combination.
  • the protein product of the human sterol regulatory component binding protein cleavage active protein 22 gene expression can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). )Wait,
  • pMSXND expression vectors expressed in mammalian cells Lee and Na thans, J Bio Chem. 263: 3521, 1988
  • baculovirus-derived vectors expressed in insect cells in short, as long as it can be replicated and stabilized in a host, any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain origins of replication, promoters, marker genes, and translation Translation of regulatory elements.
  • Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Examples include 100 to 270 base pairs of the SV40 enhancer at the late side of the origin of replication, polyoma enhancers and adenovirus enhancers at the late side of the origin of replication.
  • a polynucleotide encoding a human sterol regulatory component-binding protein cleavage active protein 22 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a polynucleotide containing the polynucleotide or the recombinant vector.
  • Genetically engineered host cells refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • Escherichia coli, Streptomyces bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells insect cells
  • fly S2 or Sf 9 animal cells
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • polynucleotide sequence of the present invention can be used to express or produce recombinant human sterol regulatory component-binding protein cleavage active protein 22 (Sc ience, 1984; 224: 1431). Generally there are the following steps:
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid
  • polypeptides of the present invention as well as antagonists, agonists and inhibitors of the polypeptides, can be directly used in the treatment of diseases, for example, they can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immune diseases.
  • Drugs that inhibit HMG CoA reductase extract the feedback system by consuming cholesterol from liver cells, and trigger an increase in LDL receptors and a decrease in plasma LDL (Goldstein et al., 1993). The drug is expected to reduce heart attacks and prolong the life of atherosclerotic patients.
  • the key molecules of the cholesterol feedback system are a pair of membrane-bound transcription factors SREBP-1 and SREBP-2, and the activity of SCAP can regulate the splicing of SREBP, which indicates that SCAP plays an important role in cholesterol metabolism (Nohturfft A et al. (1999)
  • Cardiovascular diseases Coronary atherosclerotic heart disease such as occult heart disease, angina pectoris, myocardial infarction, strangulation coronary heart disease, hypertension sterol derivative metabolic disorders: (1) Bile acid metabolic disorders such as Bile cirrhosis (2) Sexual developmental disorders during growth and development: precocious puberty, delayed sexual development, sexual differentiation disorders, other developmental defects of the external genitalia (3) Endocrine and metabolic syndromes: Hyperadrenal diseases such as Cushing syndrome Hyperaldosteronism, adrenal insufficiency, such as acute adrenal insufficiency, chronic adrenal insufficiency, tumors, such as lipoma, lipoblastoma, liposarcoma.
  • Coronary atherosclerotic heart disease such as occult heart disease, angina pectoris, myocardial infarction, strangulation coronary heart disease, hypertension sterol derivative metabolic disorders: (1) Bile acid metabolic disorders such as Bile cirrhosis (2) Sexual developmental disorders during growth and
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human sterol regulatory component binding protein cleavage active protein 22.
  • Agonists enhance human sterol regulatory components, binding proteins, cleavage-active proteins, 22 and stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to cell proliferation, such as various cancers.
  • a mammalian cell or a membrane preparation expressing a human sterol regulatory component-binding protein cleavage active protein 22 can be cultured together with a labeled human sterol regulatory component-binding protein cleavage active protein 22 in the presence of a drug. . The ability of the drug to increase or block this interaction is then determined.
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.
  • a mutated human sterol regulatory component-binding protein cleavage active protein 22 may be shortened and lack a signaling functional domain of the human sterol regulatory component-binding protein cleavage active protein 22, although it can interact with downstream Substrate binding, but lacks signaling activity. Therefore, the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of human sterol regulatory component binding protein cleavage active protein 22.
  • the polynucleotide encoding the human sterol regulatory component-binding protein cleavage active protein 22 can be used for the diagnosis of diseases related to the human sterol regulatory component-binding protein cleavage active protein 22.
  • Polynucleotides encoding human sterol regulatory component-binding protein cleavage active protein 22 can be used to detect the expression of human sterol regulatory component-binding protein cleavage active protein 22 or the binding of human sterol regulatory components in a disease state Abnormal expression of protein cleavage active protein 22.
  • the DNA sequence encoding the human sterol regulatory component-binding protein cleavage active protein 22 can be used to hybridize biopsy specimens to determine the expression of the human sterol regulatory component-binding protein cleavage active protein 22.
  • Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and related kits are commercially available.
  • a part or all of the polynucleotide of the present invention can be used as a probe to be fixed on a microarray or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in a tissue.
  • cDNA or genomic sequences between the affected and unaffected individuals need to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosome, such as deletions or translocations that are visible at the chromosomal level or detectable using cDNA sequence-based PCR. Accurately located based on the resolution capabilities of current physical mapping and gene mapping technologies The cDNA to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution and one gene per 20 kb).
  • Example 3 Cloning of a gene encoding human sterol regulatory component-binding protein cleavage active protein 22 by RT-PCR method. Using fetal brain cell total RNA as a template and ol igo-dT as a primer for reverse transcription reaction to synthesize cDNA.
  • Amplification conditions 50 ⁇ l / L KC1, 10mmol / L Tri s-
  • RNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as l-2728bp shown in SEQ ID NO: 1.
  • Example 4 Northern blot analysis of the expression of human sterol-modulated binding protein cleavage active protein 22 gene
  • Total RNA was extracted in one step [Ana l. Biochem 1987, 162, 156-159] This method includes acidic thiocyanate Guanidine phenol-chloroform extraction. The tissue is homogenized with 4M guanidine isothiocyanate-25m sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform are added. -Isoamyl alcohol (49: 1), mixed and centrifuged.
  • Pr imer4 5 '-CCCGGATCCTCACACTTGATCCCGAAGGCGG-3' (Seq ID No: 6)
  • the 5 ends of these two primers contain Ndel and BamHI digestion sites, respectively, followed by the 5 'and 3' coding sequences of the target gene , Ndel and BamHI restriction sites correspond to the expression vector plasmid pET-
  • Ndel and BamHI were used to double-digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase.
  • the ligation product was transformed into coliform bacteria DH5 a by calcium chloride method. 1 After culturing overnight on LB plates containing kanamycin (final concentration 30 g / ml), use colony PCR method to screen positive clones and perform sequencing. A positive clone (pET-0492gl2) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) using the calcium chloride method.
  • Polypeptide synthesizer (product of PE company) was used to synthesize the following peptides specific for human sterol regulatory component binding protein cleavage active protein 22:

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un nouveau polypeptide, une protéine humaine active 22 (SCAP) impliquée dans la coupure des protéines SREBP, et un polynucléotide codant pour ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment des tumeurs malignes, de l'hémopathie, de l'infection par VIH, de maladies immunitaires et de diverses inflammations. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant pour la protéine humaine active 22 (SCAP) impliquée dans la coupure des protéines SREBP.
PCT/CN2000/000389 1999-10-27 2000-10-27 Nouveau polypeptide, proteine humaine active 22 (scap) impliquee dans la coupure des proteines srebp, et polynucleotide codant pour ce polypeptide WO2001031002A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU12652/01A AU1265201A (en) 1999-10-27 2000-10-27 A novel polypeptide, a human sterol regulatory constituent binding protein cutting active protein 22 and the polynucleotide encoding the polypeptide

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN99119857.3 1999-10-27
CN99119857 1999-10-27

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WO2001031002A1 true WO2001031002A1 (fr) 2001-05-03

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PCT/CN2000/000389 WO2001031002A1 (fr) 1999-10-27 2000-10-27 Nouveau polypeptide, proteine humaine active 22 (scap) impliquee dans la coupure des proteines srebp, et polynucleotide codant pour ce polypeptide

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WO (1) WO2001031002A1 (fr)

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [online] 20 April 1999 (1999-04-20), Database accession no. AC005598 *
DATABASE GENBANK [online] 5 May 1999 (1999-05-05), Database accession no. AC005088 *
DATABASE GENBANK [online] 9 October 1999 (1999-10-09), Database accession no. AC008115 *
GENOME RES., vol. 8, no. 11, November 1998 (1998-11-01), pages 1097 - 1108 *

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