WO2000026365A1

WO2000026365A1 - NOVEL GENE AND PROTEIN ERRη ENCODED THEREBY

Info

Publication number: WO2000026365A1
Application number: PCT/JP1999/006097
Authority: WO
Inventors: Osamu Ohara; Takahiro Nagase; Nobuo Nomura; Kiyoshi Takayama; Hitoshi Toyoda; Makoto Yoshimoto
Original assignee: Kazusa Dna Research Institute Foundation; Taisho Pharmaceutical Co., Ltd.
Priority date: 1998-11-04
Filing date: 1999-11-02
Publication date: 2000-05-11
Also published as: AU3788500A

Abstract

A novel protein ERRη obtained by cloning a human brain-origin cDNA library and a gene errη encoding the same. The gene errη and the novel protein ERRη are usable as drugs or developing drugs.

Description

Specification

Novel genes and their encoded proteins

The present invention relates to a novel human brain-derived protein ERRa having nuclear receptor activity, and a gene errr encoding the protein.

The nuclear receptor is a receptor that binds to a low-molecular-weight, fat-soluble bioactive substance existing in a living body, moves into the nucleus, and becomes activated. The activated nuclear receptor binds to a specific gene sequence (nuclear receptor binding sequence) located in a region that regulates transcription of a specific gene on the chromosome, and regulates the transcription of the specific gene. It plays an important role in regulating metabolism and maintaining homeostasis in living organisms. Steroid hormones, retinoic acid, prostaglandin A, prostaglandin J, etc. have already been reported as low molecular fat-soluble bioactive substances that can bind to such nuclear receptors, and the corresponding nuclear receptors Have also been found. In addition, although the ligand is unknown, ERR (estrogen—relatatedreeceptor), R〇Rretiinoiccaidrrecceptor-reelatedoorphanereceprot, etc., have been reported as proteins that can act as nuclear receptors.

However, particularly in the brain, it is expected that unknown bioactive substances other than the above-mentioned low-molecular fat-soluble bioactive substances exist, and play an important role in the regulation of brain metabolism and maintenance of homeostasis. Is believed to be In addition, it is expected that there is a new nuclear receptor protein that can bind to these unknown physiologically active substances, but there are many unknowns such as characteristics and gene structure.

Therefore, by clarifying new biomolecules that are responsible for nuclear receptor activity in addition to the nuclear receptors that have been reported hitherto, such biomolecules can be used directly as pharmaceuticals or indirectly used as pharmaceutical compounds. the purpose of the can subjected to the search with the inferred to be _t present invention is to identify such a molecule is to use in the development of pharmaceutical, etc., or a pharmaceutical or the like. Disclosure of the invention The present inventors have conducted intensive studies to ascertain a desired protein from genes expressed in the human brain. As a result, the existence of a novel protein ERRa (estrogen-related receptor) and the gene encoding it Successful isolation of the erratum completed the present invention. That is, the present invention relates to (a) a protein consisting of the amino acid sequence of SEQ ID NO: 1, or (b) one or several amino acids in the amino acid sequence of SEQ ID NO: 1 deleted, substituted or added. The present invention relates to a protein comprising a modified amino acid sequence and having nuclear receptor activity.

Furthermore, the present invention relates to (c) a gene comprising the DNA of SEQ ID NO: 2, or (d) a protein which hybridizes with the DNA of SEQ ID NO: 2 under stringent conditions and has nuclear receptor activity. This is for the DNA that encodes

The gene of the present invention, errr, can be isolated from a human brain-derived cDNA library as a cDNA fragment containing the gene. The cDNA library used by the present inventors was prepared based on human brain-derived mRNA commercially available from Clonetech.

In the above-described cDNA library, a protein having nuclear receptor activity is identified by the method of Ohara et al. (DNA Research 4, 53-59 (1997)). An exhaustive cDNA analysis method using a cDNA library was used. Randomly selected 25,000 recombinants from one human brain-derived long-chain cDNA library prepared by the method of Ohara et al. And selected 5 'and 3' Determine the nucleotide sequence and use the DNA analysis program (BLAST and FastA) to select a clone homologous to the previously reported gene encoding ERR / 3 from the sequence on the 5 'side of all clones. So you can find it.

The presence of the protein-coding region (ORF, openread inframe) in the nucleotide sequence can be confirmed by a general-purpose method of analyzing the nucleotide sequence using a computer program. The present inventors convinced that the gene of interest was present in the cDNA sequence, found one ORF in the sequence using a computer, and identified this gene as an err. The encoded protein was named ERR. ERRr of the present invention consists of a total of 458 amino acid residues It is a protein with a molecular weight of about 50 kilodaltons (kDa).

errr is a gene consisting of base pair 1377 (bp) shown in SEQ ID NO: 2. Using this errτ, a recombinant gene can be prepared by a general gene recombination technique using an appropriate host vector system. Suitable vectors include E. coli-derived plasmids (eg, pBR322, PUC118, etc.), Bacillus subtilis-derived plasmids (eg, pUB110, pC194, etc.), yeast-derived plasmids (eg, PSH19 and others), and animal viruses such as bacteriophage II retrovirus and vaccinia virus. At the time of recombination, it is also possible to add a translation start codon and a translation stop codon using an appropriate synthetic DNA adapter. In order to further express the gene, an appropriate expression promoter is connected upstream of the gene. The promoter to be used may be appropriately selected depending on the host. For example, when the host is Escherichia coli, T7 promoter, 1 ac promoter, trp promoter, λ PL promoter, etc., and when the host is Bacillus, SΡΟ promoter, etc.酵母 5 promoter, GAP promoter, ADH promoter, etc. can be used when the host is yeast, and SV40-derived promoter, retrovirus promoter—Yuichi etc. can be used when the host is an animal cell. .

The gene can also be expressed as a fusion protein with another protein (eg, daltathione S transferase, protein A, etc.). The fused ERRa expressed in this manner can be excised using an appropriate protease (eg, thrombin or the like).

Hosts that can be used for expression of ERR include various strains of Escherichiaco 1 i, a bacterium belonging to the genus Escherichia, various strains of Bacill 1 1 u _s_ subti 1 is, a bacterium belonging to the genus Bacillus, and yeasts such as S acchar omv Various cescerevisiae strains and animal cells include COS-7 cells and CHO cells.

As a method for transforming a host cell using the above-mentioned recombinant vector, a transformation method generally used for a selected host cell can be applied.

In the present invention, in addition to the DNA sequence shown in SEQ ID NO: 2, DNAs that hybridize and encode proteins having nuclear receptor activity are also within the scope of the invention.

In other words, in the full-length sequence of the errr, the DNA sequence is partially modified by various artificial treatments, such as site-directed mutagenesis, random mutation by treatment with a mutagen, mutation, deletion, and ligation of DNA fragments by restriction enzyme cleavage. SEQ ID NO: 2 even if the DNA mutant is a DNA that hybridizes under stringent conditions with err and encodes a protein having nuclear receptor activity. Irrespective of the difference from the DNA sequence shown in the above, is within the scope of the present invention.

The extent of the above-mentioned DNA mutation is within an allowable range as long as it has a homology of 80% or more with the DNA sequence of errr. In addition, the degree of hybridization with the err is the same under normal conditions, for example, when the probe is labeled with DIG DNA Labeling kit (Boehringer's Mannheim Cat.No. 1 175033), the DIGE asyH at 32 ° C is used. Hybridized in a yb solution (Beringer's Mannheim Cat. No. 1603558) and membrane in a 50X: 0.5XS SC solution (containing 0.1% [w / v] SDS) (1 XSSC is 0.15M NaC 0.015M sodium citrate) under the condition of washing, and it is only necessary to hybridize to err.

In addition, a protein encoded by a mutant gene having high homology to errr as described above and having a nuclear receptor activity is also included in the scope of the present invention.

That is, even if a mutant has one or more amino acids in the amino acid sequence of ERRa deleted, substituted or added, if the mutant is a protein having nuclear receptor activity, The body is within the scope of the present invention.

The side chains of the amino acids that are the constituents of proteins differ in hydrophobicity, charge, size, etc., but do not substantially affect the three-dimensional structure (also called three-dimensional structure) of the entire protein In this sense, some highly conservative relationships are known empirically and by physicochemical measurements. For example, for substitution of amino acid residues, dalysin (G1y) and proline (Pro), G1y and alanine (A1a) Or valine (Val), leucine (Leu) and isoleucine (Ile), glutamic acid (G1u) and glutamine (Gin), aspartic acid (Asp) and asparagine (Asn), Cysteine (Cys) and threonine (Thr); Thr and serine (Ser) or A1a; lysine (Lys) and arginine (Arg);

Therefore, even if it is a mutant protein resulting from substitution, insertion, deletion or the like in the amino acid sequence of ERRa shown in SEQ ID NO: 1, the mutation is a highly conserved mutation in the three-dimensional structure of ERRa, if a protein that mutant proteins to have a similarly nuclear receptor activity and ERR _r, it can be said to be within the scope of the present invention. The degree of mutation is within an acceptable range when the homology with the amino acid sequence shown in SEQ ID NO: 1 is 80% or more. Industrial applicability

The sequence recognized by ERRa has been found to be present in the 5 'transcriptional regulatory region of the medi urn-cha ί nacy 1 coenzyme ad ehydo rogenase gene, which is important for 3 oxidation of intracellular fat. . Therefore, ERRa is considered to have a function as a nuclear receptor that regulates the transcription of lipid metabolism-related enzyme genes and regulates the amount of fatty acids in the living body (Fig. 1).

This medium-chainacylcoenzyme A dehydorogenase gene is said to be deeply involved in arteriosclerosis and diabetes. Therefore, ERRr itself or a compound capable of binding to ERRr and modulating nuclear receptor function is expected to be a therapeutic drug for arteriosclerosis and diabetes. In addition, by using gene errrs and protein ERRT, search and evaluation of substances having the same function as ERRr, substances that promote or inhibit the function, or substances that promote gene expression, etc. Can be performed efficiently. For example, it is possible to produce a recombinant gene in which a so-called repo overnight gene such as a luciferase gene or a phosphatase gene is linked downstream of a nucleic acid sequence recognized and bound by the ERRa. Prepare a system in which appropriate cells transformed with such a recombinant gene or the recombinant gene and ERR are coexistent in a test tube, and a test substance is prepared. By examining the expression of the reporter gene upon addition of E. coli, a substance that can regulate the nuclear receptor activity of ERRa can be efficiently detected. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail with reference to Examples, but it is needless to say that the present invention is not limited to these Examples. Unless otherwise specified, the experimental procedures used in the following Examples were performed using various standard procedures represented by Molecular Cloning 2nd. Ed. (Cold Spring Harbour Lab. Press, 1989). The procedure can be performed according to the description in a simple experiment manual or the instruction manual of a commercial kit, and under the recommended conditions for each commercial product such as a restriction enzyme. Example 1 Cloning of errrr

1) Construction of human brain-derived long chain cDNA library

Using an oligonucleotide having a Not I site (GACTAGTTCTAGAT CGCGAGCGGCCGCCC (T) 15) (Gibco BRL) as a primer, a human brain-derived mRNA (Clontech) as a type II Super Scrit II reverse transcriptase kit (Gibco BRL) to synthesize double-stranded cDNA. An adapter (Gibco BRL) having a Sail site was ligated with the cDNA. After Not I digestion, cDNA fragments of 3 kb or more were purified by low-melting agarose electrophoresis at a 1% concentration.

The purified cDNA fragment was ligated with a pBluescriptISK + plasmid that had been treated with the Sa1I-NotI restriction enzyme. The recombinant plasmid was introduced into E. coli E1ctRoMax DH10B (Gibco BRL) by electroporation.

Next, 25,000 recombinants were randomly selected from the library, the recombinant DNA was extracted, and the nucleotide sequences on the 5, 5 and 3 'sides of the cDNA portion of 15,000 clones were determined. . For sequencing, a DNA sequencer (PRI SM377, manufactured by ABI) and a reaction kit manufactured by PE Applied Biosystems were used. 2) Selection of clones containing the sequence of err

When the sequence on the 5 'side of all clones determined in 1) was compared with the previously reported ERR) 3 using a DNA analysis program (BLAST and Fast A), the clone name HJ04617 was significant. Showed homology.

3) Determination of nucleotide sequence of DNA fragment

Using a DNA sequencer manufactured by PE Applied Biosystems, the entire base sequences of both strands were determined. Most sequences were determined from shotgun clones using the dye primer method. For some of the nucleotide sequences, oligonucleotides were synthesized based on the determined nucleotide sequences and determined by the primer walking method. SEQ ID NO: 3 shows the entire nucleotide sequence of cDNA of the clone.

The cDNA contains a protein consisting of 458 residues (ORF encoding ERR T (SEQ ID NO: 3). A termination codon appeared in the same reading frame upstream of the methionine residue which is the initiation codon of the protein. Thus, it was confirmed that the amino acid sequence of the protein encoded by the cDNA fragment was the only one shown in SEQ ID NO: 3.

FIG. 2 shows the amino acid homology between ERR / 3 already reported and ERRT of the present invention. Both show high homology. FIG. 3 shows that the DNA binding region of ERRa has particularly high homology to ERRα and β already reported. Example 2 Confirmation of protein expression by invitrotranslation method of err 6 ^,

In the presence of ( ³⁵ S) methionine, the plasmid containing errra prepared in Example 1 was used in the TNT T 7 copper led reticulocyte 1 ysate system.

(Promega) was used to perform invitrotrans. A part of the reaction solution was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by BAS-2000 (Fuji Photo Kogyo). As a result, as shown in FIG. 4, a main band was confirmed at a position of about 50 kDa.

Example 3 Subcloning of err into Expression Vector for Animal Cells

From the clone HJ04617 isolated in Example 1, cDNA (2.4 kb) containing a sequence encoding ERRα was obtained using restriction enzymes Sa1I and Af1II. This was introduced into XhoI and Af1II of an expression vector pcDNA3.1 (-) / Hygro (manufactured by Invitrogen) for animal cells, and the expression vector and pcDNA3.1 were used in the usual manner. Got err-a.

Example 4 Preparation of transformant capable of expressing err

1) Transformation into CV1 cells and acquisition of transformants capable of transient expression

CV1 cells were cultured in a plastic Petri dish having a diameter of 60 mm. The medium used was a MEM medium (manufactured by Gibco, hereinafter referred to as growth medium) containing 10% fetal bovine serum (Dainippon Pharmaceutical), 50 units of Zm1 benicillin, and 50 g of Zm1 streptomycin. The cells were cultured in the presence of 5% C ° 2 at ° C. When the cell density reached 50%, the LI POFE CTAM INE reagent (manufactured by Gibco) containing the pcDNA3.1-err obtained in Example 2 was overlaid on the cells and cultured for 6 hours. Thereafter, the culture medium was replaced with a growth medium and cultured for 48 hours to isolate a transiently expressible transformant. In addition, for use as a control, only a transformant capable of transient expression was introduced into CV1 cells by introducing pcDNA3.1 alone (Vector Invitrogen) in the same manner as described above. Released.

2) Transfer to CHOk 1 cells and obtain stable transformants

CHOk 1 cells were cultured in a plastic Petri dish having a diameter of 60 mm. The medium used was HamF_1 2 (manufactured by Gibco, hereafter referred to as growth medium) containing 10% fetal bovine serum (Dainippon Pharmaceutical), 50 units of penicillin of Zm1 and 50 gZm1 of streptomycin. The cells were cultured at 5 ° C. in the presence of 5% CO 2. When the cell density reached 50%, the LI POFECTAM INE reagent (manufactured by Gibco) containing pcDNA3.1-errr obtained in Example 2 was overlaid on the cells and cultured for 6 hours. The medium was replaced with a growth medium and cultured for 48 hours. After dispersing the cells with trypsin, the cell suspension was dispensed into a 60 mm-diameter plastic petri dish and cultured for another 24 hours. After removing the medium, the medium was replaced with a growth medium containing a hygromycin reagent (manufactured by Gibco; final concentration: 400 g / m 1). The medium containing the reagent was changed every three days, and the cells were cultured for 2 weeks. When the cell colonies became visible to the naked eye, three colonies were isolated using a stainless steel cup. For control, add pc DNA3.1 to C HOk 1 cells only (Invitrogen) Was introduced in the same manner as described above, and a stable transformant was isolated.

Example 5 DNA binding test of ERRa by gel shift assay

The following nucleotides of sequence 11 were synthesized using a DNA synthesizer (380: 6, manufactured by 881).

5 '-GATCGACGCTTTCAAGGTCATATGCG -3'

3 '-CTGCGAAAGTTCCAGTATACGCGATC-5'

This nucleotide was labeled with (r- ^32P ) ATP using T4 polynucleotide kinase (promega) to prepare a labeled probe, which was purified by gel filtration. The plasmid containing errα prepared in Example 1 was subjected to invitro translation using a TNT T 7 coupled reticulocyte lysate system (Promega), and used as an ERRT protein for gel shift assay.

The binding test between the ERRa and the labeled probe was carried out using a gel shift atscore core system (Promega). Binding was detected by native polyacrylamide electrophoresis and analyzed with BAS-2000 (Fuji Photo Kogyo).

When the labeled probe and ERRa coexisted, a shift in the electrophoretic band was observed (Fig. 5, lane a). On the other hand, when a large excess of unlabeled probe was added at the same time, or in the absence of ERRa, no shift in the electrophoretic band was observed (Fig. 5, lane b, lane c). In addition, a band shift was also observed when a large excess of unlabeled probe containing a sequence different from Sequence-1 was added (Fig. 5, lane d). This proved that Sequence-1 contains a sequence that ERRa can specifically recognize and bind to.

In the above operation, the compound is added to the binding reaction solution between the ERRa and the labeled probe, and the gel shift assay is performed to increase or decrease the amount of the shifted band as compared to when no compound is added. As an index, it is possible to search for a compound that regulates the activity of ERRa.

Example 6 Construction of an ERR A binding substance screening method using repo overnight gene 1) Screening method using luciferase gene

The double-stranded oligonucleotide consisting of the sequence-1 shown in Example 5 was s Sim lex Virus Thymidine Kinase Promoter Lucifera zelepo overnight plasmid, pRL—TK Introduction into the BamHI site of Vector (Promega), and plasmid pRL—ERRE TK It was isolated according to a conventional method.

Err stably expressed CHO cells were cultured under the same conditions as in Example 5, and when the cell density reached 50%, the pRL obtained in Example 5—ERRETK2 zg and pcDNA 3.1 (+) / LI POFECTAMINE reagent (manufactured by Gibco) containing 0.2 ng of Neo (manufactured by Invitrogen) was layered on the cells and cultured for 6 hours, and then replaced with a growth medium and cultured for 48 hours. After dispersing the cells with trypsin, the cell suspension was dispensed into a 60 mm-diameter plastic petri dish and cultured for another 24 hours. After removing the medium, the medium was replaced with a growth medium containing a G418 reagent (manufactured by Gibco; final concentration 500 / X g / ml) and a hygromycin reagent (200 / gZml). The medium supplemented with the reagent was changed every three days, and the cells were cultured for 2 weeks. When cell colonies became visible to the naked eye, three colonies were isolated using a stainless steel cup. For use as a control, pRL3.1 ERRETK and PCDNA3.1 (+) ZNeo were also introduced into CHO k1 cells transfected with pcDNA3.1 / Hygro isolated in Example 1 in the same manner as described above. Stable transformants were isolated.

CHO K1 cells stably expressing errrs isolated in this manner were cultured in 96 we11 plates at 100,000 cells with Zwe11, the screening compound was added at a concentration of 10 M, and the cells were further cultured for 6 hours. . After culturing, the luciferase activity is measured using Luciferase Atsusey reagent (Promega), and the compound that has increased or decreased by 50% compared to ERR-free pRL-ERR ET transfected CHO cells was hit. A compound.

2) Screening method using phosphatase gene

The double-stranded oligonucleotide consisting of the sequence-1 shown in Example 5 was replaced with pTK which is a secreted alkaline phosphatase-zelepo overnight plasmid containing Herpes Sim1exVirusThymidineKinase promoter. — Introduced into STKAP Vector (Clontech) to construct and isolate plasmid pERRE-TK-SEAP according to a conventional method. After mixing DNA transfer reagent LF2000 (Gibco) 0.41 and Opti-MEM medium (Gibco) 251, the mixture was kept at room temperature for 5 minutes to prepare a diluted LF2000 solution. After mixing the above plasmid pERRE-TK-SEAP 0.035 g with the pc DNA3.1-err 70.19 g prepared in Example 3 in Opti-MEM medium 251, LF A 2,000-diluted solution was added, and the mixture was kept at room temperature for 20 minutes to prepare a transgenic solution. Using a MEM medium (manufactured by Gibco, hereinafter referred to as growth medium) containing 10% fetal bovine serum (Dainippon Pharmaceutical), 50 units / m1 penicillin, and 50 g / m1 streptomycin, at 37 ° C (:, 5% C0 ₂ was suspended CV 1 cells cultured in the presence to be 60,000 in growth medium 1 00 1, was added gene transfer fluid 50 / X 1, immediately 96 we 1 24 hours after the seeding, a test sample (screening compound) was added at about 10 M, and the cells were cultured for another 24 hours to recover the cell culture solution. The activity of secreted alkaline phosphatase in the liquid can be evaluated using the index CDP-Star (manufactured by Tropics) was used to measure the activity of secreted alkaline phosphatase. The compound that has increased or decreased by 50% as the hit compound.

According to this screening method, the known compound 5α-ρregnane-3a-ol-20-one (manufactured by Sigma) was found to have approximately 3-fold transcription enhancing activity at a concentration of 10 M. . BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the relationship between a sequence that can be recognized and bound by ERRr and a lipid metabolism-related gene located downstream of the sequence.

FIG. 2 shows a comparison of amino acid sequence homology between ERR / 3 and ERRa of the present invention. In the figure,: indicates a highly conserved amino acid substitution site.

FIG. 3 shows a comparison of the homology of the DNA binding region portion of ERR and ERR. 'In the figure indicates a site where amino acid residues do not match in ERR, β, and α.

FIG. 4 shows the results of SDS-PAGE of ER Rr expressed by the in vitro translation method using err 7 ". Fig. 5 shows the results of gel shift assay using ERR "T and a target probe.

Claims

The scope of the claims

1 The following (a) or (b) protein:

(a) a protein consisting of the amino acid sequence of SEQ ID NO: 1

(b) a protein consisting of the amino acid sequence of SEQ ID NO: 1 with one or several amino acids deleted, substituted or added, and having nuclear receptor activity;

2 DNA of (a) or (b) below

(a) DNA comprising the nucleotide sequence of SEQ ID NO: 2

(b) a DNA that hybridizes with the DNA of SEQ ID NO: 2 under stringent conditions and encodes a protein having nuclear receptor activity.