JPWO2003048360A1 - Novel protein having the property of binding to mammalian rapamycin target protein (mTOR) and gene thereof - Google Patents

Abstract

A novel protein having the property of binding to a mammalian rapamycin target protein (mTOR) and a gene thereof are provided. The gene according to the present invention is a gene encoding any one of the following proteins (a) to (c). (A) A protein comprising the amino acid sequence shown in SEQ ID NO: 2 or 4 in the sequence listing. (B) the amino acid sequence shown in SEQ ID NO: 2 or 4, consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added, and a mammalian rapamycin target protein (mTOR ) A protein that binds to (C) the amino acid sequence shown in SEQ ID NO: 2 or 4, consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added, and a TOS motif (mTOR signaling motif) A protein that has the property of binding to the protein it has.

Description

【図面の簡単な説明】
図１は、タンパク質合成に関与するシグナル伝達を説明する図である。
図２は、ヒト、ショウジョウバエ、線虫、出芽酵母、分裂酵母およびシロイヌナズナにおけるｒａｐｔｏｒおよびそのホモログのアミノ酸配列を示す図である。
図３は、図２に示すアミノ酸配列の続きを示す図である。
図４は、図３に示すアミノ酸配列の続きを示す図である。
図５は、図４に示すアミノ酸配列の続きを示す図である。
図６は、図５に示すアミノ酸配列の続きを示す図である。
図７は、図６に示すアミノ酸配列の続きを示す図である。
図８は、図７に示すアミノ酸配列の続きを示す図である。
図９は、図８に示すアミノ酸配列の続きを示す図である。
図１０は、ヒトおよびマウスにおけるｒａｐｔｏｒのアミノ酸配列を示す図である。
図１１は、図１０に示すアミノ酸配列の続きを示す図である。
図１２は、図１１に示すアミノ酸配列の続きを示す図である。
図１３は、ヒトのゲノム上に位置する、ｒａｐｔｏｒのエキソンとイントロンとを示す図である。
図１４は、図１３上の、エキソン２６からエキソン３４までの詳細を示す図である。
図１５は、ｍＴＯＲに対するモノクローナル抗体を用いてｍＴＯＲを免疫沈降し、ｅＩＦ４ＥＢＰ１に対するリン酸化活性を示す図である。
図１６は、新規ｍＴＯＲ結合タンパク質ｒａｐｔｏｒをｍＴＯＲとともに精製後、ｍＴＯＲを含む分画（ｆｒ．７）と含まない分画（ｆｒ．１３）とに分け、抗ｍＴＯＲモノクローナル抗体（ｍＴＯＲｉｐ）、または、ｎｏｒｍａｌ ｍｏｕｓｅ ｇｌｏｂｕｌｉｎ（ＮＭＧｉｐ）を固相化したプロテインＧセファロースビーズによる免疫沈降を行い、タンパク質を１％ＮＰ４０またはグリシン（ｐＨ＝２．８）で溶出し、ＳＤＳ−ＰＡＧＥでタンパク質を分離後、銀染色を行った結果を示す図である。
図１７は、ｒａｐｔｏｒのペプチド断片を、３０％アセトニトリル／０．１％ギ酸を用いてＺｉｐ−Ｔｉｐから溶出し、Ｑ−Ｔｏｆ２（マイクロマス社）を用いたＥＳＩ−Ｑ−ＴＯＦ−ＭＳによって、その溶出液を解析した結果得られたスペクトルを示す図である。
図１８は、ｒａｐｔｏｒのペプチド断片を、５０％アセトニトリル／０．１％ギ酸を用いてＺｉｐ−Ｔｉｐから溶出し、Ｑ−Ｔｏｆ２（マイクロマス社）を用いたＥＳＩ−Ｑ−ＴＯＦ−ＭＳによって、その溶出液を解析した結果得られたスペクトルを示す図である。
図１９は、図１７から同位体イオンを除いたスペクトルを示す図である。
図２０は、図１８から同位体イオンを除いたスペクトルを示す図である。
図２１は、ペプチドマスフィンガープリンティング法による同定の際に帰属できなかった２種のイオンのスペクトルを示す図である。
図２２は、ｍ／ｚ＝５７９．３３（２＋）のＭＳ／ＭＳ解析で得られたプロダクトイオンの情報に基づき、得られたアミノ酸配列を示す図である。
図２３は、ｍ／ｚ＝６１５．８２（２＋）のＭＳ／ＭＳ解析で得られたプロダクトイオンの情報に基づき、得られたアミノ酸配列を示す図である。
図２４は、新規に解明されたｒａｐｔｏｒのＮ末端アミノ酸配列と、そのアミノ酸配列を示すスペクトルとを示す図である。
図２５は、図２４の示す、Ｎ末端アミノ酸配列とそのアミノ酸配列を示すスペクトルとの続きを示す図である。
図２６は、ＭＨ＋＝３６６４．７１のイオンの高質量側に位置し、３６８０．７８のイオンを示す図である。
図２７は、細胞上清を用いて免疫沈降し、界面活性剤（ＮＰ４０）の有無で条件分けを行い、抗ｍＴＯＲ抗体および抗ｐ１５０抗体（抗ｒａｐｔｏｒ抗体）を用いてウエスタンブロットを行った結果を示す図である。
図２８は、ＨＡ−ｍＴＯＲ、ＦＬＡＧ−ｐ１５０、ＧＳＴ−４ＥＢＰ、ＧＳＴ−ｐ７０、ＧＳＴを形質移入し、その細胞の上清をグルタチオンセファロースビーズとインキュベートし、抗ＨＡ抗体と抗ＦＬＡＧ抗体との混合物、および抗ＧＳＴ抗体でブロットした結果を示す図である。
図２９は、ＨＡ−ｍＴＯＲ、ＦＬＡＧ−ｐ１５０、ＧＳＴ−４ＥＢＰ、ＧＳＴ−ｐ７０、ＧＳＴを形質移入し、その細胞の上清を抗ＦＬＡＧ抗体で免疫沈降し、抗ＨＡ抗体と抗ＦＬＡＧ抗体との混合物、および抗ＧＳＴ抗体でブロットした結果を示す図である。
図３０は、ＨＡ−ｍＴＯＲ、ＦＬＡＧ−ｐ１５０、ＧＳＴ−４ＥＢＰ、ＧＳＴ−ｐ７０、ＧＳＴを形質移入し、その細胞の上清を抗ＨＡ抗体でウエスタンブロットを行った結果を示す図である。
図３１は、ＨＡ−ｍＴＯＲ、ＦＬＡＧ−ｐ１５０を形質移入し、その細胞上清を抗ＨＡ抗体で免疫沈降し、ＮＰ４０を含む洗浄用緩衝液またはＮＰ４０を含まない洗浄用緩衝液で洗浄し、ＧＳＴ−４ＥＢＰ１を基質として、γ−^３２Ｐ−ＡＴＰ存在下でリン酸化アッセイを行い、解析した結果を示す図である。
図３２は、ＦＬＡＧ−ｐ１５０をトランスフェクションし、その細胞上清を抗ＦＬＡＧ抗体で免疫沈降し、ＮＰ４０を含むまたは含まない洗浄用緩衝液で沈降物を洗浄し、ＧＳＴ−４ＥＢＰ１を基質にγ−^３２Ｐ−ＡＴＰ存在下でリン酸化アッセイを行った後の解析結果を示す図である。
図３３は、予想される各エキソンについて、対応するｃＤＮＡ配列と、エキソンの塩基数と、エキソンの配列番号と、エキソンを含んでいる配列の配列番号と、エキソンを含んでいる配列におけるエキソン開始位置および終了位置とを一覧にした表である。
図３４は、ＨＥＫ２９３細胞に、ＦＬＡＧ−ｒａｐｔｏｒ（ＦＬＡＧ−ｐ１５０）と、ＧＳＴ融合ｐ７０Ｓ６キナーゼ（ＧＳＴ−ｐ７０Ｓ６ｋ）、２８番目のフェニルアラニンをアラニンに置換したＧＳＴ−ｐ７０Ｓ６ｋの変異体（ＧＳＴ−ｐ７０Ｓ６ｋ−Ｆ２８Ａ）、ＧＳＴ融合ＰＤＫ１（ＧＳＴ−ＰＤＫ１）のいずれかとの共発現を行い、それらの細胞溶解液と抗体とを用いてウェスタンブロッティングを行った結果を示す図である。
図３５は、ＨＥＫ２９３細胞に、ＦＬＡＧ−ｒａｐｔｏｒと、ＧＳＴ−ｐ７０Ｓ６ｋ、ＧＳＴ−ｐ７０Ｓ６ｋ−Ｆ２８Ａ、カルボキシル末端欠失不活化ｐ７０Ｓ６キナーゼ変異体（ＧＳＴ−ｐ７０Ｓ６ｋ−ＫＭ／ΔＣＴ）のいずれかとの共発現を行い、それらの細胞溶解液と抗体とを用いてウェスタンブロッティングを行った結果を示す図である。
図３６は、ＨＥＫ２９３細胞に、ＦＬＡＧ−ｒａｐｔｏｒと、ＧＳＴ融合４Ｅ−ＢＰ１（ＧＳＴ−４Ｅ−ＢＰ１）、１１４番目のフェニルアラニンをアラニンに変異させたＧＳＴ−４Ｅ−ＢＰ１の変異体（ＧＳＴ−４Ｅ−ＢＰ１−Ｆ１１４Ａ）、ＧＳＴ融合ＰＤＫ１（ＧＳＴ−ＰＤＫ１）のいずれかとの共発現を行い、それらの細胞溶解液と抗体とを用いてウェスタンブロッティングを行った結果を示す図である。
図３７は、ＨＥＫ２９３細胞に、ＦＬＡＧ−ｒａｐｔｏｒ、ｒａｐｔｏｒのカルボキシル末端欠失変異体（ＦＬＡＧ−ｒａｐｔｏｒ−ΔＣＴ）、ｒａｐｔｏｒのアミノ末端欠失変異体（ＦＬＡＧ−ｒａｐｔｏｒ−ΔＮＴ）、ｒａｐｔｏｒのＷＤリピートドメイン（ＦＬＡＧ−ｒａｐｔｏｒ−ＷＤ）のそれぞれと、ＧＳＴ−ｐ７０Ｓ６ｋまたはＧＳＴ−ｐ７０Ｓ６ｋ−Ｆ２８Ａとの共発現を行い、それらの細胞溶解液と抗体とを用いてウェスタンブロッティングを行った結果を示す図である。
図３８は、抗ｍＴＯＲ抗体またはノーマルマウスＩｇＧ（ＮＭＧ）とＨＥＫ２９３細胞とを用いて免疫沈降物を得て、基質としてＧＳＴ−４Ｅ−ＢＰ１−Ｆ１１４ＡまたはＧＳＴ−４Ｅ−ＢＰ１を用いたｍＴＯＲキナーゼアッセイを行い、さらに、反応液と抗体とを用いて、ウェスタンブロッティングを行った結果を示す図である。
図３９は、上記図３８におけるＧＳＴ−４Ｅ−ＢＰ１にとりこまれた^３２Ｐを測定して、その測定結果を示すグラフである。
図４０は、抗ｍＴＯＲ抗体またはノーマルマウスＩｇＧ（ＮＭＧ）とＨＥＫ２９３細胞とを用いて免疫沈降物を得て、基質としてＧＳＴ−ｐ７０Ｓ６ｋ−Ｆ２８ＡまたはＧＳＴ−ｐ７０Ｓ６ｋを用いたｍＴＯＲキナーゼアッセイを行い、さらに、反応液と抗体とを用いて、ウェスタンブロッティングを行った結果を示す図である。
図４１は、上記図４０におけるＧＳＴ−ｐ７０Ｓ６ｋにとりこまれた^３２Ｐを測定して、その測定結果を示すグラフである。
図４２は、エキソン７の塩基配列（枠で囲った部分）と、そのエキソンの前後にある塩基配列とを示す図である。
図４３は、エキソン１４の塩基配列（枠で囲った部分）と、そのエキソンの前後にある塩基配列とを示す図である。Technical field
The present invention relates to a novel protein having a property of binding to a mammalian target of rapamycin (hereinafter referred to as “mTOR”) and a gene thereof.
Background art
In mammals, the intracellular target protein mTOR of the organic compound rapamycin known as an immunosuppressant is a 290 kDa (molecular weight 290,000) serine-threonine protein phosphatase consisting of about 2500 amino acids, and is a member of the ATM kinase family. Are known. mTOR is thought to regulate cell growth (cell size) while controlling various molecules involved in transcription and translation of mRNA, regulation of cell cycle, and protein degradation. The whole picture is not clear.
In recent years, the relationship between the above-described signal transmission system via mTOR and the signal transmission system from cell growth factors is being clarified. This will be briefly described with reference to FIG. 1. First, insulin, which is one of the cell growth factors, is considered to be involved in protein synthesis through the following pathway. When insulin binds to the insulin receptor, PI-3 kinase is activated. When PI-3 kinase is activated, PDK1 and p70S6 kinase are sequentially activated, and finally protein synthesis is promoted.
On the other hand, the signal transduction system via mTOR will be described in detail below, including the history of research from the discovery of rapamycin.
In 1975, a new organic compound was isolated from the bacterium Streptomyces hygroscopicus obtained from the soil of Ister Island floating in the South Pacific and named rapamycin. Several years later, it was discovered that rapamycin had an immunosuppressive effect, and research into its mechanism of action began. In the 1990s, the mechanism of action gradually became apparent. The research results are listed below.
1) It was revealed that rapamycin binds to a small 12 kDa protein FK-506 binding protein-12 (FKBP-12) present in cells to form a complex.
2) Studies using budding yeast revealed that the intracellular target protein of the rapamycin / FKBP-12 complex was TOR (Target of Rapamycin). There are two TORs of Saccharomyces cerevisiae, and they were named TOR1 and TOR2. Both were giant proteins of 300 kDa and had a kinase domain at the carboxyl terminus.
3) This kinase domain is homologous to the ATM gene product that is the causative gene for cerebellar ataxia-telangeletasia, and TOR is classified as a member of the ATM gene product family. . This family group is considered to be a kinase involved in cell cycle checkpoint control.
4) Subsequently, the gene of the mammalian homologue of TOR was cloned and named mTOR (mammalian Target of Rapamycin).
5) For two signaling molecules p70S6 kinase and eIF-4EBP involved in translation of mRNA, p70S6 kinase is completely inactivated and eIF-4EBP is dephosphorylated by treating the cells with rapamycin. mTOR proved to be a signal regulatory molecule upstream of these p70S6 kinase and eIF-4EBP (see FIG. 1).
6) The eIF-4EBP is an eIF-4E binding protein (hereinafter referred to as eIF-4E binding protein), and may be referred to as “eIF4EBP1” or simply “4EBP1”. eIF-4EBP is a molecule involved in translation of mRNA, similar to p70S6 kinase. Most eukaryotic mRNA is 7 ^Me It has a GTP group or Cap structure at the 5 'end. The protein that binds to the Cap structure is eIF-4E. The eIF-4E, which is a scaffold protein, binds to eIF-4E bound to the Cap structure and promotes translation of mRNA. The eIF-4EBP controls the binding of eIF-4G to the Cap structure by binding to eIF-4E competitively with eIF-4G.
The ability of eIF-4EBP to bind to eIF-4E is suppressed by multiple phosphorylation of eIF-4EBP. This phosphorylation site has a serine-proline or threonine-proline motif. Phosphorylation at this site is promoted by the addition of cell growth factors and the abundance of amino acids in the cell environment. This phosphorylation is promoted by mTOR immunoprecipitated from the cell extract. Therefore, this phosphorylation is considered to be carried out by mTOR itself or an unidentified protein phosphorylase bound to mTOR.
Thus, regarding the signal transduction system via mTOR, 1) mTOR acts as a sensor of amino acid balance in the cellular environment in mammals, 2) p70S6 kinase and eIF-4EBP involved in mRNA translation It exists upstream of the molecular group and regulates the activity of each molecule by regulating its phosphorylation state. 3) For activation of the molecular group involved in translation of these mRNAs by cell growth factor, from mTOR input is essential as a priming signal 4) In other words, mTOR regulates protein synthesis and regulates cell growth (cell size) by regulating the phosphorylation state of p70S6 kinase and eIF-4EBP I understand that.
As described above, mTOR and the signal transduction system via mTOR have various physiology such as protein synthesis, transcription and translation of mRNA, regulation of cell cycle, degradation of protein, regulation of cell growth (cell size). It is thought to be involved in various diseases related to the function, and thus related to these physiological functions. Therefore, if the presence of a protein that binds to the above mTOR is clarified, this protein can be used as a candidate protein that regulates the action of the above mTOR, such as pathological analysis of such diseases, development of therapeutic agents, improvement of treatment, etc. It is expected to make an important contribution to the medical and industrial fields.
The present invention has been made in view of the above problems, and an object of the present invention is to provide a novel protein having a property of binding to a mammalian rapamycin target protein (mTOR), a gene thereof, and a method of using the same. .
Disclosure of the invention
In view of the above problems, the inventor of the present application diligently investigated the existence of a molecule that binds to mTOR. As a result, it was found that phosphorylation of eIF-4EBP by the mTOR immunoprecipitate was greatly suppressed by washing with a buffer containing a surfactant during immunoprecipitation of mTOR. The present inventor considered that such phosphorylation suppression occurs because the surfactant dissociates an important factor for phosphorylating eIF-4EBP present in the mTOR immunoprecipitate. .
As a result of further research and analysis using mass spectrometry, 5′-RACE method, etc., the inventor of the present application purifies and identifies a novel protein of about 150 kDa whose binding to mTOR is inhibited by a surfactant. The full-length gene sequence was determined, and the novel protein was found to be a molecule that binds to mTOR and plays an extremely important role in the phosphorylation of eIF-4EBP by mTOR, thereby completing the present invention. It was.
First, the gene according to the present invention is a gene encoding the following proteins (a) to (c).
(A) A protein comprising the amino acid sequence shown in SEQ ID NO: 2 or 4 in the sequence listing.
(B) the amino acid sequence shown in SEQ ID NO: 2 or 4, consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added, and a mammalian rapamycin target protein (mTOR ) A protein that binds to
(C) the amino acid sequence shown in SEQ ID NO: 2 or 4, consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added, and a TOS motif (mTOR signaling motif) A protein that has the property of binding to the protein it has.
The protein of the above (a) is a novel protein raptor that has been purified and identified by the present inventor and clarified its full-length amino acid sequence, as will be described later. SEQ ID NO: 2 shows the amino acid sequence derived from human, SEQ ID NO: 4 shows the amino acid sequences derived from mice. This raptor is a molecule that binds to mTOR and regulates the enzyme activity (kinase activity) of mTOR, and is considered to be a molecule that plays an extremely important role in the signal transduction system via mTOR. Therefore, the gene and protein according to the present invention are not only useful as research materials for research and analysis of the above-mentioned mTOR-mediated signal transduction system and its regulatory mechanism, but through such research, the signal transduction system and its There is a possibility that it can be widely used for pathological analysis of various diseases related to regulatory mechanisms, development of therapeutic agents, and improvement of treatment.
Further, the inventor of the present application (1) that the raptor binds not only to eIF-4EBP but also p70S6 kinase, and (2) eOS-4EBP and p70S6 kinase, which are mTOR substrate proteins, include a TOS motif ( a conserved sequence consisting of 5 amino acids called mTOR signaling motif), and that the TOS motif is essential for mTOR signaling, (3) binding of eIF-4EBP and p70S6 kinase to raptor, It was clarified that the above TOS motif is essential for phosphorylation of these substrate proteins by mTOR.
That is, the function of raptor is considered to be a scaffold protein that binds to mTOR and its substrate proteins, eIF-4EBP and p70S6 kinase, and provides a field for phosphorylation.
TOS of the TOS motif is an abbreviation for TOR Signaling. That is, the TOS motif is a TOR signaling motif and is an essential sequence (sequence consisting of 5 amino acids) for signal transmission from mTOR to downstream p70S6 kinase and eIF4EBP. And TOS motifs were identified on those downstream molecules. That is, the TOS motif is a sequence called FDDL (human) that exists near the amino terminus in p70S6 kinase. The TOS motif is a sequence called FEMDI (human) present at the carboxyl terminus in eIF4EBP.
The above “gene” means at least genomic DNA, cDNA, and mRNA, and the gene according to the present invention includes (1) nucleotides 183 to 4187 of the nucleotide sequence shown in SEQ ID NO: 1. CDNA derived from a human having the sequence as an open reading frame, {circle around (2)} of the nucleotide sequence shown in SEQ ID NO: 3, mouse-derived cDNA having the nucleotide sequence of positions 175 to 4179 as an open reading frame, and {circle around (3)} of these cDNAs MRNA having a base sequence corresponding to the base sequence, (4) human-derived genomic DNA having each base sequence (each exon sequence) shown in SEQ ID NOs: 5-35, and (5) consecutively in SEQ ID NOs: 36-48 An example is human-derived genomic DNA having a series of base sequences shown.
The “gene” includes not only double-stranded DNA but also single-stranded DNA and RNA such as sense strand and antisense strand constituting the DNA. Furthermore, the “gene” includes sequences such as untranslated region (UTR) sequences and vector sequences (including expression vector sequences) in addition to the sequences encoding the proteins (a) to (c) above. There may be. For example, the gene of the present invention can be amplified as desired by constructing the gene of the present invention by connecting the sequence encoding the proteins (a) to (c) above to a vector sequence and amplifying the gene in an appropriate host. be able to. Moreover, you may use the partial arrangement | sequence of the gene of this invention for a probe. Examples of the case of using the probe in this manner include a case where a partial sequence of the gene of the present invention is immobilized on a chip to constitute a DNA chip, and the DNA chip is used for various tests / diagnosis. .
Secondly, the protein according to the present invention is any one of the proteins (a) to (c) above, that is, (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or 4, (b) SEQ ID NO: 2 Or the amino acid sequence shown in 4, consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added, and has the property of binding to a mammalian rapamycin target protein (mTOR). Or (c) an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 2 or 4, and a TOS motif (mTOR A protein having a property of binding to a protein having a signaling motif).
Here, the “protein” may be in a state isolated and purified from cells, tissues, or the like, or in a state where a gene encoding the protein is introduced into a host cell and the protein is expressed in the cell. There may be. The protein according to the present invention may contain an additional polypeptide. Examples of the case where such a polypeptide is added include a case where the protein of the present invention is epitope-tagged with HA, FLAG, or the like.
In addition, the above “one or more amino acids are substituted, deleted, inserted, and / or added” means substitution, deletion, insertion, and the like by a known mutant protein production method such as site-directed mutagenesis. It means that the number of amino acids (preferably 10 or less, more preferably 7 or less, more preferably 5 or less) that can be added is substituted, deleted, inserted, and / or added. Thus, in other words, the proteins of (b) and (c) above are mutant proteins of the protein of (a), and the “mutation” mentioned here is artificially produced mainly by known methods for producing mutant proteins. Although the introduced mutation is meant, a naturally occurring similar mutant protein may be isolated and purified.
The protein according to the present invention may be further specified by the following properties and characteristics A) to D).
A) It has a property of binding to a mammalian rapamycin target protein (mTOR) and enhancing the enzyme activity of the protein.
B) It has a property of binding to a translation initiation factor binding protein (eIF-4EBP) of eukaryotic cells.
C) It has the property of binding to p70S6 kinase.
D) It has a property of binding to a protein having a TOS motif (mTOR signaling motif).
The protein according to the present invention is a variant of the proteins (a) to (c) described above, and has a property of binding to a mammalian rapamycin target protein (mTOR), a property of enhancing the enzyme activity of the protein, Mutant protein having an abnormality in any of the property of binding to a nuclear cell translation initiation factor binding protein (eIF-4EBP), the property of binding to p70S6 kinase, or the property of binding to a protein having a TOS motif (mTOR signaling motif) It may be. Such a mutated protein is useful in protein functional analysis, for example, by comparing a structure with a wild-type protein, a region essential for activity in the structure becomes clear. In addition, the gene according to the present invention includes a gene encoding such a mutant protein, and such a gene is useful for, for example, production of a new transformant.
Third, the binding inhibitor according to the present invention is a binding inhibitor that inhibits the binding of the proteins (a) to (c) above to the mammalian rapamycin target protein (mTOR), and such binding Examples of the inhibitor include a surfactant such as NP-40 and CHAPS, as well as a binding inhibitor obtained by the screening method of the present invention. Such binding inhibitors are not only useful as research materials for studying and analyzing the above-described mTOR-mediated signal transduction system and its regulatory mechanism, but also through such research, are involved in the signal transduction system and its regulatory mechanism. There is a possibility that it can be effectively used widely for pathological analysis of various diseases, development of therapeutic agents, and improvement of treatment.
Other objects, features, and advantages of the present invention will be fully understood from the following description. The benefits of the present invention will become apparent from the following description with reference to the accompanying drawings.
BEST MODE FOR CARRYING OUT THE INVENTION
(1) Protein raptor according to the present invention and the sequence, structure, etc. of the gene thereof
As described above, the present inventor determined the amino acid sequence of a novel protein of about 150 kDa that binds to mTOR using mass spectrometry, 5′-RACE method, and the like, and the full-length cDNA of the gene encoding the protein. The sequence was successfully determined and this new protein was originally designated as “p150 ^TORAP ". TORAP is an abbreviation for TOR-associated protein. Furthermore, as a result of the functional analysis of this new protein (mTOR binding protein), it has an action of regulating the activity of mTOR. Therefore, the name of this new protein is finally “raptor (regularly associated protein of mTOR)”. Named. In the following, for convenience of explanation, the novel protein of the present invention is simply referred to as “p150” or “raptor”.
As a result of functional analysis, it was confirmed that the protein raptor binds not only to mTOR but also to eIF-4EBP and p70S6 kinase. That is, the function of raptor is considered to be a scaffold protein that binds to mTOR and its substrate proteins, eIF-4EBP and p70S6 kinase, and provides a field for phosphorylation.
The amino acid sequence of the protein raptor is shown in SEQ ID NOs: 2 and 4. SEQ ID NO: 2 shows the amino acid sequence of the raptor derived from human, and SEQ ID NO: 4 shows the amino acid sequence of the raptor derived from mouse.
The base sequence of the full-length cDNA sequence of the raptor is shown in SEQ ID NOs: 1 and 3. SEQ ID NO: 1 shows the cDNA sequence of a raptor derived from human, and SEQ ID NO: 3 shows the cDNA sequence of a raptor derived from mouse.
In the nucleotide sequence of SEQ ID NO: 1, the 183rd to 4187th nucleotide sequence corresponds to an open reading frame (ORF) region encoding the human-derived protein raptor. Further, in the base sequence of SEQ ID NO: 3, the 175th to 4179th base sequence corresponds to an open reading frame (ORF) region encoding the mouse-derived protein raptor. Each cDNA sequence shown in SEQ ID NOs: 1 and 3 contained an untranslated region (UTR) on the 5 ′ side and 3 ′ side in addition to this ORF region.
The ORF region was composed of 4005 bases, both human and mouse, and the amino acid sequence deduced from this gene was 1335 residues.
Based on the above results, further database searches were performed. As a result, it was found that the raptor homologs exist across species, such as Drosophila, nematodes, budding yeast, fission yeast, and Arabidopsis thaliana. 2 to 9 are alignment data showing homology between these homologs. In the figure, 1 is human, 2 is Drosophila, 3 is nematode, 4 is budding yeast, 5 is fission yeast, and 6 is Arabidopsis thaliana. An array is shown. Further, in the figure, black regions indicate amino acids conserved in three or more species, and gray regions indicate amino acids having the same properties (similar).
Comparing the amino acid sequences of the above homologues, it was found that the region from amino acid residues 49 to 667 was conserved very well even across species such as Drosophila, nematode, yeast, and Arabidopsis. . This result suggests that the region from amino acid residues 49 to 667 is important for raptor function across species.
Furthermore, as a result of analysis, it was found that there is a region where the structure of WD-40 repeat is repeated seven times from the 1015th position to the C-terminal of the amino acid sequence of the raptor. The WD-40 repeat structure is considered to be a structure that forms a scaffold for interacting with various signaling molecules. In addition, WD-40 repeat structures can be found in many conventional signal transduction molecules. From these facts, the raptor is considered to have a function as a scaffold protein that transmits various signals.
10 to 12 show the results of examining the homology of amino acid sequences between human-derived raptor and mouse-derived raptor. As shown in the figure, a high homology of 96.55% was observed between human and mouse.
Moreover, the database search of the human genome draft sequence was performed using the cDNA sequence of the above-mentioned human raptor. As a result, it was found that the entire genome sequence of raptor derived from human exists on chromosome 17.
As a result of organizing and analyzing the data obtained by the above search, the expected number of exons is considered to be 34. However, exon 1 could not be identified. Moreover, although it was not possible to connect the full length of the genome at present, the total number of bases was considered to be at least 292258 bases.
Based on the above analysis results, FIG. 13 shows an overall image of the exon-intron structure. In FIG. 14, the figure from the exon 26 to the exon 34 was shown. In FIGS. 13 and 14, X is inserted in the portion where the intron is not continuous.
SEQ ID NOs: 5 to 35 show the base sequences (exon sequences) of exons 2 to 6, exons 8 to 13, and exons 15 to 34 among the expected number of exons 34. SEQ ID NO: 49 shows the base sequence of exon 7, and SEQ ID NO: 50 shows the base sequence of exon 14. Moreover, a series of base sequences shown continuously in SEQ ID NOs: 36 to 48 are full-length genomic DNA sequences of human-derived raptor. In FIG. 33, for each predicted exon, the corresponding cDNA sequence, the number of bases, the corresponding sequence number, and the sequence containing the exon are listed.
In the item “exon” in the table of FIG. 33, the names of exons (exons 1 to 34) are described. In the item “cDNA sequence”, each exon shown in the item “exon” corresponds to the position of the cDNA sequence shown in SEQ ID NO: 1. For example, for exon 2, “345-447” is described in the item “cDNA sequence”. This indicates that exon 2 corresponds to the 345th to 447th bases in the cDNA sequence.
In the table of FIG. 33, the “base number (bp)” item indicates the base number of each exon, and the “exon sequence number” item indicates the sequence number in which each exon is indicated.
In the item “sequence number including exons” in the table of FIG. 33, the item “SEQ ID NO” indicates the sequence number of the base sequence (human genomic DNA sequence) including exons. In the array, the exon start position is indicated in the “exon start position” item, and the exon end position is indicated in the “exon end position” item.
For example, exon 2 is included in the sequence of SEQ ID NO: 36, and corresponds to the 12143th to 12243th bases in the sequence.
In FIG. 33, for exon 7, “exon start position” and “exon end position” in the item “sequence containing exons” are described as 941 and 1000, respectively. These positions indicate the positions of exons when the base sequence shown in FIG. 42 is counted from the top. FIG. 42 shows a region where exon 7 (“E7” in the figure) in FIG. 13 is sandwiched between two X marks in a base sequence.
Similarly, in FIG. 33, for exon 14, “exon start position” is described as 806, and “exon end position” is described as 880. These positions indicate the positions of exons when the base sequence shown in FIG. 43 is counted from the top. FIG. 43 shows a region where exon 14 (“E14” in the figure) in FIG. 13 is sandwiched between two X marks in a base sequence.
(2) Usefulness of the protein raptor according to the present invention and its gene
The above mTOR is a molecule that senses the nutritional state in the cellular environment, in particular the amino acid balance, and controls various cell functions, but it ultimately controls cell growth, in other words, the central molecule that regulates cell size. Has attracted attention as. In addition, evidence that mTOR is associated with various diseases is gradually accumulating. Examples are as follows.
a) Association with diabetes: Glucose intolerance occurs due to homo knockout of p70S6 kinase, a downstream molecule of mTOR. It is possible that the signal transduction system of amino acid-mTOR-p70S6 kinase is involved in undernutrition diabetes seen in developing countries.
b) Relationship with adipocytes: Knockout of 4EBP1, a downstream molecule of mTOR, revealed that white adipocyte differentiation was suppressed, brown adipocyte differentiation was promoted, and energy metabolism was increased. Yes. This is an experimental result suggesting a relationship between mTOR-raptor and “obesity or skinny”.
c) Association with angina pectoris: Angina pectoris is a disease that causes stenosis of the coronary artery, which is the heart's nutritional blood vessel, causing the myocardial infarction when the heart becomes ischemic and worsens. There is an angioplasty treatment method that physically pushes the constricted coronary artery as an angina treatment. Furthermore, a treatment method has been developed in which a thin metallic tube called a stent is inserted into the expanded portion to delay restenosis. However, the restenosis rate is still high even in the treatment using this stent, which is a big problem in this field. However, recently, it has been reported that the rate of restenosis is greatly reduced when rapamycin is infiltrated into this stent.
d) Rapamycin itself or its derivatives have been reported to have anticancer activity, and development and clinical trials of anticancer drugs using the rapamycin skeleton are in progress.
e) Recently, there is a research technique in which mutation is randomly introduced into the genome, an animal in which an interesting phenotype appears is analyzed, and the causative gene is searched. By this method, a mouse called “flat top” in which the first half of the brain was deleted was born, and this causative gene was found to be mTOR. In addition, if rapamycin is continuously administered to mice in early pregnancy, children with similar phenotypes are born. This suggests the possibility that mTOR is involved in brain formation.
From the above situation, the usefulness of the protein raptor (p150), which is a binding molecule of mTOR and plays an important role in phosphorylation of 4EBP1, and the gene thereof include the following utilities.
A) It can be used for screening of a compound (inhibitor) that inhibits the binding between mTOR and raptor, or the binding between raptor and 4EBP1. If such an inhibitor is developed,
(1) It may be a therapeutic agent for obesity and has the potential to lead to the treatment of diabetes, hypertension, antilipidemia, heart disease, etc. caused by obesity.
(2) It may be a therapeutic agent for restenosis after treatment with angioplasty for angina.
(3) There is a possibility of clinical application as an anticancer agent.
B) If single nucleotide polymorphism (SNP) which may exist on the genome sequence of raptor is utilized, application to the following diagnosis can be expected.
▲ 1 ▼ Application to genetic diagnosis of each individual about obesity and skinnyness
(2) Application to clinical diagnosis of drug sensitivity to obesity and skinnyness
(3) Clinical diagnosis of sensitivity to restenosis treatment after angioplasty treatment of angina
(4) Application to clinical diagnosis of sensitivity to anticancer drugs
C) By examining the expression level of raptor mRNA, application to each diagnosis can be expected in the same manner as described in b) above.
D) There is a possibility that the gene therapy using the raptor gene can be used for the treatment of each disease described in the above a).
As described in detail in Examples below, the raptor is a molecule that binds to mTOR and regulates the enzyme activity (kinase activity) of mTOR, and is extremely important in the signal transduction system via the mTOR. It is considered a molecule that plays a role. Therefore, the gene and protein according to the present invention are not only useful as research materials for research and analysis of the above-mentioned mTOR-mediated signal transduction system and its regulatory mechanism, but through such research, the signal transduction system and its There is a possibility that it can be widely used for pathological analysis of various diseases related to regulatory mechanisms, development of therapeutic agents, and improvement of treatment.
The novel protein (raptor) of the present invention is considered to be a scaffold protein that binds not only to mTOR but also to its substrate proteins, eIF-4EBP and p70S6 kinase, and provides a field for phosphorylation (described later). See example).
The mTOR substrate proteins eIF-4EBP and p70S6 kinase have a conserved sequence consisting of 5 amino acids called the TOS motif (mTOR signaling motif), and the TOS motif is used for mTOR signaling. It was known to be essential.
The present inventor has revealed that the above TOS motif is essential for binding of eIF-4EBP and p70S6 kinase to raptor and phosphorylation of these substrate proteins by mTOR. This finding indicates that other proteins with a TOS motif may bind to raptor and become a substrate for mTOR. In addition, the search for substances that inhibit the binding of the raptor and the TOS motif may lead to the development of new drugs that suppress the mTOR signal and suppress the immunosuppressive effect, anticancer action, and coronary restenosis in ischemic heart disease. Showing sex.
As described in the above section a), the substance that inhibits the binding between the protein raptor of the present invention and mTOR (or a protein having a TOS motif such as eIF-4EBP or p70S6 kinase) is a drug candidate molecule. (Drug discovery target) and is medically and industrially useful. The present invention includes a screening method for such substances.
As the screening method of the present invention, various conventionally known methods for examining the presence / absence of binding between substances and the presence / absence of dissociation can be applied and are not particularly limited. For example, the above raptor binds to p70S6 kinase via its amino terminal region (amino acid residue 1 to 904), and this binding is essential for phosphorylation of p70S6 kinase (see the following examples). Therefore, in a cell-free system, any one of the following (1) to (4) is expressed, and the material and mTOR (or a TOS motif such as eIF-4EBP or p70S6 kinase) are added. And a screening method for detecting a substance that inhibits the binding to the protein from the candidate molecules by ELISA or the like.
(1) Protein of the present invention such as the above raptor
(2) A partial protein of the protein of (1) above, which contains at least the 1st to 904th sequence (binding region with p70S6 kinase) in the amino acid sequence of the full-length protein.
(3) Partial protein of the protein of (1) above, wherein the partial protein includes a binding region with mTOR (or eIF-4EBP) in the amino acid sequence of the full-length protein.
(4) Modified protein of (1) above or partial protein of (2) (3) above
Since the raptor binds to p70S6 kinase via its amino terminal region (amino acid residue 1 to 904), eIF-4EBP having the same TOS motif also passes through the amino terminal region. The possibility of binding to raptor is high.
The above-mentioned “(protein) variant” means that one or several (preferably 7 or less, more preferably 5 or less, more preferably 3 or less) amino acids of the protein are substituted, deleted or inserted. , And / or an added variant, when the protein is labeled with a tag such as His or Myc, or when the protein is fused with a fluorescent protein (GFP, luciferase, etc.) or another protein It is used in the meaning including the case where it is modified by phosphorylation or sugar chain bonding.
For mTOR (or a protein having a TOS motif such as eIF-4EBP or p70S6 kinase) used in the above screening method, not only a full-length protein but also a partial protein including a binding region with raptor may be used. Alternatively, a partial protein variant may be used.
Needless to say, the screening method of the present invention is not limited to the above-described method, and screening may be performed in cells using cultured cells or the like instead of screening in a cell-free system.
In addition, (1) a method of searching for a substance that binds to the binding region of mTOR (or a protein having a TOS motif such as eIF-4EBP or p70S6 kinase) with a raptor by fixing the binding region to the column; The screening method of the present invention employs various conventionally known methods for examining the presence or absence of binding between substances or the presence or absence of dissociation, such as a method of searching for substances that inhibit the binding between raptor and mTOR using precipitation-immunoblot. Applicable.
Furthermore, in the screening method of the present invention, screening may be performed using proteins other than humans, for example, mouse homologs, rat homologs, and other homologs of other organisms.
(3) Recombinant expression vector of the present invention
The recombinant expression vector of the present invention comprises the gene of the present invention encoding any one of the proteins (a) to (c), for example, any of the nucleotide sequences shown in SEQ ID NO: 1 or 3 And a recombinant expression vector into which a cDNA having is inserted. For the production of the recombinant expression vector, a plasmid, phage, cosmid or the like can be used, but it is not particularly limited.
The transformant of the present invention is a transformant into which the gene of the present invention encoding any one of the proteins (a) to (c) is introduced. Here, “gene introduced” means that the gene is introduced into a target cell (host cell) so as to be expressed by a known genetic engineering technique (gene manipulation technique). The “transformant” means not only cells / tissues / organs but also animals. The target animal is not particularly limited, and examples thereof include mammals such as cows, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice and rats. In particular, rodents such as mice and rats are widely used as experimental animals and pathological model animals, and many inbred strains have been created, and technologies for fertilized egg culture, in vitro fertilization, etc. are in place. Mice are preferred as experimental animals and pathological model animals, and knockout mice and the like are further analyzed for the functions of the above-mentioned protein raptor and homologs thereof, development of diagnostic methods for diseases involving these proteins, development of treatment methods, etc. Useful for.
The gene detection instrument of the present invention uses at least a part of the base sequence or its complementary sequence in the gene of the present invention as a probe. The gene detection instrument can be used for detection and measurement of the expression pattern of the gene of the present invention under various conditions. Examples of the gene detection instrument of the present invention include a DNA chip in which the probe that specifically hybridizes with the gene of the present invention is immobilized on a substrate (carrier).
The antibody of the present invention is an antibody obtained as a polyclonal antibody or a monoclonal antibody by a known method using any one of the proteins (a) to (c) or a partial peptide thereof as an antigen. The antibody of the present invention can be used for detection and measurement of the protein of the present invention, and may be used for diagnosis and treatment.
(4) Protein and gene acquisition method according to the present invention
[4-1: Gene acquisition method]
As described above, the raptor (p150), which is the protein according to the present invention, and various characteristics such as the sequence, structure, function, and utility of the gene have been described. Hereinafter, the protein and gene acquisition method according to the present invention will be described. .
The method for obtaining the gene encoding the protein raptor is not particularly limited, and a DNA fragment containing the gene sequence is isolated and cloned by various methods based on the sequence information disclosed above. be able to. For example, a probe that specifically hybridizes with a partial sequence of cDNA encoding the raptor may be prepared, and a human or mouse genomic DNA library or cDNA library may be screened. As such a probe, any probe of any sequence / length may be used as long as it specifically hybridizes to at least a part of the base sequence of cDNA encoding the raptor or its complementary sequence. Moreover, what is necessary is just to perform about each step in the said screening on the conditions used normally.
The clone obtained by the above screening can be analyzed in more detail by preparing a restriction enzyme map and determining its nucleotide sequence (sequencing). From these analyses, it can be easily confirmed whether a DNA fragment containing the gene sequence according to the present invention has been obtained.
In addition, the sequence of the probe is selected from regions considered to be important for the function of the raptor (for example, the region from the 49th to the 667th amino acid sequence), and genomic DNA of humans, mice and other organisms. If a (or cDNA) library is screened, there is a high possibility that a gene encoding a homologous molecule or a similar molecule having the same function as the raptor can be isolated and cloned.
The method for obtaining the gene (polynucleotide) according to the present invention includes a method using an amplification means such as PCR in addition to the above screening method. For example, among the cDNA sequences of the above raptor, primers are prepared from the sequences of untranslated regions on the 5 ′ side and 3 ′ side (or their complementary sequences), and human and mouse genomic DNA ( Alternatively, a large amount of DNA fragments containing the polynucleotide of the present invention can be obtained by performing PCR or the like using cDNA) as a template and amplifying the DNA region sandwiched between both primers.
[4-2: Protein acquisition method]
The method for obtaining the protein according to the present invention is not particularly limited. For example, a gene obtained as described above (such as cDNA encoding the above raptor or a homologous molecule thereof) can be obtained by a known method. The protein according to the present invention such as the above-mentioned protein raptor can be easily obtained by incorporating into a microorganism such as Escherichia coli or yeast, or animal cells, and expressing and purifying the protein encoded by the cDNA. When introducing a foreign gene into the host in this way, there are various expression vectors and hosts that incorporate a promoter that functions in the host in the recombination region of the foreign gene. do it. The method for extracting the produced protein varies depending on the host used and the nature of the protein, but the target protein can be purified relatively easily by using a tag or the like.
The method for producing the raptor mutant protein is not particularly limited. For example, site-directed mutagenesis (Hashimoto-Gotoh, Gene 152, 271-275 (1995), etc.), PCR, etc. Using a known mutant protein production method such as a method for producing a mutant protein by introducing a point mutation into a base sequence using a transposon, or a method for producing a mutant strain by inserting a transposon, the gene obtained as described above is used. It can be produced by modifying a base sequence so that one or more bases are substituted, deleted, inserted, and / or added. In addition, a commercially available kit (eg, QuikChange Site-Directed Mutagenesis Kit, manufactured by Stratagene) may be used for the production of the mutant protein.
Many examples of mutant proteins produced as described above have the same activity and function as the wild type. On the other hand, mutations are introduced so that the activity and function are abnormal. Many have already been made. For example, when a mutant protein of the above protein raptor is prepared, a property that binds to mTOR, a property that enhances the enzyme activity of mTOR by causing mutation in the binding region with mTOR or the binding region with eIF-4EBP, Alternatively, it is highly possible that a mutant protein having an abnormality in any of the properties of binding to eIF-4EBP can be produced. Similarly, the property of binding to mTOR by causing mutation in the region considered to be important for the function of raptor (for example, the region from the 49th to the 667th amino acid sequence and the WD-40 repeat region), There is a possibility that a mutant protein having an abnormality in either the property of enhancing the enzyme activity of mTOR or the property of binding to eIF-4EBP may be produced.
[4-3: Gene detection instrument]
The gene detection instrument uses a partial base sequence or its complementary sequence in the gene of the present invention as a probe. For example, a DNA chip formed by immobilizing an oligonucleotide (probe) on a substrate (carrier) can be mentioned. Here, the “DNA chip” mainly means a synthetic DNA chip using a synthesized oligonucleotide as a probe, but also includes an affixed DNA microarray using a cDNA such as a PCR product as a probe.
The sequence used as a probe can be determined by a conventionally known method for identifying a characteristic sequence from a cDNA sequence. For example, SAGE: Serial Analysis of Gene Expression Method (Science 276: 1268, 1997; Cell 88: 243, 1997; Science 270: 484, 1995; Nature 389: 300, 1997; U.S. Pat. No. 5,695,937).
In addition, what is necessary is just to employ | adopt a well-known method for manufacture of a DNA chip. For example, when a synthetic oligonucleotide is used as an oligonucleotide, the oligonucleotide may be synthesized on a substrate by a combination of a photolithography technique and a solid phase DNA synthesis technique. On the other hand, when cDNA is used as an oligonucleotide, it may be pasted on a substrate using an array machine.
Similarly to a general DNA chip, a perfect match probe (oligonucleotide) and a mismatch probe in which one perfect base substitution is performed in the perfect match probe may be arranged to further improve the accuracy of gene detection. Furthermore, in order to detect different genes in parallel, a plurality of types of oligonucleotides may be fixed on the same substrate to constitute a DNA chip.
[4-4: Recombinant expression vector and transformant]
The recombinant expression vector contains the gene of the present invention. The specific type of the vector is not particularly limited, and a vector that can be expressed in the host cell may be appropriately selected. That is, according to the type of the host cell, a promoter sequence may be appropriately selected in order to reliably express the gene, and this and the gene according to the present invention incorporated into various plasmids may be used as an expression vector.
In order to confirm whether or not the gene of the present invention has been introduced into the host cell, and whether or not it is reliably expressed in the host cell, various markers may be used. For example, a gene that is deleted in the host cell is used as a marker, and a plasmid containing this marker and the gene of the present invention is introduced into the host cell as an expression vector. Thus, the introduction of the gene of the present invention can be confirmed from the expression of the marker gene. Alternatively, the protein according to the present invention may be expressed as a fusion protein. For example, the green fluorescent protein GFP (Green Fluorescent Protein) derived from Aequorea jellyfish may be used as a marker, and the protein according to the present invention may be expressed as a GFP fusion protein. Good.
The host cell is not particularly limited, and various conventionally known cells can be suitably used. Specifically, including cells derived from humans or mice, for example, bacteria such as Escherichia coli, yeasts (budding yeast Saccharomyces cerevisiae, fission yeast Schizosaccharomyces pombe), nematodes Caenorhabditis elegans X Oocytes, cultured cells of various mammals (rats, rabbits, pigs, monkeys, etc.), cultured cells of insects such as Drosophila melanogaster, silkworm, etc., but are not particularly limited.
A method for introducing the expression vector into a host cell, that is, a transformation method is not particularly limited, and a conventionally known method such as an electroporation method, a calcium phosphate method, a liposome method, or a DEAE dextran method can be suitably used. .
[4-5: Antibody]
The antibody is an antibody obtained as a polyclonal antibody or a monoclonal antibody by a known method using the protein of the present invention or a partial peptide thereof as an antigen. Known methods include, for example, literature (Harlow et al., “Antibodies: A laboratory manual (Cold Spring Harbor Laboratory, New York (1988))”, Iwasaki et al., “Monoclonal antibody hybridoma and ELISA, Kodansha” (1991). The antibody prepared in this way is effective for detecting the protein of the present invention.
(Example)
Hereinafter, the present invention will be described in detail with reference to examples. In addition, this invention is not limited to description of a following example, A various change is possible within the scope of the present invention. Example 1: Experimental data suggesting the presence of a novel mTOR binding protein
Using a monoclonal antibody against mTOR, mTOR was immunoprecipitated from HEK293 cells, and phosphorylation activity against eIF4EBP1 co-purified with mTOR was analyzed. Specifically, using eIF4EBP1 (GST-4EBP1) with GST-tag as a substrate, γ- ³² The phosphorylation assay of mTOR was performed in the presence of P-ATP, and the sample after the reaction was analyzed by autoradiography after SDS-PAGE.
Moreover, when washing | cleaning after the said immunoprecipitation, four things from which the conditions of washing | cleaning differ were prepared. The conditions are roughly divided into those using a buffer solution containing sodium chloride (NaCl) for washing and those using a buffer solution containing CHAPS (surfactant). Buffers containing sodium chloride were prepared with concentrations of 0.5M, 1M, and 1.5M, respectively. On the other hand, CHAPS having a concentration of 1% was used. Note that CHAPS is a trade name (manufacturer: Calbiochem-Novabiochem Corporation), and 3-[(3-colamidopropyl) dimethylammonio] -1-propanesulfonate is a surfactant having a main component.
Furthermore, another test condition different from the above was prepared. This was obtained by salting out with 30% ammonium sulfate before immunoprecipitation, solubilizing it again in a buffer solution, immunoprecipitating mTOR, and analyzing the phosphorylation activity of mTOR in the same manner as described above.
The experimental results are shown in FIG. In the figure, lanes 1 to 3 are respectively washed with NaCl solutions of 0.5 M, 1 M and 1.5 M, lane 4 is salted out with 30% ammonium sulfate before immunoprecipitation, and lane 5 is This is a result of washing with a buffer containing CHAPS. In the figure, “← mTOR-P” indicates the band position of phosphorylated mTOR, and “← GST-4EBP1-P” indicates the band position of phosphorylated GST-4EBP1.
As shown in FIG. 15, in lanes 1 to 3, phosphorylated GST-4EBP1 bands were observed. That is, even when the mTOR immunoprecipitate is washed with a buffer containing NaCl, the phosphorylation activity (kinase activity) of mTOR against eIF4EBP1 is kept normal. Furthermore, it was found that the phosphorylation activity was very stable even when washed with a buffer containing a relatively high concentration of 1.5 M sodium chloride (lane 3).
In lane 4, a band of GST-4EBP1-P was also observed. That is, also in this case, it was found that the phosphorylation activity of mTOR to eIF4EBP1 was kept normal and the phosphorylation activity to the salt was very stable.
On the other hand, in lane 5, the band of GST-4EBP1-P is hardly recognized. In other words, the phosphorylation activity of mTOR is very weak against washing with surfactants such as NP-40 and CHAPS, and when washed with a buffer containing these surfactants, the kinase activity of mTOR is almost completely eliminated. Turned out to be lost.
The above experimental results suggested that a molecule that binds to mTOR and is essential for the phosphorylation activity of mTOR to eIF4EBP1 exists, and that the binding between the molecule and mTOR is inhibited by the surfactant.
(Example 2: Purification of novel mTOR binding protein raptor)
The novel mTOR binding protein raptor was purified as follows.
First, 5 x 10 frozen ⁹ Individual HeLa cells were suspended in 140 ml of buffer A. The composition of Buffer A is 20 mM Tris, 20 mM NaCl, 1 mM EDTA, 20 mM β-glycophosphate, 5 mM EGTA, 1 mM DTT, 2 μg / ml aprotinin, 20 μg / ml leupeptin, 1 mM PMSF. Next, the cells were disrupted with ultrasonic waves. After centrifugation at 700 × g for 20 minutes, the supernatant was collected, and further centrifuged at 100,000 × g for 1 hour, and the supernatant was collected. To the supernatant of this cytoplasmic fraction, ammonium sulfate was added to a final concentration of 30%, left on ice for 10 minutes, and then centrifuged at 20,000 × g to collect the precipitate.
Next, the precipitate was resuspended in 50 ml of buffer A, filtered through a 0.45 μm filter, and then applied to a Hiload 16/10 SP column (Amersham Pharmacia Biotech Co., Ltd.). The column-bound protein was eluted with a NaCl concentration gradient from 20 mM to 500 mM, and the fraction containing mTOR was detected by dot blotting using an anti-mTOR monoclonal antibody. The peak fraction containing mTOR and the fraction containing only a small amount of mTOR as a control were collected and immunoprecipitated. For immunoprecipitation, an anti-mTOR monoclonal antibody or protein G Sepharose beads on which normal mouse globulin was immobilized were used. After the reaction, protein G sepharose beads bound with protein were washed with buffer A containing 0.5 M NaCl.
After the washing, the bound protein was eluted. The elution conditions were as follows: elution with glycine (pH = 2.8) alone, elution with 1% NP-40 (surfactant), and elution at the first stage with 1% NP-40. Subsequently, the second-stage elution was performed with glycine.
Samples eluted under the above conditions were each subjected to 10% SDS-PAGE. Thereafter, the protein was stained by a negative staining method to detect the protein. The result is shown in FIG.
In the figure, “mTOR ip” represents an immunoprecipitation using an anti-mTOR monoclonal antibody, and “NMG ip” represents an immunoprecipitation using a normal mouse globulin. “Fr. “7” represents a peak fraction containing mTOR, and “fr.13” represents a fraction containing a small amount of mTOR as a control.
In the figure, one lane on the left is eluted with glycine (pH = 2.8) alone, and among the six lanes on the right, the three lanes labeled “1% NP-40” on the bottom are The three lanes eluted with 1% NP-40 (surfactant) and labeled “Glycine (pH, 2.8)” at the bottom are the first step elution with 1% NP-40. The second stage elution was performed with glycine.
Furthermore, at the left end of the figure, the positions of mTOR, p150 (raptor), p39 and IgG are indicated by arrows, respectively, and at the right end of the figure, the positions of molecular weight markers are indicated.
As shown in FIG. 16, in the third lane from the left, that is, in the case of “mTOR ip” “fr.7” and “1% NP-40”, a band having a molecular weight of about 150 kDa (between 200 kDa and 120 kDa). Band). This band was cut out from the gel, and as a result of protein identification and functional analysis as shown in the Examples below, it was found that it was a novel protein (p150 (raptor)) that binds to mTOR.
(Example 3: Identification of p150 (raptor) by mass spectrometry)
The protein (p150) contained in the gel cut out in Example 2 was digested in the gel with lysyl endopeptidase (manufactured by Wako Pure Chemical Industries). Next, the digest was extracted and desalted using Zip-Tip (Millipore). As an eluate of peptide fragments from Zip-Tip, 4% each of 30% acetonitrile / 0.1% formic acid and 50% acetonitrile / 0.1% formic acid was eluted. Each eluate was analyzed by ESI-Q-TOF-MS using Q-Tof2 (manufactured by Micromass).
From the spectra (FIGS. 17 and 18) obtained by the above analysis, four types of spectra showing main ions were selected and tandem mass spectrometry (MS / MS analysis) was performed. Furthermore, based on the MS / MS analysis, protein identification was performed by Mascot MS / MS ion search (Mascot MS / MS Ions Search).
The ions subjected to MS / MS analysis are the following four types (1) to (4).
(1) m / z = 759.89 (2+) observed with 30% acetonitrile / 0.1% formic acid eluate (FIG. 17)
(2) m / z = 813.78 (3+) observed in 30% acetonitrile / 0.1% formic acid eluate (FIG. 17)
(3) m / z = 1132.58 (3+) observed in 50% acetonitrile / 0.1% formic acid eluate (FIG. 18)
(4) m / z = 821.97 (2+) observed with 50% acetonitrile / 0.1% formic acid eluate (FIG. 18)
Mascot MS / MS Ions Search was performed based on the product ion information obtained by each MS / MS analysis and the mass number information of each parent ion. As a result, with a high score of 301, the analyzed protein (p150 (raptor)) was identified as a protein (KIAA1303 protein [Homo sapiens]) encoded by the KIAA1303 cDNA clone.
In addition, proteins were identified by peptide mass fingerprinting. However, many multivalent ions were detected in the spectrum obtained by the ESI-Q-TOF-MS analysis. Of course, the molecular ion of each peptide fragment was accompanied by an isotope ion. Therefore, even if the data obtained by the above analysis is used as it is, the protein cannot be identified by the peptide mass fingerprinting method. Therefore, data conversion was performed. Specifically, data conversion was performed using software called MaxEnt3 (manufactured by Micromass) to convert multivalent ions into monovalent ions. Furthermore, a spectrum excluding isotope ions was created using MaxEnt3. FIG. 19 shows the spectrum of the 30% acetonitrile / 0.1% formic acid eluate after treatment with MaxEnt3, and FIG. 20 shows the spectrum of the 50% acetonitrile / 0.1% formic acid eluate.
Using the mass data list of peptide fragments obtained from FIG. 19 and FIG. 20, peptide mass fingerprinting by Mascot (http://www.matrixscience.com/) was performed to identify proteins. As a result, the analyzed protein (p150 (raptor)) was identified as a protein (KIAA1303 protein [Homo sapiens]) encoded by the KIAA1303 cDNA clone with a high score of 251.
The identification using Mascot MS / MS Ions Search and the identification using Mascot mass fingerprinting are independent protein identification methods that are independent of each other. The protein (p150 (raptor)) has been identified as the same protein.
However, the base sequence of the gene registered on the database as KIAA1303 protein lacks the start codon, and the amino acid sequence of KIAA1303 protein has not been registered.
(Example 4: Analysis of amino terminal sequence of p150 (raptor) using mass spectrometry)
Since the amino terminal (N-terminal) side sequence of p150 (raptor) could not be determined from the gene information registered in the database, we tried to obtain as much information as possible by mass spectrometry. It was.
MS / MS analysis was performed on two types of ions that could not be assigned upon identification by the peptide mass fingerprinting method. The ions subjected to MS / MS analysis are the following two types (arrows in FIG. 21).
(1) Observed with 30% acetonitrile / 0.1% formic acid eluent, m / z = 579.33 (2+)
(2) m / z = 615.82 (2+) observed with 30% acetonitrile / 0.1% formic acid eluent
Mascot MS / MS Ion Search was performed based on the product ion information obtained by MS / MS analysis of m / z = 579.33 (2+). For the search, dbEST was used. As a result, Accession No. The amino acid sequence contained in the EST clones of AW752810 and AA50808038 was identified as ALETIGANLQK (FIG. 22).
Further, Mascot MS / MS Ion Search was performed based on the product ion information obtained by the MS / MS analysis of m / z = 615.82 (2+). As a result, Accession No. It was identified as QSLDPTVDEVK, the amino acid sequence contained in the EST clones of AW752810, AA50808038 and AA508904 (FIG. 23).
Further, THC database search was performed using the above three EST clones (Accession No. AW752810, AA50808038 and AA508904). As a result, all three clones were included in THC597050.
Of the amino acid sequences obtained by translating the above THC597050 base sequence, the amino acid sequence spanning 83 residues from valine (Val) at 273 residue to arginine (Arg) at 355 residue of Frame 1 is The amino acid sequence of the protein registered in (KIAA1303 protein) completely matched the sequence from Val at the third residue. Going back from the overlap to the N-terminal direction, there were 7 start codons before the stop codon appeared.
In order to determine the N-terminus of p150 (raptor), an attempt was made to assign a peak that could not be assigned when peptide mass fingerprinting of p150 (raptor) was performed with reference to the amino acid sequence of Frame 1 in THC597050. The results are shown in FIGS. 24 and 25. Assuming that the start codon closest to the upstream stop codon is the p150 start codon, the N-terminal sequence of the p150 precursor protein is MESEML-.
It is said that 60 to 80% of the intracellular proteins of eukaryotic cells are acetylated at the N-terminus. Initiation methionine (Meti), which is a peptide chain synthesis initiation signal, generally has a side chain turning radius of 1.43 × 10 6. ^-10 2 amino acids smaller than m (1.43Å) (ie, glycine (Gly), alanine (Ala), serine (Ser), cysteine (Cys), threonine (Thr), proline (Pro), and valine (Val)) When it continues as the amino acid residue of the residue, it is eliminated by the action of methionine aminopeptidase. On the other hand, when the other 13 amino acid residues having a side chain turning radius larger than 1.43 Å are present as the second residue, Meti remains as the N-terminal amino acid of the protein. Thereafter, when the amino acid residue following Meti is aspartic acid (Asp), glutamine (Glu), or asparagine (Asn), Meti is acetylated by the action of N-acetyltransferase 2 (NAT2).
Considering the above matters, when the N-terminal amino acid sequence of raptor is MESEML-, it is highly possible that the N-terminal methionine is acetylated without being eliminated. If the N-terminus of raptor has this sequence and is further acetylated, it is theoretically expected that ions of MH + = 366.71 will be observed. And on the mass spectrum, 3664.70 ions were observed (see FIG. 24). This fragment contains 3 residues of Met, in which case some methionine is expected to be oxidized and converted to methionine sulfoxide. Looking closely at the mass spectrum, 3680.78 ions were also detected on the higher mass side of the ions with MH + = 3664.71 (FIG. 26), which is a fragment ion containing one residue methionine sulfoxide. The presence of this ion also indicates that MH + = 3664.71 is derived from the N-terminal fragment of raptor. The above results are consistent with the fact that no significant PTH amino acids were obtained when raptor was subjected to Edman degradation.
From the above analysis, it was determined that the N-terminal amino acid sequence of raptor was MESEML-, and the entire amino acid sequence of raptor was as shown in SEQ ID NO: 2. In addition, from 219th, it is a part which overlaps with KIAA1303.
(Example 5: Acquisition of full-length cDNA of raptor and characteristics of its amino acid sequence)
Along with the determination of the amino acid sequence, a primer was prepared based on the identified gene sequence of KIAA1303, and 5 ′ upstream cDNA of KIAA1303 gene was obtained from a human placenta cDNA library by 5′-RACE method. . By overlapping the obtained 5′-RACE product with the gene sequence of KIAA1303, the full-length cDNA sequence of raptor consisting of 4005 bases of open reading frame was determined. The amino acid sequence deduced from this gene was 1335 residues.
Based on the above results, a database search was performed. As a result, it was found that raptor homologs exist even across species such as Drosophila, nematodes, budding yeast, fission yeast, and Arabidopsis thaliana. Comparison of amino acid sequences revealed that the region from amino acid residues 49 to 667 is very well conserved even across species such as Drosophila, nematode, yeast, and Arabidopsis thaliana (FIG. 2). (See FIG. 9). This result suggests that the region from amino acid residues 49 to 667 is important for raptor function across species.
Furthermore, it has been found that there is a region in which the structure WD-40 repeat is repeated seven times from the 1015th position to the C-terminus of the amino acid sequence. The WD-40 repeat structure is considered to be a structure that forms a scaffold for interacting with various signaling molecules. In addition, a WD-40 repeat structure can be found in many signal transduction molecules. Considering these facts, raptor is also likely to have a function as a scaffold protein that transmits various signals.
(Example 6: Experiment for examining binding between raptor (p150) and mTOR)
First, an anti-raptor rabbit polyclonal antibody (anti-p150 rabbit polyclonal antibody) using the N-terminal 17 residues of raptor (p150) as an antigen was prepared. Next, using the cell supernatant prepared from HEK293 cells, raptor was immunoprecipitated with an anti-raptor rabbit polyclonal antibody, and the immunoprecipitate was subjected to Western blotting with an anti-mTOR mouse monoclonal antibody. As a result, mTOR coimmunized with raptor. It was confirmed that sedimentation occurred (see lane 2 in FIG. 27). It was also confirmed that this binding disappeared by washing with a washing buffer containing 1% NP-40 (see lane 4 in the figure). Conversely, immunoprecipitation was carried out with an anti-mTOR mouse monoclonal antibody, and the immunoprecipitate was subjected to Western blotting with an anti-raptor rabbit polyclonal antibody. As a result, raptor was co-immunoprecipitated with mTOR (see lane 6 in the figure). Also in this case, it was confirmed that this binding disappeared by washing with a washing buffer containing 1% NP-40 (see lane 8 in the figure).
In addition, referring to FIG. 27, the above-described experiment for examining the binding between endogenous raptor and mTOR will be described in more detail. The cell supernatant prepared from HEK293 cells is treated with anti-raptor antibody (anti-p150 antibody) (lane in the same figure). Immunoprecipitation was performed with preimmune rabbit antiserum (lanes 1 and 3 in the same figure), anti-mTOR antibody (lanes 6 and 8 in the same figure), or control mouse IgG (lanes 5 and 7 in the same figure). After washing with a washing buffer containing NP-40 or a washing buffer containing no NP-40 (NP-40 + or-), the sample was separated by SDS-PAGE and then transferred to a PVDF membrane. The PVDF membrane was divided into upper and lower parts, and Western blotting was performed using an anti-mTOR antibody (upper) on the upper side and an anti-raptor antibody (lower) on the lower side. mTOR and raptor (p150) are indicated by arrows, respectively.
(Example 7: Binding of raptor, mTOR, and eIF4EBP1)
One or both of an expression plasmid encoding HA-mTOR, an expression plasmid encoding FLAG-p150, an expression plasmid encoding GST-4EBP, an expression plasmid encoding GST-p70, or GST, HEK293 Cells were transfected (transfected) and co-expressed. Note that HA-mTOR means HA-tag-attached mTOR, FLAG-p150 means FLAG-tag-attached p150 (raptor), GST-4EBP means GST-tag-attached eIF4EBP1, and GST-p70 means GST. P70S6 kinase with G-tag, GST is GST-tag.
Next, the cells were extracted without using a surfactant, and the cells were disrupted. Furthermore, the cell supernatant was divided into three.
The first supernatant was immunoprecipitated with anti-FLAG antibody. The sample was then washed. Then, it isolate | separated by SDS-PAGE and transferred to the PVDF membrane. Next, the PVDF membrane was cut into upper and lower parts. The upper side was blotted with a mixture of anti-HA and anti-FLAG antibodies. The lower side was blotted with anti-GST antibody. The result is shown in FIG.
The second supernatant was incubated with glutathione sepharose beads. The sample was then washed. Then, it isolate | separated by SDS-PAGE and transferred to the PVDF membrane. Next, the PVDF membrane was cut into upper and lower parts. The upper side was blotted with a mixture of anti-HA and anti-FLAG antibodies. The lower side was blotted with anti-GST antibody. The result is shown in FIG.
The third supernatant was directly mixed with the sample buffer and Western blotted with anti-HA antibody to confirm the expression of HA-mTOR. The result is shown in FIG.
In addition, in the table | surface of FIG.28, FIG.29, FIG.30,-shows not having transfected, + shows having transfected. In FIGS. 28 and 29, the band positions of HA-mTOR, FLAG-p150, GST-4EBP or GST-p70 are indicated by arrows, respectively. In FIG. 30, the band position of HA-mTOR is indicated by an arrow.
First, FIG. 28 will be described. In the condition of lane 5, the respective bands of HA-mTOR, FLAG-p150, and GST-4EBP can be found. That is, when FLAG-p150 was immunoprecipitated, binding to HA-mTOR was observed, and further, GST-4EBP was co-precipitated.
In lane 3 of FIG. 28, coprecipitation of FLAG-p150 and GST-4EBp is observed. In other words, mTOR and 4EBP1 were considered to be bonded via p150.
The experiment in which GST-p70 as a control was expressed in lane 6 to lane 10 of FIG. 28 is shown. In lane 9 and lane 10, a band showing binding between HA-mTOR and FLAG-p150 was confirmed. However, in lane 7 and lane 9, a significant band indicating binding between p150 (raptor) and GST-p70 could not be confirmed.
Next, FIG. 29 will be described. FIG. 28 shows the state in which the cell supernatant was immunoprecipitated with an anti-FLAG antibody. In the example shown in FIG. 29, a pull-down assay using glutathione sepharose beads was performed instead of immunoprecipitation with the anti-FLAG antibody in FIG.
In FIG. 29, compared with GST-p70 (lanes 7 and 9), GST-4EBP (lanes 3 and 5) showed significant co-precipitation of FLAG-p150 and mTOR in FIG. The observed phenomenon was reconfirmed.
Next, FIG. 30 will be described. In FIG. 30, the expression of HA-mTOR was confirmed by Western blotting the cell supernatant with an anti-HA antibody. In lanes 4, 5, 8, 9, and 10, transfected with an expression plasmid encoding HA-mTOR, the expression of HA-mTOR can be confirmed.
Moreover, although the data is not shown, the binding between FLAG-p150 and GST-4EBP was not lost by washing with a washing buffer containing 1% NP40. That is, the bond between FLAG-p150 and GST-4EBP was considered to be a stronger bond.
(Example 8: Effect of raptor (p150) on the activity of mTOR phosphorylating eIF4EBP1)
One or both of an expression plasmid encoding HA-mTOR and an expression plasmid encoding FLAG-p150 were transfected (transfected) into HEK293 cells. In the table,-indicates that it was not transfected, and + indicates that it was transfected.
Next, after disrupting the cells, the cell supernatant was immunoprecipitated with an anti-HA antibody, and washed with a washing buffer containing NP40 or a washing buffer containing no NP40. In the table, those washed with a washing buffer containing NP40 are shown as +, and those washed with a washing buffer containing no NP40 are shown as-.
Next, using GST-4EBP1 as a substrate, γ- ³² The phosphorylation assay was performed in the presence of P-ATP. Thereafter, the sample was subjected to SDS-PAGE and transferred to a PVDF membrane. It was analyzed by autoradiography (the upper part of FIG. 31). Thereafter, the PVDF membrane was subjected to Western blotting using a mixture of anti-HA antibody and anti-FLAG antibody (middle of FIG. 31). A part of the cell supernatant was subjected to Western blotting with an anti-FLAG antibody as it was to confirm the expression of FLAG-p150 (lower row in FIG. 31).
In the figure, phosphorylated HA-mTOR (HA-mTOR-P), phosphorylated FLAG-p150 (FLAG-p150-p), phosphorylated GST-4EBP1 (GST-4EBP1-P) , HA-mTOR, FLAG-p150 band positions are indicated by arrows.
The upper and middle lanes 7 and 8 in FIG. 31 are cells obtained by co-expressing HA-mTOR with FLAG-p150 in HEK293 cells. The upper and middle lanes 5 and 6 in FIG. 31 are cells in which only HA-mTOR is expressed alone.
First, the results in the upper part of FIG. 31 will be described. FIG. 31 shows the phosphorylation enzyme activity for eIF4EBP1 performed by HA-mTOR. According to the upper part of FIG. 31, it was found that the enzyme activity in lane 7 was 5 to 6 times higher than the enzyme activity in lane 5. That is, the presence of FLAG-p150 increases the enzyme activity of phosphorylation for eIF4EBP1. When washing with a washing buffer containing 1% NP40 is performed, the effect of enhancing the enzyme activity of mTOR by FLAG-p150 disappears. This can be understood by comparing the results of lanes 7 and 8. These results revealed that FLAG-p150 is an essential molecule for phosphorylation of eIF4EBP1 by mTOR.
(Example 9: Phosphorylation activity using eIF4EBP1 as a substrate by endogenous mTOR co-immunoprecipitated with raptor (p150))
First, HEK293 cells transfected with FLAG-p150 and non-transfected HEK293 cells were prepared. Next, the cells were disrupted, and then the cell supernatant was immunoprecipitated with an anti-FLAG antibody. The precipitate was washed with a washing buffer containing NP40 (shown as NP40 + in the figure) or a washing buffer without NP40 (shown as NP40- in the figure). Thereafter, GST-4EBP1 as a substrate and γ- ³² The phosphorylation assay was performed in the presence of P-ATP. Next, the sample was subjected to SDS-PAGE. Then, it transferred to the PVDF film | membrane and analyzed by autoradiography (upper stage). Thereafter, the PVDF membrane was subjected to Western blotting using an anti-FLAG antibody (middle) and an anti-mTOR antibody (lower).
In the figure, phosphorylated mTOR (mTOR-P), phosphorylated FLAG-p150 (FLAG-p150-p), phosphorylated GST-4EBP1 (GST-4EBP1-P), FLAG-p150 , And mTOR band positions are indicated by arrows.
In order to further confirm the relationship between the three molecules, in FIG. 32, only FLAG-p150 was transfected into HEK293 cells, and the relationship with endogenous mTOR was examined.
Lanes 2 and 4 are washed with a washing buffer containing NP40, and lanes 1 and 3 are washed with a washing buffer containing no NP40.
According to the western blot in the middle and lower rows of FIG. 32, the binding of FLAG-p150 to endogenous mTOR could be confirmed in the absence of NP40. And it was confirmed by the autoradiography of the upper stage that FLAG-p150 phosphorylates GST-4EBP1 only when mTOR is bound in the absence of NP40.
There is also an interesting fact shown in the upper autoradiography. This is that FLAG-p150 is phosphorylated even when mTOR is not bound to FLAG-p150 in the presence of NP40 (upper lane 4). This fact suggests that an enzyme that phosphorylates raptor (p150) exists in addition to mTOR.
(Example 10: TOS motif of p70S6 kinase is required for binding to raptor)
In order to examine whether the TOS motif of p70S6 kinase is required for binding to raptor, the following experiments 1 and 2 were performed. The result of Experiment 1 is shown in FIG. 34, and the result of Experiment 2 is shown in FIG.
Example 10-Experiment 1: In HEK293 cells, FLAG-raptor (lanes 2, 4-6), GST-fused p70S6 kinase (GST-p70S6k) (lanes 3-4), GST with 28th phenylalanine substituted with alanine. -Co-expression with either a mutant of p70S6k (GST-p70S6k-F28A) (lane 5) or GST-fused PDK1 (GST-PDK1) (lane 6).
Next, the GST fusion protein was purified from these cell lysates using glutathione sepharose beads, and then washed and eluted. Next, the eluted product was separated using SDS-PAGE and transferred to a PVDF membrane. The membrane was cut, and Western blotting was performed using an anti-FLAG antibody at the top and an anti-GST antibody at the bottom. The result of the Western blotting is shown in the item “Eluates” in FIG.
In order to confirm the expression of FLAG-raptor, the cell lysate was first separated using SDS-PAGE and transferred to a PVDF membrane. Next, immunostaining Western blotting was performed using an anti-FLAG antibody. The result is shown in the item “Lysates” in FIG.
Example 10-Experiment 2: HEK293 cells were treated with FLAG-raptor (lanes 2, 4-6), GST-p70S6k (lanes 3-4), GST-p70S6k-F28A (lane 5), carboxyl-terminal deletion inactivated. Co-expression with any of the p70S6 kinase mutants (GST-p70S6k-KM / ΔCT) (lane 6) was performed. Cell lysis, purification of the GST fusion protein, and elution were performed in the same manner as described above (Example 10-Experiment 1). The result is shown in FIG.
In FIG. 34 and FIG. 35, “+” indicates that the molecule is expressed, and “−” indicates that the molecule is not expressed (hereinafter the same).
First, Example 10-Experiment 1 will be described. In the item “Eluates” in FIG. 34, in Western blotting using an anti-FLAG antibody, bands can be confirmed only in 4 lanes, that is, only FLAG-raptor and GST-p70S6k are co-expressed. That is, raptor was shown to bind to p70S6 kinase (p70S6 kinase alphaI).
Furthermore, no band is observed in p70S6k-F28A (a mutant of p70S6 kinase in which the 28th phenylalanine is substituted with alanine), which is a mutant having the TOS motif destroyed. That is, it was shown that p70S6 kinase in which the TOS motif was destroyed and raptor do not bind.
Next, Example 10-Experiment 2 will be described. In the item “Eluates” in FIG. 35, in Western blotting using an anti-FLAG antibody, bands can be confirmed in 4 lanes and 6 lanes. These 6 lanes show the results of the p70S6k-KM / ΔCT mutant in which the amino acid 104 amino acid at the carboxyl terminus of p70S6 kinase was deleted and the enzyme activity was lost by mutating the ATP binding site of the kinase domain. That is, it is shown that the p70S6k-KM / ΔCT mutant bound to raptor, and that the phosphorylase activity of p70S6 kinase or the 104 amino acids at the carboxyl terminus of p70S6 kinase is not essential for the binding of raptor to p70S6 kinase. It has been shown.
(Example 11: TOS motif of 4E-BP1 is required for binding to raptor)
Next, the following experiment was conducted to examine whether the TOS motif of 4E-BP1 is necessary for binding with raptor. The results are shown in FIG.
First, in HEK293 cells, FLAG-raptor (lanes 2, 4 to 6), GST-fused 4E-BP1 (GST-4E-BP1) (lanes 3 to 4), and GST− in which 114th phenylalanine was mutated to alanine. Co-expression with either 4E-BP1 mutant (GST-4E-BP1-F114A) (lane 5) or GST-fused PDK1 (GST-PDK1) (lane 6) was performed. Next, the GST fusion protein was purified from these cell lysates using glutathione sepharose beads, and then washed and eluted. Next, the eluted material was separated using SDS-PAGE and transferred to a PVDF membrane. The membrane was cut, and Western blotting was performed using an anti-FLAG antibody at the top and an anti-GST antibody at the bottom. The result is shown in the item “Eluates” in FIG.
In order to confirm the expression of FLAG-raptor, the same cell lysate as described above was separated using SDS-PAGE, transferred to a PVDF membrane, and then subjected to Western blotting using an anti-FLAG antibody. The result is shown in the item “Lysates” in FIG.
In the item of “Eluates” in FIG. 36, in Western blotting using an anti-FLAG antibody, bands are observed only in 4 lanes, that is, only those in which FLAG-raptor and GST-4E-BP1 are co-expressed. This shows the binding between raptor and eIF-4EBP. On the other hand, no band is observed in eIF-4EBP-F114A (a mutant of eIF-4EBP in which the 114th phenylalanine is substituted with alanine), that is, a lane in which the TOS motif is destroyed. The result of FIG. 36 above shows that the mutant does not bind to raptor, and that a TOS motif is required for binding between raptor and eIF-4EBP.
(Example 12: The amino terminal side of raptor is required for binding to the TOS motif of P70S6 kinase)
In order to investigate which region of raptor is required for binding to the TOS motif of p70S6 kinase, the following experiment was conducted to examine the binding of raptor to full length of raptor and various partial length mutants with p70S6 kinase. The result is shown in FIG.
First, in HEK293 cells, FLAG-raptor (lanes 4 to 5), raptor carboxyl-terminal deletion mutant (FLAG-raptor-ΔCT) (lanes 6 to 7), raptor amino-terminal deletion mutant (FLAG- raptor-ΔNT) (lanes 8-9), raptor's WD repeat domain (FLAG-raptor-WD) (lanes 10-11), respectively, and GST-p70S6k (lanes 2, 4, 6, 8, 10) or Co-expression with GST-p70S6k-F28A (lanes 3, 5, 7, 9, 11) was performed. The GST fusion protein was purified from these cell lysates using glutathione sepharose beads, and then washed and eluted. Next, the eluted product was separated using SDS-PAGE and transferred to a PVDF membrane. The membrane was cut, and Western blotting was performed using an anti-FLAG antibody at the top and an anti-GST antibody at the bottom. The result is shown in the item “Eluates” in FIG.
In order to confirm the expression of FLAG-raptor, the same cell lysate as described above was separated using SDS-PAGE, transferred to a PVDF membrane, and then subjected to Western blotting using an anti-FLAG antibody. The result is shown in the item “Lysates” in FIG.
In the item “Eluates” in FIG. 37, in Western blotting using an anti-FLAG antibody, 4 lanes (indicated by “← FLAG-raptor” in the figure) and 6 lanes (indicated by “← FLAG-raptor-ΔCT” in the figure) A band is recognized.
The results shown in FIG. 37 show that p70S6 kinase binds to the full length of raptor and a carboxyl-terminal deletion mutant (FLAG-raptor-ΔCT), and the amino terminal of raptor (amino acid 1) due to the binding between p70S6 kinase and raptor. -904) indicates that it is necessary.
(Example 13: Effect of TOS motif mutation in the presence or absence of raptor on phosphorylation of 4E-BP1 or p70S6 kinase in vitro by mTOR)
The effects of the TOS motif mutation on the phosphorylation of 4E-BP1 or p70S6 kinase by mTOR in the presence or absence of raptor were examined in Experiments 1 and 2 below. The results of Experiment 1 are shown in FIGS. 38 and 39, and the results of Experiment 2 are shown in FIGS.
Example 13-Experiment 1: First, HEK293 cells were lysed with a lysis buffer without surfactant. Next, immunoprecipitation was performed using an anti-mTOR antibody (lanes 2-4, 6-7) or normal mouse IgG (NMG) (lanes 1, 5). Lysis buffer containing 0.5% NaCl containing 1% NP-40 (lanes 1-2, 5-6) or lysis buffer containing 0.5M NaCl not containing NP-40 (lanes 3-4, 7) After washing the immunoprecipitate, an mTOR kinase assay was performed.
In the mTOR kinase assay, GST-4E-BP1-F114A (lanes 1 to 3) or GST-4E-BP1 (lanes 5 to 7) was used as the substrate. The reaction solution was separated by SDS-PAGE, transferred to a PVDF membrane, and subjected to autoradiography. The result is shown in the item “Autoradiography” in FIG. Subsequently, Western blotting was performed on the membrane using an anti-mTOR antibody, an anti-raptor antibody, and an anti-GST antibody. The result is shown in FIG. The results of blotting with an anti-mTOR antibody are in the item “anti-mTOR blot”, the results of blotting with an anti-raptor antibody are in the item of “anti-raptor blot”, and the result of blotting with an anti-GST antibody is “anti-GST”. “Blot” item.
Furthermore, it was taken in by GST-4E-BP1 shown in FIG. ³² P was measured using BAS2500 and graphed. The result is shown in FIG.
As described above, mTOR kinase activity in the presence and absence of raptor was measured using eIF-4EBP and eIF-4EBP-F114A as substrates. As a result, mTOR phosphorylated eIF-4EBP only in the presence of raptor. This is particularly true for the 7 lane “← ³² This can be understood from the band (FIG. 38) at the position of “P-GST-4E-BP1” and the value of 7 lanes in the graph of FIG. 39. On the other hand, mTOR failed to phosphorylate eIF-4EBP-F114A regardless of the presence or absence of raptor.
The above results showed that the binding of raptor and eIF-4EBP via the TOS motif is essential for the phosphorylation of eIF-4EBP by mTOR.
Example 13-Experiment 2: The mTOR kinase assay was performed in the same manner as in Example 13-Experiment 1 using HEK293 cells. The substrate used was GST-p70S6k-F28A (lanes 1 to 3) or GST-p70S6k (lanes 5 to 7). The reaction solution was separated by SDS-PAGE, transferred to a PVDF membrane, and subjected to autoradiography. The result of the autoradiography is shown in the item “Autoradiography” in FIG.
Subsequently, Western blotting was performed on the membrane using an anti-mTOR antibody (anti-mTOR blot), an anti-raptor antibody (anti-raptor blot), and an anti-GST antibody (anti-GST blot). Phosphorylation of threonine at position 412 of GST-p70S6k and GST-p70S6k-F28A was identified by Western blot using an anti-phosphorylated 412 threonine antibody.
The results of these Western blots are shown in FIG. The results of Western blotting with an anti-mTOR antibody are in the item “anti-mTOR blot”, the results of Western blotting with an anti-raptor antibody are in the item “anti-raptor blot”, and the results of Western blotting with an anti-GST antibody are “ The results of Western blotting with an anti-phosphorylated 412 threonine antibody are shown in “anti-GST blot” in “anti-412-P blot”.
Furthermore, it was taken in by GST-p70S6k shown in FIG. ³² P was measured using BAS2500 and graphed. The result is shown in FIG.
As described above, mTOR kinase activity was measured using p70S6 kinase and p70S6 kinase-F28A as substrates and in the presence or absence of raptor. As a result, mTOR phosphorylated p70S6 kinase in the presence of raptor, but in the absence of raptor, the degree of phosphorylation was reduced by about 80%. This can be understood by looking at the results for 6 lanes and 7 lanes in FIG.
On the other hand, mTOR phosphorylated GST-p70S6k-F28A regardless of the presence or absence of raptor, but the degree was the same as the degree of phosphorylation of p70S6 kinase by mTOR in the absence of raptor. (Refer to the results of lanes 2 to 3 and 6 in FIG. 41).
From this result, it was revealed that when p70S6 kinase was used as a substrate, most of the mTOR kinase activity was dependent on the binding to raptor and partly independent of the binding to raptor.
The results of Examples 10 to 13 show the following (1) to (5).
(1) It supports that the function of raptor is a scaffold protein that binds mTOR and its substrate proteins eIF-4EBP and p70S6 kinase and provides a field for phosphorylation reaction.
(2) This suggests that the binding of raptor to eIF-4EBP and p70S6 kinase is essential for signal transduction from amino acids via mTOR.
(3) The TOS motif is essential for the binding of eIF-4EBP and p70S6 kinase to raptor and phosphorylation of these substrate proteins by mTOR.
(4) This suggests the possibility that other proteins having the TOS motif bind to raptor and become mTOR substrates.
(5) The search for substances that inhibit the binding of raptor and the TOS motif leads to the development of new drugs, such as suppressors of mTOR signals, immunosuppressive effects, anticancer effects, and inhibitors of restenosis of coronary arteries in ischemic heart disease. It shows the possibility.
It should be noted that the specific embodiments or examples made in the above “Best Mode for Carrying Out the Invention” are merely to clarify the technical contents of the present invention, and such specific examples are not limited to these. The present invention should not be construed as being limited to a narrow sense, but can be implemented with various modifications within the spirit of the present invention and the scope of the following claims.
Industrial applicability
As described above, the present invention relates to a novel protein having a property of binding to a mammalian rapamycin target protein (mTOR) and a gene thereof, such as pathological analysis of various diseases, development of a therapeutic agent, improvement of treatment, etc. It can be used effectively widely.
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a diagram illustrating signal transduction involved in protein synthesis.
FIG. 2 shows the amino acid sequences of raptor and its homologues in humans, Drosophila, nematodes, budding yeast, fission yeast and Arabidopsis thaliana.
FIG. 3 shows the continuation of the amino acid sequence shown in FIG.
FIG. 4 shows the continuation of the amino acid sequence shown in FIG.
FIG. 5 shows the continuation of the amino acid sequence shown in FIG.
FIG. 6 shows the continuation of the amino acid sequence shown in FIG.
FIG. 7 shows the continuation of the amino acid sequence shown in FIG.
FIG. 8 shows the continuation of the amino acid sequence shown in FIG.
FIG. 9 shows the continuation of the amino acid sequence shown in FIG.
FIG. 10 shows the amino acid sequence of raptor in human and mouse.
FIG. 11 shows the continuation of the amino acid sequence shown in FIG.
FIG. 12 shows the continuation of the amino acid sequence shown in FIG.
FIG. 13 is a diagram showing raptor exons and introns located on the human genome.
FIG. 14 is a diagram showing the details from exon 26 to exon 34 on FIG.
FIG. 15 shows the phosphorylation activity for eIF4EBP1 by immunoprecipitation of mTOR using a monoclonal antibody against mTOR.
FIG. 16 shows that a novel mTOR-binding protein raptor is purified with mTOR and then divided into a fraction containing mTOR (fr. 7) and a fraction not containing mTOR (fr. 13), and an anti-mTOR monoclonal antibody (mTORip) or normal Perform immunoprecipitation with protein G sepharose beads on which mouse globulin (NMGip) is immobilized, elute the protein with 1% NP40 or glycine (pH = 2.8), separate the protein with SDS-PAGE, and then perform silver staining. It is a figure which shows the result of having performed.
FIG. 17 shows that the peptide fragment of raptor was eluted from Zip-Tip using 30% acetonitrile / 0.1% formic acid and analyzed by ESI-Q-TOF-MS using Q-Tof2 (Micromass). It is a figure which shows the spectrum obtained as a result of analyzing an eluate.
FIG. 18 shows that the peptide fragment of raptor was eluted from Zip-Tip using 50% acetonitrile / 0.1% formic acid and analyzed by ESI-Q-TOF-MS using Q-Tof2 (Micromass). It is a figure which shows the spectrum obtained as a result of analyzing an eluate.
FIG. 19 shows a spectrum obtained by removing isotope ions from FIG.
FIG. 20 shows a spectrum obtained by removing isotope ions from FIG.
FIG. 21 is a diagram showing spectra of two types of ions that could not be assigned during identification by the peptide mass fingerprinting method.
FIG. 22 is a diagram showing an amino acid sequence obtained based on product ion information obtained by MS / MS analysis of m / z = 579.33 (2+).
FIG. 23 is a diagram showing an amino acid sequence obtained based on product ion information obtained by MS / MS analysis of m / z = 615.82 (2+).
FIG. 24 is a diagram showing a newly clarified N-terminal amino acid sequence of raptor and a spectrum showing the amino acid sequence.
FIG. 25 is a diagram showing the continuation of the N-terminal amino acid sequence and the spectrum showing the amino acid sequence shown in FIG.
FIG. 26 is a diagram showing ions of 3680.78 located on the high mass side of ions of MH + = 3664.71.
FIG. 27 shows the results of immunoprecipitation using cell supernatant, categorization with or without surfactant (NP40), and Western blotting using anti-mTOR antibody and anti-p150 antibody (anti-raptor antibody). FIG.
FIG. 28 shows transfection of HA-mTOR, FLAG-p150, GST-4EBP, GST-p70, GST, incubating the cell supernatant with glutathione sepharose beads, and a mixture of anti-HA and anti-FLAG antibodies; It is a figure which shows the result blotted with the anti- GST antibody.
FIG. 29 shows transfection of HA-mTOR, FLAG-p150, GST-4EBP, GST-p70, GST, immunoprecipitation of the supernatant of the cells with anti-FLAG antibody, and a mixture of anti-HA antibody and anti-FLAG antibody. , And the results of blotting with anti-GST antibody.
FIG. 30 shows the results of transfection of HA-mTOR, FLAG-p150, GST-4EBP, GST-p70, and GST, and western blotting of the supernatant of the cells with anti-HA antibody.
FIG. 31 shows transfection of HA-mTOR, FLAG-p150, and immunoprecipitation of the cell supernatant with an anti-HA antibody, washing with a washing buffer containing NP40 or a washing buffer containing no NP40, -4EBP1 as a substrate and γ- ³² It is a figure which shows the result of having analyzed and analyzed phosphorylation assay in P-ATP presence.
FIG. 32 shows transfection of FLAG-p150, immunoprecipitation of the cell supernatant with anti-FLAG antibody, washing of the precipitate with a washing buffer with or without NP40, and GST-4EBP1 as a substrate and γ- ³² It is a figure which shows the analysis result after performing phosphorylation assay in P-ATP presence.
FIG. 33 shows, for each predicted exon, the corresponding cDNA sequence, the number of exon bases, the exon sequence number, the sequence number of the sequence containing the exon, and the exon start position in the sequence containing the exon. And a list of end positions.
FIG. 34 shows HEK293 cells, FLAG-raptor (FLAG-p150), GST-fused p70S6 kinase (GST-p70S6k), and GST-p70S6k mutant in which 28th phenylalanine is substituted with alanine (GST-p70S6k-F28A). It is a figure which shows the result of having performed the co-expression with either of GST fusion PDK1 (GST-PDK1), and performing Western blotting using those cell lysates and an antibody.
FIG. 35 shows that HEK293 cells were co-expressed with FLAG-raptor and any of GST-p70S6k, GST-p70S6k-F28A, carboxyl-terminal deletion inactivated p70S6 kinase mutant (GST-p70S6k-KM / ΔCT). It is a figure which shows the result of having performed Western blotting using those cell lysates and antibodies.
FIG. 36 shows HEK293 cells, FLAG-raptor, GST fusion 4E-BP1 (GST-4E-BP1), and GST-4E-BP1 mutant (GST-4E-BP1) in which 114th phenylalanine is mutated to alanine. -F114A), the co-expression with either GST fusion PDK1 (GST-PDK1), and it is a figure which shows the result of having performed the Western blotting using those cell lysates and an antibody.
FIG. 37 shows HEK293 cells, FLAG-raptor, raptor carboxyl-terminal deletion mutant (FLAG-raptor-ΔCT), raptor amino-terminal deletion mutant (FLAG-raptor-ΔNT), raptor WD repeat domain ( (FLAG-raptor-WD) is co-expressed with GST-p70S6k or GST-p70S6k-F28A, and the results of Western blotting using these cell lysates and antibodies are shown.
FIG. 38 shows an mTOR kinase assay using an anti-mTOR antibody or normal mouse IgG (NMG) and HEK293 cells to obtain an immunoprecipitate and using GST-4E-BP1-F114A or GST-4E-BP1 as a substrate. It is a figure which shows the result of having performed and also performing the Western blotting using the reaction liquid and the antibody.
39 is incorporated into GST-4E-BP1 in FIG. ³² It is a graph which measures P and shows the measurement result.
FIG. 40 shows an immunoprecipitate obtained using an anti-mTOR antibody or normal mouse IgG (NMG) and HEK293 cells, and an mTOR kinase assay using GST-p70S6k-F28A or GST-p70S6k as a substrate was performed. It is a figure which shows the result of having performed western blotting using the reaction liquid and the antibody.
41 is incorporated into GST-p70S6k in FIG. 40 above. ³² It is a graph which measures P and shows the measurement result.
FIG. 42 is a diagram showing a base sequence of exon 7 (portion surrounded by a frame) and base sequences before and after the exon.
FIG. 43 is a diagram showing the base sequence of exon 14 (portion surrounded by a frame) and the base sequences before and after the exon.

Claims (21)

The gene which codes the protein as described in any of the following (a)-(c).
(A) A protein comprising the amino acid sequence shown in SEQ ID NO: 2 or 4 in the sequence listing.
(B) the amino acid sequence shown in SEQ ID NO: 2 or 4, consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added, and a mammalian rapamycin target protein (mTOR ) A protein that binds to
(C) the amino acid sequence shown in SEQ ID NO: 2 or 4, consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added, and a TOS motif (mTOR signaling motif) A protein that has the property of binding to the protein it has. The gene according to claim 1, which is cDNA. The gene according to claim 2, which has the 183rd to 4187th base sequence as an open reading frame in the base sequence represented by SEQ ID NO: 1. The gene according to claim 2, which has the 175 to 4179th nucleotide sequence as an open reading frame in the nucleotide sequence represented by SEQ ID NO: 3. The gene according to claim 1, which is genomic DNA. The gene according to claim 5, which has each base sequence shown in SEQ ID NOs: 5 to 35. The gene according to claim 5, which has a series of base sequences continuously shown in SEQ ID NOs: 36 to 48. A protein comprising the amino acid sequence shown in SEQ ID NO: 2 or 4. It consists of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence shown in SEQ ID NO: 2 or 4, and binds to a mammalian rapamycin target protein (mTOR) A protein that has the property of A protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence shown in SEQ ID NO: 2 or 4, and having a TOS motif (mTOR signaling motif) A protein that binds. The protein according to any one of claims 8 to 10, which has a property of binding to a mammalian rapamycin target protein (mTOR) and enhancing the enzyme activity of the protein. The protein according to any one of claims 8 to 11, which has a property of binding to a translation initiation factor binding protein (eIF-4EBP) of a eukaryotic cell. The protein according to any one of claims 8 to 12, which has a property of binding to p70S6 kinase. A protein according to any one of claims 8 to 13, (1) a mammalian rapamycin target protein (mTOR), (2) a eukaryotic translation initiation factor binding protein (eIF-4EBP), (3) A binding inhibitor that inhibits binding to p70S6 kinase, or (4) a protein having a TOS motif (mTOR signaling motif). 15. A method for screening for a binding inhibitor according to claim 14, wherein any one of the following substances (1) to (4) is used.
(1) The protein according to any one of claims 8 to 13, (2) a partial protein of the protein of (1) above, wherein at least the 1st to 904th sequence in the amino acid sequence of the full length protein Partial protein comprising (3) Partial protein of the protein of (1) above, wherein the partial protein comprising the binding region to mTOR or eIF-4EBP in the amino acid sequence of the full-length protein (4) Protein of (1) above Or the modified partial protein of (2) or (3) above The mutant protein according to any one of claims 8 to 13, wherein the mutant protein has an abnormality in any of the following properties (a) to (e).
(B) A property of binding to mammalian rapamycin target protein (mTOR) (b) A property of enhancing the enzymatic activity of mammalian rapamycin target protein (mTOR) (c) A translation initiation factor binding protein (eIF-4EBP) of eukaryotic cells (D) Properties binding to p70S6 kinase (e) Properties binding to a protein having a TOS motif (mTOR signaling motif) A gene encoding the mutant protein according to claim 16. An antibody against the protein according to any one of claims 8 to 13 and 16. A recombinant expression vector comprising the gene according to any one of claims 1 to 7 and 17. A transformant into which the gene according to any one of claims 1 to 7 and 17 has been introduced. A gene detection instrument using, as a probe, at least a part of a base sequence or a complementary sequence thereof in the gene according to any one of claims 1 to 7.

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