JP2018523656A - 非アルコール性脂肪肝調節因子14−3−3タンパク質 - Google Patents
非アルコール性脂肪肝調節因子14−3−3タンパク質 Download PDFInfo
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Abstract
Description
しかし、本発明が達成しようする技術的課題は、以上で言及した課題に限定されず、言及されない他の課題は、下記の記載から当業者に明確に理解され得る。
coli)や昆虫細胞を利用するよりも哺乳動物細胞を利用する場合が、タンパク質の活性度や溶解性の観点から天然型のものと類似すると認められる。
ダルベッコ変法イーグル培地(Dulbecco’s modified Eagle’s medium、DMEM)、199培地(M199)、ウシ胎児血清(fetal bovine serum、FBS)、ペニシリン、ストレプトマイシン、Opti−MEMは、Invitrogen(Carlsbad、CA)で購入した。
PPARγ2の転写活性は、肝疾患関連タンパク質の発現に重要な役割をする。したがってPPARγ2により調節されるaP2プロモーターを利用してPPARγ2の転写活性を測定し、7種類の14−3−3タンパク質の役割を確認した。このために、HEK−293T細胞を12−ウェルプレートに各ウェル当たり1×105細胞を播種(seeding)した後、0.5μgのaP2プロモーター構造物と14−3−3イソ型タンパク質(α、β、γ、δ、ε、ζ、η、θ)(0.5μg)またはsi−14−3−3βおよびsi−14−3−3γsiRNA(5nMと10nM、Santa Cruz)をE−fection plus reagent(Lugen)を利用してトランスフェクションした。DNAとE−fection plus reagentの比率は、1:2で使用し、方法は、製造社のマニュアルに沿って行った。トランスフェクションした後、ピオグリタゾン(pioglitazon;Pio、Santa Cruz)10μMを24時間処理し、当該細胞を氷冷PBSで洗浄およびレポータライシスバッファー(Promega、Madison、WI)でウェル当たり80μLを使用して細胞を溶解させた後、10,000×gに4℃で10分間遠心分離して上澄み液を収集し、ルシフェラーゼ活性を測定した。機器は、ルミノメーター20/20n(Turner Biosystems、Sunnyvale、CA)を使用した。標準化(Normalization)のために、pSV40−β−ガラクトシダーゼを共トランスフェクション(cotransfected)した。収集した上澄み液は、β−ガラクトシダーゼ活性を測定し、ルシフェラーゼ活性を補正して、値をグラフで示した。このためにβ−ガラクトシダーゼ酵素分析システム(Promega、Madison、WI)を使用し、DU530分光光度計(Beckman Instruments、Palo Alto、CA)を使用して分析した。
転写活性の実験の結果、14−3−3βと14−3−3γがPPARγ2の転写活性を調節することから見て、14−3−3βと14−3−3γがPPARγ2と結合し得ると認められる。
PPARγ2タンパク質は、活性化関数1(activation function 1;AF−1、アミノ酸1−138)、DNA結合ドメイン(DBD、アミノ酸139−203)、ヒンジ領域(アミノ酸204−310)、活性化関数2(activation function 2;AF−2、アミノ酸311−505)ドメインからなると報告されたことがある(図3A)。したがって、PPARγ2のいかなるドメインの部分に14−3−3βと14−3−3γが結合するのかを確認するために、GST−プルダウンアッセイを進めた。
14−3−3タンパク質は、ターゲットタンパク質のリン酸化した残基に結合すると知られている。PPARγ2の活性に関連したリン酸化残基は、セリン112とセリン273であると知られている。これにより、セリン112とセリン273残基がリン酸化しないPPARγ2の突然変異(PPARγ2のS112AとS273A)を製作して、14−3−3タンパク質との結合をGST−プルダウンアッセイを通じて確認した。
以前の実験結果を通じて14−3−3βと14−3−3γがリン酸化したPPARγ2のセリン273残基に結合することを確認したのであり、同じ残基における結合は、二つのタンパク質が競争的に結合すると判断された。したがって、PPARγ2との結合で14−3−3βと14−3−3γが競争的な結合をするか否かを確認するために、GST−プルダウンアッセイを進めた。
肥満マウスの肝でPPARγ2の発現および活性度が増加しており、そのターゲット遺伝子の発現が増加していると知られている。In vitro実験でこのような肥満環境を作るために、脂肪酸の一種類であるオレイン酸(OA)を処理して、PPARγ2および14−3−3βと14−3−3γのmRNAの発現量を確認した。
HepG2細胞を6−ウェルプレートに各ウェル当たり4×105細胞を播種した後、GFP−14−3−3β DNA(4μg)またはGFP−14−3−3γDNA(4μg)をE−fection plus reagent(Lugen)を利用してトランスフェクションした。DNAとE−fection plus reagentの比率は、1:2で使用し、方法は、製造社のマニュアルに沿って行った。トランスフェクションした後、オレイン酸(OA)200μMを72時間処理し、mRNAの抽出は、当該細胞をTrizol(Invitrogen)でウェル当たり1mLを使用して細胞を溶解させた後、クロロホルム200μLを添加してよく混ぜた後、常温に5分間置き、12,000×gに4℃で15分間遠心分離して、上清液をイソプロパノール500μLと混ぜた後、10分間氷に置き、12,000×gに4℃で15分間遠心分離して、上清液を除去し、ペレットをジエチルピロカルボン酸(DEPC)処理された3次蒸留水で溶かした。2μgのmRNAをSuperscript First Strand cDNA Synthesis Kit(Bioneer、Daejeon、South Korea)を利用してcDNAを合成し、これを鋳型としてヒトのPPARγ2(配列番号1、2)、14−3−3β(配列番号3、4)、14−3−3γ(配列番号5、6)、SREBP−1c、FAT/CD36、β−アクチン特異的なプライマーを利用してポリメラーゼ連鎖反応(PCR)を通じて増幅し、マウスのSREBP−1c、FAT/CD36、GAPDH特異的なプライマーを利用してリアルタイムポリメラーゼ連鎖反応を通じて増幅した。β−アクチンとGAPDHは、各細胞のmRNAを同一量で比較したものであるかを把握できる対照群として使用した。
この結果は、CD36遺伝子の発現を調節するためにプロモーター地域に結合するPPARγ2が14−3−3βにより増加し、14−3−3γにより減少することが分かる。
HEK−293T細胞を100mmのプレートに各ウェル当たり2×106細胞を播種した後、mGST−PPARγ2 DNA(4μg)とHA−14−3−3β DNA(4μg)またはHA−14−3−3γDNA(4μg)をE−fection plus reagent(Lugen)を利用してトランスフェクションした。DNAとE−fection plus reagentの比率は、1:2で使用し、方法は、製造社のマニュアルに沿って行った。トランスフェクションした後、オレイン酸(OA)200μMを72時間処理し、当該細胞を氷冷PBSで洗浄およびライシスバッファー(150mMのNaCl、1mMのEDTA、1%のノニデットP−40、5%のグリセロール、25mMのトリス−HCl、pH7.5、プロテアーゼ阻害剤入り)でウェル当たり500μLを使用して細胞を溶解させた後、10,000×gに4℃で10分間遠心分離してタンパク質を抽出した後、1000μgのタンパク質をグルタチオンセファロース4Bビーズ(GE Healthcare、Buckinghamshire、UK)20μLと6時間反応させた後、ビーズを1mLの氷冷PBSで洗浄する過程を5回繰り返して行い、ビーズを100℃で10分間沸かした後、10%のSDS−PAGEゲルで分離して、ウェスタンブロットでそれぞれ(GST、HA:Santa Cruz)の抗体を使用してタンパク質を確認したのであり、すべての抗体は、1:3000の比率で使用した。
HepG2細胞とマウス初代肝細胞を6−ウェルプレートに各ウェル当たり4×105細胞を播種した後、GFP−14−3−3β DNA(1μg)またはGFP−14−3−3γDNA(1μg)およびsi−14−3−3β(10μM、Santa Cruz)またはsi−14−3−3γ(10μM、Santa Cruz)をE−fection plus reagent(Lugen)を利用してトランスフェクションした。DNAとE−fection plus reagentの比率は、1:2で使用し、方法は、製造社のマニュアルに沿って行った。トランスフェクションした後、オレイン酸(OA)200μMを72時間処理し、当該細胞を氷冷PBSで洗浄した後、4%のパラホルムアルデヒドを利用して細胞を固定し、6時間室温で0.35%のオイルレッドO溶液で細胞を染色した。染色された細胞を1mLのイソプロパノールで脱色して、550nmの波長でDU530分光光度計(Beckman Instruments、Palo Alto、CA)を使用して分析した。
トリグリセリド(中性脂肪)は、脂肪酸の一形態であり、これは、脂肪量の指標として用いられる。トリグリセリドを測定して生化学的な脂質検査を進めた。
Claims (16)
- 14−3−3γ遺伝子を含む、非アルコール性脂肪肝の予防または治療用医薬組成物。
- 14−3−3γタンパク質を含む、非アルコール性脂肪肝の予防または治療用医薬組成物。
- 非アルコール性脂肪肝が疑われる患者の生物学的試料から14−3−3γ遺伝子のmRNAまたはそのタンパク質の水準を測定するためのプローブを含む、非アルコール性脂肪肝診断用組成物。
- 14−3−3γ遺伝子のmRNA水準を測定するためのプローブは、前記mRNAに対する核酸プローブまたはプライマーである、請求項3に記載の非アルコール性脂肪肝診断用組成物。
- 14−3−3γタンパク質の水準を測定するためのプローブは、前記タンパク質に対する抗体である、請求項3に記載の非アルコール性脂肪肝診断用組成物。
- 14−3−3γ遺伝子またはタンパク質を含む細胞を候補物質と人体外で接触させ、
前記候補物質による前記遺伝子またはタンパク質の発現量の変化を測定することを含む、非アルコール性脂肪肝の予防または治療用医薬のスクリーニング方法。 - 前記候補物質が14−3−3γ遺伝子またはタンパク質の発現を上向き調節するとき、非アルコール性脂肪肝の予防または治療用医薬であると判別する、請求項6に記載の非アルコール性脂肪肝の予防または治療用医薬のスクリーニング方法。
- 14−3−3βタンパク質および/または14−3−3γタンパク質をPPARγ2タンパク質と共に候補物質と接触させ、
前記候補物質によるPPARγ2に対する14−3−3βタンパク質および/または14−3−3γタンパク質の結合変化を測定することを含む、非アルコール性脂肪肝の予防または治療用医薬のスクリーニング方法。 - 前記候補物質によるPPARγ2に対する14−3−3βタンパク質および/または14−3−3γタンパク質の結合変化の測定は、PPARγ2のSer273残基に対する14−3−3βタンパク質および/または14−3−3γタンパク質の結合変化を測定することである、 請求項8に記載の非アルコール性脂肪肝の予防または治療用医薬のスクリーニング方法。
- 前記候補物質によるPPARγ2に対する14−3−3βタンパク質および/または14−3−3γタンパク質の結合変化の測定の結果、前記候補物質がPPARγ2に対する14−3−3βタンパク質の結合を減少させたり、PPARγ2に対する14−3−3γタンパク質の結合を増加させるとき、非アルコール性脂肪肝の予防または治療用医薬であると判別する、請求項8に記載の非アルコール性脂肪肝の予防または治療用医薬のスクリーニング方法。
- 14−3−3γ遺伝子を含むベクターを個体に導入する段階を含む、非アルコール性脂肪肝の予防または治療方法。
- 非アルコール性脂肪肝が疑われる患者から生物学的試料を収得する段階と;
前記生物学的試料に14−3−3γ遺伝子のmRNAまたはそのタンパク質の水準を測定するためのプローブを処理して14−3−3γを検出する段階と;
前記検出された14−3−3γの水準を正常対照群と比較する段階と;を含む、非アルコール性脂肪肝の診断方法。 - 14−3−3γ遺伝子のmRNA水準を測定するためのプローブは、前記mRNAに対する核酸プローブまたはプライマーである、請求項12に記載の非アルコール性脂肪肝の診断方法。
- 14−3−3γタンパク質の水準を測定するためのプローブは、前記タンパク質に対する抗体である、請求項12に記載の非アルコール性脂肪肝の診断方法。
- 前記測定された14−3−3γ遺伝子のmRNAまたはそのタンパク質の水準が正常対照群に比べて低い場合、増加した非アルコール性脂肪肝の危険度を示すことを特徴とする、請求項12に記載の非アルコール性脂肪肝の診断方法。
- 非アルコール性脂肪肝が疑われる患者から生物学的試料を収得する段階と;
前記生物学的試料に14−3−3γ遺伝子のmRNAまたはそのタンパク質の水準を測定するためのプローブを処理して14−3−3γの水準を測定する段階と;を含む、14−3−3γの検出方法。
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