JP4230294B2 - Novel adapter molecule that binds to mammalian Toll-like receptor 4 and mediates type I interferon induction and uses thereof - Google Patents

Description

【０１３６】
また、各アダプター分子のドメイン構造を図４に示す。図４において黒く塗った部分がＴＩＲドメインである。ヒトＴＩＣＡＭ−２は２３５アミノ酸残基からなり、ＴＩＲドメインは７５−２１３番目のアミノ酸残基に相当する。同様に、ＴＩＲＡＰは２３５アミノ酸残基からなり、ＴＩＲドメインは８５−２１４番目のアミノ酸残基に相当、ＭｙＤ８８は２９６アミノ酸残基からなり、ＴＩＲドメインは１６０−２９６番目のアミノ酸残基に相当、ＴＩＣＡＭ−１は７１２アミノ酸残基からなり、ＴＩＲドメインは３９４−５３２番目のアミノ酸残基に相当する。
【０１３７】
ヒトゲノムデータベース解析により、ＴＩＣＡＭ−２遺伝子は染色体５ｑ２２に位置することが明らかとなった。
【０１３８】
なお、ヒトＴＩＣＡＭ−２およびマウスＴＩＣＡＭ−２のｃＤＮＡ配列およびアミノ酸配列は、ＤＤＢＪデータベースに登録済みであり（本願出願時には未公表）、それらのGenebankアクセッション番号はそれぞれＡＢ０９１０５４およびＡＢ１００４４１である。
【０１３９】
〔実施例２：ノーザンブロッティングおよびＲＴ−ＰＣＲによるＴＩＣＡＭ−２ｍＲＮＡの検出〕
ヒト１２レーンＭＴＮ Ｂｌｏｔおよびヒト１２レーンＭＴＮ ＢｌｏｔＩＩＩ（商品名、クロンテック社製）を用いて、³²Ｐで標識した全長ヒトＴＩＣＡＭ−２ｃＤＮＡプローブでストリンジェントな条件下にハイブリダイズし、その後標識したβ−アクチンプローブで再ハイブリダイズした。ＢＡＳ２０００画像解析装置（富士写真フィルム株式会社製）を用いて４〜２４時間露光し、ｍＲＮＡのシグナルを検出した。その結果を図５（ａ）に示す。
【０１４０】
図５（ａ）から明らかなように、３．３−３．６ｋｂのｍＲＮＡがリンパ節、前立腺、胃、甲状腺、気管、心臓、骨格筋および胎盤を含む多くの組織で検出された。リンパ節、前立腺、胃、甲状腺、筋肉および肺では３．６および３．３ｋｂの２つのバンドが認められた。１．８ｋｂのバンドは胎盤のみに特異的に認められた。
【０１４１】
ｍＲＮＡの検出および半定量の目的で、ＲＴ−ＰＣＲを行った。プライマーは、ヒトＴＩＣＡＭ−２検出にはＦ１およびＲ１を用い（配列番号５、６）、β−アクチン検出にはｈＢＡＣＴ−Ｆ１およびｈＢＡＣＴ−Ｒ１を用いた（配列番号７、８）。トータルＲＮＡは各種ヒト細胞または細胞株から分離し、ＴＩＣＡＭ−２は３５サイクル、β−アクチンは２０サイクルのＲＴ−ＰＣＲを行った。その結果を図５（ｂ）に示す。
【０１４２】
図５（ｂ）から明らかなように、ヒト末梢血由来の未熟な樹状細胞（ｉＤＣ）、マクロファージおよびナチュラルキラー細胞（ＮＫ）においてＴＩＣＡＭ−２のｍＲＮＡが存在した。また、ＴおよびＢリンパ球系細胞株、線維芽細胞株にもＴＩＣＡＭ−２のｍＲＮＡが存在した。
【０１４３】
〔実施例３：酵母ツー・ハイブリッド法によるＴＩＣＡＭ−２と他の分子との会合の検討〕
酵母培地は文献（Sherman, F. et al.「Method in Yeast Genetics」Cold Spring Harbor press, NY, USA. pp.22-46. (1986)）に記載されたものを使用した。酵母ＡＨ１０９株（クロンテック社製）をｂａｉｔおよびｐｒｅｙプラスミドで形質転換した。形質転換した酵母をストリークし、３ないし５日間インキュベートした。ＢＤ−ＴＬＲ２、ＴＬＲ３、ＴＬＲ４、ＴＬＲ５、ＴＬＲ６、ＴＬＲ７、ＴＬＲ８およびＴＬＲ９はＴＬＲのＴＩＲドメインをｐＧＢＫＴ７（クロンテック社製）に挿入して作製した。ＢＤ−ＴＩＣＡＭ−２およびＡＤ−ＴＩＣＡＭ−２は全長野生型ＴＩＣＡＭ−２ｃＤＮＡをｐＧＢＫＴ７（ｂａｉｔ）またはｐＧＡＤＴ７（ｐｒｅｙ）に挿入して作製した。ＢＤ−ＴＩＣＡＭ−１はＴＩＣＡＭ−１フラグメント（開始コドンに対応する１１０２ｂｐから２１４２ｂｐの部分）をｐＧＢＫＴ７に挿入して作製した。ＡＤ−ＴＩＲＡＰおよびＡＤ−ＭｙＤ８８は全長タンパク質をコードするｃＤＮＡフラグメントをｐＧＡＤＴ７に挿入して作製した。
【０１４４】
ＳＤ−ＷＬＨはトリプトファン、ロイシンおよびヒスチジンを欠失した酵母合成デキストロース培地であり、ＳＤ−ＷＬＨＡはさらにアデニンを欠失した培地である。ＳＤ−ＷＬＨプレートは中程度の厳密度のプレートであり、ＳＤ−ＷＬＨＡプレートは高い厳密度のプレートである。すなわち、ＳＤ−ＷＬＨは弱い相互作用を反映し、ＳＤ−ＷＬＨＡは強い相互作用を反映する。上記結果を図６（ａ）〜（ｃ）に示す。
【０１４５】
図６（ａ）〜（ｃ）から明らかなように、ＳＤ−ＷＬＨＡプレートにおいてＴＩＣＡＭ−２とＴＬＲ４との間に強い会合が観察され、ＳＤ−ＷＬＨＡプレートにおいてＴＩＣＡＭ−２とＴＬＲ３間の弱い会合が観察された（図６（ａ）参照）。また、ＴＩＣＡＭ−２はホモ２量体を形成し（図６（ｂ）参照）、ＴＩＣＡＭ−１とヘテロ２量体を形成することが観察された（図６（ｃ）参照）。
【０１４６】
〔実施例４：免疫沈降法によるＴＩＣＡＭ−２と他の分子との会合の検討〕
〈実験方法〉
ＨＥＫ２９３細胞を６ウエルプレートに培養し、リポフェクタミン２０００試薬（Lipofectamine 2000 reagent、Gibco-BLR社製）を用いてｐＥＦＢＯＳ／ＴＩＣＡＭ−２（０．５μｇ）またはｐＥＦＢＯＳ／ＴＩＣＡＭ−２−ＨＡ（０．５μｇ）と共に以下に示すベクターを一過性にトランスフェクトした。なお、「ＨＡ」はＨＡタグが付いていることを表す。
【０１４７】
ｐＣＭＶ／ＴＬＲ４またはｐＣＭＶ／Ｆｌａｇ−ＴＬＲ４（２μｇ）、ｐＥＦＢＯＳ／ＴＬＲ３またはｐＥＦＢＯＳ／Ｆｌａｇ−ＴＬＲ３（３μｇ）、ｐＣＭＶ／ＴＬＲ２またはｐＣＭＶ／Ｆｌａｇ−ＴＬＲ２（２μｇ）、ｐＥＦＢＯＳ／ＴＩＣＡＭ−１−ＨＡ（０．５μｇ）、ｐＥＦＢＯＳ／ＭＤ−２（１μｇ）。これらを上記ＨＥＫ２９３細胞にトランスフェクトした。なお、「Ｆｌａｇ」はＦｌａｇタグが付いていることを表す。
【０１４８】
トランスフェクトするＤＮＡの総量（４μｇ）は、空ベクターを加えて一定にした。ここで「空ベクター」とは発現させるための遺伝子を挿入していないベクターを意味する。
【０１４９】
トランスフェクションの２４時間後に、培地のみ、またはＬＰＳで１５ないし６０分間細胞を刺激した。その後、細胞溶解用緩衝液（２５ｍＭトリス（ｐＨ７．５）、１５０ｍＭＮａＣｌ、１％ＮＰ−４０、２ｍＭＰＭＳＦ、２５ｍＭヨードアセトアミド、１０ｍＭＥＤＴＡ）で細胞を溶解した。遠心分離後、細胞溶解液に抗Ｆｌａｇモノクローナル抗体Ｍ２（シグマ社製）を加えて４℃で２時間インキュベートした。マウスＩｇＧ１をコントロールに用いた。プロテインＧセファロースと共に免疫複合体を沈降させ、０．０２％のＴｒｉｔｏｎＸ１００を含むＰＢＳ（リン酸緩衝生理食塩液）で３回、十分に洗浄した。免疫沈降したタンパク質を１％ＳＤＳ、０．２％ＮＰ−４０および５％２−メルカプトエタノールを含むＰＢＳで溶出し、煮沸後、ＳＤＳ−ＰＡＧＥで分離した。引き続き、各抗体を用いて免疫ブロッティングを行った。いくつかの実験では、細胞溶解液を直接免疫ブロッティングに使用した。
【０１５０】
〈実験結果〉
上記結果を図７（ａ）〜（ｃ）に示す。図７（ａ）、（ｂ）および（ｃ）とも、上段は抗ＨＡ抗体でプローブされた結果を示し、下段は抗Ｆｌａｇ抗体で再プローブされた結果を示す。各レーンの上部にトランスフェクションされた発現ベクターを示している。「＋^F」はＦｌａｇタグが付いていることを表す。
【０１５１】
図７（ａ）から明らかなように、ＴＩＣＡＭ−２はＴＬＲ４と結合することが確認された（レーン６）。また、ＴＩＣＡＭ−２とＴＬＲ２またはＴＬＲ３との会合は、この条件では検出されなかった（レーン１〜４）。ＴＬＲ２はＭＤ−２と共発現した場合でもＴＩＣＡＭ−２と共沈降しなかった（レーン１、２）。５０ｋＤａ付近の微量のバンドは非特異的で、すべてのレーンでみられている。図７（ａ）上段の矢印はＨＡタグを付けたＴＩＣＡＭ−２を示す。下段の括弧はＦｌａｇタグを付けたＴＬＲタンパク質を示す。図７（ａ）に示した結果は、ＴＬＲ２、ＴＬＲ３およびＴＬＲ４に対する特異的モノクローナル抗体を用いて確認した。
【０１５２】
ＴＬＲ４とＴＩＣＡＭ−２との相互作用に関して、ＬＰＳの効果を検討した結果、図７（ｂ）に示すように、ＴＬＲ４発現細胞をＬＰＳで刺激しても、ＴＬＲ４とＴＩＣＡＭ−２との会合は影響を受けなかった（レーン３、４）。（ａ）と同様に５０ｋＤａ付近のバンドは非特異的なバンドである。図７（ｂ）上段の矢印はＴＩＣＡＭ−２のバンドを示す。下段の矢頭はＴＬＲ４を示す。
【０１５３】
ＴＩＣＡＭ−２とＴＩＣＡＭ−１、ＴＩＣＡＭ−２およびＴＬＲ４との会合について検討した結果、図７（ｃ）に示すように、ＴＬＲ４はＴＩＣＡＭ−２が存在するときのみＴＩＣＡＭ−１を引き寄せることが示された（レーン６）。同一条件下で、ＴＬＲ３は直接ＴＩＣＡＭ−１を引き寄せることが知られている（非特許文献１２）。また、ＴＩＣＡＭ−２−ＨＡとＴＩＣＡＭ−２−Ｆｌａｇが共沈降したことから、ＴＩＣＡＭ−２は２量体を形成することが示された（レーン１）。ＴＩＣＡＭ−２−ＨＡのバックグランドレベルは極微量であった（レーン２）。ＴＩＣＡＭ−２とＴＬＲ４は会合した（レーン３）。ＴＩＣＡＭ−２はＴＬＲ４非存在下ではＴＩＣＡＭ−１とほとんど結合しなかった（レーン４）。ＴＩＣＡＭ−１とＴＬＲ４との間の会合は検出されなかった（レーン５）。図７（ｃ）上段の矢印はＴＩＣＡＭ−１およびＴＩＣＡＭ−２のバンドを示す。下段の矢頭（白抜き）はＴＬＲ４を示す。
【０１５４】
なお、データを示していないが、ＴＩＣＡＭ−２のドミナントネガティブ体であるＴＩＣＡＭ−２（Ｐ１１６Ｈ）およびＴＩＣＡＭ−２（Ｃ１１７Ｈ）は、ＴＩＣＡＭ−１およびＴＩＣＡＭ−２と２量体を形成する能力を失った。また、ＴＬＲ４とＴＩＣＡＭ−１との関係は、抗ＴＬＲ４モノクローナル抗体を用いて確認した。
【０１５５】
〔実施例５：レポーター遺伝子分析によるＴＩＣＡＭ−２の機能分析〕
〈実験方法〉
ＨＥＫ２９３細胞を２４ウエルプレートに培養し（２×１０⁵個／ウエル）、リポフェクタミン２０００試薬（Lipofectamine 2000 reagent、Gibco-BLR社製）を用いてルシフェラーゼに結合したＮＦ−κＢレポーター遺伝子（０．１μｇ、Stratagene社製）またはｐ−１２５ｌｕｃレポータープラスミド（０．１μｇ）と共に以下に示すベクターを一過性にトランスフェクトした。
【０１５６】
なお、〔細胞および試薬〕の項に記載したように、本ＨＥＫ２９３細胞はＴＩＣＡＭ−２のｍＲＮＡを発現しているが、ＴＬＲ３のｍＲＮＡは発現しておらず、ＴＩＣＡＭ−１のｍＲＮＡは極僅かのみ発現していた。
【０１５７】
ＴＬＲ２、ＴＬＲ３、ＴＬＲ４＋ＭＤ−２を発現させるｐＥＦＢＯＳ（以上は０．１μｇ）。ＴＩＣＡＭ−１、ドミナントネガティブ体のＴＩＣＡＭ−１[ＴＩＣＡＭ−１ ＴＩＲ（Ｐ４３４Ｈ）]、ＭｙＤ８８、ＴＩＲＡＰ、ＴＩＣＡＭ−２、ＴＩＣＡＭ−２（ＴＩＲ）、ＴＩＣＡＭ−２（Ｐ１１６Ｈ）、ＴＩＣＡＭ−２（Ｃ１１７Ｈ）、ＩＲＡＫ−１（１−２１１）、ＩＲＡＫ−１（１−９６）を発現させるｐＥＦＢＯＳ（以上は１０、１００または２００ｎｇ）。コントロールとしてベクターのみ。これらを上記ＨＥＫ２９３細胞にトランスフェクトした。
【０１５８】
なお、図８にＴＩＣＡＭ−２、ＴＩＣＡＭ−２（ＴＩＲ）（図８ではＴＩＣＡＭ−２−ＴＩＲと表している。）、ＴＩＣＡＭ−２（Ｐ１１６Ｈ）（図８ではＴＩＣＡＭ−２−ＰＨと表している。）およびＴＩＣＡＭ−２（Ｃ１１７Ｈ）（図８ではＴＩＣＡＭ−２−ＣＨと表している。）の構造を示した。黒く塗っている部分はＴＩＲドメインを示す。ＴＩＣＡＭ−２−ＴＩＲは６８〜２３５番目のアミノ酸部分を含む。ＴＩＣＡＭ−２−ＰＨは１１６番目のプロリンをヒスチジンに置換し（白丸）、ＴＩＣＡＭ−２−ＣＨは１１７番目のシステインをヒスチジンに置換した（白丸）。
【０１５９】
ドミナントネガティブ体のＴＩＣＡＭ−１、ＴＩＣＡＭ−１ＴＩＲ（Ｐ４３４Ｈ）の構築は非特許文献１２に記載されている。ｐ−１２５ｌｕｃレポーターは谷口維紹博士（東京大学）から提供を受けたもので、ピッカジーンルシフェラーゼレポータープラスミド（東洋インキ社製）に挿入されたヒトＩＦＮ−βプロモーター領域を含む。ｐＡＰ−１−ＬｕｃレポータープラスミドはStratagene社から購入した。ＥＬＡＭ−１プロモーターを含むレポータープラスミドは自製した（Lien, E. et al. (1999) J. Biol.Chem. 274, 33419-33425.）。
【０１６０】
トランスフェクトするＤＮＡの総量（０．８−１．０μｇ）は、空ベクターを加えて調整した。また、５ｎｇのｐＣＭＶβプラスミド（クロンテック社製）を内部コントロールに使用した。
【０１６１】
ドミナントネガティブ体（ＭｙＤ８８[ＭｙＤ８８（ＴＩＲ）]、ＴＩＲＡＰ[ＴＩＲＡＰ（Ｐ１２５Ｈ）]、ＴＩＣＡＭ−１[ＴＩＣＡＭ−１ＴＩＲ（Ｐ４３４Ｈ）]またはＴＩＣＡＭ−２[ＴＩＣＡＭ−２（Ｃ１１７Ｈ）]）発現ベクターを細胞に共トランスフェクトした。２４時間後に、培地のみ、ＬＰＳ（１００ｎｇ／ｍｌ）、ポリミキシンＢで処理したＭＡＬＰ−２（１００ｎＭ）またはポリミキシンＢで処理したポリ（Ｉ：Ｃ）（１０μｇ／ｍｌ）で細胞を６時間刺激した。細胞溶解用緩衝液（プロメガ社製）を用いて細胞を溶解し、製造業者の取扱説明書に従って、ルシフェラーゼおよびβ−ガラクトシダーゼ活性を測定した。数値は少なくとも３回の独立した実験（それぞれの実験はデュプリケートで行った）の平均値と標準偏差で表した。
【０１６２】
〈実験結果〉
ルシフェラーゼに結合したＮＦ−κＢレポーター、ｐ−１２５ｌｕｃレポーター（ＩＦＮ−β）またはＡＰ−１レポータープラスミドを有するＨＥＫ２９３細胞に、空ベクターまたはＴＩＣＡＭ−２発現ベクター（１０、１００または２００ｎｇ）をトランスフェクションし、レポーター活性を検討した。その結果を図９（ａ）〜（ｃ）に示す。ＴＩＣＡＭ−２タンパク質を過剰発現させたＨＥＫ２９３細胞はＩＦＮ−βプロモーター（図９（ａ）参照）およびＮＦ−κＢ（図９（ｂ）参照）をマイルドに誘導した。一方、ＡＰ−１を誘導しなかった（図９（ｃ）参照）。
【０１６３】
野生型および変異型ＴＩＣＡＭ−２をＨＥＫ２９３細胞に過剰発現させ、ＮＦ−κＢおよびＩＦＮ−βプロモーターの活性化を検討した。その結果を図１０（ａ）〜（ｃ）に示す。
【０１６４】
全長のＴＩＣＡＭ−２およびＮ末端領域を欠失したＴＩＣＡＭ−２−ＴＩＲはＩＦＮ−βプロモーターを活性化し、ホモ２量体形成を経由していることが示唆された。ＴＩＲドメインに変異を有するＴＩＣＡＭ−２−ＣＨはＩＦＮ−βプロモーターを活性化しなかった（図１０（ａ）参照）。ＮＦ−κＢについては、ＴＩＣＡＭ−２のみがＮＦ−κＢを活性化し、ＩＦＮ−βプロモーターを活性化したＴＩＣＡＭ−２−ＴＩＲはＮＦ−κＢを活性化しなかった（図１０（ｂ）参照）。
【０１６５】
ＴＬＲ４、ＣＤ１４およびＭＤ−２を発現させたＨＥＫ２９３に、さらに野生型および変異型ＴＩＣＡＭ−２を発現させ、ＩＦＮ−βプロモーター活性化に関してＬＰＳ刺激の影響を検討した（図１０（ｃ）参照）。その結果、ＴＩＣＡＭ−２およびＴＩＣＡＭ−２−ＴＩＲはＬＰＳ刺激によりＩＦＮ−βプロモーター活性が僅かに強くなった。また、野生型ＴＩＣＡＭ−２はバックグランドを増加したが、おそらく２量体形成によるものと考えられた。ＴＩＣＡＭ−２−ＣＨ（ＴＩＣＡＭ−２（Ｃ１１７Ｈ））はドミナントネガティブとして機能し、ＬＰＳ−ＴＬＲ４を介するＩＦＮ−β活性化を効果的に阻害した。
【０１６６】
ＴＬＲ３を発現させたＨＥＫ２９３、またはＴＬＲ４、ＣＤ１４およびＭＤ−２を発現させたＨＥＫ２９３に、さらにＴＩＣＡＭ−２−ＣＨを発現させた。ＴＬＲ３を発現させた細胞に対しては５０μｇのポリ（Ｉ：Ｃ）、ＴＬＲ４を発現させた細胞に対しては１００ｎｇ／ｍｌのＬＰＳで６時間刺激し、ＩＦＮ−βプロモーターおよびＮＦ−κＢ活性化について検討した。その結果を図１１（ａ）・（ｂ）に示す。
【０１６７】
図１１（ａ）・（ｂ）から明らかなように、ＴＩＣＡＭ−２−ＣＨは、ＬＰＳ−ＴＬＲ４を介するＮＦ−κＢおよびＩＦＮ−βプロモーター活性化の両方を阻害し、ドミナントネガティブな調節因子として働いた。一方、ＴＩＣＡＭ−２−ＣＨは、ｄｓＲＮＡ−ＴＬＲ３を介するＮＦ−κＢおよびＩＦＮ−βプロモーター活性化をどちらも阻害しなかった。なお、データを示していないが、ＴＩＣＡＭ−２（Ｐ１１６Ｈ）についてもＴＩＣＡＭ−２（Ｃ１１７Ｈ）と同じ結果であり、ドミナントネガティブとして機能することを確認した。
【０１６８】
ＴＬＲ３を発現させたＨＥＫ２９３、またはＴＬＲ４、ＣＤ１４およびＭＤ−２を発現させたＨＥＫ２９３に、さらにＴＩＣＡＭ−１のドミナントネガティブ体であるＴＩＣＡＭ−１ＴＩＲ（Ｐ４３４Ｈ）（図ではＴＩＣＡＭ−１ＤＮ）を発現させた。上記と同様に、ポリ（Ｉ：Ｃ）またはＬＰＳで刺激し、ＩＦＮ−βプロモーターおよびＮＦ−κＢ活性化について検討した。その結果を図１２（ａ）・（ｂ）に示す。
【０１６９】
ＴＩＣＡＭ−１ＴＩＲ（Ｐ４３４Ｈ）はＬＰＳ−ＴＬＲ４を介するＩＦＮ−β活性化を阻害したが、阻害の効力はポリ（Ｉ：Ｃ）−ＴＬＲ３を介するＩＦＮ−β活性化の阻害より小さかった（図１２（ａ）参照）。一方、ＴＩＣＡＭ−１ＴＩＲ（Ｐ４３４Ｈ）はポリ（Ｉ：Ｃ）−ＴＬＲ３を介するＮＦ−κＢ活性化を阻害したが、ＬＰＳ−ＴＬＲ４を介するＮＦ−κＢ活性化を阻害しなかった（図１２（ｂ）参照）。
【０１７０】
ＴＩＣＡＭ−１とＴＩＣＡＭ−２の効果が相乗的か否かを評価するため、ＴＩＣＡＭ−１とＴＩＣＡＭ−２の両方を過剰発現させた細胞とＴＩＣＡＭ−２のみを過剰発現させた細胞におけるＩＦＮ−βプロモーターおよびＮＦ−κＢ活性化について検討した。その結果を図１３（ａ）・（ｂ）に示す。ＩＦＮ−βプロモーターは高用量のＴＩＣＡＭ−２で相乗的に活性化されたが、ＴＩＣＡＭ−２はＴＩＣＡＭ−１より効果が小さかった（図１３（ａ）参照）。この結果から、ＴＬＲ４を介したＩＦＮ−β誘導機能においてＴＩＣＡＭ−1が主に関与していることが示唆された。一方、ＮＦ−κＢ活性化には相乗効果が認められなかった（図１３（ｂ）参照）。
【０１７１】
ＴＩＣＡＭ−２過剰発現細胞に、ＴＩＣＡＭ−１、ＭｙＤ８８およびＴＩＲＡＰのそれぞれのドミナントネガティブ体（図ではＤＮと表示）を共発現させ、ＩＦＮ−βプロモーターおよびＮＦ−κＢ活性化について検討した。その結果を図１４（ａ）・（ｂ）に示す。
【０１７２】
図１４（ａ）・（ｂ）から明らかなように、ＩＦＮ−βプロモーターおよびＮＦ−κＢの活性化はＴＩＣＡＭ−１ＴＩＲ（Ｐ４３４Ｈ）により強く抑制された。このＴＩＣＡＭ−１ＴＩＲ（Ｐ４３４Ｈ）の効果はＴＩＣＡＭ−２機能の妨害に対して特異的であり、ＭｙＤ８８およびＴＩＲＡＰのドミナントネガティブ体は、ＩＦＮ−βプロモーターまたはＮＦ−κＢのＴＩＣＡＭ−２を介した活性化を妨害しなかった。
【０１７３】
ＴＩＣＡＭ−１過剰発現細胞に、ＴＩＣＡＭ−２のドミナントネガティブ体である。ＴＩＣＡＭ−２（Ｃ１１７Ｈ）（図ではＴＩＣＡＭ−２−ＣＨ）を共発現させ、ＩＦＮ−βプロモーターおよびＮＦ−κＢ活性化について検討した。その結果を図１５（ａ）・（ｂ）に示す。図１５（ａ）・（ｂ）から明らかなように、ＴＩＣＡＭ−２（Ｃ１１７Ｈ）はＴＩＣＡＭ−１を介するＩＦＮまたはＮＦ−κＢの活性化を阻害しなかった。
【０１７４】
上記、図１４（ａ）・（ｂ）および図１５（ａ）・（ｂ）の結果から、ＴＩＣＡＭ−２自体よりむしろＴＩＣＡＭ−１がＴＩＣＡＭ−２に近づくことがアダプター機能として重要であると考えられた。
【０１７５】
〔実施例６：ＴＩＣＡＭ−２によるＩＲＦ−３の活性化およびＴＩＣＡＭ−２とＩＲＡＫとの関係の検討〕
ＮＦ−κＢ、ＡＰ−１およびＩＲＦ−３はＩＦＮ−βを誘導する転写因子であることが知られている（Taniguti, T. et al. (2001) Annu. Rev. Immunol. 19, 623-655.）。また、ＩＲＦ−３はＨｅＬａ細胞でＴＬＲ３アゴニストに対する反応において活性化されることが示された（非特許文献１２、およびIwamura, T. (2001) Genes Cells 6, 375-388.）。そこで以下の実験を行った。
【０１７６】
ＨｅＬａ細胞に、空ベクター、ＭｙＤ８８プラスミド、ＴＩＲＡＰプラスミド、ＴＩＣＡＭ−１プラスミドおよびＴＩＣＡＭ−２プラスミド（各１μｇ）をトランスフェクトした。２４時間後に、培地のみ、または２０μｇ／ｍｌのＬＰＳで１時間処理し、細胞溶解液を調製してｎａｔｉｖｅＰＡＧＥに供した。その結果を図１６（ａ）に示す。
【０１７７】
図１６（ａ）から明らかなように、ＴＩＣＡＭ−１のトランスフェクションによりＩＲＦ−３の活性化が認められたが、ＴＩＣＡＭ−２、ＭｙＤ８８またはＴＩＲＡＰのトランスフェクションでは認められなかった。ＡＰ−１活性化はＴＩＣＡＭ−２のみがトランスフェクションされた細胞においては観察されなかったが（図９（ａ）〜（ｃ）参照）、ＴＩＣＡＭ−１がトランスフェクションされた場合はＡＰ−１活性化が認められた（非特許文献１２）。しかし、ＩＦＮ−βプロモーターはＴＩＣＡＭ−２のみのトランスフェクションによってＮＦ−κＢ活性化と同時に活性化された（図９（ａ）〜（ｃ）参照）。したがって、ＩＦＮ−βプロモーターはＴＩＣＡＭ−２過剰発現の場合にはＮＦ−κＢを経由して活性化された。
【０１７８】
ＴＩＣＡＭ−２のトランスフェクションは弱いＮＦ−κＢ活性を誘導するので、次にＩＲＡＫ−１および−２がアダプターを介したＮＦ−κＢ活性化に貢献するかどうかを検討した。すなわち、ＨＥＫ２９３細胞にｐ−１２５ｌｕｃレポータープラスミドをトランスフェクトし、さらに空ベクター、ＴＩＣＡＭ−２プラスミド（１００ｎｇ）またはＴＩＣＡＭ−２プラスミド＋ＩＲＡＫ−１（１−２１１）プラスミドを同時にトランスフェクトした。２４時間後にルシフェラーゼレポーター活性を測定した。その結果を図１６（ｂ）に示す。なお、図中のControl（斜線のバー）は空ベクターのみをトランスフェクトした細胞、黒塗りのバーはＴＩＣＡＭ−２のみをトランスフェクトした細胞を表す。値は３回の独立した実験の代表値である。
【０１７９】
図１６（ｂ）から明らかなように、ＩＲＡＫ−１のドミナントネガティブ体（ＩＲＡＫ−１（１−２１１））はＴＩＣＡＭ−２によるＩＦＮ−βプロモーター活性化を阻害した。また、ＮＦ−κＢの活性化を阻害した（示していない）。同様の実験において、ＩＲＡＫ−２のドミナントネガティブ体はＴＩＣＡＭ−２を介するＮＦ−κＢおよびＩＦＮ−βプロモーター活性化をほとんど阻害しなかった（示していない）。このように、ＩＲＡＫ−１はＴＩＣＡＭ−２の下流で作用するようであり、ＮＦ−κＢを介するＩＦＮ−βプロモーター活性化に関連がある。
【０１８０】
〔実施例７：ＲＮＡ干渉（ＲＮＡｉ）および定量ＰＣＲ法を用いた、ＴＬＲ４を介するＩＦＮ−β誘導におけるＴＩＣＡＭ−２およびＴＩＣＡＭ−１の関与の検討〕
ＴＩＣＡＭ−１／ＴＩＣＡＭ−２を介するＩＦＮ−β産生への関与を調べるために、マクロファージ様細胞であるマウスＲＡＷ２６４．７を用いてｓｉＲＮＡ（small interfering RNA）のトランスフェクションによりＴＩＣＡＭ−２ノックダウン細胞を作製し、ＬＰＳに対する反応におけるＩＦＮ−β誘導を検討した。
【０１８１】
〈実験方法〉
マウスＲＡＷ２６４．７細胞を６ウエルプレートに播種し（５×１０⁶細胞／ウエル）２４時間培養した。細胞に、バッファーのみ、コントロールｓｉＲＮＡ（ヒトＴＬＲ３またはヒトＬａｍｉｎＡ／Ｃに対するもの、配列番号２７〜３０、非特許文献１１参照）、マウスＴＩＣＡＭ−２に対するｓｉＲＮＡ（配列番号２１〜２４）またはマウスＴＩＣＡＭ−１に対するｓｉＲＮＡ（配列番号２５、２６）をオリゴフェクタミン試薬（Oligofectamine reagent、Invitrogen社製）を用いてトランスフェクトした。トランスフェクトの２４時間後に、５ｍＭのＥＤＴＡを含む生理食塩液で処理して細胞を集め、２４ウエルプレートに播種した。翌日、細胞は３０−５０％のコンフルエント状態であった。再度オリゴフェクタミンを用いてｓｉＲＮＡをトランスフェクトし、２日間培養した。培養上清を除去し、１００ｎｇ／ｍｌのＬＰＳを含む培地を各ウエルに加えた。刺激前および刺激６時間後にＴＲＩＺＯＬ試薬を用いて細胞からトータルＲＮＡを抽出した。
【０１８２】
ヒトＴＬＲ３に対するｓｉＲＮＡをコントロールとして用いた。標的のＴＬＲ３の塩基配列はヒトとマウスの間で保存されていないので、コントロールｓｉＲＮＡはマウスＴＬＲ３をダウンレギュレートしないからである。ＲＮＡ干渉に使用したオリゴヌクレオチドはＪｂｉｏＳ社から購入した。
【０１８３】
定量ＰＣＲ分析はiCycler iQ Real-Time detection system with SYBR Green Supermix reagents（Bio-Rad社製）を用いて実施した。定量ＰＣＲのプライマーには、ＴＩＣＡＭ−２用にｍＦ１、ｍＲ２、ｍＦ３およびｍＲ３を用い、ＴＩＣＡＭ−１用にｍＴ−１−Ｆ１およびｍＴ−１−Ｒ１、ＩＦＮ−β用にｍＩＦＮ−Ｆ１、ｍＩＦＮ−Ｒ１およびｍＩＦＮ−Ｒ２を用い、β―アクチン用にｍＢＡＣＴ−Ｆ１およびｍＢＡＣＴ−Ｒ１を用いた。（配列番号９、１１〜２０）ＰＣＲのサイクル数は、ＴＩＣＡＭ−２が２７サイクル、ＩＦＮ−βが３０サイクル、β−アクチンが２２サイクルとした。
【０１８４】
マウスＴＩＣＡＭ−２に対する２種類のｓｉＲＮＡの標的部位を図１７（ａ）に示す。図中サイトＡはＮ末端側に位置し、サイトＢはＴＩＲドメイン内に位置する。
【０１８５】
〈実験結果〉
図１７（ｂ）に、定量ＰＣＲ産物の１％アガロースゲル電気泳動像（エチジウムブロマイド染色）を示す。左がＬＰＳ刺激前のトータルＲＮＡを試料として用いた場合、右がＬＰＳ刺激後のトータルＲＮＡを試料として用いた場合の結果である。使用したｓｉＲＮＡを上部に表示した。「Ｍｏｃｋ」はｓｉＲＮＡのトランスフェクト時にバッファーのみを用いたものである。ＴＩＣＡＭ−２のｍＲＮＡレベルは、サイトＡを標的としたＲＮＡ干渉により約６０％まで抑制され、サイトＢを標的としたＲＮＡ干渉により約２６％まで抑制された。
【０１８６】
ＩＦＮ−β用プライマーを用いて定量ＰＣＲを行ったときのＰＣＲ産物の相対的コピー数を図１７（ｃ）に示す。数値はβ−アクチンにより補正した。Ｍｏｃｋの平均値を基準に（１．００として）、各値を計算した。なお、数値は３回の実験の平均値である。また、使用したｓｉＲＮＡを下部に示した。ＴＩＣＡＭ−２のノックダウンはＩＦＮ−βの誘導を特異的に阻害した。ＩＦＮ−βｍＲＮＡレベルは、サイトＡを標的としたＲＮＡ干渉により約６０％まで抑制され、サイトＢを標的としたＲＮＡ干渉により約３５％まで抑制された。
【０１８７】
マウスＴＩＣＡＭ−１に対するＲＮＡ干渉によりＬＰＳ刺激後のＩＦＮ−β誘導を調べた。その結果を図１７（ｄ）に示す。数値は、図１７（ｃ）の実験と同様に計算した。また、使用したｓｉＲＮＡを下部に示した。ＬａｍｉｎＡ／ＣおよびヒトＴＬＲ３のｓｉＲＮＡｓはコントロールとして使用した。ＴＩＣＡＭ−１のノックダウンによっても、ＩＦＮ−βｍＲＮＡレベルは抑制された。なお、データを示していないが、ＴＩＣＡＭ−１のｍＲＮＡレベルは約７０％まで抑制された。
【０１８８】
以上の結果より、ＴＩＣＡＭ−１およびＴＩＣＡＭ−２の両方ともマクロファージ様細胞においてＬＰＳを介してＩＦＮ−βを最大限に誘導するために必要ナ分子であることが明らかとなった。また、実施例４に示した免疫沈降の実験結果（図７（ａ）〜（ｃ）参照）と合わせて、ＴＩＣＡＭ−２は物理的にＭｙＤ８８非依存性経路の一部としてＬＰＳを介するＩＦＮ−β誘導のための、ＴＬＲ４のＴＩＲドメインとＴＩＣＡＭ−１を架橋するアダプターであることが明らかとなった。
【０１８９】
【発明の効果】
本発明は、哺乳動物のＴｏｌｌ様受容体４（ＴＬＲ４）に結合し、ＴＬＲ４とＴＩＣＡＭ−１を架橋することによりＬＰＳ／ＴＬＲ４のシグナルをＴＩＣＡＭ−１に伝え、Ｉ型インターフェロン（Ｉ型ＩＦＮ）産生を誘導する機能を有する新規アダプター分子ＴＩＣＡＭ−２を同定したことにより完成されたものである。新規アダプター分子ＴＩＣＡＭ−２は、Ｉ型ＩＦＮ誘導を司るアダプターであり、選択的にＬＰＳ／ＴＬＲ４のシグナル系の中からＩ型ＩＦＮ誘導系だけを調節できる。
【０１９０】
本発明はＩ型ＩＦＮ誘導を増強することによりＩ型ＩＦＮが有効なウイルス感染症の予防・治療薬剤、Ｉ型ＩＦＮが有効なガン治療薬剤等等に有用である。一方Ｉ型ＩＦＮ誘導を抑制することにより、エンドトキシンショック、自己免疫疾患、アトピー性疾患等の予防・治療薬剤等にも有用である。したがって、広く創薬、医療の分野での応用が大いに期待できる。
【０１９１】
【配列表】

【図面の簡単な説明】
【図１】ヒトＴＬＲ２、３、４および５と各ＴＬＲ特異的シグナル伝達経路を決定するアダプターとの会合の形態を示す模式図である。
【図２】ヒトＴＩＣＡＭ−２のアミノ酸配列と、マウスＴＩＣＡＭ−２のアミノ酸配列のアライメントを示す図である。
【図３】ヒトＴＩＣＡＭ−２、マウスＴＩＣＡＭ−２、ヒトＴＩＣＡＭ−１、マウスＴＩＣＡＭ−１、ヒトＭｙＤ８８、ヒトＴＩＲＡＰの各ＴＩＲドメイン、およびヒトＴＬＲ２のＴＩＲのアライメントを示す図である。
【図４】４種類のヒトアダプター分子のドメイン構造を示す模式図である。
【図５】（ａ）は、特定の組織由来のＴＩＣＡＭ−２ｍＲＮＡのノーザンブロット結果を示す図であり、（ｂ）は、様々なヒト細胞およびヒト細胞株におけるＴＩＣＡＭ−２ ｍＲＮＡをＲＴ−ＰＣＲにより検出した結果を示す図である。
【図６】（ａ）〜（ｃ）は、酵母ツー・ハイブリッド法によるＴＩＣＡＭ−２とＴＬＲｓまたはアダプターとの間の会合実験の結果を示す図である。
【図７】（ａ）〜（ｃ）は、免疫沈降法によるＴＩＣＡＭ−２とＴＬＲｓまたはアダプターとの間の会合実験の結果を示す図である。
【図８】野生型および変異型ＴＩＣＡＭ−２の構造を示す模式図である。
【図９】（ａ）〜（ｃ）は、ＴＩＣＡＭ−２発現によるＮＦ−κＢ、ＩＦＮ−βプロモーターおよびＡＰ−１の活性化を検討した結果を示すグラフである。
【図１０】（ａ）〜（ｃ）は、野生型および変異型ＴＩＣＡＭ−２発現によるＮＦ−κＢおよびＩＦＮ−βプロモーターの活性化を検討した結果を示すグラフである。
【図１１】（ａ）・（ｂ）は、ＴＬＲ３およびＴＬＲ４のシグナル伝達に対する変異型ＴＩＣＡＭ−２の影響を検討した結果を示すグラフである。
【図１２】（ａ）・（ｂ）は、ＴＬＲ３およびＴＬＲ４のシグナル伝達に対する変異型ＴＩＣＡＭ−１の影響を検討した結果を示すグラフである。
【図１３】（ａ）・（ｂ）は、ＴＩＣＡＭ−１とＴＩＣＡＭ−２の相乗効果を検討した結果を示すグラフである。
【図１４】（ａ）・（ｂ）は、ＴＩＣＡＭ−２と、ＴＩＣＡＭ−１、ＭｙＤ８８およびＴＩＲＡＰのそれぞれのドミナントネガティブ体とを共発現させた場合の、ＩＦＮ−βプロモーターおよびＮＦ−κＢ活性化について検討した結果を示すグラフである。
【図１５】ＴＩＣＡＭ−１とＴＩＣＡＭ−２のドミナントネガティブ体とを共発現させた場合の、ＩＦＮ−βプロモーターおよびＮＦ−κＢ活性化について検討した結果を示すグラフである。
【図１６】（ａ）は、各アダプター分子を発現させたＨｅＬａ細胞をＬＰＳで処理し、その細胞溶解液をｎａｔｉｖｅＰＡＧＥに供した結果を示す図であり、（ｂ）は、ＴＩＣＡＭ−２またはＴＩＣＡＭ−２＋ＩＲＡＫ−１ドミナントネガティブ体を発現させた細胞におけるＩＦＮ−βプロモーター活性化を検討した結果を示すグラフである。
【図１７】（ａ）は、マウスＴＩＣＡＭ−２のＲＮＡ干渉に用いたｓｉＲＮＡの部位を示す模式図であり、（ｂ）は、ＲＮＡ干渉後のＩＦＮ−βｍＲＮＡおよびＴＩＣＡＭ−２ ｍＲＮＡを定量ＰＣＲ法で増幅したＰＣＲ産物の電気泳動結果を示す図であり、（ｃ）は、ＴＩＣＡＭ−２に対するＲＮＡ干渉後、ＩＦＮ−βｍＲＮＡを定量ＰＣＲ法により定量した結果を示すグラフであり、（ｄ）は、マウスＴＩＣＡＭ−１に対するＲＮＡ干渉によりＬＰＳ刺激後のＩＦＮ−βｍＲＮＡを定量ＰＣＲ法で定量した結果を示すグラフである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to novel adapter molecule proteins and variants thereof that bind to mammalian Toll-like receptor 4 and mediate induction of type I interferon, genes encoding them, and uses thereof.
[0002]
[Prior art]
Conventionally, the biological defense mechanism against bacterial infection has been discussed from the acquired immune system via specific T cells and B cells. However, since the discovery of a Toll-like receptor (hereinafter abbreviated as “TLR”), the role of the innate immune system in the defense of infection has attracted attention.
[0003]
TLR is a signal receptor on the cell membrane involved in the recognition of microbial components in the innate immune system, which is an important biological defense mechanism that exists universally from flies to humans, and has attracted attention as a second immune discrimination system . Currently, 10 receptors (human TLR1-10) belonging to the TLR family have been confirmed in humans, and these mouse homologs (mouse TLR1-10) have also been confirmed.
[0004]
TLR is composed of leucine rich repeat (LRR) and C-terminal flanking region in the extracellular region, and TIR (Toll-interleukin-1 receptor) having homology with interleukin 1 receptor in the cell. ) Consists of domains. Studies on this TLR are revealing signal transduction pathways involved in the recognition of microbial components in the innate immune system.
[0005]
Lipopolysaccharide (hereinafter abbreviated as “LPS”) is a cell wall constituent of Gram-negative bacteria, also called endotoxin (endotoxin), and is highly toxic to mammals. Endotoxin shock caused by infection with gram-negative bacteria is caused by activation of the coagulation system, induction of iNOS (inducible nitric oxide synthase) expression, peripheral vasodilation, high cardiac output, and fever. In the end stage, the patient is in a low cardiac output and dies. Examples of host responses that cause endotoxin shock include activation of a MAPK (mitogen-activated protein kinase) system, activation of transcription factors, and production of inflammatory cytokines by intracellular signal transduction via the TLR / CD14 receptor.
[0006]
LPS is an agonist of TLR4 and activates macrophages and dendritic cells in the innate immune system. TLR4 recognizes LPS on the cell membrane together with MD-2 (myeloid differentiation factor-2) and CD14, and NF-κB (nuclear factorκB) and IRF-3 (interferon-regulatory factor3) Is activated (Non-patent Document 1). In the cell, IRF-3 is activated following induction of interferon (hereinafter abbreviated as “IFN”)-β (Non-patent Document 2). TLR4 signaling activates NF-κB through the adapter molecule MyD88, but MyD88-dependent signaling pathway does not cause LPS-induced dendritic cell maturation or IFN-β induction (Non-patent Document 2). 3). This was confirmed by analysis of cells derived from MyD88-deficient mice (Non-patent Document 4). The cell response not induced by MyD88 was called MyD88-independent response and was expected to involve an unknown adapter molecule.
[0007]
TIRAP (also called Toll-interleukin-1 receptor domain-containing adapter protein, Mal) was originally thought to be necessary for the MyD88-independent pathway (Non-patent Documents 5 and 6). Indeed, dominant negative forms of TIRAP and synthetic TIRAP peptides were reported to inhibit IFN-β induction and PKR phosphorylation (Non-Patent Documents 5 and 7). However, dendritic cell maturation and type I IFN induction are almost independent of TIRAP in TIRAP knockout studies, and TIRAP ^-/- The cell phenotype is MyD88. ^-/- It was similar to the cell phenotype (Non-Patent Documents 8 and 9). Therefore, TIRAP-mediated activation of the IFN-β-inducing pathway appears to be minimal, if any, in cultured mouse cells and many human cells.
[0008]
TLR3 recognizes polyinosinic acid-polycytidylic acid (hereinafter abbreviated as “poly (I: C)”), activates the IFN-β promoter, and activates NF-κB to a lower extent (Non-Patent Documents 10 and 11). 12). Type I IFN production and dendritic cell maturation are typical cellular responses induced by dendritic cell activation via TLR3. Recently, an adapter molecule named TIR domain containing adapter molecule-1 (hereinafter abbreviated as “TICAM-1”) directly binds to TIR of TLR3 and activates IRF-3 independently of MyD88. It became clear that. However, in the yeast two-hybrid system and direct immunoprecipitation derived from mammalian cells, TICAM-1 hardly bound to the TIR of TLR4 (Non-patent Document 12). This result predicted that another factor or mechanism was involved in the MyD88-independent pathway of TLR4.
[0009]
By the way, type I IFN (IFNα and IFNβ) induced by TLR is known to exhibit antiviral action and anticancer action. Specifically, type I IFN is known to have the following actions.
[0010]
It is known that type I IFN exerts an antiviral action by a plurality of action mechanisms as described below.
[0011]
(1) Deactivate viral mRNA and activate intracellular genes that suppress host protein translation. This suppresses virus replication and suppresses virus growth.
[0012]
(2) It induces the expression of MHC class I molecules and induces resistance to natural killer (NK) cells. Cytotoxic CD8 ⁺ Increases sensitivity to T cells. Furthermore, it is also involved in suppression of T cell activation and enhancement of T cell suppressor activity.
[0013]
(3) Activate natural killer (NK) cells that selectively damage virus-infected cells, and induce apoptosis by natural killer (NK) cells in the virus.
[0014]
In addition, it is known that type I IFN exerts an antitumor effect by the following plural mechanisms of action.
[0015]
1) Destabilize mRNA in tumor cells and activate intracellular genes that suppress host protein translation. Thereby, the synthesis | combination of the protein in a tumor cell is suppressed and the proliferation of a tumor cell is suppressed.
[0016]
2) Activates anti-tumor effectors such as macrophages, natural killer (NK) cells, natural killer T (NKT) cells, etc., and induces apoptosis in tumor cells by damaging tumor cells with these anti-tumor effectors.
[0017]
3) Activate natural killer (NK) cells that selectively damage virus-infected cells, and induce apoptosis by natural killer (NK) cells in tumor cells.
[0018]
Furthermore, since type I IFN is also involved in suppression of T cell activation and enhancement of T cell suppressor activity as described above, it is considered that certain types of autoimmune diseases can be improved.
[0019]
Since type I IFN has antiviral and antitumor effects as described above, conventionally, IFNα preparations and IFNβ preparations have been developed from hepatitis B, hepatitis C, hepatitis C, liver cancer, kidney It is used as a therapeutic agent for cancer. For example, “Sumiferon (registered trademark)” manufactured by Sumitomo Pharmaceutical Co., Ltd., which is a natural type IFNα preparation, has shown good results in clinical application.
[0020]
[Non-Patent Document 1]
Medzhitov, R. (2001) Toll-like receptors and innate immunity. Nat. Rev. Immunol. 1: 135-145.
[0021]
[Non-Patent Document 2]
Kawai, T., Takeuchi, O., Fujita, T., Inoue, J., Muhlradt, PF, Sato, S., Hoshino, K., and Akira, S. (2001) Lipopolysaccharide stimulates the MyD88-independent pathway and results in activation of IFN-regulatory factor 3 and the expression of a subset of lipopolysaccharide-inducible genes.J. Immunol. 167: 5887-5894.
[0022]
[Non-Patent Document 3]
Kaisho, T., Takeuchi, o., Kawai, T., Hoshino, K., and Akira, S. (2001) Endotoxin-induced maturation of MyD88-deficient dendritic cells. J. Immunol. 166: 5688-5694.
[0023]
[Non-Patent Document 4]
Kawai, T., Adachi, O., Ogawa, T., Takeda, K., and Akira, S. (1999) Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity 11: 115-122.
[0024]
[Non-Patent Document 5]
Horng, T., Barton, GM, and Medzhitov, R. (2001) TIRAP: an adapter molecule in the Toll signaling pathway. Nat. Immunol. 2: 835-841.
[0025]
[Non-Patent Document 6]
Fitzgerald, KA, Palsson-McDermott, EM, Bowie, AG, Jefferies, CA, Mansell, AS, Brady, G., Brint, E., Dunne, A., Gray, P., Harte, MT, McMurray, D. , Smith, DE, Sims, JE, Bird, TA, and O'Neill, LA (2001) Mal (MyD88-adapter-like) is required for Toll-like receptor-4 signal transduction.Nature 413: 78-83.
[0026]
[Non-Patent Document 7]
Toshchakov, V., Jones, BW, Perera, PY, Thomas, K., Cody, MJ, Zhang, S., Williams, BR, Major, J., Hamilton, TA, Fenton, MJ, and Vogel, SN (2002 ) TLR4, but not TLR2, mediates IFN--induced STAT1α / β-dependent gene expression in macrophages. Nat. Immunol. 3: 392-398.
[0027]
[Non-Patent Document 8]
Yamamoto, M., Sato, S., Hemmi, H., Sanjo, H., Uematsu, S., Kaisho, T., Hoshino, K., Takeuti, O., Kobayashi, M., Fujita, T., Takeda, K., and Akira, S. (2002) Essential role for TIRAP in activation of the signaling cascade shared by TLR2 and TLR4.Nature 420: 324-329.
[0028]
[Non-patent document 9]
Horng, T., Barton, GM, Flavell, RA, and Medzhitov, R. (2002) The adapter molecule TIRAP provides signaling specificity for Toll-like receptors. Nature 420: 329-333.
[0029]
[Non-Patent Document 10]
Alexopoulou, L., Holt, AC, Medzhitov, R., and Flavell, RA (2001) Recognition of double-stranded RNA and activation of NF-κB by Toll-like receptor 3. Nature 413: 732-738.
[0030]
[Non-Patent Document 11]
Matsumoto, M., Kikkawa, S., Kohase, M., Miyake, K., and Seya, T. (2002) Establishment of a monoclonal antibody against human Toll-like receptor3 that blocks double-stranded RNA-mediated signaling. Biophys Res Commun. 293: 1364-1369.
[0031]
[Non-Patent Document 12]
Oshiumi, H., Matsumoto, M., Funami, K., Akazawa, T., and Seya, T. (2003) TICAM-1, an adaptor molecule that participates in Toll-like receptor3-mediated interferon β-induction. Nature Immunol. 4: 161-167.
[0032]
[Problems to be solved by the invention]
As described above, there is another pathway that does not depend on MyD88 in signaling of TLR4 that recognizes LPS, and this MyD88-independent pathway causes maturation of dendritic cells and induction of type I IFN caused by LPS. It was reported. From a comprehensive evaluation of these known facts, it was predicted that an unknown adapter molecule was involved in the MyD88-independent pathway of TLR4, but the adapter molecule has not yet been identified.
[0033]
If a molecule that directly binds to TLR4 and transmits a signal downstream to induce type I IFN is clarified, it is possible to elucidate a signal transduction pathway and a signal transduction regulatory mechanism that induce type I IFN in an innate immune response via TLR4. It is thought that it can be used effectively for elucidating the pathology of various diseases related to the innate immune system and for developing therapeutic agents by regulating the innate immune response.
[0034]
In addition, the above-mentioned interferon preparations used for the treatment of viral infections and tumors are systemically administered. Therefore, at concentrations where therapeutic effects can be obtained, psychological / neurological symptoms (such as depression), autoimmune diseases (thyroid dysfunction, Side effects such as worsening of autoimmune hepatitis, hemolytic anemia, ulcerative colitis, rheumatoid arthritis, etc. are strong. The side effect of interferon preparations is that interferon is introduced even into normal host cells that do not originally produce interferon, so the presentation of self-antigens to T cells that should be originally performed during an immune response to external antigens is the introduction of interferon. It is thought that this is because it is induced throughout the site and autoimmune phenomenon is likely to occur.
[0035]
Thus, since the interferon preparation has a strong side effect at a concentration at which a therapeutic effect can be obtained, it is difficult to maintain a sufficient anticancer action by systemic administration. Moreover, even if the interferon preparation is administered locally, it is considered difficult to completely avoid side effects.
[0036]
When a molecule that directly binds to TLR4 and induces type I IFN by transmitting a signal downstream is clarified, treatment of viral infections, tumors, etc. by enhancing local type I interferon production in vivo It is believed that new therapeutic drugs can be developed. Furthermore, by providing a substance that inhibits the production of type I interferon in vivo through the functional analysis of this novel protein, and by inhibiting the production of type I interferon in vivo by this substance, autoimmune diseases, atopic diseases, etc. It is considered that a new therapeutic agent for treating can be provided.
[0037]
Furthermore, since TLR4 is a receptor that recognizes LPS, endotoxin shock can be prevented and treated with the substance that inhibits the production of type I interferon in vivo.
[0038]
Endotoxin shock caused by infection with gram-negative bacteria exhibits peripheral vasodilation, high cardiac output, fever, etc. through the activation of the coagulation system and induction of iNOS due to the production of inflammatory cytokines. Since there is a low cardiac output and death, there is currently no effective treatment, and there is an urgent need to develop an effective treatment.
[0039]
The present invention has been made in view of the above problems, and an object of the present invention is to provide a novel adapter molecule that directly binds to TLR4 and mediates type I IFN induction, a gene thereof, a drug using the same, and the like. It is in.
[0040]
[Means for Solving the Problems]
As a result of intensive studies in view of the above problems, the present inventors have identified a novel adapter molecule that directly binds to TLR4, and this molecule crosslinks TLR4 and TICAM-1, thereby allowing a MyD88-independent pathway via TLR4. Was found to induce IRF-3 activation due to LPS, and the present invention was completed.
[0041]
That is, the novel adapter molecule according to the present invention is an adapter that controls induction of type I IFN, and is characterized in that it can selectively regulate only the induction system of type I IFN from the LPS / TLR4 signal system.
[0042]
Since the novel adapter molecule has a TIR domain having a high homology with the TIR domain of TIR-containing adapter molecule-1 (TICAM-1), the TIR domain-containing adapter molecule 2 (TIR-containing adapter molecule-2). Hereinafter, the novel adapter molecule according to the present invention is referred to as “TICAM-2” as appropriate.
[0043]
The adapter molecule TICAM-2 is a novel adapter protein that was first identified by the present inventors and whose full-length amino acid sequence was clarified. SEQ ID NO: 2 shows a human-derived amino acid sequence, and SEQ ID NO: 4 shows a mouse-derived amino acid sequence.
[0044]
Therefore, the protein according to the present invention is a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or 4 or a variant thereof. Specifically, the following proteins are included in the present invention.
(1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or 4.
(2) The amino acid sequence shown in SEQ ID NO: 2 consists of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added, and 75 of the sequence shown in SEQ ID NO: 2 A protein having the amino acid sequence of −213.
(3) The amino acid sequence shown in SEQ ID NO: 4 consists of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added, and 72 of the sequences shown in SEQ ID NO: 4 A protein having the amino acid sequence at position 210.
(4) 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 the mammalian Toll-like receptor 4 A protein that has both the property of directly binding to and the property of mediating type I IFN induction.
(5) A property of the amino acid sequence shown in SEQ ID NO: 2 or 4, comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added, and mediates induction of type I IFN On the other hand, a protein having an abnormality in the property of directly binding to the mammalian Toll-like receptor 4.
(6) 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 the mammalian Toll-like receptor 4 A protein that has the property of directly binding to I, but having an abnormality in the property of mediating induction of type I IFN.
(7) The protein according to (6) above, wherein in the amino acid sequence shown in SEQ ID NO: 2, at least the 116th amino acid is substituted or deleted.
(8) The protein according to (6) above, wherein at least the 117th amino acid is substituted or deleted in the amino acid sequence shown in SEQ ID NO: 2.
[0045]
The above proteins are useful not only as research materials for research and analysis of TIR4-mediated signal transduction system and its regulatory mechanism, but also through these studies to elucidate the pathology of various diseases related to the signal transduction system and its regulatory mechanism. There is a possibility that it can be used effectively.
[0046]
Among the above proteins, (1) to (5) are proteins that normally have a function of mediating type I IFN induction of TICAM-2. As will be described later, it was confirmed that TICAM-2 activates the IFN-β promoter when overexpressed alone. Therefore, the proteins (1) to (5) are effective for various diseases that can be improved by the production of type I IFN. For example, it can be used as a drug for preventing / treating viral infections, or a drug for treating cancers (such as renal cancer, cervical cancer, and hepatic cancer after hepatitis) in which type I IFN is effective. That is, the present invention includes a virus infection preventive / therapeutic agent and a cancer therapeutic agent characterized by containing the TICAM-2 protein or TICAM-2 protein variant of (1) to (5) above.
[0047]
The above (6) to (8) are dominant negative mutant proteins having an abnormality in the function of mediating type I IFN induction. While these dominant negative mutants have the property of directly binding to TLR4, they have an abnormality in the property of mediating type I IFN induction and block the signal transmission of TLR4. Therefore, the proteins (6) to (8) can suppress the production of type I IFN, and can be used as drugs for treating adverse events caused by the production of type I IFN, such as autoimmune diseases and atopic diseases. . Furthermore, since TLR4 specifically recognizes LPS and its signal transduction becomes a trigger for endotoxin shock, the above proteins (6) to (8) can be used as drugs for preventing and treating endotoxin shock caused by LPS. . That is, the present invention includes an autoimmune disease therapeutic agent, an atopic disease therapeutic agent, and an endotoxin shock preventive / therapeutic agent characterized by including a variant of the TICAM-2 protein of (6) to (8) above It is.
[0048]
The genes according to the present invention include the following genes.
(A) A gene encoding the protein of (1) to (8) above.
(B) A gene having a base sequence represented by SEQ ID NO: 1 or 3.
(C) Hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 1 or 3, and directly binds to a mammalian Toll-like receptor 4 A gene that encodes a protein that has both properties and mediates the induction of type I interferon induction.
(D) While hybridizing under stringent conditions with DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 1 or 3, and having the property of mediating type I interferon induction, A gene encoding a protein having an abnormality in the property of directly binding to a mammalian Toll-like receptor 4.
(E) Hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 1 or 3, and directly binds to a mammalian Toll-like receptor 4 A gene that encodes a protein having an abnormality in the property of mediating induction of type I interferon while having properties.
[0049]
Among the above genes, (a) a gene encoding the protein (1) to (5) and (b) to (d) are proteins having a function of mediating type I IFN induction of TICAM-2 normally. It is a gene that encodes. In addition, the genes encoding the proteins (6) to (8) in (a) above, and (e) are the genes encoding dominant negative mutant proteins having an abnormality in the function of mediating type I IFN induction. .
[0050]
The above genes are not only useful as research materials for research and analysis of TIR4-mediated signal transduction system and its regulatory mechanism, but through such research, the pathology of various diseases related to the signal transduction system and its regulatory mechanism. It may be used effectively for elucidation.
[0051]
The recombinant expression vector according to the present invention comprises the genes (a) to (e) above.
[0052]
When the recombinant expression vector is used, the target cell can be transformed by a known genetic engineering technique to introduce the gene into the target cell. The transformant obtained by transformation is not only useful as a research material for studying and analyzing the signal transduction system via TIR4 and its regulatory mechanism, but through such research, the signal transduction system and its regulatory mechanism are also used. There is a possibility that it can be effectively used to elucidate the pathology of various diseases related to the disease.
[0053]
Among the above recombinant expression vectors, the recombinant expression vector containing the gene (1) to (5) encoding the protein (a) and the gene (b) to (d) prevents viral infection. -It can be used as a drug to treat or a cancer to treat type I IFN. That is, the present invention includes a viral infection characterized in that it comprises a gene encoding the protein (1) to (5) of (a) above and a recombinant expression vector containing the gene (b) to (d). Prophylactic / therapeutic drugs and cancer therapeutic drugs are included.
[0054]
Among the above recombinant expression vectors, the recombinant expression vector comprising the gene encoding the protein (6) to (8) of (a) and the gene of (e) treats autoimmune diseases and atopic diseases. It can be used as an agent for preventing or treating endotoxin shock caused by LPS. That is, the present invention includes a therapeutic agent for an autoimmune disease comprising a gene encoding the protein (6) to (8) of (a) above and a recombinant expression vector containing the gene (e). , Drugs for the treatment of atopic diseases, and drugs for the prevention and treatment of endotoxin shock.
[0055]
The polynucleotide according to the present invention is a polynucleotide containing a part of the 87-791st nucleotide sequence in the nucleotide sequence shown in SEQ ID NO: 1, or the 120-815th nucleotide in the nucleotide sequence shown in SEQ ID NO: 3. It is a polynucleotide containing a part of the base sequence.
[0056]
The polynucleotide can be used as siRNA (small interfering RNA) for RNA interference. By conducting RNA interference experiments using these siRNAs, not only is it useful as a research material for studying and analyzing the signal transduction system via TIR4 and its regulatory mechanism, but through such research, the signal transduction system and It may be used effectively to elucidate the pathology of various diseases related to the regulatory mechanism.
[0057]
In addition, by transfecting these siRNAs into target cells, the expression of TICAM-2 protein can be suppressed, whereby the polynucleotide can be used as a drug for treating autoimmune diseases and atopic diseases, and LPS. It can be used as a drug for preventing and treating the endotoxin shock caused by it. That is, the present invention includes an autoimmune disease therapeutic agent, an atopic disease therapeutic agent, and an endotoxin shock preventive / therapeutic agent characterized by containing the above-mentioned polynucleotide.
[0058]
The antibodies according to the present invention include the following antibodies.
(I) An antibody against the proteins (1) to (8) above.
(Ii) An antibody against the protein of any one of (1) to (5) above, which inhibits the property of mediating the induction of type I IFN by binding to the protein.
[0059]
The above antibody is considered useful for detection of TICAM-2 protein expression, purification of TICAM-2 protein, and the like.
[0060]
Since the antibody of (ii) inhibits the property of TICAM-2 that mediates type I IFN induction, it is used as a drug for treating autoimmune diseases and atopic diseases, and as a drug for preventing and treating endotoxin shock caused by LPS. Available. That is, the present invention includes an autoimmune disease therapeutic agent, an atopic disease therapeutic agent, and an endotoxin shock preventive / therapeutic agent characterized by including the antibody of (ii) above.
[0061]
The present invention includes a method of screening for a substance that suppresses or inhibits the function of TICAM-2 that mediates type I IFN induction. In this screening method, the above proteins (1) to (5) may be used. In addition, substances that suppress or inhibit the function of TICAM-2 that mediates type I IFN induction obtained by using this screening method include drugs that treat autoimmune diseases and atopic diseases, and endotoxin shock caused by LPS Can be used as a drug to prevent or treat That is, the present invention includes an agent for treating an autoimmune disease and atopic disease treatment characterized in that it contains a substance that suppresses or inhibits a function that mediates type I IFN induction of TICAM-2 obtained by using the above screening method. Drugs and endotoxin shock prevention and treatment drugs are included.
[0062]
DETAILED DESCRIPTION OF THE INVENTION
An embodiment of the present invention will be described as follows. Note that the present invention is not limited to this.
[0063]
[1] Novel adapter molecule TICAM-2 protein and gene thereof according to the present invention
Based on previous research on the LPS / TLR4 signaling pathway, there are MyD88-dependent and MyD88-independent pathways in the LPS / TLR4 signaling pathway, and unknown adapter molecules in the MyD88-independent pathway Was expected to be involved.
[0064]
The present inventors tried to identify the unknown adapter molecule, physically cross-links TLR4 and TICAM-1, and functionally transmits LPS / TLR4 signals to TICAM-1 to sequentially produce IRF-3. A novel adapter molecule, TICAM-2, that activates and identified human and mouse TICAM-2 genes.
[0065]
FIG. 1 outlines the association of human TLRs 2, 3, 4 and 5 with adapters that determine each TLR-specific signaling pathway. In the figure, M-1 represents MyD88, M-2 represents TIRAP (Mal), T-1 represents TICAM-1, and T-2 represents TICAM-2. In TLR5, MyD88 binds directly and activates NF-κB. TLR2 transmits a signal to MyD88 via TIRAP and activates NF-κB. In TLR3, NF-κB is activated by a MyD88-mediated pathway (MyD88-dependent pathway), and an IFN-β promoter is activated by a TICAM-1-mediated pathway (MyD88-independent pathway). In TLR4, TLR4 attracts two adapters TIRAP and TICAM-2 by the LPS signal and links them to MyD88 and TICAM-1, respectively, thereby forming a MyD88-dependent pathway and a MyD88-independent pathway.
[0066]
SEQ ID NO: 1 shows the base sequence of human TICAM-2 full-length cDNA, and SEQ ID NO: 3 shows the base sequence of mouse TICAM-2 full-length cDNA. In the base sequence of SEQ ID NO: 1, the 87th to 791st base sequences correspond to an open reading frame (ORF) region encoding human TICAM-2, and in the base sequence of SEQ ID NO: 3, the 120th to 815th base sequences are Corresponds to the open reading frame (ORF) region encoding mouse TICAM-2. In addition to this ORF region, each cDNA sequence shown in SEQ ID NOs: 1 and 3 contains untranslated regions on the 5 ′ side and 3 ′ side, respectively.
[0067]
SEQ ID NO: 2 shows the amino acid sequence of human TICAM-2 protein, and SEQ ID NO: 4 shows the amino acid sequence of mouse TICAM-2 protein. These amino acid sequences are the amino acid sequences of the proteins encoded by the ORFs of the base sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3, respectively.
[0068]
The protein according to the present invention is not limited to the two proteins whose amino acid sequences are shown in SEQ ID NO: 2 and SEQ ID NO: 4, and may be a mutant protein in which a part thereof is modified. That is, the protein of the present invention includes not only (i) a protein comprising any one of the amino acid sequences represented by SEQ ID NO: 2 or 4, but also (ii) any amino acid sequence represented by SEQ ID NO: 2 or 4, Also included are proteins consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added.
[0069]
The above “one or several amino acids are substituted, deleted, inserted, and / or added” means substitution, deletion, insertion, or the like by a known mutant protein production method such as site-directed mutagenesis. And / or a number that can be added (preferably 10 or less, more preferably 7 or less, and even more preferably 5 or less) amino acids are substituted, deleted, inserted, and / or added. Thus, the protein of (ii) above is a mutant protein of the protein of (i) above, and the “mutation” referred to here means a mutation artificially introduced mainly by a known mutant protein production method. However, it may be a product obtained by isolating and purifying a similar mutant protein that exists in nature.
[0070]
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-labeled by His, Myc, Flag, or the like.
[0071]
Furthermore, the protein of (ii) includes a protein that normally has a property of mediating type I IFN induction among a property of directly binding to TLR4 possessed by TICAM-2 and a property of mediating type I IFN induction; And proteins having an abnormality in the property of mediating IFN induction.
[0072]
Examples of the former protein include: (1) TIR domainin (the amino acid sequence at positions 75 to 213 in the sequence shown in SEQ ID NO: 2 in humans, and the 72nd to 210th positions in the sequence shown in SEQ ID NO: 4 in mice) (2) a protein having the above mutation in a portion other than (amino acid sequence part), (2) a protein having the above mutation and having both the property of directly binding to TLR4 and the property of mediating type I IFN induction, (3) having the above mutation However, there is a protein having an abnormality in the property of directly binding to TLR4 while having a property of mediating induction of type I IFN. The phrase “having an abnormality in the property of directly binding to TLR4” means that the property of binding to TLR4 has been lost, that is, it does not bind to TLR4.
[0073]
Examples of the latter protein include (4) a protein having the above mutation and having the property of directly binding to TLR4 while having an abnormality in the property of mediating type I IFN induction. “Having an abnormality in the property of mediating induction of type I IFN” means that the IFN-β promoter is not activated, as shown in FIGS. 10 (a) and 11 (a) in [Example 5]. To do. Specifically, a protein in which the 116th proline is replaced with histidine, or a protein in which the 117th cysteine is replaced with histidine has the property of directly binding to TLR4, while the property of mediating type I IFN induction. It is known to be a protein with an abnormality.
[0074]
The gene according to the present invention includes (I) a gene encoding the protein of (i) or (ii) above, (II) a gene having any one of the nucleotide sequences shown in SEQ ID NO: 1 or 3, and (III) sequence A gene that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to DNA consisting of the base sequence shown in No. 1 or 3 is included.
[0075]
“Stringent conditions” in (III) above is hybridization only when at least 90% identity, preferably at least 95% identity, most preferably at least 97% identity exists between sequences. Means that happens.
[0076]
Hybridization can be performed by a conventionally known method such as the method described in J. Sambrook et al. Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory (1989). Usually, the higher the temperature and the lower the salt concentration, the higher the stringency (the more difficult it becomes to hybridize).
[0077]
The gene of (III) above includes a gene encoding a protein normally having the property of mediating type I IFN induction among the properties of TICAM-2 directly binding to TLR4 and the properties of mediating type I IFN induction And a gene encoding a protein having an abnormality in the property of mediating type I IFN induction.
[0078]
The “gene” of the present invention includes DNA and RNA. DNA includes, for example, cDNA and genomic DNA obtained by cloning, chemical synthesis techniques, or a combination thereof. The DNA may be double-stranded or single-stranded, and the single-stranded DNA may be a coding DNA that serves as a sense strand or an anti-coding DNA that serves as an antisense strand (the antisense strand serves as a probe). Or it can be used as an antisense drug).
[0079]
Furthermore, the “gene” of the present invention may include sequences such as untranslated region (UTR) sequences and vector sequences (including expression vector sequences), in addition to sequences encoding proteins.
[0080]
[2] Methods for obtaining genes and proteins according to the present invention
The method for obtaining the gene according to the present invention is not particularly limited, and a DNA fragment comprising the above SEQ ID NO: 1 or 3 is isolated and cloned by various methods based on the disclosed sequence information and the like. can do.
[0081]
For example, a probe that specifically hybridizes with a partial sequence of the nucleotide sequence shown in SEQ ID NO: 1 or 3 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 shown in SEQ ID NO: 1 or 3 or its complementary sequence. . Moreover, what is necessary is just to perform about each step in the said screening on the conditions used normally.
[0082]
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.
[0083]
In addition, if the sequence of the probe is selected from TIR domains considered to be important for the function of TICAM-2 and a cDNA library of human, mouse or other mammal is screened, the same function as TICAM-2 is obtained. Genes encoding homologous and related molecules can be isolated and cloned.
[0084]
As a method for obtaining the gene according to the present invention, there is a method using an amplification means such as PCR in addition to the above screening method. For example, in the base sequence shown in SEQ ID NO: 1 or 3, primers are prepared from the 5 ′ side and 3 ′ side sequences (or their complementary sequences), and human or mouse genomic DNA is used using these primers. By performing PCR or the like using (or cDNA) or the like as a template and amplifying a DNA region sandwiched between both primers, a large amount of DNA fragments containing the gene of the present invention can be obtained.
[0085]
The method for obtaining the protein according to the present invention is not particularly limited. For example, a recombinant expression vector into which the gene obtained as described above is introduced is prepared, and Escherichia coli, yeast, etc. are prepared by a well-known method. The protein according to the present invention can be easily obtained by expressing and purifying the protein encoded by the gene as a transformant by incorporating it into a microorganism or animal cell.
[0086]
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 for the expression 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.
[0087]
The method for producing the mutant protein is not particularly limited. For example, site-directed mutagenesis (Hashimoto-Gotoh, Gene 152, 271-275 (1995), etc.), PCR method is used to determine the nucleotide sequence. One or more bases are substituted in the base sequence of each cDNA using a known mutant protein production method such as a method for producing a mutant protein by introducing a point mutation, or a mutant strain production method by inserting a transposon. , Deletions, insertions, and / or modifications can be made.
[0088]
[3] Recombinant expression vector
The recombinant expression vector according to the present invention contains a gene encoding the above-described TICAM-2 or a mutant thereof. 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.
[0089]
When the TICAM-2 protein or a variant thereof is introduced into a human using this vector, the recombinant expression vector according to the present invention is preferably a viral vector. Although it does not specifically limit as this viral vector, For example, an adenoviral vector and a retroviral vector can be mentioned. When used for medical applications such as gene therapy, it is advantageous to select a vector that has been used since it is possible to predict the safety and effectiveness of gene therapy.
[0090]
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.
[0091]
The host cell is not particularly limited, and various conventionally known cells can be suitably used. Specifically, including cells derived from human or mouse, for example, bacteria such as Escherichia coli, yeast (budding yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe), nematode (Caenorhabditis elegans), Xenopus laevis) oocytes, cultured cells of various mammals (rats, rabbits, pigs, monkeys, etc.), cultured cells of insects such as Drosophila melanogaster, Bombyx mori, etc., but are not particularly limited. Absent.
[0092]
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. .
[0093]
[4] Polynucleotide
The polynucleotide according to the present invention is the nucleotide sequence of the 87th to 791th base sequence (ORF region) in the base sequence shown in SEQ ID NO: 1, or the base sequence of the 120th to 815th in the base sequence shown in SEQ ID NO: 3. It is a polynucleotide containing a part of (ORF region).
[0094]
The polynucleotide according to the present invention includes DNA and RNA, and can be obtained by amplification techniques such as cloning, chemical synthesis, and PCR. It may be single-stranded or double-stranded, and the length (number of bases) is not particularly limited. This polynucleotide can be used for probes for detecting TICAM-2 gene, antisense methods, RNA interference methods and the like.
[0095]
When the polynucleotide is used as siRNA (small interfering RNA) for RNA interference (RNAi), it is preferably a double-stranded RNA, preferably 21 to 23 bases. Here, RNA interference (RNAi) is a phenomenon in which double-stranded RNA (siRNA) introduced into a cell suppresses the expression (protein synthesis) of a gene having the same sequence as the target gene (mRNA). It is a method of suppressing the expression by destroying.
[0096]
In order to suppress the expression of the TICAM-2 gene by RNAi, it is necessary to design an siRNA having an ORF base sequence. The siRNA is not particularly limited as long as it has an ORF base sequence, but preferably has a base sequence that is 75 bases or more downstream from the start codon, and more preferably a TIR domain base sequence.
[0097]
[5] Antibody
The antibody according to the present invention is an antibody obtained as a polyclonal antibody or a monoclonal antibody by a known method using the protein according to the present invention or a partial peptide thereof as an antigen. Known methods are described, for example, in 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 method is mentioned. The antibody thus prepared is effective for detecting the protein of the present invention.
[0098]
In addition, the antibody according to the present invention is the above-described (i) protein consisting of any one of the amino acid sequences shown in SEQ ID NO: 2 or 4, or (ii) any one of the amino acid sequences shown in SEQ ID NO: 2 or 4, An antibody against a protein having a property of mediating type I IFN induction among proteins consisting of amino acid sequences in which one or several amino acids are substituted, deleted, inserted, and / or added, It is an antibody that inhibits the property of mediating type I IFN induction.
[0099]
Such an antibody can also be obtained as a polyclonal antibody or a monoclonal antibody by a known method using the target protein or a partial peptide thereof as an antigen. Whether the obtained antibody has the property of inhibiting the property of mediating type I IFN induction is determined by, for example, mediating TICAM-2 type I IFN induction using cultured cells expressing TICAM-2. What is necessary is just to confirm whether the property to do is inhibited by adding an antibody.
[0100]
[6] Screening method and substance obtained by the screening method
The screening method according to the present invention is a method for screening for a substance that suppresses or inhibits the function of TICAM-2 that mediates type I IFN induction, and is performed using the protein according to the present invention. Examples of the protein to be used include (i) a protein consisting of any one of the amino acid sequences shown in SEQ ID NO: 2 or 4, or (ii) any one of the amino acid sequences shown in SEQ ID NO: 2 or 4, or a number Among proteins consisting of amino acid sequences in which a single amino acid is substituted, deleted, inserted, and / or added, a protein having a normal property of mediating type I IFN induction can be mentioned.
[0101]
As a screening method, a conventionally known method for examining the presence or absence of the property of suppressing or inhibiting the function that mediates type I IFN induction can be applied, and it is not particularly limited.
[0102]
Substances that suppress or inhibit the function of mediating type I IFN induction obtained by screening are also included in the present invention and can be used for the uses described below.
[0103]
[7] Drugs for preventing and treating viral infections and drugs for cancer treatment
TICAM-2 is an adapter that controls the induction of type I IFN, and is characterized in that it can selectively regulate only the type I IFN induction system from the LPS / TLR4 signal system. TICAM-2 can also activate the IFN-β promoter when it alone is overexpressed alone.
[0104]
Therefore, TICAM-2 protein and a mutant protein of TICAM-2 having the property of mediating induction of type I IFN can be used for the prevention or treatment of various diseases for which type I IFN is effective. For example, it can be used as a drug for preventing / treating viral infections, or a drug for treating cancers in which type I IFN is effective (eg, renal cancer, cervical cancer, liver cancer after hepatitis). Specifically, prevention / treatment of viral infectious disease containing any one of the protein (1), (2) or (3) of (i) or (ii) shown in the above-mentioned embodiment [1] It can be implemented as a drug or a cancer treatment drug.
[0105]
In addition, a recombinant expression vector containing a gene encoding either the TICAM-2 protein or a TICAM-2 mutant protein that normally has a property of mediating induction of type I IFN, a viral infection site, its peripheral site, or By introducing the above type I IFN into an effective cancer lesion, it becomes possible to prevent or treat viral infection and to treat cancer.
[0106]
Therefore, the above recombinant expression vector can be used as a viral infection prevention / treatment drug or a cancer treatment drug. Specifically, the gene encoding any one of the proteins (1), (2), and (3) of (i) or (ii) shown in the above embodiment [1], SEQ ID NO: 1 or Type I of genes having any of the nucleotide sequences shown in 3 and genes that hybridize under stringent conditions with DNA having a nucleotide sequence complementary to the DNA having the nucleotide sequence shown in SEQ ID NO: 1 or 3 It can be implemented as a prophylactic / therapeutic agent for viral infections or a cancer therapeutic agent comprising a recombinant vector containing any of the genes encoding proteins that normally have the property of mediating IFN induction.
[0107]
The diseases to which the above protein or recombinant expression vector can be applied are not limited to these, and can be widely applied to diseases that can be improved by production of type I IFN.
[0108]
[8] Endotoxin shock prevention / treatment drug, autoimmune disease treatment drug, and atopic disease treatment drug
LPS is a cell wall constituent of Gram-negative bacteria, also called endotoxin (endotoxin), and is highly toxic to mammals. Endotoxin shock caused by infection with gram-negative bacteria exhibits peripheral vasodilation, high cardiac output, fever, etc. through activation of the coagulation system, induction of iNOS expression, and low cardiac output at the end, resulting in death It reaches. Host responses that cause endotoxin shock include activation of the MAPK system and activation of transcription factors by intracellular signaling through the TLR / CD14 receptor, and production of inflammatory cytokines. There is currently no effective treatment, and the development of an effective treatment is expected.
[0109]
Since TICAM-2 is an adapter molecule that mediates LPS / TLR4 signal and induces the production of type I IFN, endotoxin shock can be prevented or treated by suppressing or inhibiting the signal-mediated function of TICAM-2. Is possible. Further, by suppressing or inhibiting the signal-mediated function of TICAM-2 to suppress the production of type I IFN, it is effective in improving adverse events caused by the production of type I IFN. Examples of adverse events caused by the production of type I IFN include autoimmune diseases and atopic diseases.
[0110]
Therefore, a mutant protein of TICAM-2 having an abnormality in the property of mediating type I IFN induction can be used as an endotoxin shock preventive / therapeutic agent, an autoimmune disease therapeutic agent, or an atopic disease therapeutic agent. Specifically, it can be carried out as an endotoxin shock preventive / therapeutic agent, an autoimmune disease therapeutic agent or an atopic disease therapeutic agent containing the protein (4) of (ii) shown in the aforementioned embodiment [1]. it can.
[0111]
Similarly, if a recombinant expression vector containing a gene encoding a mutant protein of TICAM-2 having an abnormality in the property of mediating the induction of type I IFN is applied to humans to express the protein in vivo, I It is effective in improving adverse events caused by production of type IFN.
[0112]
Therefore, a recombinant expression vector containing a gene encoding a mutant protein of TICAM-2 having an abnormality in the property of mediating the induction of type I IFN is a drug for preventing or treating endotoxin shock, a drug for treating an autoimmune disease or a drug for treating atopic disease Can be used as Specifically, from the base sequence complementary to the gene encoding the protein (4) of (ii) shown in the above embodiment [1], the DNA consisting of the base sequence shown in SEQ ID NO: 1 or 3 Endotoxin shock prophylactic / therapeutic agent comprising a recombinant vector containing any of the genes encoding a protein abnormally having the property of mediating type I IFN induction among the genes that hybridize with DNA under stringent conditions, self It can be implemented as an immune disease therapeutic agent or an atopic disease therapeutic agent.
[0113]
If the expression of TICAM-2 gene (TICAM-2 protein synthesis) is suppressed by RNA interference (RNAi) using the polynucleotide of the present invention as siRNA, it is effective in improving adverse events caused by the production of type I IFN It is.
[0114]
Therefore, the polynucleotide (siRNA) can be used as an endotoxin shock preventive / therapeutic agent, an autoimmune disease therapeutic agent, or an atopic disease therapeutic agent. Specifically, among the base sequence shown in SEQ ID NO: 1, a polynucleotide containing a part of the 87-791th base sequence, or in the base sequence shown in SEQ ID NO: 3, the 120-815th base sequence It can be implemented as an endotoxin shock preventive / therapeutic agent, an autoimmune disease therapeutic agent or an atopic disease therapeutic agent containing any one of the polynucleotides comprising a part of
[0115]
When an antibody that inhibits the property of TICAM-2 that mediates type I IFN induction according to the present invention is applied to humans, it is effective in improving adverse events caused by type I IFN production.
[0116]
Therefore, an antibody that inhibits the above-described property of TICAM-2 that mediates type I IFN induction can be used as an agent for preventing or treating endotoxin shock, a drug for treating an autoimmune disease, or a drug for treating atopic disease. Specifically, it is an antibody against the protein (1), (2), or (3) of (i) or (ii) shown in the above embodiment [1], wherein I It can be carried out as an endotoxin shock preventive / therapeutic agent, an autoimmune disease therapeutic agent or an atopic disease therapeutic agent comprising an antibody that inhibits the property of mediating type IFN induction.
[0117]
When a substance that suppresses or inhibits the function of TICAM-2 that mediates type I IFN induction obtained by the screening method according to the present invention is applied to humans, it is effective in improving adverse events caused by type I IFN production. It is.
[0118]
Therefore, the substance obtained by the above screening method that suppresses or inhibits the function that mediates type I IFN induction of TICAM-2 is used as an endotoxin shock preventive / therapeutic agent, an autoimmune disease therapeutic agent, or an atopic disease therapeutic agent. can do. Specifically, an endotoxin shock preventive / therapeutic agent, an autoimmune disease therapeutic agent or an atopy comprising a substance that suppresses or inhibits the function of mediating type I IFN induction of TICAM-2 obtained by the screening method according to the present invention. It can be implemented as a drug for treating sexual diseases.
[0119]
The adverse events caused by the production of type I IFN are not limited to endotoxin shock, autoimmune diseases and atopic diseases.
[0120]
The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims, and embodiments obtained by appropriately combining technical means disclosed in different embodiments. Is also included in the technical scope of the present invention.
[0121]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to this. The cell lines and specific experimental methods used in each example will be described first.
[0122]
[Cells and reagents]
Human lung fibroblast cell line MRC-5 was obtained from Riken Cell Bank (Tsukuba City, Ibaraki Prefecture). The epithelial cell line HeLa was obtained from the Tokyo Metropolitan Institute of Clinical Medicine. MRC-5 and HeLa were cultured in minimum essential medium (MEM) supplemented with 5% and 10% heat-inactivated fetal bovine serum (JRH Bioscience) and antibiotics, respectively.
[0123]
The mouse macrophage cell line RAW267.4 was cultured in RPMI 1640 supplemented with 10% fetal bovine serum (see Hirono, A. et al. (2002) J. Biochem (Tokyo). 132, 83-91).
[0124]
HEK293 cell line (obtained from RIKEN) was cultured in Dulbecco's modified minimal essential medium (DMEM) supplemented with 10% fetal bovine serum and antibiotics. The HEK293 cell line used expresses TICAM-2 mRNA, but does not express TLR3 mRNA, and only very little TICAM-1 mRNA is expressed.
[0125]
Poly (I: C) was purchased from Amersham Pharmacia Biotech. Polymyxin B, LPS (derived from E. coli, serotype 0111: B4) and mouse IgG1 were purchased from Sigma. Mycoplasma lipopeptide MALP-2 was prepared by the method described in the literature (Nishiguti, M. et al. (2001) J. Immunol. 166, 2610-2616.). Reagents other than LPS were treated with polymyxin B (10 μg / ml) at 37 ° C. for 1 hour before cell stimulation.
[0126]
Monoclonal antibodies against human TLR3 (TLR3.7) and monoclonal antibodies against human TLR2 (TLR2.45) are described in the literature (Non-Patent Document 10 and Uehori, J. et al. (2003) Infect. Immun. (In press)). Prepared by the described method. Monoclonal antibody against human TLR4 was provided by Dr. Kensuke Miyake of the Institute of Medical Science, University of Tokyo (For details on the production method, see Shimazu, R. et al. (1999) J. Exp. Med. 189, 1777-1782 ). Anti-IRF-3 antibody was used for the quantification of IRF-3 activity described in the literature (Iwamura, T. et al. (2001) Genes Cells 6, 375-388.).
[0127]
[Expression vector]
CDNA encoding full-length human TICAM-2 was inserted into the XhoI-NotI site of the pEFBOS plasmid. CDNA corresponding to the 68-235th amino acid portion of TICAM-2 was inserted into the XhoI-NotI site downstream of the Kozak sequence of the pEFBOS plasmid to prepare pEFBOS-TICAM-2 (TIR). pEFBOS-TICAM-2 (P116H) and pEFBOS-TICAM-2 (C117H) convert 116th proline to histidine and 117th cysteine to histidine by site-directed mutagenesis. It was produced by. The plasmid was prepared using an endotoxin-free Plasmid Maxi kit (trade name, manufactured by Qiagen).
[0128]
[Example 1: Cloning of TICAM-2 molecule]
Sequences containing a TIR similar to that of TICAM-1 were searched in an EST (expressed sequence tag) database to identify related partial cDNAs for human and mouse. That is, a BLAST search was performed on the human TICAM-1 protein sequence with NCBI's BLAST server using the tblastn program, and an EST of TICAM-2 was found. Human ESTs were searched in the DDBJ database to predict the TICAM-2 cDNA sequence. As a result, it was found that two EST clones, BQ438847 and AW080879, cover the entire deduced sequence of TICAM-2. Further, BLAST search was performed to find a mouse cDNA sequence very similar to human TICAM-2 (accession number AK080879).
[0129]
Full-length cDNA of human TICAM-2 was obtained by RT-PCR using MRC5 cells. F1 and R1 were used as PCR primers (SEQ ID NOs: 5 and 6). In addition, full-length cDNA of mouse TICAM-2 was obtained by RT-PCR using NIH3T3 cells. MF1 and mR1 were used as PCR primers (SEQ ID NOs: 9, 10).
[0130]
Alignment between the amino acid sequence of human TICAM-2 and the amino acid sequence of mouse TICAM-2 was performed using ClustalW. The result is shown in FIG. The upper line in the figure shows the TIR-like motif of TICAM-2, the asterisk (*) is the same residue, 2 points (:) are substitutions with the same amino acid, 1 point (•) is substitution with a similar amino acid, hu Represents human and mu represents mouse.
[0131]
As shown in FIG. 2, the human TICAM-2 protein is composed of 235 amino acids and has a short (about 70 amino acids) N-terminal serine / threonine rich domain, a TIR motif and a C-terminal 20 amino acid extension. The N-terminal and C-terminal domains of TICAM-2 are not similar to TICAM-1. The TIR motif portion and C-terminal portion are very well conserved between human and mouse TICAM-2.
[0132]
Human TICAM-2 (amino acids 75-213), mouse TICAM-2 (amino acids 72-210), human TICAM-1 (amino acids 394-532), mouse TICAM-1 (amino acids 396-534), human MyD88 (amino acids 160- 296, accession number U70451), human TIRAP (amino acids 85-214, accession number AF406652) TIR domains and human TLR2 TIR (amino acids 640-784, accession number U88878) were aligned using ClustalW. The result is shown in FIG. In the figure, an asterisk (*) indicates the same residue, 2 points (:) indicate substitution with the same amino acid, 1 point (•) indicates substitution with a similar amino acid, hu indicates human, and mu indicates mouse.
[0133]
As shown in FIG. 3, although the similarity between the TIR motif of TICAM-2 and the TIR motif of known adapters TIRAP and MyD88 is low, the basic amino acid and serine arranged in tandem are contained in the N-terminal domain of TICAM-2. This is also seen in TIRAP. TICAM-2, like TICAM-1, lacks conserved sequences present in other TIR-containing proteins. Conservative sequences are Box 1 (F / Y) D, Box 2 RD and Box 3 FW in FIG. 3 (Xu, Y. et al. (2000) Nature 408: 111-115). As seen in MyD88, TIRAP, and TICAM-1, the proline of the BB loop, which is essential for TIR-mediated signal transduction, is located in front of the conserved location in TICAM-2 or replaced with cysteine Has been.
[0134]
Table 1 shows the amino acid identity between each adapter molecule in the TIR domain alignment of the TLR adapter molecule.
[0135]
[Table 1]

[0136]
Moreover, the domain structure of each adapter molecule is shown in FIG. In FIG. 4, the black part is the TIR domain. Human TICAM-2 consists of 235 amino acid residues, and the TIR domain corresponds to amino acid residues 75-213. Similarly, TIRAP consists of 235 amino acid residues, TIR domain corresponds to amino acid residues 85-214, MyD88 consists of 296 amino acid residues, TIR domain corresponds to amino acid residues 160-296, TICAM -1 consists of 712 amino acid residues, and the TIR domain corresponds to amino acid residues 394 to 532.
[0137]
Analysis of the human genome database revealed that the TICAM-2 gene is located on chromosome 5q22.
[0138]
The cDNA sequences and amino acid sequences of human TICAM-2 and mouse TICAM-2 have been registered in the DDBJ database (unpublished at the time of filing this application), and their Genebank accession numbers are AB091054 and AB100441, respectively.
[0139]
[Example 2: Detection of TICAM-2 mRNA by Northern blotting and RT-PCR]
Using human 12 lane MTN Blot and human 12 lane MTN Blot III (trade name, manufactured by Clontech), ³² Hybridization was performed under stringent conditions with a full-length human TICAM-2 cDNA probe labeled with P, and then rehybridized with a labeled β-actin probe. Using a BAS2000 image analyzer (Fuji Photo Film Co., Ltd.), exposure was performed for 4 to 24 hours, and mRNA signals were detected. The result is shown in FIG.
[0140]
As is clear from FIG. 5 (a), 3.3-3.6 kb mRNA was detected in many tissues including lymph node, prostate, stomach, thyroid, trachea, heart, skeletal muscle and placenta. Two bands of 3.6 and 3.3 kb were observed in the lymph node, prostate, stomach, thyroid, muscle and lung. A 1.8 kb band was specifically observed only in the placenta.
[0141]
RT-PCR was performed for the purpose of mRNA detection and semi-quantification. As primers, F1 and R1 were used for detecting human TICAM-2 (SEQ ID NOs: 5 and 6), and hBACT-F1 and hBACT-R1 were used for detecting β-actin (SEQ ID NOs: 7 and 8). Total RNA was isolated from various human cells or cell lines, and 35 cycles of TICAM-2 and 20 cycles of β-actin were performed. The result is shown in FIG.
[0142]
As is clear from FIG. 5 (b), TICAM-2 mRNA was present in immature dendritic cells (iDC), macrophages and natural killer cells (NK) derived from human peripheral blood. TICAM-2 mRNA was also present in T and B lymphocyte cell lines and fibroblast cell lines.
[0143]
[Example 3: Examination of association between TICAM-2 and other molecules by yeast two-hybrid method]
A yeast medium described in the literature (Sherman, F. et al. “Method in Yeast Genetics”, Cold Spring Harbor Press, NY, USA. Pp. 22-46. (1986)) was used. Yeast AH109 strain (Clontech) was transformed with bait and prey plasmids. The transformed yeast was streaked and incubated for 3 to 5 days. BD-TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8 and TLR9 were prepared by inserting the TIR domain of TLR into pGBKT7 (Clontech). BD-TICAM-2 and AD-TICAM-2 were prepared by inserting full-length wild type TICAM-2 cDNA into pGBKT7 (bait) or pGADT7 (prey). BD-TICAM-1 was prepared by inserting a TICAM-1 fragment (part from 1102 bp to 2142 bp corresponding to the start codon) into pGBKT7. AD-TIRAP and AD-MyD88 were prepared by inserting a cDNA fragment encoding the full-length protein into pGADT7.
[0144]
SD-WLH is a yeast synthetic dextrose medium lacking tryptophan, leucine and histidine, and SD-WLHA is a medium lacking adenine. The SD-WLH plate is a medium stringency plate and the SD-WLHA plate is a high stringency plate. That is, SD-WLH reflects a weak interaction and SD-WLHA reflects a strong interaction. The said result is shown to Fig.6 (a)-(c).
[0145]
As is clear from FIGS. 6 (a) to (c), strong association between TICAM-2 and TLR4 is observed in the SD-WLHA plate, and weak association between TICAM-2 and TLR3 is observed in the SD-WLHA plate. Observed (see FIG. 6A). Moreover, it was observed that TICAM-2 forms a homodimer (see FIG. 6B) and forms a heterodimer with TICAM-1 (see FIG. 6C).
[0146]
[Example 4: Examination of association between TICAM-2 and other molecules by immunoprecipitation]
<experimental method>
HEK293 cells are cultured in a 6-well plate and pEFBOS / TICAM-2 (0.5 μg) or pEFBOS / TICAM-2-HA (0.5 μg) using Lipofectamine 2000 reagent (Gibco-BLR). In addition, the following vectors were transiently transfected. Note that “HA” indicates that an HA tag is attached.
[0147]
pCMV / TLR4 or pCMV / Flag-TLR4 (2 μg), pEFBOS / TLR3 or pEFBOS / Flag-TLR3 (3 μg), pCMV / TLR2 or pCMV / Flag-TLR2 (2 μg), pEFBOS / TICAM-1H0.5A-TICAM-1HA ), PEFBOS / MD-2 (1 μg). These were transfected into the HEK293 cells. Note that “Flag” indicates that a Flag tag is attached.
[0148]
The total amount of DNA to be transfected (4 μg) was made constant by adding empty vector. Here, “empty vector” means a vector into which a gene for expression is not inserted.
[0149]
Twenty-four hours after transfection, cells were stimulated with medium alone or LPS for 15-60 minutes. Thereafter, the cells were lysed with a cell lysis buffer (25 mM Tris (pH 7.5), 150 mM NaCl, 1% NP-40, 2 mM PMSF, 25 mM iodoacetamide, 10 mM EDTA). After centrifugation, anti-Flag monoclonal antibody M2 (manufactured by Sigma) was added to the cell lysate and incubated at 4 ° C. for 2 hours. Mouse IgG1 was used as a control. The immune complex was precipitated together with protein G sepharose and washed thoroughly three times with PBS (phosphate buffered saline) containing 0.02% Triton X100. The immunoprecipitated protein was eluted with PBS containing 1% SDS, 0.2% NP-40 and 5% 2-mercaptoethanol, boiled, and separated by SDS-PAGE. Subsequently, immunoblotting was performed using each antibody. In some experiments, cell lysates were used directly for immunoblotting.
[0150]
<Experimental result>
The said result is shown to Fig.7 (a)-(c). 7 (a), (b) and (c), the upper part shows the result of probing with the anti-HA antibody, and the lower part shows the result of reprobing with the anti-Flag antibody. The transfected expression vector is shown at the top of each lane. “+ ^F "Indicates that a Flag tag is attached.
[0151]
As is clear from FIG. 7 (a), it was confirmed that TICAM-2 binds to TLR4 (lane 6). Moreover, the association between TICAM-2 and TLR2 or TLR3 was not detected under these conditions (lanes 1 to 4). TLR2 did not co-precipitate with TICAM-2 even when co-expressed with MD-2 (lanes 1 and 2). A trace band around 50 kDa is non-specific and is seen in all lanes. The arrow at the top of FIG. 7A shows TICAM-2 with an HA tag. The lower brackets indicate TLR proteins with a Flag tag. The results shown in FIG. 7 (a) were confirmed using specific monoclonal antibodies against TLR2, TLR3 and TLR4.
[0152]
As a result of examining the effect of LPS on the interaction between TLR4 and TICAM-2, as shown in FIG. 7 (b), the association between TLR4 and TICAM-2 is affected even when TLR4-expressing cells are stimulated with LPS. (Lanes 3 and 4). As in (a), the band near 50 kDa is a non-specific band. The arrow at the top of FIG. 7B shows the TICAM-2 band. The lower arrowhead indicates TLR4.
[0153]
As a result of examining the association of TICAM-2 with TICAM-1, TICAM-2 and TLR4, it was shown that TLR4 attracts TICAM-1 only when TICAM-2 is present, as shown in FIG. 7 (c). (Lane 6). Under the same conditions, TLR3 is known to attract TICAM-1 directly (Non-patent Document 12). Moreover, since TICAM-2-HA and TICAM-2-Flag co-precipitated, it was shown that TICAM-2 forms a dimer (lane 1). The background level of TICAM-2-HA was very small (lane 2). TICAM-2 and TLR4 were associated (lane 3). TICAM-2 hardly bound to TICAM-1 in the absence of TLR4 (lane 4). No association between TICAM-1 and TLR4 was detected (lane 5). The arrow at the top of FIG. 7 (c) shows the bands of TICAM-1 and TICAM-2. The lower arrowhead (outlined) indicates TLR4.
[0154]
Although data are not shown, TICAM-2 (P116H) and TICAM-2 (C117H), which are dominant negative forms of TICAM-2, have lost the ability to form dimers with TICAM-1 and TICAM-2. It was. The relationship between TLR4 and TICAM-1 was confirmed using an anti-TLR4 monoclonal antibody.
[0155]
[Example 5: Functional analysis of TICAM-2 by reporter gene analysis]
<experimental method>
HEK293 cells are cultured in 24-well plates (2 × 10 ^Five NF-κB reporter gene (0.1 μg, Stratagene) or p-125luc reporter plasmid (0.1 μg) bound to luciferase using Lipofectamine 2000 reagent (Gibco-BLR). ) And the following vectors were transiently transfected.
[0156]
As described in the [Cells and Reagents] section, the HEK293 cells express TICAM-2 mRNA, but do not express TLR3 mRNA, and there is very little TICAM-1 mRNA. It was expressed.
[0157]
PEFBOS that expresses TLR2, TLR3, TLR4 + MD-2 (more than 0.1 μg). TICAM-1, dominant negative TICAM-1 [TICAM-1 TIR (P434H)], MyD88, TIRAP, TICAM-2, TICAM-2 (TIR), TICAM-2 (P116H), TICAM-2 (C117H), IRAK-1 (1-211), pEFBOS that expresses IRAK-1 (1-96) (10, 100 or 200 ng above). Vector only as a control. These were transfected into the HEK293 cells.
[0158]
In FIG. 8, TICAM-2, TICAM-2 (TIR) (represented as TICAM-2-TIR in FIG. 8), TICAM-2 (P116H) (represented as TICAM-2-PH in FIG. 8). ) And TICAM-2 (C117H) (represented as TICAM-2-CH in FIG. 8). The part painted black indicates the TIR domain. TICAM-2-TIR contains the 68th to 235th amino acid moiety. TICAM-2-PH substituted the 116th proline with histidine (white circle), and TICAM-2-CH substituted the 117th cysteine with histidine (white circle).
[0159]
Non-patent document 12 describes the construction of dominant negative TICAM-1 and TICAM-1TIR (P434H). The p-125luc reporter was provided by Dr. Issho Taniguchi (University of Tokyo) and contains the human IFN-β promoter region inserted into the Picker Gene Luciferase Reporter Plasmid (manufactured by Toyo Ink). pAP-1-Luc reporter plasmid was purchased from Stratagene. A reporter plasmid containing the ELAM-1 promoter was produced in-house (Lien, E. et al. (1999) J. Biol. Chem. 274, 33419-33425.).
[0160]
The total amount of DNA to be transfected (0.8-1.0 μg) was adjusted by adding empty vector. In addition, 5 ng of pCMVβ plasmid (Clontech) was used as an internal control.
[0161]
Dominant negative form (MyD88 [MyD88 (TIR)], TIRAP [TIRAP (P125H)], TICAM-1 [TICAM-1 TIR (P434H)]) or TICAM-2 [TICAM-2 (C117H)]) expression vector Transfected. After 24 hours, cells were stimulated for 6 hours with medium alone, LPS (100 ng / ml), MALP-2 treated with polymyxin B (100 nM), or poly (I: C) treated with polymyxin B (10 μg / ml). Cells were lysed using a cell lysis buffer (Promega), and luciferase and β-galactosidase activities were measured according to the manufacturer's instructions. Numerical values were expressed as the mean and standard deviation of at least three independent experiments (each experiment was performed in duplicate).
[0162]
<Experimental result>
HEK293 cells with NF-κB reporter, p-125luc reporter (IFN-β) or AP-1 reporter plasmid conjugated to luciferase are transfected with empty vector or TICAM-2 expression vector (10, 100 or 200 ng), Reporter activity was examined. The results are shown in FIGS. HEK293 cells overexpressing TICAM-2 protein induced mildly the IFN-β promoter (see FIG. 9 (a)) and NF-κB (see FIG. 9 (b)). On the other hand, AP-1 was not induced (see FIG. 9 (c)).
[0163]
Wild type and mutant TICAM-2 were overexpressed in HEK293 cells, and activation of NF-κB and IFN-β promoters was examined. The results are shown in FIGS.
[0164]
It was suggested that the full length TICAM-2 and the TICAM-2-TIR lacking the N-terminal region activate the IFN-β promoter and go through homodimer formation. TICAM-2-CH having a mutation in the TIR domain did not activate the IFN-β promoter (see FIG. 10 (a)). As for NF-κB, only TICAM-2 activated NF-κB, and TICAM-2-TIR that activated the IFN-β promoter did not activate NF-κB (see FIG. 10B).
[0165]
Wild-type and mutant-type TICAM-2 were further expressed in HEK293 in which TLR4, CD14 and MD-2 were expressed, and the effect of LPS stimulation on IFN-β promoter activation was examined (see FIG. 10 (c)). As a result, TICAM-2 and TICAM-2-TIR were slightly enhanced in IFN-β promoter activity by LPS stimulation. Wild-type TICAM-2 also increased the background, probably due to dimer formation. TICAM-2-CH (TICAM-2 (C117H)) functioned as a dominant negative and effectively inhibited IFN-β activation via LPS-TLR4.
[0166]
TICAM-2-CH was further expressed in HEK293 in which TLR3 was expressed or HEK293 in which TLR4, CD14 and MD-2 were expressed. Stimulation of cells expressing TLR3 with 50 μg of poly (I: C) and cells expressing TLR4 with 100 ng / ml LPS for 6 hours to activate IFN-β promoter and NF-κB Was examined. The results are shown in FIGS. 11 (a) and 11 (b).
[0167]
As is clear from FIGS. 11 (a) and 11 (b), TICAM-2-CH inhibits both NF-κB and IFN-β promoter activation via LPS-TLR4 and acts as a dominant negative regulator. It was. On the other hand, TICAM-2-CH did not inhibit both NF-κB and IFN-β promoter activation via dsRNA-TLR3. Although data is not shown, TICAM-2 (P116H) also has the same result as TICAM-2 (C117H), and was confirmed to function as a dominant negative.
[0168]
HEK293 in which TLR3 was expressed, or HEK293 in which TLR4, CD14 and MD-2 were expressed, was further expressed with TICAM-1TIR (P434H) (TICAM-1DN in the figure), which is a dominant negative form of TICAM-1. In the same manner as above, stimulation with poly (I: C) or LPS was performed, and IFN-β promoter and NF-κB activation were examined. The results are shown in FIGS. 12 (a) and 12 (b).
[0169]
TICAM-1TIR (P434H) inhibited IFN-β activation via LPS-TLR4, but the potency of inhibition was less than inhibition of IFN-β activation via poly (I: C) -TLR3 (FIG. 12 ( a)). On the other hand, TICAM-1TIR (P434H) inhibited NF-κB activation via poly (I: C) -TLR3, but did not inhibit NF-κB activation via LPS-TLR4 (FIG. 12 (b)). reference).
[0170]
To evaluate whether the effects of TICAM-1 and TICAM-2 are synergistic, IFN-β in cells overexpressing both TICAM-1 and TICAM-2 and cells overexpressing only TICAM-2 Promoter and NF-κB activation were examined. The results are shown in FIGS. 13 (a) and (b). The IFN-β promoter was synergistically activated with high doses of TICAM-2, but TICAM-2 was less effective than TICAM-1 (see FIG. 13 (a)). From these results, it was suggested that TICAM-1 is mainly involved in the IFN-β induction function via TLR4. On the other hand, no synergistic effect was observed in NF-κB activation (see FIG. 13 (b)).
[0171]
TICAM-2 overexpressing cells were coexpressed with TICAM-1, MyD88, and TIRAP, respectively, and examined for activation of IFN-β promoter and NF-κB. The results are shown in FIGS. 14 (a) and 14 (b).
[0172]
As is clear from FIGS. 14 (a) and (b), the activation of the IFN-β promoter and NF-κB was strongly suppressed by TICAM-1TIR (P434H). This effect of TICAM-1 TIR (P434H) is specific for disruption of TICAM-2 function, and MyD88 and TIRAP dominant negatives activate IFN-β promoter or NF-κB via TICAM-2 Did not disturb.
[0173]
It is a dominant negative form of TICAM-2 in TICAM-1 overexpressing cells. TICAM-2 (C117H) (in the figure, TICAM-2-CH) was co-expressed, and IFN-β promoter and NF-κB activation were examined. The results are shown in FIGS. 15 (a) and 15 (b). As is clear from FIGS. 15A and 15B, TICAM-2 (C117H) did not inhibit the activation of IFN or NF-κB via TICAM-1.
[0174]
From the results shown in FIGS. 14 (a) and 14 (b) and FIGS. 15 (a) and 15 (b), it is considered that it is important as an adapter function that TICAM-1 approaches TICAM-2 rather than TICAM-2 itself. It was.
[0175]
[Example 6: Activation of IRF-3 by TICAM-2 and examination of relationship between TICAM-2 and IRAK]
NF-κB, AP-1 and IRF-3 are known to be transcription factors that induce IFN-β (Taniguti, T. et al. (2001) Annu. Rev. Immunol. 19, 623-655 .). It was also shown that IRF-3 is activated in response to TLR3 agonists in HeLa cells (Non-Patent Document 12, and Iwamura, T. (2001) Genes Cells 6, 375-388.). Therefore, the following experiment was conducted.
[0176]
HeLa cells were transfected with empty vector, MyD88 plasmid, TIRAP plasmid, TICAM-1 plasmid and TICAM-2 plasmid (1 μg each). After 24 hours, the cells were treated with medium alone or 20 μg / ml LPS for 1 hour, and a cell lysate was prepared and subjected to native PAGE. The result is shown in FIG.
[0177]
As is clear from FIG. 16 (a), activation of IRF-3 was observed by transfection of TICAM-1, but not by transfection of TICAM-2, MyD88 or TIRAP. AP-1 activation was not observed in cells transfected with TICAM-2 only (see FIGS. 9 (a)-(c)), but AP-1 activity was observed when TICAM-1 was transfected. Was confirmed (Non-patent Document 12). However, the IFN-β promoter was activated simultaneously with NF-κB activation by transfection with TICAM-2 alone (see FIGS. 9A to 9C). Therefore, the IFN-β promoter was activated via NF-κB in the case of TICAM-2 overexpression.
[0178]
Since transfection of TICAM-2 induces weak NF-κB activity, we next examined whether IRAK-1 and -2 contribute to adapter-mediated NF-κB activation. That is, HE-293 cells were transfected with p-125luc reporter plasmid, and further transfected with an empty vector, TICAM-2 plasmid (100 ng) or TICAM-2 plasmid + IRAK-1 (1-211) plasmid. After 24 hours, the luciferase reporter activity was measured. The result is shown in FIG. In the figure, Control (hatched bars) represents cells transfected with only the empty vector, and black bars represent cells transfected with only TICAM-2. Values are representative of 3 independent experiments.
[0179]
As is clear from FIG. 16 (b), the dominant negative form of IRAK-1 (IRAK-1 (1-211)) inhibited IFN-β promoter activation by TICAM-2. It also inhibited activation of NF-κB (not shown). In similar experiments, the dominant negative form of IRAK-2 hardly inhibited TICAM-2 mediated NF-κB and IFN-β promoter activation (not shown). Thus, IRAK-1 appears to act downstream of TICAM-2 and is associated with IFN-β promoter activation via NF-κB.
[0180]
[Example 7: Examination of involvement of TICAM-2 and TICAM-1 in IFN-β induction via TLR4 using RNA interference (RNAi) and quantitative PCR]
In order to investigate the involvement in IFN-β production via TICAM-1 / TICAM-2, TICAM-2 knockdown cells were obtained by transfection of siRNA (small interfering RNA) using mouse RAW264.7, which is a macrophage-like cell. We prepared and examined IFN-β induction in response to LPS.
[0181]
<experimental method>
Mouse RAW264.7 cells were seeded in 6 well plates (5 × 10 5 ⁶ Cells / well) The cells were cultured for 24 hours. In cells, only buffer, control siRNA (for human TLR3 or human LaminA / C, SEQ ID NO: 27-30, see Non-Patent Document 11), siRNA for mouse TICAM-2 (SEQ ID NO: 21-24) or mouse TICAM-1 SiRNA (SEQ ID NOs: 25 and 26) was transfected using Oligofectamine reagent (Oligofectamine reagent, manufactured by Invitrogen). Twenty-four hours after transfection, cells were collected by treatment with physiological saline containing 5 mM EDTA and seeded in 24-well plates. The next day, the cells were 30-50% confluent. Again, siRNA was transfected with oligofectamine and cultured for 2 days. The culture supernatant was removed and a medium containing 100 ng / ml LPS was added to each well. Total RNA was extracted from cells using TRIZOL reagent before and 6 hours after stimulation.
[0182]
SiRNA against human TLR3 was used as a control. This is because control siRNA does not down-regulate mouse TLR3 because the target TLR3 base sequence is not conserved between human and mouse. Oligonucleotides used for RNA interference were purchased from JbioS.
[0183]
Quantitative PCR analysis was performed using iCycler iQ Real-Time detection system with SYBR Green Supermix reagents (Bio-Rad). As primers for quantitative PCR, mF1, mR2, mF3 and mR3 were used for TICAM-2, mT-1-F1 and mT-1-R1 for TICAM-1, and mIFN-F1, mIFN- for IFN-β. R1 and mIFN-R2 were used and mBACT-F1 and mBACT-R1 were used for β-actin. (SEQ ID NO: 9, 11-20) The number of PCR cycles was 27 for TICAM-2, 30 for IFN-β, and 22 for β-actin.
[0184]
FIG. 17 (a) shows two siRNA target sites for mouse TICAM-2. In the figure, site A is located on the N-terminal side, and site B is located in the TIR domain.
[0185]
<Experimental result>
FIG. 17B shows a 1% agarose gel electrophoresis image (ethidium bromide staining) of the quantitative PCR product. The left is the result when the total RNA before LPS stimulation is used as a sample, and the right is the result when the total RNA after LPS stimulation is used as a sample. The siRNA used is indicated at the top. “Mock” uses only buffer at the time of siRNA transfection. TICAM-2 mRNA levels were suppressed to about 60% by RNA interference targeting site A, and to about 26% by RNA interference targeting site B.
[0186]
FIG. 17 (c) shows the relative copy number of the PCR product when quantitative PCR was performed using the IFN-β primer. Numerical values were corrected by β-actin. Each value was calculated based on the average value of Mock (as 1.00). The numerical value is an average value of three experiments. The siRNA used is shown at the bottom. TICAM-2 knockdown specifically inhibited the induction of IFN-β. IFN-β mRNA levels were suppressed to about 60% by RNA interference targeting site A, and to about 35% by RNA interference targeting site B.
[0187]
IFN-β induction after LPS stimulation was examined by RNA interference with mouse TICAM-1. The result is shown in FIG. Numerical values were calculated in the same manner as in the experiment of FIG. The siRNA used is shown at the bottom. Lamin A / C and human TLR3 siRNAs were used as controls. TICAM-1 knockdown also suppressed IFN-β mRNA levels. Although not shown, the TICAM-1 mRNA level was suppressed to about 70%.
[0188]
From the above results, it was revealed that both TICAM-1 and TICAM-2 are necessary molecules for maximally inducing IFN-β through LPS in macrophage-like cells. In addition to the experimental results of immunoprecipitation shown in Example 4 (see FIGS. 7 (a) to (c)), TICAM-2 physically acts as an IFN-mediated LPS as part of the MyD88-independent pathway. It was revealed that it is an adapter that crosslinks the TIR domain of TLR4 and TICAM-1 for β induction.
[0189]
【The invention's effect】
The present invention transmits LPS / TLR4 signal to TICAM-1 by binding to mammalian Toll-like receptor 4 (TLR4) and cross-linking TLR4 and TICAM-1 to produce type I interferon (type I IFN) It was completed by identifying a novel adapter molecule TICAM-2 having a function of inducing. The novel adapter molecule TICAM-2 is an adapter that controls type I IFN induction, and can selectively regulate only the type I IFN induction system from the LPS / TLR4 signal system.
[0190]
The present invention is useful as a preventive / therapeutic agent for viral infections in which type I IFN is effective by enhancing induction of type I IFN, a cancer therapeutic agent in which type I IFN is effective, and the like. On the other hand, by suppressing the induction of type I IFN, it is also useful for prophylactic / therapeutic agents for endotoxin shock, autoimmune diseases, atopic diseases and the like. Therefore, it can be expected to be widely applied in the fields of drug discovery and medicine.
[0191]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a schematic diagram showing the form of association of human TLRs 2, 3, 4, and 5 with adapters that determine each TLR-specific signal transduction pathway.
FIG. 2 is a diagram showing an alignment between the amino acid sequence of human TICAM-2 and the amino acid sequence of mouse TICAM-2.
FIG. 3 shows alignments of TIR domains of human TICAM-2, mouse TICAM-2, human TICAM-1, mouse TICAM-1, human MyD88, human TIRAP, and human TLR2.
FIG. 4 is a schematic diagram showing domain structures of four types of human adapter molecules.
FIG. 5 (a) shows the northern blot results of TICAM-2 mRNA derived from a specific tissue, and FIG. 5 (b) shows TICAM-2 mRNA in various human cells and human cell lines by RT-PCR. It is a figure which shows the detected result.
FIGS. 6A to 6C are diagrams showing the results of an association experiment between TICAM-2 and TLRs or adapters by the yeast two-hybrid method.
FIGS. 7A to 7C are diagrams showing the results of an association experiment between TICAM-2 and TLRs or adapters by immunoprecipitation.
FIG. 8 is a schematic diagram showing the structures of wild-type and mutant TICAM-2.
9 (a) to (c) are graphs showing the results of examining the activation of NF-κB, IFN-β promoter and AP-1 by TICAM-2 expression.
FIG. 10 (a) to (c) are graphs showing the results of examining the activation of NF-κB and IFN-β promoters by expression of wild type and mutant TICAM-2.
FIGS. 11A and 11B are graphs showing the results of examining the effect of mutant TICAM-2 on TLR3 and TLR4 signaling. FIGS.
FIGS. 12A and 12B are graphs showing the results of examining the effect of mutant TICAM-1 on TLR3 and TLR4 signaling. FIGS.
FIGS. 13A and 13B are graphs showing the results of examining the synergistic effect of TICAM-1 and TICAM-2. FIGS.
FIGS. 14A and 14B show activation of IFN-β promoter and NF-κB when TICAM-2 and TICAM-1, MyD88, and TIRAP, respectively, are negatively expressed. It is a graph which shows the result of having examined about.
FIG. 15 is a graph showing the results of examining IFN-β promoter and NF-κB activation when co-expressing TICAM-1 and a dominant negative form of TICAM-2.
FIG. 16 (a) is a diagram showing the results of treating HeLa cells expressing each adapter molecule with LPS and subjecting the cell lysate to native PAGE, and FIG. 16 (b) shows TICAM-2 or TICAM. It is a graph which shows the result of having examined the IFN- (beta) promoter activation in the cell which expressed -2 + IRAK-1 dominant negative body.
FIG. 17A is a schematic diagram showing the site of siRNA used for RNA interference of mouse TICAM-2, and FIG. 17B is a quantitative PCR method for IFN-β mRNA and TICAM-2 mRNA after RNA interference. (C) is a graph showing the results of quantifying IFN-β mRNA by quantitative PCR after RNA interference with TICAM-2, (d) It is a graph which shows the result of having quantified IFN- (beta) mRNA after LPS stimulation by quantitative PCR method by RNA interference with mouse | mouth TICAM-1.

Claims (8)

The amino acid sequence shown in SEQ ID NO: 2 consists of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added, and binds directly to a mammalian Toll-like receptor 4 A protein having an abnormality in the property of mediating induction of type I interferon ,
A protein in which at least the 116th amino acid has been substituted or deleted . The amino acid sequence shown in SEQ ID NO: 2 consists of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added, and binds directly to a mammalian Toll-like receptor 4 A protein having an abnormality in the property of mediating induction of type I interferon ,
A protein in which at least the 117th amino acid has been substituted or deleted . A gene encoding the protein according to claim 1 or 2 . A method for screening a substance that suppresses or inhibits a function of mediating induction of type I interferon of a novel adapter molecule that binds to a mammalian Toll-like receptor 4.
A screening method using the protein according to any one of 1) to 4) below .
1) Protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or 4
2) a protein comprising at least the 75-213th amino acid sequence of the sequence shown in SEQ ID NO: 2
3) a protein comprising at least the 72-210th amino acid sequence of the sequence shown in SEQ ID NO: 4
4) The amino acid sequence according to any one of 1) to 3) is composed of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added, and enhances the production of type I interferon. Protein with properties An IFN-β promoter activator that enhances the production of type I interferon by the function of mediating type I interferon induction of a novel adapter molecule that binds to mammalian Toll-like receptor 4 ,
An IFN-β promoter activator comprising the protein according to any one of 1) to 4) below or a gene encoding the protein .
1) Protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or 4
2) a protein comprising at least the 75-213th amino acid sequence of the sequence shown in SEQ ID NO: 2
3) a protein comprising at least the 72-210th amino acid sequence of the sequence shown in SEQ ID NO: 4
4) The amino acid sequence according to any one of 1) to 3) is composed of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added, and enhances the production of type I interferon. Protein with properties An IFN-β promoter inhibitor that suppresses the production of type I interferon by suppressing or inhibiting the function that mediates type I interferon induction of a novel adapter molecule that binds to mammalian Toll-like receptor 4 ,
An IFN-β promoter inhibitor comprising the protein according to any one of 5) to 7) below or a gene encoding the protein.
5) A protein in which at least the 116th amino acid has been substituted or deleted in the amino acid sequence shown in SEQ ID NO: 2
6) A protein in which at least the 117th amino acid has been substituted or deleted in the amino acid sequence shown in SEQ ID NO: 2
7) The amino acid sequence described in 5) or 6) is composed of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added, and has the property of suppressing the production of type I interferon. protein A type I interferon production enhancer comprising the IFN-β promoter activator according to claim 5. A production inhibitor of type I interferon comprising the IFN-β promoter inhibitor according to claim 6.

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