JP3993439B2 - New genes whose expression changes by AIP treatment - Google Patents

Description

【００３７】
本発明のAIP処理によって起こる細胞の増殖阻害時に増加する遺伝子hRUPP-1の組織分布の結果を図７に示す。hRUPP-1遺伝子はヒトの脳、心臓、腎臓、肝臓、肺、膵臓、胎盤、筋肉、大腸、卵巣、白血球、前立腺、小腸、脾臓、精巣及び胸線などに発現が見られ、特に肺、膵臓、大腸、白血球、前立腺、脾臓、精巣及び胸線に高い発現が観察された。
【００３８】
（２）ヒト各組織におけるhRRCP-1の発現
以下のhRRCP-1遺伝子特異的プライマーを用いて、上述のhRUPP-1遺伝子特異的プライマーを用いた場合と同様の方法に従って、hRUPP-1の組織発現を分析した。本発明のAIP処理によって起こる細胞の増殖阻害時に増加する遺伝子hRRCP-1の組織分布の結果を図８に示す。hRRCP-1遺伝子はヒトの脳、心臓、腎臓、肝臓、肺、膵臓、胎盤、大腸、卵巣、白血球、前立腺、小腸、脾臓、精巣及び胸線等に発現が見られ、特に肺、膵臓、前立腺、脾臓、精巣及び胸線に高い発現が観察された。

【００３９】
（３）ヒト各組織におけるhERRj-1の発現
以下のhERRj-1遺伝子特異的プライマーを用いて、上述のhRUPP-1遺伝子特異的プライマーを用いた場合と同様の方法に従って、hERRj-1の組織発現を分析した。本発明のAIP処理によって起こる細胞の増殖阻害時に増加する遺伝子hERRj-1の組織分布の結果を図９に示す。hERRj-1遺伝子はヒトの脳、心臓、腎臓、肝臓、肺、膵臓、胎盤、大腸、卵巣、白血球、前立腺、小腸、脾臓、精巣及び胸線等に発現が見られ、特に肝臓、肺、膵臓、前立腺、脾臓及び精巣に高い発現が観察された。

【００４０】
４．組換発現系を用いた蛋白質発現
(１)hRUPP-1の製造
hRUPP-1の全長をコードするcDNA(配列番号１)を、GSTをコードする配列を含む大腸菌用発現ベクターpGEX-2T(Amersham Pharmacia Biotech)、及び、pQE-30(QUIAGEN)のBamHI/SmaI領域中にクローニングし発現プラスミドを得た。得られたプラスミドを大腸菌M15(pREP4)、または、XL1 Blue中に形質転換し、37℃、NZCYM培地中で生育させた。培養物のOD₆₀₀が約0.5に達した時点で、最終濃度1mMとなるようイソプロピル-β-チオガラクトピラノシド(IPTG)を添加し、さらに5時間培養した後、細胞を回収した。
【００４１】
組換え蛋白質の精製は、The QIAexpressionist(QIAGEN)及びGST Gene Fusion System(Amersham Pharmacia Biotech)の説明書に従って行った。次にpGEX組換え転写ユニットより得られたGST融合蛋白質からGSTを除去するために、調製したGST融合蛋白質を約2mg/mlまで濃縮し、20mM Hepes-NaOH(pH8.0)、150mM NaClに対して4℃で、透析した。透析物に最終濃度2mMになるようにCaCl₂を加え、GST融合蛋白質との重量比で約1/200量のトロンビンを加え、氷上で5時間インキュベートした。その後、グルタチオン-Sepharose、及び、ベンズアミジン-Sepharose(Amersham Pharmacia Biotech)と混合した。4℃で一晩、回転しながら混合した後、カラムに充填し、素通り画分と20mM HEPES-NaOH(pH8.0)、150mM NaClの洗浄画分を集め親和性に基づき所望の組換え蛋白質を得る。
【００４２】
(２)hRRCP-1の製造
hRRCP-1の全長をコードするcDNA(配列番号３)を、GSTをコードする配列を含む大腸菌用発現ベクターpGEX-2T(Amersham Pharmacia Biotech)、及び、pQE-30(QUIAGEN)のBamHI/SmaI領域中にクローニングし発現プラスミドを得た。得られたプラスミドを大腸菌M15(pREP4)、または、XL1 Blue中に形質転換し、37℃、NZCYM培地中で生育させた。培養物のOD₆₀₀が約0.5に達した時点で、最終濃度1mMとなるようイソプロピル-β-チオガラクトピラノシド(IPTG)を添加し、さらに5時間培養した後、細胞を回収した。
【００４３】
組換え蛋白質の精製は、The QIAexpressionist(QIAGEN)及びGST Gene Fusion System(Amersham Pharmacia Biotech)の説明書に従って行った。次にpGEX組換え転写ユニットより得られたGST融合蛋白質からGSTを除去するために、調製したGST融合蛋白質を約2mg/mlまで濃縮し、20mM Hepes-NaOH(pH8.0)、150mM NaClに対して4℃で、透析した。透析物に最終濃度2mMになるようにCaCl₂を加え、GST融合蛋白質との重量比で約1/200量のトロンビンを加え、氷上で5時間インキュベートした。その後、グルタチオン-Sepharose、及び、ベンズアミジン-Sepharose(Amersham Pharmacia Biotech)と混合した。4℃で一晩、回転しながら混合した後、カラムに充填し、素通り画分と20mM HEPES-NaOH(pH8.0)、150mM NaClの洗浄画分を集め、親和性に基づき所望の組換え蛋白質を得る。
【００４４】
(３)hERRj-1の製造
成熟hERRj-1(配列番号５のアミノ酸35〜793)をコードするcDNAをGSTをコードする配列を含む大腸菌用発現ベクターpGEX-2T(Amersham Pharmacia Biotech)、及びpQE-30(QUIAGEN)のBamHI/SmaI領域中にクローニングし発現プラスミドを得た。得られたプラスミドを大腸菌M15(pREP4)またはXL1 Blue中に形質転換し37℃、NZCYM培地中で生育させた。培養物のOD₆₀₀が約0.5に達した時点で、最終濃度1mMとなるようイソプロピル-β-チオガラクトピラノシド(IPTG)を添加し、さらに5時間培養した後、細胞を回収した。
【００４５】
組換え蛋白質の精製は、The QIAexpressionist(QIAGEN)及びGST Gene Fusion System(Amersham Pharmacia Biotech)の説明書に従って行った。次にpGEX組換え転写ユニットより得られたGST融合蛋白質からGSTを除去するために、調製したGST融合蛋白質を約2mg/mlまで濃縮し、20mM Hepes-NaOH(pH8.0)、150mM NaClに対して4℃で、透析した。透析物に最終濃度2mMになるようにCaCl₂を加え、GST融合蛋白質との重量比で約1/200量のトロンビンを加え、氷上で5時間インキュベートした。その後、グルタチオン-Sepharose、及び、ベンズアミジン-Sepharose(Amersham Pharmacia Biotech)と混合した。4℃で一晩、回転しながら混合した後、カラムに充填し、素通り画分と20mM HEPES-NaOH(pH8.0)、150mM NaClの洗浄画分を集め、親和性に基づき所望の組換え蛋白質を得る。
【００４６】
５．機能解析
（１）hRUPP-1の細胞における発現
特異的モノクローナル抗体と可逆的に結合するエピトープを提供し、そして発現された蛋白質の検出及び容易な精製を可能にするため、全長をコードするhRUPP-1遺伝子のC末端にペプチドAsp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys(DYKDDDDK)をコードするヌクレオチドよりなるC末端融合蛋白質として発現されるようにし、動物細胞発現ベクターpcDNA3.1(CMVプロモーター)(Invitrogen社)に組み込み、組換発現ベクターとし以下のように各動物細胞に導入した。ミュータント蛋白質をコードする遺伝子はPCR法で作製し、エピトープ融合蛋白質として発現されるようにし、動物細胞発現ベクターpCMVに組み込み、組換発現ベクターとし動物細胞に導入した。
【００４７】
細胞形態の変化
Hela及びNIH3T3細胞にhRUPP-1遺伝子とlacZ遺伝子を共発現させ、Xgalを用いて染色することによりhRUPP-1が発現されると青くなるようにした。Hela及びNIH3T3細胞に全長のhRUPP-1遺伝子を導入した時の細胞の形態を図１０に示す。コントロールとして、hRUPP-1遺伝子を持たない空のベクターを用いた。NIH3T3細胞にhRUPP-1遺伝子を導入すると細胞が丸くなったが、Hela細胞にhRUPP-1遺伝子を導入しても細胞の形態に何の変化も見られなかった。すなわち、hRUPP-1は細胞を丸くする活性（細胞を丸くする活性をRU(Round-Up)と略す）を持ち、その性質は細胞選択的であり、生体内では組織特異的または時期特異的に機能することが予測された。
【００４８】
機能ドメイン解析
hRUPP-1の機能ドメインを解析するため、ミュータント蛋白質をコードする遺伝子をエピトープ融合蛋白質として発現されるようにし、動物細胞発現ベクターpCMVに組み込み、組換発現ベクターとし動物細胞に導入した。ミュータント蛋白質をコードする遺伝子は、hRUPP-1遺伝子よりPCR法により作製した。ミュータント蛋白質の模式図を図１１に示す。hRUPP-1遺伝子、或いは、ミュータント遺伝子をCHO細胞及びHela細胞に導入した時の細胞の形態は、lacZ遺伝子と共発現させ、Xgalを用いて染色することにより、導入した遺伝子が発現すると細胞が青く染色されるようにして検出した（図１２）。コントロールとして、hRUPP-1遺伝子を持たない空のベクターを用いた。図１１中のａは1148アミノ酸残基からなる全長hRUPP-1を表し、その下のｂ〜ｇは、該全長ｈRUPP-1を欠失させた各断片を表す。図１２は、CHO細胞では全長a、d、e、fは弱いRUを示すが、ミュータントb、c、gは強いRUを持ち、Hela細胞では、a、d、eはRUがなく、ミュータントb、c、f、gはRUを伴う細胞の増殖阻害が観察されたことを示す。
【００４９】
以上のことから、配列番号２のアミノ酸残基1〜305までからなるドメイン、または751〜1148までからなるドメインが強いRU及び細胞増殖抑制活性を有し、該ドメインを利用し強いRU及び細胞増殖抑制活性を持つミュータントhRUPP-1を作製することができる。ミュータントの作製はこのドメインに制限されるものではなく、ミュータントgのようにドメイン間の組み合わせ、または一定のドメインを短くすることによっても作製することができる。
【００５０】
hRUPP-1 の分解
シグナル依存的に活性化または発現誘導される転写因子群Jun、Fos、Myc、p53及びNF-κBの抑制性因子I-κB、多くのサイクリン関連蛋白質がユビキチン-プロテアソームによる制御を受け分解される。NIH3T3細胞でのhRUPP-1の分解について検証するため、NIH3T3細胞にhRUPP-1遺伝子を発現させ、10μMのMG132で10時間処理後、細胞を溶解し、SDS-PAGEに供した。その後、PVDF膜に転写し、各々の蛋白質を免疫染色した（図１３）。図１３中、＋は遺伝子導入、または、MG132処置を、−はhRUPP-1遺伝子を持たない空のベクターの導入、または、MG132未処置を表す。
【００５１】
NIH3T3細胞にhRUPP-1を導入された蛋白質はよく分解される（図１３第1レーン）が、プロテアソーム阻害剤であるMG132で処理すると分解が抑制された（図１３第2レーン）。対照として加えた、ユビキチンープロテアソームにより分解される癌抑制蛋白質p53もプロテアソーム阻害剤であるMG132で処理すると分解が抑制された（図１３第1〜4）。
【００５２】
hRUPP-1 のポリユビキチン化
シグナル伝達依存的に分解される蛋白質の多くは分解に先立ちポリユビキチン化され、蛋白質分解装置プロテアソームによって速やかに分解されることが知られていたので、hRUPP-1が細胞内でポリユビキチン化されることを確認するため、hRUPP-1遺伝子とユビキチン遺伝子を293細胞に共発現させ、10μMのMG132で6時間処理するか、処理をしなかった。その後、細胞を溶解し、hRUPP-1をエピトープ抗体で特異的に免疫沈降し、SDS-PAGEに付した後、PVDF膜に転写し、導入したユビキチンに対する抗体を用いて免疫染色しhRUPP-1のユビキチン化を調べた（図１４Ａ）。また、細胞溶解後、溶解液を直接SDS-PAGEに供し、PVDF膜に転写した後、hRUPP-1を直接免疫染色した(図１４Ｂ)。＋はhRUPP-1遺伝子導入またはMG132処理を、−はhRUPP-1遺伝子を持たない空のベクターの導入、または、MG132未処理を表す。
【００５３】
その結果、hRUPP-1は細胞内でポリユビキチン化されることが示された(図１４レーン４及び８)。また、293細胞でhRUPP-1はポリユビキチン化されるが、著しい分解は見られず、プロテオソーム阻害剤MG132処理によっても著しい増加は観察されなかった（図１４レーン８）。以上のことからhRUPP-1は細胞内でポリユビキチン化され、プロテアソームによる分解を受けることが分かった。また、他のシグナル依存的に活性化または発現誘導される転写因子群Jun、Fos、Myc、p53及びNF-κBの抑制性因子I-κB、多くのサイクリン関連蛋白質のようにhRUPP-1もシグナル依存的或いは細胞（組織）選択的な分解制御を受けることが判明した。
【００５４】
（２）hRRCP-1の発現
特異的モノクローナル抗体と可逆的に結合するエピトープを提供し、そして発現された蛋白質の検出及び容易な精製を可能にするため、全長をコードするhRRCP-1遺伝子のC末端にペプチドAsp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys(DYKDDDDK）またはGlu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu(EQKLISEEDL）をコードするヌクレオチドよりなるC末端融合蛋白質として発現されるようにし、動物細胞発現ベクターpME18S（SRαプロモーター）(Hara,T.及びMiyama,A.,EMBO J. 11,1875〜1884,1992年)に組み込み、組換発現ベクターとし以下のように各動物細胞に導入した。ミュータント蛋白質をコードする遺伝子はPCR法で作製し、エピトープ融合蛋白質として発現されるようにし、動物細胞発現ベクターpME18Sに組み込み、組換発現ベクターとし動物細胞に導入した。
【００５５】
結果
hRRCP-1及び既に報告されているMayven[Soltysik-Espanola,M.et al.,Mol.Biol.Cell 10,2361-2375,1999]、Keap 1[Itoh,K.et al.,Gene and Dev. 13,76-86,1999]を各々Hela細胞で発現させ、各々の蛋白質をhRRCP-1遺伝子のC末端に付加したペプチドに対する蛍光標識抗体を用いて染色した（緑色が各々の蛋白質の局在を表す、図１５）。hRRCP-1はドロソフィラkelch[Xue,F.et al.,Cell 72,681-693,1993]と非常に類似したRing-canal構造を形成し、この構造は他の哺乳類kelch因子群であるMayvenやKeap 1には観察されなかった。これは、hRRCP-1が真のドロソフィラkelchの哺乳類ホモログの可能性を強く示唆するものであり、ドロソフィラkelchのように細胞質輸送やアクチン フィラメントの構築などに深く関わっていると予測された。
【００５６】
hRRCP-1 の Ring-canal 構造形成に関わる内部ドメインの特定
hRRCP-1の持つRing-canal構造形成に関わる内部ドメインを特定するため、ミュータント蛋白質をコードする遺伝子をPCR法で作製し、エピトープ融合蛋白質として発現されるようにしてHela細胞に導入し、各々の局在を調べた(図１６)。用いたミュータント蛋白質の模式図を図１７に示す。POZドメインとIVRドメインからなるミュータント蛋白質のみRing-canal構造を形成し、この２つのドメインはRing-canal構造形成に必須不可欠なドメインであることが判明した。POZ及びIVRドメインのみでは細胞全体に、kelchドメインは核内に斑点様に、POZドメインとkelchドメインからなるミュータント蛋白質は主に細胞質に、IVRドメインとkelchドメインからなるミュータント蛋白質は細胞質または核内に局在性を示した。以上より、hRRCP-1はPOZドメインとIVRドメインを介してRing-canal構造を形成し、細胞質輸送やアクチン フィラメントの構築などに関わっていると予測された。各々のミュータント蛋白質の利用は局在についての情報を提供するのみならず、POZドメインとIVRドメインからなるミュータント蛋白質のようにRing-canal構造の分子設計の道具にもなる。各々のミュータント蛋白質はhRRCP-1の正または負の調節分子として利用することもできる。
【００５７】
hRRCP-1 のアクチンとの結合
hRRCP-1が真のドロソフィラkelchの哺乳類ホモログとして機能するためにはhRRCP-1のアクチンとの結合を前提とする。それを証明するために、hRRCP-1、Mayven及びKeap 1を各々の293細胞に発現させた後、細胞を溶解し、hRRCP-1、Mayven及びKeap 1を各々免疫沈降し、SDS-PAGEに付し、PVDF膜に転写した。各々の免疫沈降画分を蛋白質に対する抗体(図１８上)、及び、アクチンに対する抗体(図１８下)で免疫染色し、アクチンとの結合能を調べた。コントロールとしてhRUPP-1遺伝子を持たない空のベクターを用いた(ｐME-Empty)。hRRCP-1はアクチンと結合できることが判明し、ドロソフィラkelchのように細胞質輸送やアクチン フィラメントの構築などに関わっていることが強く示唆された。
【００５８】
hRRCP-1 による Mayven や Keap の機能の調節
実験により、hRRCP-1だけでなくMayvenやKeap 1もアクチン結合能を有することが示された(図１８下)。このことは、hRRCP-1はアクチンとの結合或いはMayvenやKeapとの結合を介してMayvenやKeapの機能を調節できることを意味し、それを証明するために次のような実験を行った。293細胞に各々、hRRCP-1/MycエピトープとMayven/Flagエピトープ、ｈRRCP-1/MycエピトープとKeap 1/Flagエピトープ、及び、Keap 1/FlagエピトープとMayven/Mycエピトープ遺伝子を共導入し、細胞を溶解した後、各々の細胞溶解物を抗Mycエピトープ抗体で免疫沈降し、免疫沈澱物をSDS-PAGEに供した。その後、PVDF膜に転写し、抗Flag抗体を用いて(図１９Ａ)、または、抗Myc抗体を用いて(図１９Ｂ)免疫染色した。図中、＋は各々の遺伝子導入を、−は遺伝子を持たない空のベクターの導入を表す。図１９の免疫沈降の結果よりhRRCP-1はMayvenと結合できるが、Keap1とは結合できないことが示された。
【００５９】
さらに、Hela細胞に各々、hRRCP-1/MycエピトープとMayven/Flagエピトープ、hRRCP-1/MycエピトープとKeap 1/Flagエピトープ、及び、Keap 1/FlagエピトープとMayven/Mycエピトープを共導入し、各々の細胞を抗Mycエピトープ抗体(赤)、及び、抗Flagエピトープ抗体(緑)で免疫染色した(図２０)。その結果、hRRCP-1はMayvenの局在を完全に壊すだけでなく、Mayvenを取り込むことが判明した。以上より、hRRCP-1アクチン或いはMayvenとの結合を介し、ドロソフィラkelchのような細胞質輸送やアクチン フィラメントの構築等に関わるだけでなく、Mayvenの機能の負または正の調節因子としても機能することが示唆された。
【００６０】
hRRCP-1 のアポトーシスへの関与
hRRCP-1はAIP処理により起こる細胞増殖阻害時に発現誘導される遺伝子としてクロニーングされたものである。細胞増殖阻害は細胞のアポトーシス誘導シグナルにもなる。hRRCP-1のアポトーシスへの関与を調べるため、Fas分子を恒常的に発現している細胞293/Fas細胞にhRRCP-1を導入し、抗Fas抗体刺激による選択的分解を受けるかを調べた。
【００６１】
293/Fas細胞にhRRCP-1を導入し、抗Fas抗体で一定時間刺激した後、細胞を溶解した。溶解物をSDS-PAGE後、PVDF膜に転写し、hRRCP-1(図２１Ａ)及びカスパーゼ3(図２１Ｂ)で免疫染色した。その結果、hRRCP-1は抗Fas抗体刺激により特異的に分解されることが判明した。対照として、アポトーシス実行分子カスパーゼ3（アポトーシス誘導に関与する蛋白分解酵素）の活性化も観察した。Fasによるアポトーシス誘導シグナルはプロテアーゼ（カスパーゼ）カスケードへ移行し、カスパーゼ基質の選択的分解を介し、細胞を死へと導くと考えられている。アポトーシスに関与するカスパーゼの基質については種々の候補が報告されているが、いまだ最終的な基質は分かっていない。従って、Fasシグナル(Fasリガンド、及びFas抗体を含む)によるhRRCP-1の特異的分解はアポトーシス研究の新しい分野を提供し、hRRCP-1が自己免疫疾患や癌などに関与する可能性を示唆する。また、hRRCP-1の特異的分解はアポトーシスのマーカーとして利用可能である。
【００６２】
（３）hERRj-1蛋白質の発現
特異的モノクローナル抗体と可逆的に結合するエピトープを提供し、そして発現された蛋白質の検出及び容易な精製を可能にするため、全長をコードするhERRj-1遺伝子の内部にペプチドAsp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys(DYKDDDDK)をコードするヌクレオチドよりなる内部融合蛋白質として発現されるようにし、動物細胞発現ベクターpME18S（SRαプロモーター）またはpCMV（CMVプロモーター）に組込み、組換発現ベクターとし動物細胞に導入した。ミュータント蛋白質をコードする遺伝子はPCR法で作製し、エピトープ融合蛋白質として発現されるようにし、動物細胞発現ベクターpME18SまたはpCMVに組み込み、組換発現ベクターとし動物細胞に導入した。
【００６３】
hERRj-1 の局在
hERRj-1をHela細胞で発現させ、小胞体内マーカー蛋白質であるBipとの共局在の様子を表す細胞の写真を図２２に示す。図６に示すドメイン解析の結果と一致し、hERRj-1は小胞体内に存在することが証明された。
【００６４】
hERRj-1 による細胞形態の変化
Hela細胞、及び、CHO細胞にhERRj-1遺伝子とlacZ遺伝子を共発現させ、Xgalを用いて染色し、hERRj-1が発現されると青くなるようにし、Hela及びCHO細胞にhERRj-1遺伝子を導入した時の細胞の形態を図２３に示した。コントロールとしてhERRj-1遺伝子を持たない空のベクターを用いた。hERRj-1の細胞への導入は細胞死を誘導することが分かった。小胞体ストレスは細胞死（アポトーシス）を引き起こすことが知られている。従って、hERRj-1は小胞体ストレスによる細胞死の原因分子の１つであると予測される。小胞体ストレスに対する応答機構には小胞体膜上に存在する２つのキナーゼ分子IRE 1とPERKが関わっていることが知られている。IRE 1はUPRを介して小胞体分子シャペロンを誘導させて、異常蛋白質の蓄積を防ぐ。また、IRE 1は小胞体ストレスの付加に伴うJNK（c-Jun N末端キナーゼ）の活性化に必須の分子としても同定されている。PERKはeIF2αのSer51をリン酸化してmRNAから蛋白質への翻訳を抑制し、異常蛋白質が小胞体内で蓄積することを防ぐ。これらのキナーゼは小胞体ストレス依存的に活性化され、小胞体ストレス対する防御機構として機能すると考えられている。一方、小胞体ストレスを引き起こす刺激が過剰な場合あるいは長時間持続する場合あるいは何らかの原因で小胞体ストレスに対する抵抗性が弱くなった場合、細胞はアポトーシス様の形態変化を起こし死滅するが、家族性アルツハイマー病の原因遺伝子プレセニリンーのミスセンス変異はこれらの小胞体ストレスを介した神経細胞死の原因となっていると考えられている。従って、hERRj-1は、IRE 1若しくはPERKを介した過剰の小胞体ストレスを誘導するか、または、小胞体ストレスに対する抵抗性を弱めることが予測される。
【００６５】
hERRj-1 による IRE 1 、または、 PERK の活性化
hERRj-1が、IRE 1またはPERKの活性化を誘導できることを証明するため、hERRj-1を293細胞に導入した後、小胞体ストレスを引き起こす薬剤（ツニカマイシン、DTT、タプスガルジン）を処理し、GST-ｃJunを用いて免疫沈降し、免疫沈降物をイン・ヴィトロ キナーゼ分析に付した。イン・ヴィトロ キナーゼ分析産物をSDS-PAGEに付し、PVDF膜に転写後、c-Junのリン酸化Ser63を特異的に認識する抗体を用いて免疫染色し(図２４(Ａ)上)、コントロールとして同じ細胞溶解物をJNK(c-Jun N-terminal kinase)を用いて免疫染色し(図２４(Ａ)下)、内在性JNKの活性及びeIF2αのリン酸化を調べた。また、293細胞にhERRj-1遺伝子を導入し、小胞体ストレスを引き起こす薬剤で処理し、細胞を溶解した後、溶解物を直接SDS-PAGEに付し、PVDF膜に転写後、eIF2αまたはeIF2αのリン酸化Ser51を特異的に認識する抗体を用いて免疫染色した(図２４(Ｂ))。図中、−はhERRj-1遺伝子を持たない空のベクターの導入、または薬剤処理なしを表す。Tmは、2.5μｇ/mlのツニカマイシンでの3時間にわたる処理を、DTTは5ｍMのジチオスレイトールでの3時間にわたる処理を、Tgは1μMのthapsigarginでの1時間にわたる処理を表す。
【００６６】
それにより、hERRj-1の細胞への導入により、小胞体ストレス対するc-Jun及びeIF2αのSer51のリン酸化が亢進されることが判明し、hERRj-1はIRE 1及び PERKの活性化を誘導することが分かった。以上の結果より、hERRj-1は過剰に、または、持続的にIRE 1及び PERKの活性化を誘導し、細胞死を引き起こすことが示唆された。
【００６７】
hERRj-1 による UPR 誘導
hERRj-1がIRE 1を活性化することは、hERRj-1はUPRを誘導できることを意味する。それを証明するため、hERRj-1を293細胞あるいはCHO細胞に導入し、UPRにより誘導される遺伝子BipやCHOPの発現をRT-PCR法で調べた。まず、293細胞、及び、CHO細胞にhERRj-1、または、Bip遺伝子を導入し、5μg/mlのツニカマイシンで6時間処理した細胞より、全RNAをtotal RNA Isolation Reagent(GIBCO BRL)を用いて抽出した。各々の全RNA 5μgよりProSTAR First-Strand RT-PCR Kit(STRATAGENE)を用いてcDNAを作製し、内在性hERRj-1、Bip及びCHOP遺伝子を遺伝子特異的プライマーを使用してPCR法で検出した。反応はTaqポリメラーゼの存在下、96度25秒、60度20秒、72度1分を28サイクル繰り返した。
【００６８】
結果を図２５に示す。hERRj-1は小胞体ストレスを引き起こすツニカマイシン処理により誘導され、また、hERRj-1の細胞への導入はUPRにより誘導される遺伝子BipやCHOPの発現を引き起こすことも判明した。以上のことより、hERRj-1は小胞体ストレスにより誘導され、IRE 1及び PERKの活性化とUPRを誘導する新規の小胞体分子シャペロンであることが判明した。
【００６９】
hERRj-1 の IRE 1alpha 、 IRE 1beta 及び Bip との結合
hERRj-1がUPRを活性化することを示した。BipはIRE 1またはPERKと結合し、IRE 1またはPERKの活性を阻害していることが報告されている（Bertolotti, A. et al., Nature Cell Biol. 2, 326-332, 2000）。hERRj-1によるUPRの活性はhERRj-1とIRE 1もしくはBipとの結合を介して起こっている可能性を検討するため、hERRj-1とIRE 1をCHO細胞に共発現させ、hERRj-1を免疫沈降し、免疫沈降物をSDS-PAGEに付し、PVDF膜に転写した後、IRE 1もしくはBipに対する抗体で免疫染色し、hERRj-1との結合能を調べた。その結果を図２６に示す。hERRj-1はIRE 1または内在性Bipと結合し、小胞体ストレスはその結合を増強させることが明らかとなった。以上のことより、hERRj-1はIRE 1またはBipとの結合を介し、UPRを調節することが判明した。
【００７０】
hERRj-1 の細胞死誘導ドメインの同定
CHO細胞にhERRj-1あるいはhERRj-1の各々のミュータント遺伝子をlacZ遺伝子と共発現させ、Xgalを用いて染色し、hERRj-1が発現される細胞を青くなるようにした。細胞死による細胞の形態の変化を指標にして細胞死誘導活性を調べ、その結果を図２７に示した。コントロールとしてhERRj-1遺伝子を持たない空のベクターを用いた（生細胞１００％）。図６で示したように、hERRj-1はDnajドメインと４つのチオレドキシン（TRX１から４）ドメインを持っている。hERRj-1では生細胞２５％、Dnajドメインの６３番目のアミノ酸HisをGlnに変えたミュータント（Dnajミュータント）は生細胞５２％、TRX１ドメインの活性部位の１５８番目と１６１番目のCysをSerにかえたミュータント（TRX 1ミュータント）は生細胞６０％、TRX２ドメインの活性部位の４８０番目と４８３番目のCysをSerにかえたミュータント（TRX 2ミュータント）は生細胞６２％、TRX３ドメインの活性部位の５８８番目と５９１番目のCysをSerにかえたミュータント（TRX 3ミュータント）は生細胞５５％、TRX４ドメインの活性部位の７００番目と７０３番目のCysをSerにかえたミュータント（TRX 4ミュータント）は生細胞９０％であった。さらに、TRX１から４の全ての活性部位のCysをSerに変えたミュータント（TRX-allミュータント）は生細胞９５％、DnajドメインとTRX１から４までのドメインの全てのミュータント（Dnaj/TRXミュータント）は生細胞１００％の活性を示した。以上のことから、hERRj-1の活性は主にTRXドメインにより誘導され、Dnajドメインは調節ドメインとして機能していることが予測された。
【００７１】
hERRj-1 による細胞内での IRE 1 の局在の変化
IRE 1はN-末端を小胞体のルーメン側に露出して小胞体ストレスを感知し、C-末端のキナーゼドメインとエンドヌクレアーゼドメインの機能ドメインを細胞質側に露出している小胞体膜タンパク質として機能していると考えられている。しかし、JNKの活性にはキナーゼドメインを必要とし（Urano, F. el al., Science 287, 664-666, 2000）、UPRにはエンドヌクレアーゼドメインのみを必要としている（Tirasophon, W. et al., Genes Dev. 14, 2725-2736, 2000）と予測されているが、IRE 1による小胞体ストレスに対する応答に関する両ドメインの協調性などの詳しいことは未知である。NIH3T3細胞にhERRj-1またはIRE 1を単独または共発現させ、IRE 1の局在を調べ、その結果を図２８に示した。IRE 1単独ではIRE 1は小胞体の局在性を示すが、hERRj-1を共発現させると、IRE 1の局在は大きく変化して凝集体として顕われた。しかし、hERRj-1のDnajミュータントまたはTRXミュータントはIRE 1の凝集体の形成を誘導しないことから、IRE 1の凝集体の形成にはhERRj-1の機能が必須であることが判明された。さらに、IRE 1のキナーゼドメインミュータントまたはエンドヌクレアーゼドメインミュータントは凝集体を形成しないことから、hERRj-1によるIRE 1の凝集体形成にはIRE 1のキナーゼドメインとエンドヌクレアーゼドメインの両方を必要とすることも明らかとなった。以上から、hERRj-1によるIRE 1の凝集体形成誘導を新しく同定し、IRE 1の機能にhERRj-1が深く関わっていることを示唆した。
【００７２】
マウスの臓器での mERRj-1 （ hERRj-1 のマウス相同体）の局在
以上のことから、hERRj-1の過剰発現は細胞死を誘導し、IRE 1またはBipとの結合を介し、あるいはIRE 1の凝集体の形成を介し、小胞体の機能を調節していることが判明した。hERRj-1の持つ体内での役割を探るため、hERRj-1の内部配列（合成ペプチド）に対するウサギポリクローナル抗体を作製し、膵臓、脳及び精巣の固定切片を用いて組織染色を行った。その結果を図２９に示した。膵臓のランゲルハンス島のbeta細胞、脳及び精細管の中の特種の細胞が選択的に染色された。膵臓のbeta細胞は非常に発達した小胞体を持つインスリン産生細胞で、PERKの異常による小胞体の機能異常はbeta細胞の死を伴う膵臓異常と糖尿病を引き起こす（Delepine, M. et al., Nat. Genet. 25, 406-409, 2000; Harding, H. P. et al., Mol. Cell 7, 1153-1163, 2001）。また、Bipの発現低下などの小胞体の機能異常による神経細胞死がアルツハイマー病の原因の一つと考えられている（Katayama, T. et. al., Nature Cell Biol. 1, 479-485, 1999）。さらに、糖尿病やアルツハイマー病は変性蛋白質の蓄積により起こるアミロイドーシスの一つでもある。以上のことから、hERRj-1は膵臓のbeta細胞、神経細胞及び精細胞の機能維持に必要であり、その発現異常は膵臓異常や神経変性疾患または精細胞などの分化異常の原因となることが示唆された。
【００７３】
EST 検索による hERRj-1 及び mERRj-1 の発現様式
ESTデータベースは任意のcDNAライブラリーから無作為に選ばれたcDNAの部分的配列を集めたデータベースであり、そのデータベースを検索することで、ある遺伝子のある組織での発現頻度を予測することができる。hERRj-1の持つ体内での役割を探る手段として、EST検索によるhERRj-1及びmERRj-1の発現様式を検討し、その結果を図３０Aに示した。ヒトの１３２個のcDNAの中に６８個（５１％）、マウスの８８個の中の３０個（３５％）が癌組織由来であり、さらに癌組織中のヒトの５３個（７８％）、マウスの３０個（１００％）が腺癌由来である。さらに、腺癌の中のヒトの２３個（４３％）が子宮腺癌由来であり、マウスの２５個（８３％）が乳腺癌由来であった。この結果は、ERRj-1がヒトまたはマウスの腺癌、特に、子宮腺癌及び乳腺癌に非常に高く発現していることを表している。腺は非常に発達した小胞体を持つ分泌器官であり、腺細胞でのERRj-1の発現異常による小胞体の機能異常が腺癌の発生の原因の一つになることが示唆された。また、乳腺癌と子宮腺癌はホルモン、特にエストロゲンの分泌と応答異常により発生する場合も多くある。ERRj-1と同じくTRXドメインを二つ持つ小胞体シャペロンPDIはエストロゲンと結合し、細胞内のエストロゲンの濃度を調節していると予測されている（Primm, T and Gilbert, H., JBC, 276, 281-286, 2001）。したがって、ERRj-1はホルモンとの結合またはPDIの機能を調節することにより、細胞内でのホルモンの濃度を調節している可能性も考えられる。
【００７４】
また、乳腺癌と子宮腺癌は互いに転移しやすい性質があり、乳腺癌と子宮腺癌でのERRj-1の異常発現は癌の転移に関わっている可能性もある。癌細胞は主要組織適合抗原（MHC）クラスIによる癌抗原提示が正常に行われると、細胞傷害性Tリンパ球（CTL）によりその癌細胞は排除されるが、免疫グロブリンH鎖とbeta2ミクログロブリンの２量体から成るMHCクラスIのフォールディング異常やそれに伴う分解などの原因で癌抗原提示が正常に行われないとCTLからの攻撃から逃れて、癌細胞の転移が成立する。hERRj-1がMHCクラスIによる癌抗原提示に関与可能性を証明するため、MHCクラスIのフォールディングに必須の分子であり、TRXドメインを持つ小胞体シャペロンERp57のhERRj-1による酸化還元状態の変化（図３０B）とhERRj-1による免疫グロブリンH鎖（mu鎖）の発現量（図３０C）を調べた。アルキル化試薬AMSは蛋白質の還元状態のCysと反応するし、500ダルトンの分子量の上昇をもたらす。したがって、AMS反応後の蛋白質の分子量の上昇を目安に蛋白質の酸化還元状態を推測することが可能になる。図３０Bに示したように、細胞でのERp57は完全酸化フォームと部分酸化フォームと完全還元フォームの混合状態で存在することが分かる。しかし、hERRj-1が存在するとERp57の完全酸化フォームと部分酸化フォームが完全還元フォームに変化することが判明した。さらに、図３０Cに示したように、hERRj-1は免疫グロブリンH鎖（mu鎖）に結合でき、その発現量を低下させることも判明した。ERp57はMHCクラスIの免疫グロブリンH鎖と結合し、免疫グロブリンH鎖の分解を防ぐことによりMHCクラスIによる抗原提示に寄与する。以上により、hERRj-1は免疫グロブリンH鎖と直接な結合を介し、またはERp57の機能を調節することにより、MHCクラスIによる抗原提示に関与することが分かり、hERRj-1の異常発現はMHCによる癌細胞癌抗原の低下を引き起こし、癌の転移に深く関わっている可能性が示唆された。
【００７５】
【発明の効果】
本発明のhRUPP-1は細胞を丸くする(RU)活性、細胞増殖抑制活性、及び、細胞死誘導活性を有する。細胞が丸くなる現象は細胞分裂時、接着阻害時または細胞移動時に良く見られことから、これらの現象に機能すると予測された。全長またはミュータントhRUPP-1を細胞に導入することにより細胞にRU活性及び増殖抑制活性を付与して細胞死を誘導することができる。細胞内のhRUPP-1の持つRU活性を制御することにより細胞の運動能を制御が可能であると考えられ、例えばhRUPP-1に対するアンチセンス遺伝子を投与することにより癌細胞の原発巣からの転移や癌細胞の浸潤を抑制できると考えられる。
【００７６】
また、hRUPP-1、hRUPP-1をコードする遺伝子、それに対するアンチセンスポリヌクレオチドを含むスクリーニング系を用いることにより、該蛋白質の活性を阻害、または、活性化する化合物をスクリーニングすることも可能である。そのような化合物は、細胞の増殖、転移及び分裂、並びに、細胞死を調節する薬剤として役立つと考えられる。
【００７７】
また、hRUPP-1は本明細書において示したようにポリユビキチン化され分解されるが、hRUPP-1をポリユビキチン化されないように改変することにより、hRUPP-1の生体内における分解を調節することが可能になると考えられる。
【００７８】
本発明のhRRCP-1は、Fasシグナルにより特異的に分解されるので、従来のカスパーゼ等に代えてアポトーシス（細胞死）のマーカーとして利用することができる。また、従来知られていないhRRCP-1のRing-canal構造を介した生理現象や疾病について基礎的知識を提供することができる。
【００７９】
hERRj-1はチオレドキシン ドメインとDnajドメインを持つ新規の小胞体内シャペロンである。本発明の遺伝子あるいは蛋白質の細胞内での発現は細胞死（アポトーシス）を引き起こす。本発明のhERRj-1は、c-Jun及びeIF2aのSer51をリン酸化（転写活性化）する。本発明の遺伝子あるいは蛋白質はUPRを誘導する。本発明のhERRj-1はIRE 1、Bip、免疫グロブリンと結合する。本発明のhERRj-1はIRE 1による凝集体の形成を誘導する。本発明のhERRj-1はERp57を還元させる。本発明のhERRj-1は免疫グロブリンの発現を調節する。本発明のhERRj-1は膵臓のランゲルハンス島のbeta細胞、脳及び精細胞などの小胞体の機能維持に必須である。本発明のhERRj-1は腺癌、特に、子宮腺癌及び乳腺癌に高発現している。本発明の遺伝子または蛋白質がUPRを誘導するという知見は、本発明の遺伝子または蛋白質が小胞体ストレス対する防御分子として利用できることを意味する。
【００８０】
また、hERRj-1は細胞死を誘導する活性を有するので、hRUPP-1と同様に、hERRj-1を投与することにより腫瘍細胞等において、細胞死を誘導することができるかもしれない。さらに、hERRj-1、hERRj-1をコードする遺伝子、それに対するアンチセンスポリヌクレオチドを含むスクリーニング系を用いることにより、該蛋白質の活性を阻害、または、活性化する化合物をスクリーニングすることも可能である。そのような化合物は細胞死を調節する薬剤として役立つと考えられる。
【００８１】
JNKの活性化はストレスによるアポトーシス、特に神経細胞死に必須である。hERRj-1の持つc-Junリン酸化活性(JNKの活性化)を制御することにより細胞死を制御することが可能となるかも知れない。例えば、hERRj-1に対するアンチセンス遺伝子を投与することにより、ERストレスにより起こる細胞死を抑制でき、ERストレスによる神経細胞死が原因とされるアルツハイマー病の治療や治療薬の開発に利用可能であると期待される。
【００８２】
eIF2αリン酸化の起こらないミュータントは低血糖症を引き起こし、eIF2αリン酸化は血糖値調節に必須であることから、hERRj-1遺伝子を投与することにより低血糖症の治療や治療薬の開発に利用可能であると期待される。
【００８３】
hERRj-1はIRE 1と結合でき、IRE 1の凝集体形成などを介しIRE 1の機能を調節する分子であると予測されるので、hERRj-1を利用することにより、UPRなどのIRE 1を介する信号を調節し、B細胞の分化誘導や神経変性疾患の治療に利用できると期待される。
【００８４】
hERRj-1は膵臓のランゲルハンス島のbeta細胞に高発現し、その機能維持に必須であると予測されるので、hERRj-1を利用することにより、糖尿病の治療や糖尿病の治療のための薬剤の開発に利用できる。
【００８５】
家族性アルツハイマー病(FAD)の原因遺伝子PS1の遺伝子変異は小胞体分子シャペロンBipの転写誘導の減弱を引き起こし、Bipの転写誘導阻害が神経細胞死の原因の一つとされている。hERRj-1はBipの転写誘導を活性化でき、さらにBipとの結合を介してBipの機能を調節すると予測できることから、hERRj-1遺伝子またはアンチセンス遺伝子を投与することによりこれらの病気の治療や治療薬の開発に利用可能であると期待される。
【００８６】
腺細胞でのERRj-1の発現異常は腺癌、特に、子宮腺癌及び乳腺癌発生の原因の一つになることが示唆されるので、hERRj-1を利用することにより、腺癌の治癒や治療のための薬剤の開発が期待できる。
【００８７】
hERRj-1は免疫グロブリンH鎖と直接な結合を介し、またはERp57の機能を調節することにより、MHCクラスIによる抗原提示に関与すると予測されるので、hERRj-1を利用することにより、癌の治癒が期待でき、さらに癌の転移を防ぐことが可能になると期待できる。
【００８８】
【配列表】

【図面の簡単な説明】
【図１】 AIP処理によって起こる細胞の増殖阻害時に増加する遺伝子のサブトラクション法によるクローニング法の流れを示す。
【図２】 AIP処理によって起こる細胞の増殖阻害時に減少する遺伝子のサブトラクション法によるクローニング法の流れを示す。
【図３】 サブトラクション ライブラリーのスクリーニングにより（cDNA 2-cDNA 1）プローブのみと反応するhRUPP-1のcDNA断片を持つクローン。
【図４】 サブトラクション ライブラリーのスクリーニングにより（cDNA 2-cDNA 1）プローブのみと反応するhRRCP-1のcDNA断片を持つクローン。
【図５】 サブトラクション ライブラリーのスクリーニングにより（cDNA 2-cDNA 1）プローブのみと反応するhERRj-1のcDNA断片を持つクローン。
【図６】 hERR-1のドメイン構造と各々のドメイン配列の比較図。
【図７】 hRUPP-1の組織分布。
【図８】 hRRCP-1の組織分布。
【図９】 hERRj-1の組織分布。
【図１０】 Hela及びNIH3T3細胞に全長のhRUPP-1遺伝子を導入した時の細胞の形態。
【図１１】 hRUPP-1遺伝子の機能ドメイン解析のために作成したミュータント蛋白質の模式図。
【図１２】 図１１に示したミュータント蛋白質をCHO細胞、及び、Hela細胞に導入した時の細胞の形態。
【図１３】 NIH3T3細胞でのhRUPP-1、及び、p53の分解。
【図１４】 hRUPP-1遺伝子及びユビキチン遺伝子の293細胞での共発現による、hRUPP-1の細胞内でのポリユビキチン化の確認。
【図１５】 hRRCP-1、Mayven、Keap 1のHela細胞における局在を示す。
【図１６】 hRRCP-1の持つRing-canal構造形成に関わる内部ドメインを特定するため、ミュータント蛋白質をコードする遺伝子はPCR法で作製し、エピトープ融合蛋白質として発現されるようにしてHela細胞に導入し、各々の局在を調べた。
【図１７】 hRRCP-1のミュータント蛋白質の模式図。
【図１８】 hRRCP-1、Mayven及びKeap 1を各々の293細胞に発現させた後、細胞を溶解し、hRRCP-1、Mayven及びKeap 1を各々免疫沈降し、SDS-PAGEに付し、PVDF膜に転写した。各々の免疫沈降画分を蛋白質に対する抗体(図１７上)、及び、アクチンに対する抗体(図１７下)で免疫染色し、アクチンとの結合能を調べた。
【図１９】 293細胞に各々、hRRCP-1/MycエピトープとMayven/Flagエピトープ、ｈRRCP-1/MycエピトープとKeap 1/Flagエピトープ、及び、Keap 1/FlagエピトープとMayven/Mycエピトープ遺伝子を共導入し、細胞を溶解した後、各々の細胞溶解物を抗Mycエピトープ抗体で免疫沈降し、免疫沈澱物をSDS-PAGEに供した。その後、PVDF膜に転写し、抗Flag抗体を用いて(Ａ)、または、抗Myc抗体を用いて(Ｂ)免疫染色した。
【図２０】 Hela細胞に各々、hRRCP-1/MycエピトープとMayven/Flagエピトープ、hRRCP-1/MycエピトープとKeap 1/Flagエピトープ、及び、Keap 1/FlagエピトープとMayven/Mycエピトープを共導入し、各々の細胞を抗Mycエピトープ抗体(赤)、及び、抗Flagエピトープ抗体(緑)で免疫染色した。
【図２１】 293/Fas細胞にhRRCP-1を導入し、抗Fas抗体で一定時間刺激した後、細胞を溶解し、SDS-PAGE後、PVDF膜に転写し、hRRCp-1(Ａ)及びcaspase3(Ｂ)で免疫染色した。
【図２２】 hERRj-1をHela細胞で発現させ、小胞体内マーカー蛋白質であるBipとの共局在の様子を表す細胞の写真。
【図２３】 Hela及びCHO細胞にhERRj-1遺伝子を導入した時の細胞の形態。
【図２４】 hERRj-1のIRE 1、または、PERKの活性化。
【図２５】 hERRj-1を293細胞あるいはCHO細胞に導入し、UPRにより誘導される遺伝子BipやCHOPの発現。
【図２６】 CHO細胞に各々、IRE1-alpha/T7エピトープあるいはIRE1-beta/Mycエピトープあるいは空ベクターとhERRj-1/Flagエピトープ遺伝子を共導入した後、Tm（2.5μｇ/mlのツニカマイシン）、DTT（5ｍMのジチオスレイトールで）、Tg（1μMのthapsigargin）で細胞を処理した。細胞を溶解した後、各々の細胞溶解物を抗Flagエピトープ抗体で免疫沈降し、免疫沈澱物をSDS-PAGEに供した。その後、PVDF膜に転写し、抗T７抗体と抗Flag抗体を用いて(Ａ)、抗Myc抗体と抗Flag抗体を用いて(Ｂ)、抗Bip抗体と抗Flag抗体を用いて（C）免疫染色した。
【図２７】 CHO細胞に空ベクター導入時の生細胞の割合を１００％とした時の各々のミュータント導入時の生細胞の割合を表した。
【図２８】 NIH細胞における図で表した各々の遺伝子を共発現させた時のIRE 1-alphaの局在を示す。
【図２９】 マウスの各々の固定組織切片を抗hERRj-1ペプチド抗体を用いて免疫染色した写真。矢印で囲んだ部分あるいは矢印で指した部分がmERRj-1の存在場所を表す。
【図３０】 A: hERRj-1のESTデータベース検索の結果。B: CHO細胞におけるhERRj-1遺伝子導入によるERp57蛋白の酸化還元状態。C: CHO細胞に空ベクターあるいは免疫グロブリンH（mu）鎖あるいは免疫グロブリンH（mu）鎖とhERRj-1/Flagエピトープ遺伝子を導入した後、細胞を溶解し、各々の細胞溶解物をそのまま（Total）あるいは抗免疫グロブリンH（mu）鎖抗体で免疫沈降し、SDS-PAGEに供した。PVDF膜に転写し、図で示した各々の抗体で免疫染色した。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel gene group whose expression is changed in human cells by treatment with an apoptosis-inducing protein AIP obtained from a parasite-infected fish.
[0002]
[Prior art]
AIP is a novel factor that induces apoptosis specifically in proliferating cells and is a novel apoptosis-inducing protein purified and cloned by the present inventors from parasite-infected fish [Jung, S-K. Et al.,J.Immunol.165, 1491-1497, 2000; Murakawa, M. et al.,Cell death Differ., in press]. AIP induces the production of hydrogen peroxide and depletion of amino acid lysine in the medium at 10 ng / ml and above, and causes cell death very efficiently in various highly proliferative human cancer cells by these two different mechanisms. . In addition, the growth of cancer cells can be inhibited at a low concentration of 1 ng / ml or less.
[0003]
Recently, cell surface receptor Fas antigen that induces apoptosis, protease (caspase) essential for apoptosis induction, oncogene that inhibits apoptosisbcl-2, suppressc-mycThe mechanism of intracellular information transmission related to apoptosis is becoming clear. Fas is a representative molecule of apoptotic signal, functions as a receptor that induces cell death by stimulation with Fas ligand and anti-Fas antibody, and is involved in autoimmune diseases and cancer. Apoptosis is an important process for maintaining physiological homeostasis, such as development / differentiation, normal tissue and cell turnover, etc., lymphocytes due to cancer regression, action of radiation and anticancer drugs, AIDS and other viral infections It is thought to be involved in diseases such as decrease in inflammation and inflammation. The development of drugs that regulate apoptosis is considered to have the potential to create new drugs in a wide range of fields such as the cranial nervous system, cancer, and aging.
[0004]
Markers for detecting apoptosis are also known. For example, a method of detecting a protein (PARP, Lamin, DFF45, Caspase, etc.) that is specifically degraded by Fas signal is used, and a kit that detects apoptosis by detecting a protein that is specifically degraded, Alternatively, antibodies are commercially available (MBL, Santa Cruz Biotechnology, Promega, Chemicon International, Affinity Bioreagents, Stressgen Biotechnologies, etc.).
[0005]
Recently, irreversible proteolytic control by ubiquitin / proteasome has been shown to play an important role in signal transduction pathways. The ubiquitin / proteasome system is highly conserved among eukaryotes and is considered to be one of the systems responsible for basic cellular functions. It is known that many of the proteins that are degraded depending on signal transduction are polyubiquitinated prior to degradation. Polyubiquitinated proteins are rapidly degraded by the proteasome proteosome, and the dynamic equilibrium state of proteins in the living body is changed. It is thought to keep. Transcription factors Jun, Fos, Myc, p53 and NF-κB inhibitory factor I-κB, many cyclin-related proteins that are activated or expressed in a signal-dependent manner, are regulated by ubiquitin / proteasome. Proteolysis is thought to be involved in various physiological phenomena such as cell carcinogenesis (differentiation), proliferation, inflammation, and stress response.
[0006]
For example, the tumor suppressor gene p53 that is subject to degradation control by the proteosome is well-known as a cancer regulatory gene that is frequently mutated in most human tumors such as colorectal cancer, brain tumor, liver cancer, bladder cancer, and esophageal cancer. When p53 is stabilized or activated by DNA damage, cell death accompanied by cell growth-inhibiting activity is induced. Therefore, cells with mutations in the p53 gene will continue to proliferate without causing cell growth suppression or apoptosis induction even if DNA abnormalities occur, and cells with abnormal chromosomes (cancer cells) accumulate. Become.
[0007]
Ring-canal protein kelch is a protein obtained from mutant analysis of Drosophila, which acts as an actin-binding protein. In Drosophila, it is an indispensable molecule for cytoplasmic transport and actin filament construction [Xue, F. et al.,Cell 72681-693, 1993]. Actin filaments are used in cell polarity and migration [Lauffenburger, D.A. et al.,Cell 84, 359-369, 1996], mRNA transport [Agutter, P.S et al.,.Biochem.Soc.Trans. 19, 1094-1098, 1991], neurite formation [Riederer, B.M.,Eur.J.Morphol. 28, 347-378, 1990] and the like, actin filament formation is controlled by various actin-binding proteins. Drosophila kelch-deficient mutants are known to lose their cytoplasmic transport function during oocyte development due to the inability to form Ring canals, resulting in female fertilization. The Ring canal structure in mammals also acts on the transport of substances inside or between cells, and may maintain the normal function of cells.
[0008]
Keap1 having actin binding ability has been identified as a sensor molecule that responds to oxidative stress. Oxidative stress is thought to damage biomolecules such as DNA and proteins, causing cell dysfunction and cell death, leading to carcinogenesis, aging, arteriosclerosis, and dementia.
[0009]
In order for a protein that is a functional molecule in a living body to perform a normal function, it is necessary that the protein is synthesized based on genetic information and then folded into a correct structure (folding). The endoplasmic reticulum is a place where folding of newly synthesized secreted proteins and membrane proteins takes place. Accumulation of denatured proteins in the endoplasmic reticulum is caused by external factors such as stress, cell differentiation and proliferation, cell response to the external environment, This is considered to occur when the amount of proteins that pass through the endoplasmic reticulum increases with viral infection. The cell senses the accumulation of denatured protein in the endoplasmic reticulum, and maintains homeostasis by inducing an endoplasmic reticulum chaperone to increase the folding capacity in the endoplasmic reticulum (UPR). Therefore, UPR has universal importance for cell function, but it is still unclear how cells sense, adapt and respond to the accumulation of denatured proteins in the endoplasmic reticulum. There are many.
[0010]
Endoplasmic reticulum stress is known to cause cell death (apoptosis), and the response mechanism to endoplasmic reticulum stress is known to involve two kinase molecules IRE 1 and PERK present on the endoplasmic reticulum membrane. Yes. IRE 1 induces the endoplasmic reticulum molecular chaperone via UPR and prevents accumulation of abnormal proteins. IRE 1 has also been identified as an essential molecule for the activation of JNK (c-Jun N-terminal kinase) accompanying the addition of endoplasmic reticulum stress. PERK phosphorylates Ser51 of eIF2α and suppresses translation from mRNA to protein, preventing abnormal proteins from accumulating in the endoplasmic reticulum. These kinases are activated depending on endoplasmic reticulum stress and are thought to function as a defense mechanism against endoplasmic reticulum stress. On the other hand, if the stimulus that causes endoplasmic reticulum stress is excessive, persists for a long time, or if resistance to endoplasmic reticulum stress is weakened for some reason, cells undergo apoptotic changes and die, but familial Alzheimer Disease-causing gene presenilin-1 (PS 1) missense mutation affects amyloid β protein production, and cells expressing mutant PS 1 are less resistant to endoplasmic reticulum stress, causing neuronal cell death It is thought that has become.
[0011]
[Means for Solving the Problems]
In the present invention, for the purpose of obtaining a gene related to apoptosis, a gene whose expression is changed by AIP treatment of human cells is cloned by a subtraction method, and hRUPP-1 (human homolog 1 of Round) is obtained as a gene increased by AIP treatment. -Up Phenotype Protein) and hRRCP-1 (human homolog 1 of Real Ring-Canal Protein) and endoplasmic reticulum stress sensor protein hERRj-1 (human homolog 1 of Endoplasmic) The gene encoding Reticulum Rumenal Dnaj) was cloned. Each protein sequence was deduced based on the sequence of the cloned cDNA.
[0012]
That is, the present invention relates to a protein (hRUPP-1) containing the amino acid sequence shown in SEQ ID NO: 2, a protein (hRRCP-1) containing the amino acid sequence shown in SEQ ID NO: 4, and the amino acid sequence shown in SEQ ID NO: 6. Containing protein (hERRj-1). Each of these fragments and a polypeptide having an amino acid sequence in which one or more amino acids of each of them are modified by deletion, addition, or substitution, and the same organism as each corresponding protein It relates to a polypeptide having activity. Furthermore, this invention relates to the polynucleotide which codes these.
[0013]
HRUPP-1 of the present invention is a protein consisting of 1148 amino acid residues having the amino acid sequence set forth in SEQ ID NO: 2. The biological activity of hRUPP-1 is the activity of rounding cells to induce cell death and the activity of suppressing cell growth. hRUPP-1 is also subject to degradation control by the ubiquitin / proteasome system.
[0014]
hRRCP-1 is a protein consisting of 568 amino acids having the amino acid sequence set forth in SEQ ID NO: 4. Since it has a structure very similar to the Drosophila ring-canal structure that has not been observed in mammalian kelch proteins found so far, it is an indispensable molecule for cytoplasmic transport and actin filament construction through binding to actin. Predicted to be. Examples of the biological activity of hRRCP-1 include actin binding activity. The gene encoding hRRCP-1 has homology with Keap1 identified as a sensor molecule that responds to oxidative stress.
[0015]
hERRj-1 is a novel endoplasmic reticulum chaperone having a 793 amino acid thioredoxin domain and a Dnaj domain having the amino acid sequence set forth in SEQ ID NO: 6. Expression of the gene or protein of the present invention in cells causes cell death (apoptosis). The gene or protein of the present invention phosphorylates (transcribes activation) Ser51 of c-Jun and eIF2a, and increases the folding capacity in the endoplasmic reticulum (UPR). That is, the biological activity of hERRj-1 is an activity that induces apoptosis by overexpression, a Ser51 phosphorylation activity of c-Jun and eIFα, and an activity that induces UPR.
[0016]
hRUPP-1, hRRCP-1, and hERRj-1 can be obtained by cloning according to the method of the following examples. However, those skilled in the art will prepare appropriate probes based on the sequence information and prepare appropriate probes. It can also be obtained by screening from a gene pool or by chemical synthesis based on sequence information.
[0017]
The protein of the present invention may have an additional structure or a sequence different from its natural sequence, that is, a deletion, substitution, or addition, as long as the function of the protein is not impaired. Methods for obtaining polypeptides in which part of the amino acid sequence has been deleted, substituted, or added are well known techniques (e.g., site-directed mutagenesis (Kramer, W. and Frits, H.J.,Meth.in Enzymol.,154, 350 (1987); Kunkel, T.A. et al.,Meth.in Enzymol.,154, 367 (1987))). In particular, conservative amino acid substitutions are predicted, which are substitutions made within a family of amino acids whose side chains are contiguous. Genetically encoded amino acids are generally divided into four families: (1) acidic = aspartic acid, glutamic acid; (2) basic = lysine, arginine, histidine; (3) nonpolar = alanine, valine, leucine. , Isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polarity = glycine, asparagine, glutamine, cystine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified collectively as aromatic amino acids. For example, isolated substitutions such as substitution of leucine with isoleucine or valine, substitution of aspartic acid with glutamic acid, substitution of threonine with serine, or similar conservative substitutions with structurally related amino acids are important for biological activity. Naturally, it is predicted that this will not have a significant impact. Polypeptide molecules having minor amino acid substitutions that have substantially the same amino acid sequence as a protein but do not substantially affect the functional aspects are included in the protein.
[0018]
Each of the nucleic acid sequences described in SEQ ID NOs: 1, 3, and 5 encodes the above hRUPP-1, hRRCP-1, and hERRj-1, but due to the degenerate nature of the genetic code, It will be appreciated that many different nucleic acid sequences encoding 2, 4, and 6 amino acid sequences can be constructed. The nucleic acid sequences set forth in SEQ ID NOs: 1, 3 and 5 are only one of many possible sequences encoding the protein.
[0019]
Further, based on the gene and amino acid sequence information of the present invention, using a well-known technique such as hybridization, a gene encoding a protein having a structure and function similar to those of the protein of the present invention derived from different organs and species, In addition, it is easy for those skilled in the art to obtain a protein encoded by the gene.
[0020]
It is known that a part of a protein may bear a part of a certain biological activity of the protein, and a protein fragment having such a desired activity when a full-length protein is obtained. Can be easily fragmented by chemical or enzymatic cleavage of the full-length protein, or produced by recombinant technology based on the sequence information of the full-length protein to obtain a fragment exhibiting the desired activity. It can be done. In “3. Protein expression in animal cells using recombinant expression system” in the Examples, a method for obtaining such a fragment of hRUPP-1 was exemplified. For hRCPP-1 and hERRj-1, fragments having the desired activity can be obtained in the same manner. Therefore, a fragment of the protein of the present invention and a polynucleotide encoding the fragment are also one aspect of the present invention.
[0021]
“Polynucleotide” as used herein refers to a polynucleotide comprising genomic, cDNA, DNA and RNA of semi-synthetic or synthetic origin, and refers to a polymeric form of nucleotides of any length. This term refers only to the primary structure of the molecule. Thus, this term includes double- and single-stranded DNA and antisense polynucleotides. This further includes known types of modifications such as the presence of labels known in the art, methylation, end cap structures, substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications (e.g. A certain type of uncharged bond (eg, methyl phosphonate, phosphotriester, phosphoamidate, carbamate, etc.) or substitution with a charged bond (eg, phosphothioate, phosphorodithioate, etc.)), Introduction of pendant moieties (eg, proteins (nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalating agents (eg, acridine, psoralen), chelators (eg, metals, radioactive substances, boron, oxidizing components, etc.), Alkylating agents (eg, alpha anomeric nucleic acids) are included.
[0022]
hRUPP-1, hRRCP-1, and hERRj-1 can be purified from biological samples by known methods such as chromatography based on their properties, but can also be produced by recombinant techniques. is there. Recombinant as used herein means that the protein is derived from prokaryotic and eukaryotic recombinant expression systems (eg, microorganisms, insects, plants, animals, preferably mammals or in vitro systems) This system includes any kind of cell transformation or in vitro expression system according to commonly known techniques.
[0023]
For recombinant expression, the polynucleotide of the present invention can be incorporated into a known vector such as a plasmid, cosmid, virus, bacteriophage and the like. If necessary, the polynucleotide can be inserted into a vector for the purpose of storage or the like. These vectors include, for example, Sambrook "Molecular Cloning-A Laboratory Manual" (Cold Spring Harbor Laboratory, NY) (1989) and Ausubel "Current Protocols in Molecular Biology" (Green Publishing Associates and Wiley Interscience, NY) ( (1989) (1994) can be easily constructed by those skilled in the art. In some cases, the polynucleotide of the present invention and a vector having the polynucleotide may be introduced into a liposome for delivery to a target cell.
[0024]
For expression of the polynucleotide in a cell, the polynucleotide of the present invention is preferably operably linked to a regulatory DNA sequence that allows expression. It is known that the type of control sequence to be used varies depending on the type of host cell to be selected, and those skilled in the art can easily obtain necessary promoters, terminators, transactivators, transcription factors, etc. according to the selected host cell. Factors can be selected. Further, if necessary, a gene encoding a detectable marker can be incorporated into the vector.
[0025]
When introducing a polynucleotide into a cell, it may be introduced so as to be retained outside the chromosome, or may be integrated into the host genome by homologous recombination or the like. In some cases, a plurality of polynucleotides of the present invention may be introduced into cells. The polynucleotides of the present invention can be used to transform or transfect hosts using techniques well known to those skilled in the art (Sambrook "Molecular Cloning: A Laboratory Manual" Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (See 1989). Thereafter, it is within the technical scope of those skilled in the art to set necessary culture conditions according to the type of host cell selected.
[0026]
For the purpose of detecting the protein of the present invention and the like, it is also possible for those skilled in the art to easily produce a polyclonal antibody or a monoclonal antibody against the protein based on well-known techniques (Kohler and Milstein,Nature 256: 495 (1975)). By the way, when a gene encoding an antigen protein is known, a method for analyzing hydrophobicity / hydrophilicity on the amino acid sequence of the protein (Kyte-Doolittle's method; J. Mol. Biol. 157 (1982) 105-122), a method for analyzing secondary structure (Chou-Fasman's method; Ana. Rev. Biochem. 47 (1978) pp. 251-276) etc. In addition, a computer program based on these methods (Anal. Biochem. Vol. 151 (1985), 540, p. 546) or a peptide as short as 6-9 is synthesized and its antigenicity is confirmed. The PEPSCAN method (Japanese Patent Publication No. 60-500684), which is a simple method, is a well-known technique, determines the antigenic determinant of the protein of the present invention, includes the peptide containing the portion, the antibody against the peptide, and the peptide Is known to those skilled in the art to obtain a polynucleotide encoding It is that can easily form.
[0027]
The antibody that recognizes the polynucleotide of the present invention also includes serum, monoclonal antibody, polyclonal antibody, synthetic antibody, Fab fragment, Fv fragment, and scFv fragment.
[0028]
The following examples describe the cloning method of each gene, the tissue distribution of each gene, and the expression of the protein in animal cells.
[0029]
【Example】
1. Cloning
(1) Generation of subtraction products
The AGPC (Acid Guanidinium-Phenol-Chloroform) method using human leukemia cells HL-60 (tester) treated with 0.1 ng / ml AIP for 72 hours and HL-60 (driver) without AIP treatment [total RNA preparation]Anal.Biochem. 162, 156-159]. The total RNA obtained above was treated at 65 ° C. for 5 minutes and quenched. Insoluble matter was removed by centrifugation, and 0.2 ml of 3M NaCl solution was added to adjust the salt concentration to 0.5M. The RNA solution was loaded on an oligo (dT) cellulose column (Pharmacia), and the flow-through fraction was loaded on the column again. Washing was performed with 10 mM Tri-HCl (pH 7.4), 1 mM EDTA, 0.5 M NaCl solution (several column volumes), and 10 mM Tri-HCl (pH 7.4), 1 mM EDTA, 0.1 mM NaCl solution (several column volumes). The adsorbed RNA having poly (A) was eluted with an elution buffer. The above operation was repeated again for the obtained RNA, and 1/10 volume of 3M sodium acetate (pH 5.1) and 2 volumes of ethanol were added and precipitated at −20 ° C. to obtain mRNA having poly (A). cDNA synthesis and subtraction were performed using 1.5 μg of each poly (A) RNA according to the manual attached to the PCR-Selected cDNA subtraction Kit (CLONTECH Laboratories, Inc.). The gene population that increases by AIP treatment (cDNA 2-cDNA 1) is attached to the tester cDNA (cDNA 2), and the gene population that decreases by AIP treatment (cDNA 1-cDNA 2) is attached to the driver cDNA (cDNA 1). Obtained. As a subtraction control, the cDNA attached to the kit was used according to the manual. Each of the subtraction products (cDNA 2-cDNA 1) and (cDNA 1-cDNA 2) obtained from the above was amplified by PCR and used for preparation of a subtraction library and a probe for cloning the library.
[0030]
(2) Subtraction library creation
After digestion with the restriction enzyme Eag I, the PCR amplification product of (cDNA 2-cDNA 1) is digested with the AIP treatment, and the PCR amplification product of (cDNA 1-cDNA 2) is digested with the AIP treatment. The product was purified using a QIAquick PCR purification Kit (QIAGEN). Make a mixed solution of 30 ng of cDNA and 20 ng of Not I digested pZErO-2 plasmid vector (Invitrogen) 7 ml with distilled water, 250 mM Tris-HCl (pH 7.6), 50 mM MgCl.₂, 5 mM ATP, 5 mM DTT, 2 μl of buffer solution consisting of 25% (w / v) PEG8000, 1 μl of T4 DNA ligase (1 unit / ml, Gibco BRL) was added and reacted at 16 ° C. for 4 hours to ligate the cDNA into the vector. Yeast tRNA (1mg / ml, Gibco BRL) 5μl, 7.5M NH_Four12.5 μl of OAc and 70 μl of cold ethanol were added, ethanol precipitated, and dissolved in 5 μl of distilled water. The vector cDNA was introduced into E. coli for electroporation (Electro MAX DH10B Cell, Gibco BRL) using the Biorat electroporation system (1.8 kV, 25 mF), and plated on a 15 cm LB / agar plate containing kanamycin. Incubated at 37 ° C for about 12 hours. Transfer a single colony from the above plate to a 96-well microplate containing LB / kanamycin medium and incubate at 37 ° C for about 3 hours, then transfer to a nylon filter (Amersham) on two LB / agar plates. The solution was lysed and fixed to obtain a screening filter.
[0031]
(3) Subtraction library screening
The PCR amplification products of the subtraction products (cDNA 2-cDNA 1) and (cDNA 1-cDNA 2) are digested with restriction enzymes Sma I and Eag I, and then purified using the QIAquick PCR purification kit (QIAGEN). Using a probe obtained by labeling with [α-32P] dCTP according to the instructions of a kit supplied by Pharmacia by the random prime method (Ready To Go DNA Labeling Kit), a normal colony hybridization method [Proc.Natl.Acad.Sci.USA 72: 3961,1975]. The clone obtained by this method is a fragment having a length of about 500 bp.
[0032]
(A) Gene population increased by AIP treatment
Using (cDNA 2-cDNA 1) and (cDNA 1-cDNA 2) as probes, hybridizing the same replica filter prepared from the (cDNA 2-cDNA1) library, and reacting only with (cDNA 2-cDNA 1) probes A clone was obtained (FIGS. 3 and 4). The circled clone in FIG. 3 has a cDNA fragment encoding hRUPP-1, and the circled clone in FIG. 4 has a cDNA fragment encoding hRRCP-1. FIG. 1 shows a flow chart of the cloning method by the subtraction method of a gene that increases when cell proliferation is inhibited by the AIP treatment of the present invention.
[0033]
(B) Gene population decreased by AIP treatment
Using (cDNA 2-cDNA 1) and (cDNA 1-cDNA 2) as probes, hybridizing the same replica filter prepared from (cDNA 1-cDNA 2) library, and (cDNA 1-cDNA 2) with probe only A reacting clone was obtained (FIG. 5). The circled clone in FIG. 5 has a cDNA fragment encoding hERRj-1. FIG. 2 shows a flow chart of the cloning method by the subtraction method of the gene that decreases when cell proliferation is inhibited by the AIP treatment of the present invention.
[0034]
A full-length cDNA was obtained by screening a human fetal brain cDNA library (Gibco BRL) using the fragment obtained by the subtraction method as a probe, and the entire base sequence was determined by the dye terminator method in which dideoxynucleotides were fluorescently labeled. The cDNA sequences of the genes encoding hRUPP-1 and hRRCP-1 that increase upon cell growth inhibition caused by the AIP treatment of the present invention are shown in SEQ ID NO: 1 and SEQ ID NO: 3, respectively, and the amino acid sequences of the proteins encoded by them Are shown in SEQ ID NO: 2 and SEQ ID NO: 4, respectively. In addition, the cDNA sequence encoding hERRj-1 that decreases upon cell growth inhibition caused by the AIP treatment of the present invention is shown in SEQ ID NO: 5, and the amino acid sequence of hERRj-1 is shown in SEQ ID NO: 6.
[0035]
2. Structural analysis
FIG. 6A shows a schematic diagram of the domain structure of hERRj-1 that decreases when cell proliferation is inhibited by the AIP treatment of the present invention, FIG. 6B shows a comparative diagram of the Dnaj-like domain, and FIG. 6C shows a comparative diagram of the thioredoxin-like domain. . As described above, hERRj-1 has a thioredoxin-like domain and a Dnaj domain, and thus has been found to be an endoplasmic reticulum chaperone. However, no molecule having such a domain structure has been reported so far.
[0036]
3. Tissue distribution
(1) Expression of hRUPP-1 in human tissues
Various human cell-derived cDNA libraries (Multiple Tissue cDNA Panel Human I and II) (CLONTECH Laboratories, Inc.) were amplified by PCR using the following hRUPP-1 gene-specific primers and analyzed for tissue expression. As a control, a G3PDH gene specific primer was used. The reaction was repeated 28 cycles of 95 ° C. for 20 seconds, 60 ° C. for 20 seconds, and 72 ° C. for 1 minute in the presence of Taq polymerase.

[0037]
FIG. 7 shows the result of the tissue distribution of the gene hRUPP-1, which increases when cell proliferation is inhibited by the AIP treatment of the present invention. The hRUPP-1 gene is expressed in human brain, heart, kidney, liver, lung, pancreas, placenta, muscle, large intestine, ovary, leukocyte, prostate, small intestine, spleen, testis and chest line, especially lung, pancreas High expression was observed in the large intestine, leukocytes, prostate, spleen, testis and thoracic line.
[0038]
(2) Expression of hRRCP-1 in human tissues
Using the following hRRCP-1 gene-specific primers, tissue expression of hRUPP-1 was analyzed in the same manner as in the case of using the above-described hRUPP-1 gene-specific primers. FIG. 8 shows the result of tissue distribution of the gene hRRCP-1, which increases when cell proliferation is inhibited by the AIP treatment of the present invention. hRRCP-1 gene is expressed in human brain, heart, kidney, liver, lung, pancreas, placenta, large intestine, ovary, leukocyte, prostate, small intestine, spleen, testis and thoracic line, especially lung, pancreas, prostate High expression was observed in the spleen, testis and thoracic line.

[0039]
(3) Expression of hERRj-1 in human tissues
Using the following hERRj-1 gene-specific primers, tissue expression of hERRj-1 was analyzed in the same manner as in the case of using the above-described hRUPP-1 gene-specific primers. FIG. 9 shows the result of tissue distribution of the gene hERRj-1 that increases when cell proliferation is inhibited by the AIP treatment of the present invention. hERRj-1 gene is expressed in human brain, heart, kidney, liver, lung, pancreas, placenta, large intestine, ovary, leukocyte, prostate, small intestine, spleen, testis and thoracic line, especially liver, lung, pancreas High expression was observed in prostate, spleen and testis.

[0040]
4). Protein expression using recombinant expression system
(1) Manufacture of hRUPP-1
In the BamHI / SmaI region of the expression vector pGEX-2T (Amersham Pharmacia Biotech) for E. coli containing the sequence encoding GST and the cDNA encoding the full length of hRUPP-1 (SEQ ID NO: 1) and pQE-30 (QUIAGEN) To obtain an expression plasmid. The resulting plasmid was transformed into E. coli M15 (pREP4) or XL1 Blue and grown in NZCYM medium at 37 ° C. OD of culture₆₀₀When the value reached about 0.5, isopropyl-β-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and the cells were further cultured for 5 hours, and then the cells were collected.
[0041]
Purification of the recombinant protein was performed according to the instructions of The QIAexpressionist (QIAGEN) and GST Gene Fusion System (Amersham Pharmacia Biotech). Next, in order to remove GST from the GST fusion protein obtained from the pGEX recombinant transcription unit, the prepared GST fusion protein was concentrated to about 2 mg / ml, and then against 20 mM Hepes-NaOH (pH 8.0), 150 mM NaCl. And dialyzed at 4 ° C. CaCl2 to a final concentration of 2 mM in the dialysate₂Was added and about 1/200 thrombin was added at a weight ratio to the GST fusion protein, and the mixture was incubated on ice for 5 hours. Thereafter, it was mixed with glutathione-Sepharose and benzamidine-Sepharose (Amersham Pharmacia Biotech). After mixing overnight at 4 ° C while rotating, the column is packed, and the flow-through fraction, 20 mM HEPES-NaOH (pH 8.0), and 150 mM NaCl wash fraction are collected to obtain the desired recombinant protein based on affinity. obtain.
[0042]
(2) Production of hRRCP-1
The cDNA encoding the full length of hRRCP-1 (SEQ ID NO: 3) was added to the BamHI / SmaI region of E. coli expression vector pGEX-2T (Amersham Pharmacia Biotech) containing the sequence encoding GST and pQE-30 (QUIAGEN). To obtain an expression plasmid. The resulting plasmid was transformed into E. coli M15 (pREP4) or XL1 Blue and grown in NZCYM medium at 37 ° C. OD of culture₆₀₀When the value reached about 0.5, isopropyl-β-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and the cells were further cultured for 5 hours, and then the cells were collected.
[0043]
Purification of the recombinant protein was performed according to the instructions of The QIAexpressionist (QIAGEN) and GST Gene Fusion System (Amersham Pharmacia Biotech). Next, in order to remove GST from the GST fusion protein obtained from the pGEX recombinant transcription unit, the prepared GST fusion protein was concentrated to about 2 mg / ml, and then against 20 mM Hepes-NaOH (pH 8.0), 150 mM NaCl. And dialyzed at 4 ° C. CaCl2 to a final concentration of 2 mM in the dialysate₂Was added and about 1/200 thrombin was added at a weight ratio to the GST fusion protein, and the mixture was incubated on ice for 5 hours. Thereafter, it was mixed with glutathione-Sepharose and benzamidine-Sepharose (Amersham Pharmacia Biotech). After mixing overnight at 4 ° C while rotating, the column is packed, and the flow-through fraction, 20 mM HEPES-NaOH (pH 8.0) and 150 mM NaCl washing fractions are collected, and the desired recombinant protein based on affinity is collected. Get.
[0044]
(3) Manufacture of hERRj-1
A cDNA encoding mature hERRj-1 (amino acids 35 to 793 of SEQ ID NO: 5), an expression vector pGEX-2T (Amersham Pharmacia Biotech) for E. coli containing a sequence encoding GST, and BamHI / SmaI of pQE-30 (QUIAGEN) Cloning into the region gave an expression plasmid. The resulting plasmid was transformed into E. coli M15 (pREP4) or XL1 Blue and grown in NZCYM medium at 37 ° C. OD of culture₆₀₀When the value reached about 0.5, isopropyl-β-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and the cells were further cultured for 5 hours, and then the cells were collected.
[0045]
Purification of the recombinant protein was performed according to the instructions of The QIAexpressionist (QIAGEN) and GST Gene Fusion System (Amersham Pharmacia Biotech). Next, in order to remove GST from the GST fusion protein obtained from the pGEX recombinant transcription unit, the prepared GST fusion protein was concentrated to about 2 mg / ml, and then against 20 mM Hepes-NaOH (pH 8.0), 150 mM NaCl. And dialyzed at 4 ° C. CaCl2 to a final concentration of 2 mM in the dialysate₂Was added and about 1/200 thrombin was added at a weight ratio to the GST fusion protein, and the mixture was incubated on ice for 5 hours. Thereafter, it was mixed with glutathione-Sepharose and benzamidine-Sepharose (Amersham Pharmacia Biotech). After mixing overnight at 4 ° C while rotating, the column is packed, and the flow-through fraction, 20 mM HEPES-NaOH (pH 8.0) and 150 mM NaCl washing fractions are collected, and the desired recombinant protein based on affinity is collected. Get.
[0046]
5. Functional analysis
(1) Expression of hRUPP-1 in cells
The peptide Asp-Tyr-Lys at the C-terminus of the full-length hRUPP-1 gene to provide an epitope that reversibly binds to a specific monoclonal antibody and allows detection and easy purification of the expressed protein -Asp-Asp-Asp-Asp-Lys (DYKDDDDK) is expressed as a C-terminal fusion protein consisting of nucleotides, incorporated into the animal cell expression vector pcDNA3.1 (CMV promoter) (Invitrogen) and recombined An expression vector was introduced into each animal cell as follows. A gene encoding a mutant protein was prepared by PCR, expressed as an epitope fusion protein, incorporated into an animal cell expression vector pCMV, and introduced into an animal cell as a recombinant expression vector.
[0047]
Changes in cell morphology
The hRUPP-1 gene and the lacZ gene were co-expressed in Hela and NIH3T3 cells, and stained with Xgal so that when hRUPP-1 was expressed, it turned blue. FIG. 10 shows the cell morphology when full-length hRUPP-1 gene is introduced into Hela and NIH3T3 cells. As a control, an empty vector having no hRUPP-1 gene was used. When the hRUPP-1 gene was introduced into NIH3T3 cells, the cells became rounded. However, when the hRUPP-1 gene was introduced into Hela cells, no change in cell morphology was observed. That is, hRUPP-1 has the activity of rounding cells (the activity of rounding cells is abbreviated as RU (Round-Up)), and its properties are cell-selective. In vivo, it is tissue-specific or time-specific. Expected to work.
[0048]
Functional domain analysis
In order to analyze the functional domain of hRUPP-1, a gene encoding a mutant protein was expressed as an epitope fusion protein, incorporated into an animal cell expression vector pCMV, and introduced into an animal cell as a recombinant expression vector. A gene encoding a mutant protein was prepared from the hRUPP-1 gene by PCR. A schematic diagram of the mutant protein is shown in FIG. When hRUPP-1 gene or mutant gene is introduced into CHO cells and Hela cells, the cell morphology is co-expressed with lacZ gene and stained with Xgal. Detection was performed as stained (FIG. 12). As a control, an empty vector having no hRUPP-1 gene was used. In FIG. 11, a represents a full-length hRUPP-1 consisting of 1148 amino acid residues, and b to g below it represent each fragment from which the full-length hRUPP-1 has been deleted. FIG. 12 shows that full lengths a, d, e, and f have weak RU in CHO cells, but mutants b, c, and g have strong RU, and in Hela cells, a, d, and e have no RU and mutant b. , C, f, and g indicate that cell growth inhibition with RU was observed.
[0049]
From the above, the domain consisting of amino acid residues 1 to 305 of SEQ ID NO: 2 or the domain consisting of 751 to 1148 has strong RU and cell growth inhibitory activity, and strong RU and cell growth using this domain Mutant hRUPP-1 having inhibitory activity can be produced. Mutant production is not limited to this domain, and it can also be produced by combining combinations between domains as in mutant g or shortening certain domains.
[0050]
hRUPP-1 Decomposition
The transcription factors Jun, Fos, Myc, p53 and NF-κB inhibitory factor I-κB, which are activated or induced in a signal-dependent manner, and many cyclin-related proteins are degraded under the control of the ubiquitin-proteasome. In order to verify the degradation of hRUPP-1 in NIH3T3 cells, the hRUPP-1 gene was expressed in NIH3T3 cells, treated with 10 μM MG132 for 10 hours, lysed, and subjected to SDS-PAGE. Then, it transferred to the PVDF membrane and immunostained each protein (FIG. 13). In FIG. 13, + represents gene introduction or MG132 treatment, and − represents introduction of an empty vector having no hRUPP-1 gene, or MG132 non-treatment.
[0051]
A protein in which hRUPP-1 was introduced into NIH3T3 cells was well degraded (FIG. 13, first lane), but treatment with the proteasome inhibitor MG132 suppressed the degradation (FIG. 13, second lane). As a control, the tumor suppressor protein p53 degraded by ubiquitin-proteasome was also inhibited by treatment with MG132, a proteasome inhibitor (FIGS. 13 1-4).
[0052]
hRUPP-1 Polyubiquitination
Many of the proteins that are degraded depending on signal transduction are polyubiquitinated prior to degradation and are known to be rapidly degraded by the proteasome proteasome, so that hRUPP-1 is polyubiquitinated in the cell. In order to confirm this, the hRUPP-1 gene and the ubiquitin gene were coexpressed in 293 cells and treated with 10 μM MG132 for 6 hours or not. Thereafter, the cells were lysed and hRUPP-1 was specifically immunoprecipitated with an epitope antibody, subjected to SDS-PAGE, transferred to a PVDF membrane, and immunostained with an antibody against the introduced ubiquitin, and hRUPP-1 Ubiquitination was examined (FIG. 14A). Further, after cell lysis, the lysate was directly subjected to SDS-PAGE, transferred to a PVDF membrane, and then directly stained with hRUPP-1 (FIG. 14B). + Represents introduction of hRUPP-1 gene or treatment with MG132, − represents introduction of an empty vector having no hRUPP-1 gene, or no treatment with MG132.
[0053]
As a result, it was shown that hRUPP-1 is polyubiquitinated in the cells (FIG. 14, lanes 4 and 8). In addition, hRUPP-1 was polyubiquitinated in 293 cells, but no significant degradation was observed, and no significant increase was observed by treatment with the proteosome inhibitor MG132 (FIG. 14, lane 8). From the above, it was found that hRUPP-1 is polyubiquitinated in the cell and undergoes degradation by the proteasome. In addition, other signal-dependent activation or expression-induced transcription factors Jun, Fos, Myc, p53 and NF-κB inhibitory factor I-κB, and many cyclin-related proteins, hRUPP-1 also signals. It has been found that it is subject to dependent or cell (tissue) selective degradation control.
[0054]
(2) Expression of hRRCP-1
Peptide Asp-Tyr-Lys at the C-terminus of the full-length hRRCP-1 gene to provide an epitope that reversibly binds to a specific monoclonal antibody and allows detection and easy purification of the expressed protein -Asp-Asp-Asp-Asp-Lys (DYKDDDDK) or Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu (EQKLISEEDL) C-terminal fusion protein consisting of nucleotides The animal cell expression vector pME18S (SRα promoter) (Hara, T. and Miyama, A.,EMBO J. 11, 1875-1884, 1992) and introduced into each animal cell as a recombinant expression vector as follows. A gene encoding a mutant protein was prepared by PCR, expressed as an epitope fusion protein, incorporated into an animal cell expression vector pME18S, and introduced into an animal cell as a recombinant expression vector.
[0055]
result
hRRCP-1 and the previously reported Mayven [Soltysik-Espanola, M. et al.,Mol.Biol.Cell Ten, 2361-2375,1999], Keap 1 [Itoh, K. et al.,Gene and Dev. 13, 76-86,1999] were each expressed in Hela cells, and each protein was stained with a fluorescently labeled antibody against the peptide added to the C-terminus of the hRRCP-1 gene (green indicates the localization of each protein) , FIG. 15). hRRCP-1 is derived from Drosophila kelch [Xue, F. et al.,Cell 72, 681-693, 1993] formed a ring-canal structure that was not observed in other mammalian kelch factor groups, Mayven and Keap 1. This strongly suggests that hRRCP-1 is a true mammalian homologue of Drosophila kelch, and was predicted to be deeply involved in cytoplasmic transport and actin filament construction like Drosophila kelch.
[0056]
hRRCP-1 of Ring-canal Identification of internal domains involved in structure formation
In order to identify the internal domain involved in Ring-canal structure formation of hRRCP-1, a gene encoding a mutant protein was prepared by PCR, introduced into Hela cells to be expressed as an epitope fusion protein, Localization was examined (Figure 16). A schematic diagram of the mutant protein used is shown in FIG. Only a mutant protein consisting of a POZ domain and an IVR domain forms a Ring-canal structure, and these two domains were found to be indispensable domains for Ring-canal structure formation. The POZ and IVR domains alone are the whole cell, the kelch domain is speckled in the nucleus, the mutant protein consisting of the POZ domain and the kelch domain is mainly in the cytoplasm, and the mutant protein consisting of the IVR domain and the kelch domain is in the cytoplasm or nucleus. Localization was demonstrated. Based on the above, it was predicted that hRRCP-1 forms a Ring-canal structure via the POZ domain and the IVR domain, and is involved in cytoplasmic transport and actin filament construction. The use of each mutant protein not only provides information on localization, but also serves as a molecular design tool for a ring-canal structure like a mutant protein consisting of a POZ domain and an IVR domain. Each mutant protein can also be used as a positive or negative regulatory molecule of hRRCP-1.
[0057]
hRRCP-1 Binding to actin
In order for hRRCP-1 to function as a mammalian homolog of true Drosophila kelch, it is assumed that hRRCP-1 binds to actin. To prove this, hRRCP-1, Mayven and Keap 1 were expressed in each 293 cell, the cells were lysed, and hRRCP-1, Mayven and Keap 1 were each immunoprecipitated and subjected to SDS-PAGE. And transferred to a PVDF membrane. Each immunoprecipitate fraction was immunostained with an antibody against protein (upper part of FIG. 18) and an antibody against actin (lower part of FIG. 18), and the binding ability with actin was examined. An empty vector having no hRUPP-1 gene was used as a control (pME-Empty). hRRCP-1 was found to be able to bind to actin and strongly suggested that it was involved in cytoplasmic transport and actin filament construction like Drosophila kelch.
[0058]
hRRCP-1 by Mayven And Keap Function adjustment
Experiments showed that not only hRRCP-1 but also Mayven and Keap 1 have actin binding ability (bottom of FIG. 18). This means that hRRCP-1 can regulate the functions of Mayven and Keap through binding to actin or binding to Mayven and Keap, and the following experiment was conducted to prove it. Co-introducing hRRCP-1 / Myc epitope and Mayven / Flag epitope, hRRCP-1 / Myc epitope and Keap 1 / Flag epitope, and Keap 1 / Flag epitope and Mayven / Myc epitope gene respectively into 293 cells, After lysis, each cell lysate was immunoprecipitated with anti-Myc epitope antibody, and the immunoprecipitate was subjected to SDS-PAGE. Thereafter, the cells were transferred to a PVDF membrane and immunostained using an anti-Flag antibody (FIG. 19A) or an anti-Myc antibody (FIG. 19B). In the figure, + represents the introduction of each gene, and-represents the introduction of an empty vector having no gene. The results of immunoprecipitation in FIG. 19 indicate that hRRCP-1 can bind to Mayven but cannot bind to Keap1.
[0059]
Furthermore, co-introduction of hRRCP-1 / Myc epitope and Mayven / Flag epitope, hRRCP-1 / Myc epitope and Keap 1 / Flag epitope, and Keap 1 / Flag epitope and Mayven / Myc epitope respectively into Hela cells, The cells were immunostained with an anti-Myc epitope antibody (red) and an anti-Flag epitope antibody (green) (FIG. 20). As a result, it was found that hRRCP-1 not only completely destroyed the localization of Mayven but also incorporated Mayven. Based on the above, through binding to hRRCP-1 actin or Mayven, it is not only involved in cytoplasmic transport such as Drosophila kelch and actin filament construction, but also functions as a negative or positive regulator of Mayven function. It was suggested.
[0060]
hRRCP-1 Involvement in apoptosis
hRRCP-1 has been cloned as a gene that is induced to express when cell growth is inhibited by AIP treatment. Inhibition of cell proliferation also serves as a signal for inducing apoptosis of cells. In order to examine the involvement of hRRCP-1 in apoptosis, hRRCP-1 was introduced into cells 293 / Fas cells that constitutively express Fas molecules and examined whether they were selectively degraded by anti-Fas antibody stimulation.
[0061]
HRRCP-1 was introduced into 293 / Fas cells, stimulated with anti-Fas antibody for a certain period of time, and then lysed. The lysate was transferred to a PVDF membrane after SDS-PAGE and immunostained with hRRCP-1 (FIG. 21A) and caspase 3 (FIG. 21B). As a result, it was found that hRRCP-1 was specifically degraded by anti-Fas antibody stimulation. As a control, we also observed activation of the apoptotic molecule caspase 3 (proteolytic enzyme involved in apoptosis induction). It is thought that the apoptosis-inducing signal by Fas is transferred to the protease (caspase) cascade and leads to cell death through selective degradation of the caspase substrate. Various candidates have been reported for caspase substrates involved in apoptosis, but the final substrate is still unknown. Therefore, specific degradation of hRRCP-1 by Fas signal (including Fas ligand and Fas antibody) provides a new field of apoptosis research, suggesting that hRRCP-1 may be involved in autoimmune diseases and cancer . In addition, specific degradation of hRRCP-1 can be used as a marker for apoptosis.
[0062]
(3) Expression of hERRj-1 protein
In order to provide an epitope that reversibly binds to a specific monoclonal antibody and allows the detection and easy purification of the expressed protein, the peptide Asp-Tyr-Lys- It is expressed as an internal fusion protein consisting of nucleotides encoding Asp-Asp-Asp-Asp-Lys (DYKDDDDK), and is integrated into an animal cell expression vector pME18S (SRα promoter) or pCMV (CMV promoter), and a recombinant expression vector And introduced into animal cells. A gene encoding a mutant protein was prepared by PCR, expressed as an epitope fusion protein, incorporated into an animal cell expression vector pME18S or pCMV, and introduced into an animal cell as a recombinant expression vector.
[0063]
hERRj-1 Localization of
FIG. 22 shows a photograph of cells expressing hERRj-1 in Hela cells and showing co-localization with Bip, an endoplasmic reticulum marker protein. Consistent with the results of the domain analysis shown in FIG. 6, it was proved that hERRj-1 exists in the endoplasmic reticulum.
[0064]
hERRj-1 Changes in cell morphology
Co-express hERRj-1 gene and lacZ gene in Hela cells and CHO cells, stain with Xgal, turn blue when hERRj-1 is expressed, and transfer hERRj-1 gene to Hela and CHO cells The cell morphology when introduced is shown in FIG. An empty vector without hERRj-1 gene was used as a control. It was found that introduction of hERRj-1 into cells induces cell death. Endoplasmic reticulum stress is known to cause cell death (apoptosis). Therefore, hERRj-1 is predicted to be one of the causative molecules of cell death due to endoplasmic reticulum stress. It is known that two kinase molecules IRE 1 and PERK present on the endoplasmic reticulum membrane are involved in the response mechanism to endoplasmic reticulum stress. IRE 1 induces the endoplasmic reticulum molecular chaperone via UPR and prevents the accumulation of abnormal proteins. IRE 1 has also been identified as an essential molecule for the activation of JNK (c-Jun N-terminal kinase) accompanying the addition of endoplasmic reticulum stress. PERK phosphorylates Ser51 of eIF2α and suppresses translation from mRNA to protein, preventing abnormal proteins from accumulating in the endoplasmic reticulum. These kinases are activated depending on endoplasmic reticulum stress and are thought to function as a defense mechanism against endoplasmic reticulum stress. On the other hand, if the stimulus that causes endoplasmic reticulum stress is excessive, persists for a long time, or if resistance to endoplasmic reticulum stress is weakened for some reason, cells undergo apoptotic changes and die, but familial Alzheimer Missense mutations in the disease-causing gene presenilin are thought to be responsible for these neuronal cell deaths through ER stress. Thus, hERRj-1 is expected to induce excessive ER stress via IRE 1 or PERK, or attenuate resistance to ER stress.
[0065]
hERRj-1 by IRE 1 Or PERK Activation
In order to prove that hERRj-1 can induce IRE 1 or PERK activation, after introducing hERRj-1 into 293 cells, treatment with drugs that cause endoplasmic reticulum stress (tunicamycin, DTT, thapsgargin) and GST- Immunoprecipitation was performed using cJun and the immunoprecipitate was subjected to in vitro kinase analysis. The in vitro kinase analysis product was subjected to SDS-PAGE, transferred to a PVDF membrane, and then immunostained with an antibody that specifically recognizes c-Jun phosphorylated Ser63 (FIG. 24 (A) top) and control The same cell lysate was immunostained using JNK (c-Jun N-terminal kinase) (FIG. 24 (A) bottom), and the activity of endogenous JNK and phosphorylation of eIF2α were examined. In addition, the hERRj-1 gene was introduced into 293 cells, treated with an agent that causes endoplasmic reticulum stress, and the cells were lysed.The lysate was directly subjected to SDS-PAGE, transferred to a PVDF membrane, and then eIF2α or eIF2α. Immunostaining was performed using an antibody that specifically recognizes phosphorylated Ser51 (FIG. 24B). In the figure,-represents the introduction of an empty vector having no hERRj-1 gene or no drug treatment. Tm represents treatment for 3 hours with 2.5 μg / ml tunicamycin, DTT represents treatment for 3 hours with 5 mM dithiothreitol, and Tg represents treatment for 1 hour with 1 μM thapsigargin.
[0066]
As a result, the introduction of hERRj-1 into cells revealed that Ser51 phosphorylation of c-Jun and eIF2α against endoplasmic reticulum stress was enhanced, and hERRj-1 induced activation of IRE 1 and PERK I understood that. From the above results, it was suggested that hERRj-1 excessively or continuously induces activation of IRE 1 and PERK to cause cell death.
[0067]
hERRj-1 by UPR Guidance
That hERRj-1 activates IRE 1 means that hERRj-1 can induce UPR. To prove this, hERRj-1 was introduced into 293 cells or CHO cells, and the expression of genes Bip and CHOP induced by UPR was examined by RT-PCR. First, total RNA was extracted using total RNA Isolation Reagent (GIBCO BRL) from cells treated with 5μg / ml tunicamycin for 6 hours after introduction of hERRj-1 or Bip gene into 293 and CHO cells. did. From 5 μg of each total RNA, cDNA was prepared using ProSTAR First-Strand RT-PCR Kit (STRATAGENE), and endogenous hERRj-1, Bip and CHOP genes were detected by PCR using gene-specific primers. The reaction was repeated for 28 cycles of 96 degrees 25 seconds, 60 degrees 20 seconds, 72 degrees 1 minute in the presence of Taq polymerase.
[0068]
The results are shown in FIG. It was also found that hERRj-1 is induced by tunicamycin treatment that causes endoplasmic reticulum stress, and that introduction of hERRj-1 into cells causes the expression of genes Bip and CHOP induced by UPR. These results indicate that hERRj-1 is a novel endoplasmic reticulum molecular chaperone that is induced by endoplasmic reticulum stress and induces activation of IRE 1 and PERK and UPR.
[0069]
hERRj-1 of IRE 1alpha , IRE 1beta as well as Bip Combined with
It was shown that hERRj-1 activates UPR. Bip has been reported to bind to IRE 1 or PERK and inhibit the activity of IRE 1 or PERK (Bertolotti, A. et al., Nature Cell Biol. 2, 326-332, 2000). In order to examine the possibility that the activity of UPR by hERRj-1 occurs through the binding of hERRj-1 and IRE 1 or Bip, hERRj-1 and IRE 1 are co-expressed in CHO cells, After immunoprecipitation, the immunoprecipitate was subjected to SDS-PAGE, transferred to a PVDF membrane, and then immunostained with an antibody against IRE 1 or Bip to examine the binding ability to hERRj-1. The result is shown in FIG. hERRj-1 binds to IRE 1 or endogenous Bip, and endoplasmic reticulum stress was found to enhance the binding. From the above, it was found that hERRj-1 regulates UPR through binding to IRE 1 or Bip.
[0070]
hERRj-1 Of cell death-inducing domains in rice
CHO cells were coexpressed with each mutant gene of hERRj-1 or hERRj-1 together with the lacZ gene, and stained with Xgal so that the cells expressing hERRj-1 turned blue. Cell death-inducing activity was examined using changes in cell morphology due to cell death as an index, and the results are shown in FIG. An empty vector without the hERRj-1 gene was used as a control (live cells 100%). As shown in FIG. 6, hERRj-1 has a Dnaj domain and four thioredoxin (TRX1 to 4) domains. In hERRj-1, 25% of living cells, a mutant in which the 63rd amino acid His of the Dnaj domain was changed to Gln (Dnaj mutant) were 52% of living cells, and the 158th and 161st Cys of the TRX1 domain were replaced with Ser. Mutant (TRX 1 mutant) is 60% of live cells, TRX2 domain active site 480th and 483th Cys is replaced by Ser (TRX 2 mutant) is 62% live cell, TRX3 domain active site 588 Mutants with the 5th and 591st Cys replaced with Ser (TRX 3 mutant) are 55% of live cells, and those with the 700th and 703th Cys of the TRX4 domain replaced with Ser are live cells (TRX 4 mutant). 90%. Furthermore, mutants in which Cys in all active sites of TRX1 to 4 are changed to Ser (TRX-all mutants) are 95% of living cells, and all mutants in the domain from Dnaj and TRX1 to 4 (Dnaj / TRX mutants) are It showed 100% live cell activity. Based on the above, it was predicted that hERRj-1 activity was mainly induced by the TRX domain, and that the Dnaj domain functions as a regulatory domain.
[0071]
hERRj-1 In the cell by IRE 1 Change of localization
IRE 1 senses endoplasmic reticulum stress by exposing the N-terminus to the lumen side of the endoplasmic reticulum and functions as an endoplasmic reticulum membrane protein with the functional domains of the kinase domain and endonuclease domain at the C-terminus exposed to the cytoplasm It is believed that However, the activity of JNK requires a kinase domain (Urano, F. el al., Science 287, 664-666, 2000) and the UPR requires only an endonuclease domain (Tirasophon, W. et al. , Genes Dev. 14, 2725-2736, 2000), but details such as the coordination of both domains regarding the response to ER stress by IRE 1 are unknown. NIH3T3 cells were allowed to express hERRj-1 or IRE 1 alone or co-expressed, and the localization of IRE 1 was examined. The results are shown in FIG. IRE 1 alone showed the localization of the endoplasmic reticulum when IRE 1 was used alone, but when hERRj-1 was co-expressed, the localization of IRE 1 changed greatly and appeared as an aggregate. However, the hERRj-1 Dnaj or TRX mutants did not induce the formation of IRE 1 aggregates, indicating that the function of hERRj-1 is essential for the formation of IRE 1 aggregates. Furthermore, since IRE 1 kinase domain mutants or endonuclease domain mutants do not form aggregates, ERR 1 aggregate formation by hERRj-1 requires both the IRE 1 kinase domain and the endonuclease domain. It became clear. Based on the above, we newly identified the induction of IRE 1 aggregate formation by hERRj-1 and suggested that hERRj-1 is deeply involved in the function of IRE 1.
[0072]
In mouse organs mERRj-1 ( hERRj-1 Of mouse homologues)
Based on the above, hERRj-1 overexpression induces cell death and regulates endoplasmic reticulum function through binding to IRE 1 or Bip or through the formation of IRE 1 aggregates. found. In order to investigate the role of hERRj-1 in the body, a rabbit polyclonal antibody against the internal sequence (synthetic peptide) of hERRj-1 was prepared, and tissue staining was performed using fixed sections of the pancreas, brain and testis. The results are shown in FIG. Pancreatic islets of Langerhans were selectively stained for specific cells in the brain, brain and seminiferous tubules. Pancreatic beta cells are insulin-producing cells with highly developed endoplasmic reticulum, and abnormal functioning of the endoplasmic reticulum due to PERK abnormalities causes pancreatic abnormalities with death of beta cells and diabetes (Delepine, M. et al., Nat Genet. 25, 406-409, 2000; Harding, HP et al., Mol. Cell 7, 1153-1163, 2001). In addition, neuronal cell death caused by dysfunction of the endoplasmic reticulum such as decreased expression of Bip is considered to be one of the causes of Alzheimer's disease (Katayama, T. et. Al., Nature Cell Biol. 1, 479-485, 1999). ). Diabetes and Alzheimer's disease are also amyloidosis caused by accumulation of denatured proteins. Based on the above, hERRj-1 is necessary for maintaining the functions of pancreatic beta cells, neurons and sperm cells, and abnormal expression of these cells may cause abnormal pancreas, neurodegenerative diseases or abnormal differentiation of sperm cells. It was suggested.
[0073]
EST By search hERRj-1 as well as mERRj-1 Expression pattern
The EST database is a database that collects partial sequences of cDNA randomly selected from an arbitrary cDNA library. By searching the database, the expression frequency of a certain gene can be predicted. . As a means for exploring the role of hERRj-1 in the body, the expression pattern of hERRj-1 and mERRj-1 by EST search was examined, and the results are shown in FIG. 30A. In 132 human cDNAs, 68 (51%), 30 out of 88 mice (35%) are derived from cancer tissue, and 53 human (78%) in cancer tissue, Thirty mice (100%) are derived from adenocarcinoma. In addition, 23 (43%) of the adenocarcinomas were from uterine adenocarcinoma and 25 (83%) of the mice were from mammary adenocarcinoma. This result indicates that ERRj-1 is very highly expressed in human or mouse adenocarcinoma, especially uterine adenocarcinoma and breast adenocarcinoma. The gland is a secretory organ with a very developed endoplasmic reticulum, suggesting that dysfunction of the endoplasmic reticulum due to abnormal expression of ERRj-1 in gland cells may be one of the causes of adenocarcinoma. Breast cancer and uterine adenocarcinoma often occur due to secretion of hormones, particularly estrogen, and abnormal responses. As with ERRj-1, the endoplasmic reticulum chaperone PDI, which has two TRX domains, is predicted to bind to estrogen and regulate intracellular estrogen levels (Primm, T and Gilbert, H., JBC, 276 , 281-286, 2001). Therefore, it is possible that ERRj-1 regulates the concentration of hormone in cells by regulating the binding of hormone or the function of PDI.
[0074]
Breast cancer and uterine adenocarcinomas tend to metastasize to each other, and abnormal expression of ERRj-1 in breast and uterine adenocarcinoma may be involved in cancer metastasis. When cancer cells are successfully presented with major histocompatibility antigen (MHC) class I, the cancer cells are eliminated by cytotoxic T lymphocytes (CTLs), but immunoglobulin heavy chains and beta2 microglobulin. If the cancer antigen presentation is not performed normally due to MHC class I folding abnormality consisting of the dimer of the above, or degradation associated therewith, escape from the attack from CTL and metastasis of cancer cells is established. In order to prove the possibility that hERRj-1 is involved in cancer antigen presentation by MHC class I, it is an essential molecule for MHC class I folding, and the redox state change by hERRj-1 of endoplasmic reticulum chaperone ERp57 with TRX domain (FIG. 30B) and the expression level of immunoglobulin H chain (mu chain) by hERRj-1 (FIG. 30C) were examined. The alkylating reagent AMS reacts with the reduced Cys of the protein, resulting in an increase in molecular weight of 500 Daltons. Therefore, it becomes possible to estimate the redox state of the protein with reference to the increase in the molecular weight of the protein after the AMS reaction. As shown in FIG. 30B, it can be seen that ERp57 in cells exists in a mixed state of a fully oxidized form, a partially oxidized form, and a fully reduced form. However, in the presence of hERRj-1, it was found that the fully oxidized and partially oxidized forms of ERp57 were converted to fully reduced forms. Furthermore, as shown in FIG. 30C, it was also found that hERRj-1 can bind to an immunoglobulin H chain (mu chain) and reduce its expression level. ERp57 binds to MHC class I immunoglobulin heavy chains and contributes to antigen presentation by MHC class I by preventing the degradation of immunoglobulin heavy chains. Based on the above, it is found that hERRj-1 is involved in antigen presentation by MHC class I through direct binding to immunoglobulin H chain or by regulating the function of ERp57, and abnormal expression of hERRj-1 is caused by MHC It was suggested that cancer cell cancer antigens may decrease and may be deeply involved in cancer metastasis.
[0075]
【The invention's effect】
The hRUPP-1 of the present invention has a cell rounding (RU) activity, a cell growth inhibitory activity, and a cell death inducing activity. The phenomenon of cell rounding is often seen during cell division, adhesion inhibition, or cell migration, and is expected to function in these phenomena. By introducing full-length or mutant hRUPP-1 into cells, cell death can be induced by imparting RU activity and growth inhibitory activity to the cells. It is considered that cell motility can be controlled by controlling the RU activity of hRUPP-1 in the cell. For example, administration of an antisense gene for hRUPP-1 can metastasize from the primary lesion of cancer cells. It is thought that invasion of cancer cells can be suppressed.
[0076]
It is also possible to screen for a compound that inhibits or activates the activity of the protein by using a screening system containing hRUPP-1, a gene encoding hRUPP-1, and an antisense polynucleotide thereto. . Such compounds are believed to be useful as agents that modulate cell proliferation, metastasis and division, and cell death.
[0077]
In addition, hRUPP-1 is polyubiquitinated and degraded as shown in this specification, but by modifying hRUPP-1 so that it is not polyubiquitinated, it regulates degradation of hRUPP-1 in vivo Will be possible.
[0078]
Since hRRCP-1 of the present invention is specifically decomposed by Fas signal, it can be used as a marker for apoptosis (cell death) in place of conventional caspases and the like. In addition, it is possible to provide basic knowledge about physiological phenomena and diseases through the ring-canal structure of hRRCP-1 that has not been conventionally known.
[0079]
hERRj-1 is a novel endoplasmic reticulum chaperone with a thioredoxin domain and a Dnaj domain. Expression of the gene or protein of the present invention in cells causes cell death (apoptosis). HERRj-1 of the present invention phosphorylates (transcribes activation) Ser51 of c-Jun and eIF2a. The gene or protein of the present invention induces UPR. HERRj-1 of the present invention binds to IRE 1, Bip and immunoglobulin. HERRj-1 of the present invention induces the formation of aggregates by IRE 1. HERRj-1 of the present invention reduces ERp57. The hERRj-1 of the present invention regulates immunoglobulin expression. The hERRj-1 of the present invention is essential for maintaining the function of endoplasmic reticulum such as beta cells, brain and sperm cells of the islet of Langerhans in the pancreas. The hERRj-1 of the present invention is highly expressed in adenocarcinoma, particularly uterine adenocarcinoma and breast adenocarcinoma. The finding that the gene or protein of the present invention induces UPR means that the gene or protein of the present invention can be used as a defense molecule against endoplasmic reticulum stress.
[0080]
Moreover, since hERRj-1 has an activity to induce cell death, it may be possible to induce cell death in tumor cells and the like by administering hERRj-1 in the same manner as hRUPP-1. Furthermore, it is also possible to screen for compounds that inhibit or activate the activity of the protein by using a screening system comprising hERRj-1, a gene encoding hERRj-1, and an antisense polynucleotide thereto. . Such compounds are believed to be useful as agents that modulate cell death.
[0081]
JNK activation is essential for stress-induced apoptosis, particularly neuronal cell death. It may be possible to control cell death by controlling c-Jun phosphorylation activity (activation of JNK) of hERRj-1. For example, by administering an antisense gene for hERRj-1, cell death caused by ER stress can be suppressed, and it can be used to treat Alzheimer's disease and develop therapeutics caused by neuronal cell death caused by ER stress. It is expected.
[0082]
Mutants that do not cause eIF2α phosphorylation cause hypoglycemia, and eIF2α phosphorylation is essential for blood glucose level regulation. Therefore, administration of the hERRj-1 gene can be used for the treatment of hypoglycemia and the development of therapeutic agents. Expected to be
[0083]
Since hERRj-1 can bind to IRE 1 and is predicted to be a molecule that regulates the function of IRE 1 through the formation of IRE 1 aggregates, etc., by using hERRj-1, IRE 1 such as UPR It is expected that it can be used for the induction of B cell differentiation and the treatment of neurodegenerative diseases.
[0084]
Since hERRj-1 is highly expressed in the beta cells of pancreatic islets of Langerhans and is predicted to be essential for maintaining its function, hERRj-1 can be used to treat drugs for the treatment of diabetes and diabetes. Can be used for development.
[0085]
Mutations in PS1 that cause familial Alzheimer's disease (FAD) cause a decrease in transcriptional induction of the endoplasmic reticulum molecule chaperone Bip, and inhibition of Bip transcriptional induction is one of the causes of neuronal cell death. Since hERRj-1 can activate Bip transcription induction and can be expected to regulate Bip function through binding to Bip, administration of hERRj-1 gene or antisense gene can be used to treat these diseases. It is expected to be usable for the development of therapeutic drugs.
[0086]
Abnormal expression of ERRj-1 in glandular cells is suggested to be one of the causes of adenocarcinoma, especially uterine adenocarcinoma and breast adenocarcinoma. The development of drugs for treatment can be expected.
[0087]
Since hERRj-1 is predicted to be involved in antigen presentation by MHC class I through direct binding to the immunoglobulin heavy chain or by regulating the function of ERp57, the use of hERRj-1 enables cancer It can be expected that it will be cured, and it will be possible to prevent cancer metastasis.
[0088]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows the flow of a cloning method using a subtraction method for genes that increase when cell proliferation is inhibited by AIP treatment.
FIG. 2 shows the flow of a cloning method using a subtraction method for genes that decrease when cell growth is inhibited by AIP treatment.
FIG. 3 is a clone having a cDNA fragment of hRUPP-1 that reacts only with the (cDNA 2-cDNA 1) probe by screening the subtraction library.
[FIG. 4] A clone having a cDNA fragment of hRRCP-1 that reacts only with a (cDNA 2-cDNA 1) probe by screening a subtraction library.
FIG. 5: A clone having a cDNA fragment of hERRj-1 that reacts only with the (cDNA 2-cDNA 1) probe by screening the subtraction library.
FIG. 6 shows a comparison of the domain structure of hERR-1 and each domain sequence.
FIG. 7: HRUPP-1 tissue distribution.
FIG. 8 shows tissue distribution of hRRCP-1.
[Figure 9] HERRj-1 tissue distribution.
FIG. 10 shows cell morphology when full-length hRUPP-1 gene is introduced into Hela and NIH3T3 cells.
FIG. 11 is a schematic diagram of a mutant protein prepared for functional domain analysis of the hRUPP-1 gene.
FIG. 12 shows cell morphology when the mutant protein shown in FIG. 11 is introduced into CHO cells and Hela cells.
FIG. 13: hRUPP-1 and p53 degradation in NIH3T3 cells.
FIG. 14 shows confirmation of polyubiquitination of hRUPP-1 in cells by co-expression of hRUPP-1 gene and ubiquitin gene in 293 cells.
FIG. 15 shows localization of hRRCP-1, Mayven, and Keap 1 in Hela cells.
[Fig. 16] In order to identify the internal domain of hRRCP-1 involved in ring-canal structure formation, a gene encoding a mutant protein is prepared by PCR and introduced into Hela cells so as to be expressed as an epitope fusion protein. Then, the localization of each was examined.
FIG. 17 is a schematic diagram of hRRCP-1 mutant protein.
FIG. 18: After expressing hRRCP-1, Mayven and Keap 1 in each 293 cell, the cells were lysed, hRRCP-1, Mayven and Keap 1 were each immunoprecipitated, subjected to SDS-PAGE, PVDF Transferred to the membrane. Each immunoprecipitate fraction was immunostained with an antibody against the protein (upper part of FIG. 17) and an antibody against actin (lower part of FIG. 17), and the binding ability with actin was examined.
FIG. 19 Co-introduction of hRRCP-1 / Myc epitope and Mayven / Flag epitope, hRRCP-1 / Myc epitope and Keap 1 / Flag epitope, and Keap 1 / Flag epitope and Mayven / Myc epitope gene into 293 cells, respectively. After cell lysis, each cell lysate was immunoprecipitated with an anti-Myc epitope antibody, and the immunoprecipitate was subjected to SDS-PAGE. Thereafter, the cells were transferred to a PVDF membrane, and immunostained with an anti-Flag antibody (A) or with an anti-Myc antibody (B).
FIG. 20: Co-introduction of hRRCP-1 / Myc epitope and Mayven / Flag epitope, hRRCP-1 / Myc epitope and Keap 1 / Flag epitope, and Keap 1 / Flag epitope and Mayven / Myc epitope into Hela cells, respectively. Each cell was immunostained with anti-Myc epitope antibody (red) and anti-Flag epitope antibody (green).
FIG. 21: Introducing hRRCP-1 into 293 / Fas cells, stimulating with anti-Fas antibody for a certain period of time, lysing the cells, transferring to PVDF membrane after SDS-PAGE, hRRCp-1 (A) and caspase3 Immunostaining was performed with (B).
FIG. 22 is a photograph of cells expressing hERRj-1 in Hela cells and showing co-localization with Bip, an endoplasmic reticulum marker protein.
FIG. 23 shows cell morphology when hERRj-1 gene is introduced into Hela and CHO cells.
FIG. 24. Activation of IRE 1 or PERK of hERRj-1.
FIG. 25 shows the expression of genes Bip and CHOP induced by UPR when hERRj-1 is introduced into 293 cells or CHO cells.
FIG. 26: CHO cells were co-introduced with IRE1-alpha / T7 epitope, IRE1-beta / Myc epitope or empty vector and hERRj-1 / Flag epitope gene, respectively, Tm (2.5 μg / ml tunicamycin), DTT Cells were treated with Tg (1 μM thapsigargin) (with 5 mM dithiothreitol). After cell lysis, each cell lysate was immunoprecipitated with anti-Flag epitope antibody and the immunoprecipitate was subjected to SDS-PAGE. After that, it is transferred to a PVDF membrane, using anti-T7 antibody and anti-Flag antibody (A), using anti-Myc antibody and anti-Flag antibody (B), and using anti-Bip antibody and anti-Flag antibody (C) Stained.
FIG. 27 shows the ratio of viable cells at the time of introduction of each mutant when the ratio of viable cells at the time of introduction of the empty vector to CHO cells is 100%.
FIG. 28 shows the localization of IRE 1-alpha when each gene represented in the figure in NIH cells is co-expressed.
FIG. 29 is a photograph in which each fixed tissue section of a mouse is immunostained with an anti-hERRj-1 peptide antibody. The part surrounded by the arrow or the part pointed by the arrow represents the location of mERRj-1.
FIG. 30 A: Result of hERRj-1 EST database search. B: Redox state of ERp57 protein by hERRj-1 gene transfer in CHO cells. C: After introducing empty vector or immunoglobulin H (mu) chain or immunoglobulin H (mu) chain and hERRj-1 / Flag epitope gene into CHO cells, the cells were lysed, and each cell lysate was left intact (Total ) Or anti-immunoglobulin H (mu) chain antibody, and subjected to SDS-PAGE. It was transferred to a PVDF membrane and immunostained with each antibody shown in the figure.

Claims (3)

  A protein (hRUPP-1) comprising the amino acid sequence shown in SEQ ID NO: 2 or an amino acid in which one or more amino acids are modified by deletion, addition or substitution with respect to the amino acid sequence shown in SEQ ID NO: 2 A polypeptide having a sequence, which has a cell death-inducing activity. i ) A polypeptide consisting of a portion from 1 to 305 of the amino acid sequence of SEQ ID NO: 2;
ii ) A polypeptide consisting of portions 751-1148 of the amino acid sequence of SEQ ID NO: 2; and
iii ) A polypeptide obtained by directly connecting portions 1 to 305 and 751 to 1148 of the amino acid sequence of SEQ ID NO: 2;
A protein having cell death-inducing activity selected from the group consisting of:   A polynucleotide encoding the protein according to claim 1 or 2.

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