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ILF2 enhances the DNA cytosine deaminase activity of tumor mutator APOBEC3B in multiple myeloma cells

Kazuma, Yasuhiro 京都大学 DOI:10.14989/doctor.k24187

2022.09.26

概要

APOBEC3(apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like)ファミリー分子APOBEC3B(A3B)は、シトシン脱アミノ化(CDA)活性を有し、様々な癌腫で過剰発現してAPOBECに特徴的なパターンの変異を腫瘍細胞のゲノムにもたらす。A3Bの制御機構には、転写調節や選択的スプライシング、リン酸化などの報告があるが、A3Bとの蛋白間相互作用によるCDA活性の調節に関しては明らかでない。他のAPOBECファミリー分子であるAIDは、共に高分子量複合体を構成しているhnRNP蛋白によりその機能が調節される。同様にA3Bも核内で高分子量複合体を形成していることから、その構成蛋白がCDA活性を制御している可能性を検証するため、A3B相互作用蛋白の解析を計画した。

本研究ではまず骨髄腫細胞株AMO1、RPMI8226を用いてA3Bの共免疫沈降サンプルについて質量分析を行い、hnRNP、スプライシング因子、リボソーム蛋白、RNAヘリカーゼなど30種類の相互作用蛋白の候補を同定した。

共免疫沈降サンプルのウエスタンブロッティングにより、これら候補蛋白との結合を確認できたが、SAFB以外の蛋白はRNase処理によりA3Bとの結合が失われた。次に密度勾配遠心分離法を用いて、骨髄腫細胞の細胞溶解液を高分子量分画と低分子量分画に分離し、各相互作用蛋白の分布を確認した。A3Bと同様にSAFB、SRSF7、Matrin-3はRNase処理の有無に関わらず高分子量分画に認められたが、ILF2、DHX9、RBMXはRNase処理により高分子量分画から低分子量分画に分布が移動した。以上よりSAFBを除く多くの相互作用蛋白はRNAを介してA3Bと高分子量複合体を構成していると考えられた。

この中で、A3BとRNAを介さず直接結合するSAFB、および1q21増幅を有する骨髄腫の薬剤抵抗性や予後との関連が既に知られているILF2に着目して検討を進めた。まず、蛍光免疫染色を行い、共にA3Bと核内で共局在することを確認した。さらに、蛍光標識したオリゴヌクレオチドを用いたinvitro CD Aassayを用いて、これらの蛋白がA3BのCDA活性に与える影響を評価した。精製したILF2蛋白の混合により、ILF2濃度依存性にA3BのCDA活性が増強した。また、ILF2過剰発現させた細胞溶解液のCDA活性は過剰発現していない場合に比較し36％増強し、RNAiによりILF2をノックダウンした細胞溶解液では、していない場合に比較し30％低下した。一方でSAFBはいずれの系でもCDA活性に影響を与えなかった。

以上より、A3Bの高分子量複合体を構成する蛋白の一つであるILF2がCDA活性を調節している可能性が示された。ILF2はRNA依存性にA3Bと結合する一方で、DNA結合ドメインも有することからCDAの基質となる一本鎖DNAを隣接するA3Bに提供している可能性が考えられる。内因性蛋白がA3BのCDA活性を変化させる報告はこれまでになく、今回の研究の成果はA3BによるゲノムDNAへの変異の制御機構を理解する上で重要な知見と考えられる。

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