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SEL1Lの分解中間体はポリグルタミンタンパク質の細胞質での凝集を促進する

服部, 徳哉 京都大学 DOI:10.14989/doctor.k23600

2022.01.24

概要

小胞体は、分泌タンパク質や膜タンパク質などの生合成を行い、これらのタンパク質の高次構造形成の場としての役割を担っている。そのため、小胞体の恒常性の維持は生体の生存にとって非常に重要である。小胞体関連分解(ERAD)は、小胞体に蓄積した異常なタンパク質を分解する機構のひとつである。またERADは、タンパク質の調節的分解を介した生体反応の調節も行っている。

ERADを担う因子SEL1Lは、小胞体膜に存在するI型膜タンパク質で、ユビキチンリガーゼであるHRD1と1:1の複合体を形成し、ERADにおいて中心的な役割を担っている。先行研究から、複合体を形成できないSEL1Lはubiquitin-proteasome系で分解されることが示された。また、proteasome阻害剤を加えてSEL1Lの分解を阻害すると、SEL1L分解中間体の蓄積が観察された。通常は、ERADでの分解過程でこのような分解中間体は検出されない。そこで今回、SEL1Lの分解メカニズム及び、SEL1Lの分解中間体が細胞に及ぼす影響を解明するため、この分解中間体の解析を行った。

まず、N末端およびC末端にタグをつけたSEL1Lを用いた解析から、SEL1L分解中間体は、SEL1Lタンパク質が内部で切断された結果生じたものと考えられた。細胞分画を行って、SEL1L分解中間体は細胞質に蓄積することを確認した。また、SEL1Lの分解中間体は還元条件下に存在しており、脱糖鎖型であった。ERADにおける基質の引っ張り出しに関与するp97の阻害剤を加えると分解は抑制された。これらの結果からSEL1L分解中間体は、全長のSEL1Lが一旦細胞質に出てきたのちに切断されることが明らかになった。次に過剰発現系やsiRNAを用いたノックダウン実験から、ERADを制御する小胞体レクチンであるOS-9やXTP3-Bは、SEL1Lと複合体を形成し、SEL1Lを安定化させることを見出した。また、SEL1LのC末端側に存在するプロリンに富んだ領域が分解中間体の形成に関与することを明らかにした。さらに、小胞体ストレスやパートナータンパク質の欠乏などによってSEL1Lは分解され、分解中間体が生成されることを見いだした。

伸長したポリグルタミン鎖をもつタンパク質は細胞内で凝集体を形成し、ポリグルタミン病と呼ばれる神経変性疾患を引き起こすことが知られている。今回、モデルタンパク質としてHtt-polyQ76-GFPを用いて解析を行ったところ、SEL1Lの分解中間体は、Htt- polyQ76-GFPの細胞質での凝集体形成を促進することが明らかになった。また、免疫細胞染色の結果、SEL1L分解中間体は凝集体を取り囲むように局在していた。凝集体形成に重要なSEL1Lの領域を調べたところ、タンパク質間の相互作用に関わると考えられているSE L1Lリピートの重要性が示された。この領域が、凝集体を形成しやすいタンパク質と相互作用した結果凝集体形成が促進されたと考えられた。

以上の結果から、SEL1L複合体の小胞体膜上での分解基質認識に関わる新たなサーベイランス機構の存在が示唆され、また、小胞体と細胞質におけるタンパク質品質管理機構は密接に関連していることが示された。

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