在非逆境和砷暴露下蛋白酶體與溶體途徑參與Skp2降解之比較

Comparison of Skp2 degradation via proteasomal and lysosomal pathways in non-stressed and arsenite-treated cells

吳南瑩(Nan-Ying Wu)

指導教授 ： 楊嘉鈴

國立清華大學/生命科學院/生物科技研究所/碩士(2008年)

摘要

Skp2 是一個具有 F-box 的蛋白質，屬於 Skp1-Cullin-F-box 泛素連接酶複合體 (ubiquitin ligase complex) 的次單元，它可以促使許多細胞週期激酶抑制分子 (Cyclin-dependent kinase inhibitors) 進行蛋白質降解，藉此適時調控細胞週期的運轉。目前已知在 G0-G1 期 Skp2會透過 APCCdh1 和 26S 蛋白酶體 (proteasome) 來進行降解。先前我們發現砷誘發Skp2降解需要溶體蛋白酶 (lysosomal proteases) 的參與。本篇研究主要探討在非逆境 (non-stressed) 和砷刺激情況，蛋白酶體與溶體參與 Skp2 降解的機制。利用氯化銨 (NH4Cl) 和氯奎寧 (chloroquine) 抑制溶體活性，可減緩砷誘發內生性 Skp2 的降解，而在非逆境情況溶體抑制劑則不會影響 Skp2 的含量。此外，氯化銨也可以減少砷造成外源性 Flag-tagged Skp2 的蛋白降解，其中包含 wild-type 以及 Cdh1-interaction domain 缺失之 Skp2；表示砷誘發 Skp2 之溶體降解過程中 Cdh1 不是絕對必要的。在砷處理下利用 MG132 抑制蛋白酶體活性會更增強 Skp2 蛋白的降解，並且降低氯化銨緩解的效果；表示蛋白酶體會抑制砷刺激下 Skp2 透過溶體降解的作用。有趣地，在非逆境情況抑制細胞內 CHIP 泛素連接酶的表現會顯著增加 Skp2 的含量，而這個現象是需要 Skp2 本身的 Cdh1-interaction domain 或 D-box 存在。同樣地，抑制 CHIP 表現也可以減緩砷誘發 Skp2 降解的能力。此外，隨著砷的劑量增加會誘發 Chk1 磷酸化，且在處理 1 小時有最高量的表現。利用 siRNA 技術或 caffeine 來抑制 Chk1 的表現或活性，可以增強砷誘發 Skp2 的降解作用。在 caffeine 與砷共同作用下，氯化銨仍可以減少砷造成 Skp2 蛋白降解的情況。此外單獨抑制 Chk1 蛋白表現即會造成 Skp2 的降解。綜合以上結果，顯示細胞在正常生長情況，Skp2 是藉由 CHIP 和 Cdh1 調控，促使 Skp2 透過 26S 蛋白酶體進行降解，而 Chk1 蛋白則會抑制此降解的發生。相反地，砷造成 Skp2 降解主要是透過溶體的作用，且可能與 Chk1 激酶活性無關。

關鍵字

砷 ； 溶體 ； 蛋白酶體 ； 泛素連接酶複合體 ； 氯化銨 ； 氯奎寧 ； HASH(0x1c64b6b0) ； HASH(0x1c64b750) ； HASH(0x1c64b7f0) ； HASH(0x1c64b890) ； HASH(0x1c64b930) ； HASH(0x1c64b9d0) ； HASH(0x1c64ba40) ； HASH(0x1c64bab0)

並列摘要

Skp2 is an F-box protein of the Skp1-Cullin-F-box ubiquitin ligase complex that is responsible for degradation of Cyclin-dependent kinase inhibitors to timely control cell cycle progression. Skp2 degradation is known to be mediated via the APCCdh1-26S proteasome pathway during G0/G1 stage. Additionally, our previous study suggests that Skp2 degradation requires the activities of lysosomal proteases following arsenite stress. Here I explored the mechanisms of Skp2 degradation via proteasomal and lysosomal pathways in non-stressed and arsenite-treated cells. Two lysosomal inhibitors NH4Cl and chloroquine could rescue the endogenous Skp2 levels reduced in arsenite-treated cells, whereas the inhibitors did not significantly affect those in untreated cells. NH4Cl also lessened the destruction of exogenous Flag-tagged Skp2 by arsenite during expression of wild-type or a mutant Skp2 lacking the Cdh1-interaction domain, suggesting Cdh1 is not essential for Skp2 lysosomal degradation by arsenite. Co-administering MG132, a proteasome inhibitor, further enhanced the arsenite-induced Skp2 degradation and abolished the recovery effect of NH4Cl, suggesting proteasome has an inhibitory effect on Skp2 lysosomal degradation during stress. Intriguingly, in untreated cells, depletion of an E3 ubiquitin ligase CHIP (C-terminus of Hsc70-interacting protein) using small interfering RNA (siRNA) markedly increased Skp2 levels dependent on its Cdh1-interaction domain or D-box. Similarly, CHIP depletion lowered the Skp2 lysosomal degradation induced by arsenite. Moreover, arsenite dose-dependently induced Chk1 phosphorylation and the highest levels achieved during 1 h exposure. Knockdown of Chk1 by siRNA or inhibition of Chk1 kinase activity using caffeine decreased Skp2 levels in arsenite-treated cells. Under caffeine co-treatment, NH4Cl can still prevent the arsenite-induced Skp2 degradation. Chk1 depletion, but not caffeine, decreased the Skp2 levels in untreated cells. Together, results showed here suggest that during normal growth the Skp2 proteolysis is mainly achieved via the 26S proteasome pathway mediated by the CHIP and Cdh1 E3 ubiquitin ligases, while Chk1 protein may prevent this process. Conversely, following arsenite stress the lysosomal activity is essential for Skp2 destruction and is independent of Chk1 signaling.

並列關鍵字

APC ； Chk1 ； Lysosome ； CHIP ； Autophagy ； D-box ； MAPK ； NH4Cl ； SCF ； Skp2 ； Arsenite ； Cdh1 ； Choroquine ； Proteasome

參考文獻

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