探討BIK之泛素化降解機制

The mechanism of BIK proteasomal degradation

詹智鈞(Chih-Chun Chan)

指導教授 ： 陳瑞華

國立臺灣大學/生命科學院/生化科學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.10837

摘要

BIK (Bcl-2-interacting killer) 為促進凋亡之BH3-only家族中的一員，在癌症中扮演了腫瘤抑制者的角色。最近發現，BIK可作為抗癌藥物之有效標的。抗癌藥物Bortezomib處理下會導致BIK的堆積，暗示著BIK的降解可能參與了腫瘤的生成。為了找出蛋白酶體降解BIK之機制，我們想找出負責降解BIK的E3接合酶。目前已知Cullin-RING接合酶為最普遍的E3接合酶。透過表現各種不同Cullin的顯性失活突變體在293T細胞中，我們發現Cul2和Cul5可能影響BIK表現。已知Cul2和Cul5必須透過受質辨識單元 (substrate recognition subunit; SRS) 來辨認受質，為了找出可辨識BIK、並與Cul2或Cul5以及Elongin BC結合之受質辨識單元，我們建構了51個受質辨識單元，並將其個別表現在293T細胞中。由GST pull-down實驗發現有6個受質辨識單元可與BIK結合，當中KLHDC3、ASB1及ASB7在後續實驗證明能抑制BIK蛋白表現。然而，這三個蛋白質中，只有剔除ASB7的表現會增加BIK的蛋白質量，以及延長BIK降解之半衰期。細胞中若同時表現Cul5接合酶複合體及ASB7時，會大幅促進BIK泛素化修飾程度。總而言之，我們發現Cul5-ROC2-Elongin BC與ASB7形成之泛素接合酶複合體，能促進BIK之泛素化進而降解。生物資訊學的分析發現，不論是ASB7蛋白質及其mRNA，在某些癌症有較高的表現，後續我們會去確認在這些癌症中ASB7是否會抑制BIK的表現。

關鍵字

BIK ； 蛋白酶體 ； 泛素化 ； E3接合酶Cul5 ； 受質辨識單元 ； ASB7

並列摘要

BIK (Bcl-2-interacting killer), a BH3-only pro-apoptotic protein, functions as a tumor suppressor in human cancer. Recently, BIK has emerged as a prominent target of anti-cancer drugs. Treatment of certain cancer cells with the proteasome inhibitor-Bortezomib leads to BIK accumulation, implying the importance of BIK proteasomal degradation pathway in cancer biology. To demonstrate the underlying mechanisms of BIK degradation, we intend to identify the ubiquitin E3 ligase of BIK. Cullin-RING ligases represent the largest ubiquitin ligase family. By overexpressing various dominant-negative forms of cullins in 293T cells, we identified Cullin2 (Cul2)- and Cullin5 (Cul5)- ligases as candidate regulators of BIK protein level. Both Cul2 and Cul5 are Elongin BC-based E3 ligases and require a substrate recognition subunit (SRS) for substrate recruitment. To identify the SRS that links Cul/Elongin BC to BIK, we cloned and overexpressed 51 SRSs in 293T cells. Among them, we identified six SRSs that could interact with BIK by GST pull-down assay. By overexpressing these SRSs individually into 293T cells, we showed that KLHDC3, ASB1 and ASB7 could downregulate BIK expression. However, only knockdown of ASB7 increased BIK protein level and prolonged BIK half-life. Cotransfection of Cul5-based complex and ASB7 together led to a great induction of BIK polyubiquitination. Together, our study identified an ubiquitin E3 ligase complex containing ROC2, Cul5, Elongin BC, and ASB7, which is responsible for BIK ubiquitination and degradation. Bioinformatics analysis revealed that ASB7 mRNA and protein are upregulated in certain types of cancers. Future study will aim to determine whether ASB7 contributes to BIK downregulation in these tumors.

並列關鍵字

BIK ； proteasome ； ubiquitination ； E3 ligase ； Cul5 ； SRS ； ASB7

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