SUMO E3 連接酶 RanBP2在DNA損害反應中之角色

The role of SUMO E3 ligase RanBP2 in the DNA damage response

涂芷瑄(Jyy-Shiuan Tu)

指導教授 ： 吳青錫

國立臺灣大學/醫學院/藥理學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201902047

摘要

SUMOylation 是一種蛋白質後轉譯修飾，小泛素修飾分子蛋白會經由SUMO E1、E2和E3的協助而結合到目標蛋白質上，進而調控蛋白質的穩定性、活性、蛋白質與蛋白質之間的相互作用。小泛素修飾分子蛋白對於細胞內的調控是非常重要的，小泛素修飾分子能夠調控許多細胞內的功能，像是轉錄、複製、染色體分離和DNA的修復。當細胞受到傷害後會啟動一連串的機制去維持基因的完整性，這些機制稱為DNA損傷反應 ，簡稱DDR。DDR會受到許多後轉譯修飾的調節而控制參與在DDR內的蛋白質的活化。研究發現有非常多參與在DDR內的蛋白質會被小泛素修飾分子修飾，像是RPA、ATRIP和BRCA1等等…，但是這些蛋白質的E3 ligase只有少數有被報導參與在DNA損傷反應。是否其他的E3 ligase亦參與調控龐大又複雜的DDR則成了未解的課題。我經由干擾RNA快篩發現了RanBP2，這個細胞核孔上的蛋白質，會影響ATR-Chk1 訊息路徑的活化。在我的實驗中，我發現在不同的DNA損傷的情況下，沈默 RanBP2表現會顯著的減弱ATR-Chk1 訊息路徑的磷酸化而非ATM-Chk2 訊息路徑，但是細胞的S phase比例 、DNA合成比例和雙股斷裂的形成並未顯著地受到影響。免疫螢光染色與蛋白質西方墨點法分析顯示γH2AX和RPA的焦點形成與蛋白質總量，沒有因為缺乏RanBP2而有顯著的減少。除此之外，我也確認了RanBP2不是經由改變其他參與在ATR-Chk1訊息路徑的蛋白質的表現量或是在細胞核內的數量來影響ATR-Chk1 訊息路徑。由於ATR-Chk1 訊息路徑中最上游的蛋白質為ATR，而缺乏RanBP2也有看到ATR的活化減少，因此我測試ATR是否會與RanBP2有相互作用。結果顯示，在細胞週期間期時，並未觀察到RanBP2與ATR的相互作用，但在有絲分裂時，RanBP2與ATR有相互作用。以上結果暗示，RanBP2很有可能是經由與ATR相互作用而去影響ATR-Chk1訊息路徑，但是RanBP2是否是經由SUMOylation的方式去影響ATR-Chk1 訊息路徑，或者RanBP2是否會幫助ATR的小泛素修飾分子修飾，還需要更深入的探討。

關鍵字

小泛素修飾分子 ； DNA損傷反應

並列摘要

SUMOylation is one of post-translational modification, and SUMO conjugation to substrates occurs through a process involving an E1 activating enzyme, an E2 conjugating enzyme and an E3 protein ligase. Protein modification by SUMOylation can change the stability and activity of proteins and alter protein-protein interactions. Protein SUMOylation is important for several cellular processes, including transcription, DNA replication, chromosome segregation and DNA repair. In response to DNA damage, cells activate a series of mechanisms to protect genomic integrity termed the DNA damage response (DDR). Several types of post-translational modifications are known to regulate the DDR. Previous studies have shown that hundreds of the DDR proteins including RPA, ATRIP, and BRCA1 are SUMO conjugated in response to DNA damage. However, only few SUMO E3 ligases have been implicated in the DDR. It is not clear if other SUMO E3 ligases are also involved in the DDR. With a small scale siRNA screen, I find that RanBP2, a component of nuclear pore complex, is important for activation of the ATR-Chk1 signaling pathway. I find that knockdown of RanBP2 drastically reduces damage-induced phosphorylation of the ATR-Chk1 pathway but not the ATM-Chk2 pathway. Knockdown of RanBP2 does not significantly alter cell cycle progression, DNA synthesis, and formation of damage-induced DNA double-stranded breaks. Immunofluorescence imaging and Western blotting show that the foci formation and protein levels of γH2AX and RPA are not affected. Moreover, levels of several key factors of the ATR-Chk1 pathway remain unchanged after knockdown of RanBP2. Given the activation of ATR is the upstream event in the ATR-Chk1 pathway, I test whether RanBP2 interacts and regulates ATR directly. Immunoprecipitation of RanBP2 shows that ATR associates with RanBP2 during mitosis but not in interphase. The result suggests RanBP2 may affect the ATR-Chk1 pathway by interacting with ATR. However, it needs more work to test if RanBP2 affects the ATR-Chk1 pathway via its SUMO E3 function.

並列關鍵字

RanBP2 ； SUMOylation ； DNA damage response

參考文獻

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