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研究生:張乃文
研究生(外文):Nai-Wen Zhang
論文名稱:探討hnRNPK和DDX23的交互作用在DNA受損時對hnRNPK所調控之細胞凋亡的抑制作用
論文名稱(外文):Interaction between hnRNPK and DDX23 suppresses the hnRNPK-mediated apoptosis enhancement upon DNA damage
指導教授:林照雄林照雄引用關係
指導教授(外文):Chao-Hsiung Lin
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生命科學系暨基因體科學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:英文
論文頁數:39
中文關鍵詞:hnRNPKDDX23DNA受損細胞凋亡交互作用
外文關鍵詞:hnRNPKDDX23DNA damageapoptosisInteraction
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HnRNPK是一個核酸結合蛋白質,負責調控染色體重建、轉錄、RNA剪接、穩定mRNA、轉譯以及去氧核醣核酸損傷反應。研究表明在不同癌症病人中,會有累積hnRNPK的情形。至今的研究顯示,hnRNPK與癌症的惡性轉移、病人不好的預後,甚至是癌症幹細胞的發展有相當大的關連。已知hnRNPK有不同的轉譯後修飾來影響其功能,包括甲基化、磷酸化、泛素化(ubiquitination)、小泛素化(sumoylation)。本實驗室先前研究發現hnRNPK的296與299精氨酸甲基化會抑制癌細胞的凋亡,反之減少甲基化則提高細胞凋亡。而hnRNPK的296與299精氨酸位於負責hnRNPK與其他蛋白交互作用的區域(KI domain),因此分析以KI domain與hnRNPK交互作用蛋白種類,先前結果顯示一群RNA解旋酶(DDX/DHX family proteins)與KI domain有交互作用。在我的研究題目中,我採用的人類骨肉瘤細胞使用CRISPR/Cas9技術去除內生性hnRNPK並置換成外生性野生型(wild type)或缺乏296與299精氨酸甲基化的hnRNPK。有趣的是,我的研究結果發現296與299精氨酸甲基化會提高hnRNPK和DDX23的交互作用。進一步研究證明hnRNPK和DDX23的交互作用會抑制hnRNPK所調控之細胞凋亡,並在增加DDX23表現量時看到抑制細胞凋亡的作用,減少DDX23則反之增加細胞凋亡。另外,我將DDX23分割成N端及C端兩段部分蛋白,以重組的蛋白來分析DDX23由哪段部分與hnRNPK交互作用,結果顯示hnRNPK較傾向與N端DDX23交互作用。然而,於細胞表現N端DDX23也會抑制細胞凋亡。最後,我亦以增量DDX23表現量證明了已知的DDX3與hnRNPK的交互作用會受到DDX23的影響。綜合以上結果,我可以推論hnRNPK和DDX23的交互作用在DNA受損時會抑制hnRNPK所調控之細胞凋亡,且透過干擾DDX3與hnRNPK的交互作用的方式。
Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is a nucleic acid-binding protein that participates in diverse cell events including chromatin remodeling, transcription regulation, RNA splicing, mRNA stabilization, translation regulation and DNA damage response. Remarkably, amplifications of hnRNPK in diverse human cancers have been reported, regardless of the fact that this protein is required for cell maintenance. Up to date, hnRNPK has been shown to be associated with aggressive metastasis of tumor and poor prognosis of patients as well as the induction of cancer stem cells. Moreover, several post-translational modifications, such as methylation, phosphorylation, sumoylation and ubiquitination, of hnRNPK have been shown to affect its protein function. For example, our lab previously showed that the Arg296/Arg299 methylation within the protein-interactive region (KI) of hnRNPK is able to suppress the apoptosis of U2OS cells upon DNA damage, whereas a decrease of Arg296/Arg299 methylation hnRNPK enhances cell apoptosis. In addition, the following interactome analysis revealed that several DDX/DHX family proteins interact with the KI of hnRNPK. In my thesis study, I first verified the in vivo hnRNPK-DDX23 interaction using the CRISPR/Cas9-engineered U2OS cells in which the endogenous hnRNPK was replaced with the exogenous wild type (WT) or Arg296/Arg299 methylation-defective (MD) hnRNPK. Interestingly, my results indicated that Arg296/Arg299 methylation of hnRNPK promotes the DDX23-hnRNPK interaction. Subsequently, I further showed that hnRNPK-DDX23 interaction reduces the degree of apoptosis enhancement mediated by MD-hnRNPK in U2OS cells. Meanwhile, overexpression of DDX23 in U2OS cell further suppressed apoptosis, whereas knockdown of DDX23 promoted apoptosis. Alternatively, I have also prepared the recombinant GST-tagged N-terminal-half or C-terminal-half region of DDX23 to investigate the hnRNPK-binding domain of DDX23. Currently, my pull-down assays showed that hnRNPK prefers to bind with N-terminal-half DDX23 rather than C-terminal-half. Next, I further examined whether the exogenously expressed N-terminal DDX23 affects the hnRNPK-mediated apoptosis regulation in U2OS cells. The addition of N-terminal-half DDX23 significantly reduces hnRNPK-mediated apoptosis. Finally, the interaction between DDX3 and MD-hnRNPK was interfered by DDX23. In conclusion, DDX23 seems to suppress the hnRNPK-mediated apoptosis upon DNA damage through interrupt DDX3-hnRNPK interaction in cancer cells.
Abstract i
中文摘要 ii
Table of contents iii
Abbreviation vi
Introduction 1
Cellular functions of hnRNPK and post-translational modifications of hnRNPK 1
Cancer association of hnRNPK 2
Present interactome analysis revealed DDX/DHX family is the novel cluster for hnRNPK interaction 3
Molecular functions of DDX23 in cancer 3
Specific aims of study 5
Materials and Methods 6
Results 10
Arg296/Arg299 methylation-defective hnRNPK promotes the apoptosis upon DNA damage in U2OS CRISPR cells 10
Arg296/Arg299 methylation of hnRNPK promotes the DDX23-hnRNPK interaction in cells 11
DDX17-hnRNPK interaction is not influenced by Arg296/Arg299 methylation of hnRNPK whereas DHX57 doesn’t interact with neither WT-hnRNPK nor MD-hnRNPK in cells 11
DDX23 is required for suppression of hnRNPK-mediated apoptosis upon DNA damage 11
HnRNPK prefers to bind with N-terminal-half of DDX23 rather than C-terminal-half in vitro 12
hnRNPK-DDX23 interaction reduces the degree of apoptosis enhancement mediated by MD-hnRNPK in U2OS cells 13
The interaction between DDX3 and MD-hnRNPK was interfered by DDX23 13
Discussion 14
References 17
Figures 20
Figure 1. Arg296/Arg299 methylation-defective hnRNPK didn’t affect the morphology and growth rate in U2OS (CRISPR) cells 21
Figure 2. MD-hnRNPK promotes the apoptosis upon DNA damage in U2OS CRISPR cells 22
Figure 3. Basal expression of DDX/DHX family proteins in U2OS cells carrying WT- or MD-hnRNPK 23
Figure 4. Arg296/Arg299 methylation of hnRNPK promotes the DDX23-hnRNPK interaction in cells 25
Figure 5. hnRNPK-DDX23 interaction reduces the degree of apoptosis enhancement mediated by MD-hnRNPK 27
Figure 6. HnRNPK prefers to bind with N-terminal-half of DDX23 rather than C-terminal-half in vitro 29
Figure 7. DDX23 suppresses hnRNPK-mediated apoptosis upon DNA damage 30
Figure 8. The interaction between DDX3 and MD-hnRNPK was interfered by DDX23 31
Figure 9. Interaction between hnRNPK and DDX23 suppresses the hnRNPK-mediated apoptosis upon DNA damage through interrupting hnRNPK-DDX23 interaction 33
Appendix 34
Appendix 1. Functional domains of hnRNPK 34
Appendix 2. HnRNPK-interacting DDX/DHX proteins from present interactome analysis 35
Appendix 3. Functional domains of DDX proteins 36
Appendix 4. The interaction between Arg296/Arg299 methylation-defective hnRNPK and DDX3 enhances the apoptosis upon DNA damage 37
Appendix 5. The plasmid map of diverse form of myc-DDX23s 38
Appendix 6. The plasmid map of diverse form of GST-DDX23s 39
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14 Messias, A. C., Harnisch, C., Ostareck-Lederer, A., Sattler, M. & Ostareck, D. H. The DICE-binding activity of KH domain 3 of hnRNP K is affected by c-Src-mediated tyrosine phosphorylation. Journal of molecular biology 361, 470-481, doi:10.1016/j.jmb.2006.06.025 (2006).
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