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標題: | 探討KLHL20在腫瘤進程與囊泡運送之調控分析 Functional characterization of KLHL20 in tumor progression and vesicular trafficking |
作者: | Wei-Chein Yuan 袁維謙 |
指導教授: | 陳瑞華(Reuy-Hwa Chen) |
關鍵字: | 泛素化,KLHL20,PML,缺氧,Crn7,Eps15,後高基氏體囊泡運送,肌動蛋白骨架, ubiquitination,KLHL20,PML,hypoxia,Crn7,Eps15,post-Golgi trafficking,actin, |
出版年 : | 2013 |
學位: | 博士 |
摘要: | KLHL20 是一個BTB-Kelch蛋白,在我們實驗室中發現它可以做為一個Cul3-based E3接合酶的受質接收器。KLHL20主要位於高基氏體中,但是也有一小部分的KLHL20會分布於PML-NB,因此這暗示著在細胞中不同位置的KLHL20可能具有不同的功能。在這本論文的第一個部分,我們發現KLHL20可以促進PML的泛素化。PML是一個腫瘤抑制因子,它的表現量在各式人類癌症中被抑制。在我們的研究中發現PML必須被Pin1於其S518位置進行異構化後才能被KLHL20所辨認並且促進PML的降解,進而抑制PML所參與的各式功能,包括促進細胞凋亡和老化以及抑制細胞形變及癌化。因此,KLHL20可以透過促進PML的降解,進而幫助細胞存活及癌化。除此之外,我們也發現在缺氧的情況下HIF-1會增加KLHL20的表現,進而促進PML的降解。此降解路徑在缺氧情況下以雙負回饋機制誘發HIF-1a表現,進而促進許多腫瘤缺氧反應。在人類前列腺癌中,HIF-1a、KLHL20及Pin1為過量表現,而PML的表現量則降低,後者表現量與前三者為負相關,PML降解路徑的高度活化則與腫瘤之進程息息相關,於晚期腫瘤細胞中更為顯著。我們的研究顯示KLHL20所參與的PML降解與HIF-1a的自動調控機制在腫瘤進程中扮演舉足輕重的角色。
在本論文的第二個部分,我們發現KLHL20也可以藉著幫助後高基氏體囊泡的生成進而促進從TGN至細胞膜以及核內體的貨物運送。Cul3-KLHL20泛素接合酶可以催化Crn7的K33類型泛素化作用,此類型的泛素化並不會促進降解作用,而是幫助Crn7與Eps15結合進而促進Crn7坐落於TNG。當抑制Crn7的泛素化或是阻斷它與Eps15的作用時,會減少位於TGN的肌動蛋白點(actin foci)形成,因此抑制管狀囊泡的產生。我們的研究發現KLHL20所參與的Crn7上K33類型的泛素化,在後高基氏體囊泡運送中扮演著舉足輕重的角色,因此解開了K33類型泛素鍊的重要性,可以在後高基氏體囊泡形成的過程中用來連接clathrin接受器Eps15與位於TGN的肌動蛋白改建(actin-remodeling)過程。 KLHL20, a BTB-Kelch protein, has been shown to function as a substrate adaptor of Cul3-based E3 ubiquitin ligase in our laboratory. Intriguingly, KLHL20 mainly localizes at the Golgi apparatus, while a small portion localizes at PML-NBs, thus suggesting the multiple functions of KLHL20 in different subcellular compartment. In the first part of this dissertation, we show that KLHL20 is responsible for the ubiquitination of PML, a tumor suppressor downregulated in various human cancers. Targeting PML to KLHL20 requires Pin1-mediated prolyl cis/trans isomerization of PML at the S518-P519. The Pin1/KLHL20 pathway induces PML proteolysis, thereby attenuating multiple PML biological functions, including promotion of apoptosis and senescence and suppression of cell transformation and tumorigenesis. Accordingly, KLHL20 potentiates cell survival and tumorigenesis partly through PML degradation. Additionally, we also show that KLHL20, which is induced by HIF-1, coordinates with the action of Pin1 to mediate hypoxia-induced PML proteasomal degradation. Furthermore, this PML destruction pathway constitutes a double negative feedback mechanism to amplify HIF-1a expression, thereby triggering multiple tumor hypoxia responses. In human prostate cancer, overexpression of HIF-1a, KLHL20, and Pin1 correlates with PML downregulation, and hyperactivation of the PML destruction pathway is associated with disease progression. Our study identifies a key pathway that controls PML protein stability and suggests a contribution of this pathway to aberrant PML downregulation in human cancers. In the second part of this dissertation, we show that KLHL20 is also crucial for the cargo trafficking from trans-Golgi network (TGN) to plasma membrane and endosomes by promoting the biogenesis of post-Golgi carriers on the basis of its Golgi localization. The Cul3-KLHL20 complex catalyzes a non-degradable, K33-linked polyubiquitination on coronin 7 (Crn7), which contributes to Crn7 TGN localization through an interaction with Eps15. Blockage of Crn7 ubiquitination or its interaction with Eps15 impairs the formation of F-actin foci at TGN, thereby inhibiting the generation of tubular carriers. Our study reveals a role of KLHL20-mediated K33-ubiquitination of Crn7 in post-Golgi transport, provides a cellular decoding mechanism for this ubiquitin chain type, and reveals the importance of this ubiquitination in linking clathrin adaptor Eps15 to the TGN actin-remodeling process during post-Golgi carrier biogenesis. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/15525 |
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顯示於系所單位: | 生化科學研究所 |
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