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標題: | 探討FAP-1去磷酸酶對角蛋白8與18的功能性影響 The Functional Effect of Fas-Associated Phosphatase(FAP-1) on Keratin 8/18 in Hepatocytes |
作者: | Ya-Yun Lai 賴亞筠 |
指導教授: | 葉秀慧 |
關鍵字: | FAP-1去磷酸酶, Fas-associated phosphatase-1, |
出版年 : | 2010 |
學位: | 碩士 |
摘要: | Fas-associated phosphatase-1 (FAP-1)屬於非受器型酪胺酸去磷酸酶(non-receptor protein tyrosine phosphastase)的一員,本實驗室先前利用positional cloning的方法發現FAP-1可能是一個位於chromosome 4q21-23上,參與肝癌形成的putative candidate tumor suppressor gene (TSG),但目前仍不清楚FAP-1在肝癌形成過程中的功能性影響為何。因為FAP-1是一個具有多個蛋白質交互作用功能域 (protein-protein interaction domain)的巨大骨架蛋白(scaffold protein),因此有可能藉由和肝細胞中特定分子的交互作用進一步發揮其功能性影響。先前實驗室利用yeast-two hybrid的方式發現一個與FAP-1有交互作用的新蛋白質-角蛋白18 (Keratin 18);並發現FAP-1會與細胞凋亡時不正常堆疊的角蛋白18或8有交互作用。因此本論文研究目的為釐清FAP-1是否參與不正常堆疊的角蛋白18或8後續進行降解過程之調控及分子機制。
我們採取的實驗策略是在高度表現FAP-1的HEK293T細胞株建立一個artificial system,藉由降低FAP-1的表現,並單獨外送角蛋白18或8製造出不正常堆疊的角蛋白,以利我們探討FAP-1如何調控不正常堆疊的角蛋白18或8進行蛋白質降解。本研究一開始採取lentivirus- based RNAi 降低FAP-1的表現量後,發現角蛋白18和8的表現量都有增加的現象。進一步利用cyclohexamide抑制蛋白新生後發現FAP-1會透過降低角蛋白18或8的蛋白質穩定度,影響角蛋白18或8的表現量。接著分別利用MG132或3MA這兩種蛋白質降解抑制劑的處理,發現不正常堆疊的角蛋白18和8會經由proteasome或autophagy進行降解。之後利用siRNA降低FAP-1的表現量再處理兩種蛋白質降解抑制劑,初步結果顯示FAP-1可能參與角蛋白8或18走向proteasome或autophagy的蛋白質降解路徑。 有鑑於長時間lentivirus- based RNAi和蛋白質降解抑制劑處理,可能會使兩條蛋白質降解路徑發生cross-talk,讓我們觀察到的是兩條蛋白質降解路徑互相影響後的secondary effect。因此接下來我們縮短實驗流程,分析轉染24小時內角蛋白18和8走proteasome和autophagy降解的情形,實驗結果發現蛋白18和8進行蛋白質降解的模式不盡相同,蛋白18主要走autophagy的降解途徑,角蛋白18則是兩條蛋白質降解途徑都會走。降低FAP-1的表現量後,發現FAP-1似乎主要參與調控不正常堆積的角蛋白18走向autophagy降解,對不正常堆積的角蛋白8的降解之影響則相當有限。此結果暗示角蛋白18可能藉由與FAP-1的專一性交互作用,使其能被帶往autophagy降解,走一條與角蛋白8不同的autophagy降解路徑,這將是我們未來繼續研究的重點。此外目前所有的實驗結果都是來自在HEK293T建立的artificial system,因此未來必須再利用另一個具有高度表現的FAP-1以及內生性角蛋白18和8的assay system來驗證此論文之發現,並進一步探討FAP-1調控角蛋白進行autophagy降解的生理意義。 Fas-associated phosphatase-1 (FAP-1) is a member of nonreceptor protein tyrosine phosphastase. We previously identified FAP-1 as a putative tumor suppressor gene at chromosome 4q21-23 in hepatocellular carcinoma by positional cloning. However, the functional effect of FAP-1 in hepatocarcinogenesis still remains unknown. FAP-1 is a large scaffold protein with multiple protein- protein interaction domains, we thus hypothesized that FAP-1 could through its association with specific factors to exert its function in hepatocytes. In our previous yeast-two- hybrid analysis, keratin18 was found as a novel interacting protein of FAP-1. Moreover, we found FAP-1 mainly associated with abnormally aggregated keratin18/ 8 during apoptosis. The specific aim of this study is to study if FAP-1 is involved in the degradation of abnormally aggregated keratin18/ 8 and the underlying molecular mechanisms. We approached this by establishing an artificial system using HEK293T cell line, which expresses high level of FAP-1 without endogenous K8 /K18 protein expression. We tried to generate the abnormally aggregated K8 or K18 proteins by transfection of K8 or K18 expression construct alone. In this artificial system, knockdown of FAP-1 by lentivirus- based RNAi increased the protein level of both K8 and K18. Aided by cycloheximide treatment, si-FAP-1 could increase the protein stability of K8 and K18, suggesting the involvement of FAP-1 in the degradation of K8 or K18 proteins. The treatment of specific degradation inhibitors of MG132 and 3MA demonstrated that K8 and K18 degraded through both proteasome and autophagy pathways in this assay system. The results of si-FAP1 suggested that FAP-1 might regulate the degradation of K8 and K18 through both degradation pathways. According to that long-term lenti-virus infection and degradation inhibitor treatment might result in secondary effects caused by a crosstalk between both degradation pathways, we decided to determine degradation kinetics of both keratins through both pathways. The preliminary data showed that K8 might degrade through both proteasome and autophagy pathways while K18 seems to be degraded mainly through autophagy pathway in this artificial system. The results from si-FAP-1 revealed that it might preferentially regulate the degradation of K18 though autophagy but has little effect on the degradation of K8. All of the current findings are derived from the artificial system in HEK293T cell line, which warrants further confirmation in some other r cell lines expressing high level of FAP-1 and also the endogenous K8 and K18 proteins. Moreover, the biological significance of FAP-1 in regulating Keratin degradation through autophagy would be the next issue worthy to be extensively investigated. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/10585 |
全文授權: | 同意授權(全球公開) |
顯示於系所單位: | 微生物學科所 |
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