DC 欄位 | 值 | 語言 |
dc.contributor | 指導教授:羅翊禎 | - |
dc.contributor | 臺灣大學:食品科技研究所 | zh_TW |
dc.contributor.author | 劉子寧 | zh_TW |
dc.contributor.author | Liu, Tzu-Ning | en |
dc.creator | 劉子寧 | zh_TW |
dc.creator | Liu, Tzu-Ning | en |
dc.date | 2014 | - |
dc.date.accessioned | 2014-11-27T03:46:57Z | - |
dc.date.accessioned | 2018-06-29T07:46:06Z | - |
dc.date.available | 2014-11-27T03:46:57Z | - |
dc.date.available | 2018-06-29T07:46:06Z | - |
dc.date.issued | 2014 | - |
dc.identifier.uri | http://ntur.lib.ntu.edu.tw//handle/246246/262466 | - |
dc.description.abstract | 出芽酵母(Saccharomyces cerevisiae)為分子生物學中重要的模式生物之一。因其 生活史短,基因又具保守性,故適合進行行老老化機制之研究。本實驗以驗證 Ubp8 及 Ubp10 調控 Snf1 的蛋白質降降解並影響老老化性狀狀的路路徑為目標,進行行研究。根據遺 傳資料料庫分析的結果,推測與高等動物具高度度保守性之酵母菌腺苷酸活化蛋白激 酶(AMPK) Snf1 可能為 Ubp8 及 Ubp10 此兩兩個去泛素化酵素 (deubiquitinases, DUBs) 的目標蛋白質。Snf1/AMPK 在細胞中扮演能量量調控的主要角色。正常情況下,Snf1 並不不活化;當環境中葡萄糖糖耗盡,Snf1 被上游激酶活化,透過改變染色質結構、 轉錄錄因子活化、轉錄錄作用調控等方式,進而增加細胞對其他碳源的利利用及對逆境 的耐受性。由西方墨點法的結果,發現 ubp8Δubp10Δ 細胞中的 Snf1 總量量因為 DUBs 的缺乏而有明顯減少的現象,但其 mRNA 表現並無受到阻斷;而在兩兩個 DUBs中, 又以 Ubp8 為主要影響 Snf1 蛋白質穩定性的酵素;在老老化相關路路徑上,此調控主要影響對氧化壓力力以及繼代存活壽命(CLS)的性狀狀,在複製存活壽命(RLS)上則沒有明顯差異異;此外,此突變株的再生能力力異異於目前所知的由基因不不穩定造成的癌化突變。進一步探討 Snf1 的泛素化修飾影響其磷酸化的結果顯示:在 ubp8Δubp10Δ 突變細胞的 Snf1總量量雖然減少,但有高度度磷酸化的現象,而此現象可能導致 Snf1對於壓力力反應序列列(Stress responsive element, STRE)相關之轉錄錄作用的調控,增加 ubp8Δubp10Δ 突變細胞對於逆境的耐受性。透過已知的 Snf1 調控機制,我們歸納出數數個影響 Snf1 高度度磷酸化可能的機制,以及其下游調控的蛋白影響逆境耐受的可能性。 | zh_TW |
dc.description.abstract | Ubiquitin is a small regulatory protein that is expressed ubiquitously in eukaryoticorganisms. Conjugation of ubiquitins to proteins directs them to compartments in the cell, including the proteasome, which destroys and recycles proteins. Based on the genetic interaction analysis, I purposed that Snf1, highly conserved yeast AMPK, may be a target by both of Ubp8 and Ubp10, two highly conserved ubiquitin-specific proteases (DUBs). Snf1 plays an important role in modulating energy status in the cell, which is usually inactive. Only when glucose depleted can Snf1 be activated by its upstream kinases. Via changing chromatin structures, activating transcription factors and modulating transcription, Snf1 enhances the abilities of the cell to use alternative carbon sources and resist stresses. In this study, I aimed to reveal the pathway of the ubiquitin-mediated Snf1 regulation. Western blot analyses demonstrated that the level of Snf1 was dramatically decreased in ubp8∆ubp10∆ cells, but the mRNA level detection did not change significantly. Snf1 has been shown to be involved in aging processes; I first examined the possible roles of Ubp8 and Ubp10 in yeast aging. The deletion of UBP8 and UBP10 affected the cellular resistance to oxidative stresses and chronological life span (CLS) phenotype, possibly due to the decreased Snf1 protein level; while there was no significant change in replicative life span (RLS). In addition, the ubp8∆ubp10∆ cells exhibited regrowth phenotype which is known to be induce accumulated ROS and mutagenesis; however, in those cells I found no evidence of ROS-mediated genome instabilities. Further investigation revealed that despite the protein level was decreased, Snf1 was hyperphosphorylated in ubp8∆ubp10∆ cells. The ubp8∆ubp10∆ cells was able to grow on non-fermentable carbon sources but was not the SNF1 deleted cells. Taken together, my results suggest that Snf1 is likely protected
by both Ubp8 and Ubp10 from proteasome-mediated degradation, which diminishes thecellular level of Snf1. Interestingly, the remaining Snf1 in in ubp8∆ubp10∆ cells is hyperphosphorylated via an unknown mechanism. I propose that the hyperphosphorylated Snf1 in ubp8∆ubp10∆ cells are able to activate the stress-responsive (STRE) transcription to maintain the cellular resistance to stresses. The potential pathways that may participate in the hyperphosphorylaiton of Snf1 are discussed. | en |
dc.description.tableofcontents | 口試委員審定書 ............................................................................................ i 謝誌 ............................................................................................................... ii 摘要 ..............................................................................................................iii
Abstract ....................................................................................................... iv Contents....................................................................................................... vi List of Figures ............................................................................................. ix List of Tables ................................................................................................ x
Chapter I: Introduction .............................................................................. 1
1.1 Budding yeast as a model organism.......... ..........................................1 1.2 Ubiquitin-mediated regulation .............................................................1 1.2.1 The ubiquitin-proteasome system...................... .............................1 1.2.2 Deubiquitinases: Ubp8 and Ubp10......................................................2 1.3 Structure and regulation of Snf1............................................................3 1.3.1 AMPK..................................................................................................3 1.3.2 Yeast AMPK: Snf1 complex..................................................................4 1.3.3 The regulation of Snf1 complex ....................................... ..................4 1.4 Aging in yeast ........................................................................................5 1.4.1 Replicative life span ............................................................................5 1.4.2 Chronological life span.......................................................................6
1.4.3 Adaptive regrowth .............................................................................7 1.4.4 The UPS in aging................................................................................7 1.4.5 The role of Snf1 complex in RLS.........................................................7 1.4.6 The role of Snf1 complex in CLS........................................................8
1.5 Objective of this study..........................................................................9
Chapter II: Materials and methods.........................................................10
2.1 Materials..........................................................................................10
2.1.1 Yeast strains .................................................................................10 2.1.2 Plasmids.........................................................................................10 2.1.3 Medium..........................................................................................11
2.2 Methods .............................................................................................13
2.2.1 The stability of Snf1-TAP at exponential stage ................................13 2.2.2 The mRNA expression levels of SNF1 in the mutants .......................14 2.2.3 The levels of FLAG-Snf1 protein turn-over .......................................14 2.2.4 The purification of Ub-conjugated proteins .....................................15 2.2.5 Spotting assay for alternative carbon sources and oxidative stresses.........15
2.2.6 Replicative lifespan measuremnt.......................................................15 2.2.7 Chronological lifespan measurement ..............................................16
Chapter III: Results .................................................................................. 17
3.1 Snf1 is the potential target of both Ubp8 and Ubp10...........................17 3.2 Functional interaction between Snf1 and Ubp8/Ubp10 .......................19
3.3 To determine the effect of decreased Snf1 on physiological functions...22
3.3.1 The ability of using alternative carbon sources ................................22 3.3.2 Replicative aging .............................................................................24 3.3.3 Chronological aging ........................................................................24
3.4 The role of Snf1 during aging ..............................................................25
3.4.1 The Snf1 protein levels during CLS ...................................................25 3.4.2 The effect of Snf1 on calorie restriction ............................................27 3.4.3 The stress resistance of aging cells in the absence of Ubp8/Ubp10 ..27 3.4.4 The regrowth phenotype .................... ...........................................28 3.4.5 The phosphorylation of Snf1 in DUB-deficient cells ................ .........31
Chapter IV: Discussion ............................................................................. 33
4.1 The decreased level of Snf1............................. ....................................33 4.2 The decreased Snf1 protein level and aging phenotype .......................34 4.3 AMPK activity in CR pathway.................. ..............................................35 4.4 Hyperphosphorylation of AMPK ...........................................................36 4.5 Furture work........................... ............................................................37
Chapter V: References .............................................................................. 39 Appendix ................................................................................................. 44 | zh_TW |
dc.format.extent | 3029842 bytes | - |
dc.format.mimetype | application/pdf | - |
dc.language | en_US | - |
dc.rights | 論文使用權限:不同意授權 | - |
dc.subject | Snf1 | zh_TW |
dc.subject | AMPK | zh_TW |
dc.subject | 複製存活壽命 | zh_TW |
dc.subject | 繼代存活壽命 | zh_TW |
dc.subject | 去泛素化酵素 | zh_TW |
dc.subject | 壓力力反應序列 | zh_TW |
dc.title | 腺苷酸活化蛋白激酶之泛素媒介調節影響酵母菌老老化之機制研究 | zh_TW |
dc.title | The Ubiquitin-mediated Regulation of AMPK in Chronological Aging | en |
dc.type | thesis | en |
dc.identifier.uri.fulltext | http://ntur.lib.ntu.edu.tw/bitstream/246246/262466/1/ntu-103-R00641023-1.pdf | - |
item.openairetype | thesis | - |
item.fulltext | with fulltext | - |
item.openairecristype | http://purl.org/coar/resource_type/c_46ec | - |
item.grantfulltext | open | - |
item.cerifentitytype | Publications | - |
顯示於: | 食品科技研究所
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