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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 鄧述諄(Shu-Chun Teng) | |
dc.contributor.author | Chang-Han Chen | en |
dc.contributor.author | 陳昶翰 | zh_TW |
dc.date.accessioned | 2021-06-16T09:52:27Z | - |
dc.date.available | 2022-02-24 | |
dc.date.copyright | 2017-02-24 | |
dc.date.issued | 2017 | |
dc.date.submitted | 2017-01-12 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/60043 | - |
dc.description.abstract | 老化為具有生理功能的逐漸喪失相關聯之複雜的現象。其本身是由內在因素和外在因素所調節。以前的研究顯示,利用熱量限制延長壽命是透許多磷酸化激酶的參與下,以磷酸化蛋白間的訊息傳遞所調控。然而,這些信號的磷酸化途徑是如何協調的詳細分子機制仍然知之甚少。在這裡,我們實驗室利用質譜儀的技術大規模地尋找在限制熱量攝入的酵母磷酸化和去磷酸化位點。質譜總共定量出1244磷酸化/去磷酸化位點,共632磷酸化蛋白有受到調控的變化。 從發現的磷酸化蛋白選出135個進行功能篩選,最後篩選出一個受到PKA磷酸化的有絲分裂相關的未知功能蛋白Ids2,在老化進程作出一定貢獻的一個調控。Ids2可能扮演著一個輔助性熱休克蛋白,與熱休克蛋白Hsc82和冗餘的Hsp82形成複合物。Ids2和Hsc82之間有交互作用,且Ids2模擬磷酸化的時候它與Hsc82間的交互作用產生減弱,可能進而造成Ids2模擬磷酸化菌株產生熱敏性和壽命縮短的現象。此外,我們也初步地分析Ids2以及Hsc82之間是利用各自何處的胜肽區域來產生交互作用。由結果推論,酵母菌偵測熱量攝入的變化,進而透過PKA的活化與否去影響到HSP90伴侶相關的折疊的活性,並由此影響個體的壽命。
甲基轉移酶SMYD3多發現於癌細胞中,目前相關研究以及報導所執行的功能為組蛋白甲基酶。在細胞核中,它甲基化染色體上的組蛋白H3K4、H4K5、H4K20和我們實驗室所發現的H2A.Z。在G0/G1期,SMYD3存在於細胞質當中,並甲基化MAP3K2、VEGFR1以及,過表現促進細胞生長、分化以及產生癌化。總結之前文獻發現,SMYD3為一個具有可甲基化不同蛋白進而影響細胞功能的蛋白。為了探索SMYD3新的可能性功能,我們利用酵母菌雙雜合系統,將SMYD3做為餌來找尋產生交互作用的可能蛋白。整理、分類以及功能確認這些蛋白後,統整出一列表。這列表往後將與我們實驗室另外利用串聯式質譜法所找尋出可能與SMYD3交互作用的可能蛋白列表進行合併,來探討或是找尋它目前尚未知的功能,以及與這些可能產生交互作用的蛋白當中,SMYD3所扮演的角色,以及是否會影響細胞功能、甚至導致癌化的可能。 | zh_TW |
dc.description.abstract | Aging, an intricate phenomenon associated with the gradual loss of physiological functions, is regulated by intrinsic and extrinsic factors. Previous studies indicate that calorie restriction extends the life span of a variety of species due to the involvement of multiple kinase regulations; however, the molecular mechanism of how these signaling phosphorylation pathways are coordinated is still poorly understood. Here we globally identify yeast phosphorylation and dephosphorylation sites under calorie restriction. Quantitative mass spectrometry identified a total of 1244 phosphorylation/ dephosphorylation sites on 632 proteins. Functional screen of 135 potential regulators further revealed a mitotically functional unknown protein Ids2 phosphorylated by PKA under rich medium, contributing to a key regulation in aging process. Ids2 may serve as a cochaperone to form a complex with Hsc82 and the redundant Hsp82. Ids2 and Hsc82 may interact with each other, and ids2 or ids2 phosphor-mimicking would reduce such interaction. ids2 or ids2 phosphor-mimicking cells displayed heat sensitivity and life span shortening. Furthermore, we use domain map to analyze interacting motifs between Ids2 and Hsc82. Taken together, yeast cells sense calorie intake and compromise HSP90 chaperone folding activity through PKA, and thereby influence the lifespan.
The methyltransferase SMYD3 is highly expressed in a variety of cancer cells and is found to aid in several oncogenic events. According to previous studies, SMYD3 is a universal methyltransferase in cells. It mediates the methylation of histone H3K4, H4K5, H4K20 and H2A.Z in the nucleus. SMYD3 is in the cytoplasm in G0/G1 phase, and recent studies identified that it can methylate MAP3K2 and VEGFR1 to facilitate metastasis. SMYD3 overexpression enhances cell growth and cancer invasion. To extend our knowledge based on how this methyltransferase may mediate oncogenesis, we have established a yeast two-hybrid system to hunt novel SMYD3 interacting proteins, classified those proteins based on their cellular functions, and listed out potential substrates. We may combine this list with the data derived by IP-mass, and test its functional significance by performing phenotypic or functional assays to unravel a new role of SMYD3 in directing cancer cell proliferation. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T09:52:27Z (GMT). No. of bitstreams: 1 ntu-106-R03445129-1.pdf: 2884745 bytes, checksum: bbaa09e5946321f734f76750d1b4c34a (MD5) Previous issue date: 2017 | en |
dc.description.tableofcontents | 口試委員審定書. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i
致謝. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .ii 摘要. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v TABLE OF CONTENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .vii PART1: Identification of interaction between Ids2 and Hsc82 in regulating life span in yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2 MATERIALS & METHODS Plasmids and Yeast Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Tandem Affinity Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Samples Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 LC-MS/MS Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12 Bioinformatics Analysis - Gene Ontology Enrichment Analysis & Kegg’s Othology . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14 Stress Resistance Assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . .15 Co- immunoprecipitation (Co-IP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15 Yeast Two-Hybrid analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16 Gel electrophoresis and Western blot analysis . . . . . . . . . . . . . . . . . . .. . . . . . . . .16 Choronlogical Life Span. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 In-vitro Kinase Assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . 18 RESULTS Identification of (de)Phosphorylated Proteins and Their Sites under Calorie Restriction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20 Specification of Mass Spectrometry-derived Candidates with Bioinformatics, Databases and Functional Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 PKA Regulates Ids2 S148 under CR condition. . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Interaction Between Ids2 and the Yeast Chaperone Protein HSP90. . . . . . . . . . . . 25 Interaction between Ids2 and the yeast chaperone protein HSP90 may regulate heat shock, life span in yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Ids2 may participate in the coordination of HSP90-regulated chaperone protein folding response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . 28 Domain Mapping of Interacting Motif in Ids2 and Hsc82. . . . . . . . . . . . . . . . . . . .29 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 PART2: Hunting for SMYD3 interacting proteins. . . . . . . . . . . . . . . . . . . . 43 INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 MATERIALS & METHODS Yeast Strains and Plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 cDNA library kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 Transformation efficiency and cDNA library coverage . . . . . . . . . . . . . . . . . . . . . 47 Yeast Two-Hybrid analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 RESULTS List of SMYD3 interacting proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 DISCUSSION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Fig.1: Integrated experimental and computational pipeline to determine in vivo phosphorylation or dephosphorylation substrates under CR condition. Fig.2: Schematic Representation of Mass Spectrometry-derived Candidates Specified with Bioinformatic Databases Fig.3: Interaction between Ids2 and the yeast HSP90 Fig.4: Interaction between Ids2 and the yeast HSP90 may regulate heat shock and life span in yeast. Fig.5: Domain mapping of interacting motif in Ids2 and Hsc82. Fig.6: List of SMYD3 interacting proteins TABLES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 Table.1 Yeast Strains and Plasmids Used in This Study Table.2 Oligonucleotides Used in this Study REFERENCES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .69 APPENDIX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .85 App.1: PKA regulates Ids2 S148 under CR condition App.2: Interaction between Ids2 and the yeast HSP90 App.3: Interaction between Ids2 and the yeast HSP90 may regulate heat shock and life span in yeast. App4: Interaction between Ids2 and the yeast HSP90 may regulate heat shock, life span in yeast and Ids2 may participate in the coordination of HSP90-regulated chaperone protein folding response App5: Ids2 may play an important role which partcipates in proteostasis in aging in yeast App6: Interaction between Ids2 and the HSP90. App7: Interaction between Ids2 and the yeast HSP90 may regulate heat shock, life span in yeast and Ids2 may participate in the coordination of HSP90-regulated chaperone protein folding response App8: Interaction between Ids2 and the yeast HSP90 may regulate heat shock, life span in yeast and Ids2 may participate in the coordination of HSP90-regulated chaperone protein folding response | |
dc.language.iso | en | |
dc.title | 探討Ids2與熱休克蛋白Hsc82之間的結合進而調節酵母菌之壽命以及尋找與人類甲基轉移酶SMYD3結合之可能蛋白 | zh_TW |
dc.title | Identification of interaction between Ids2 and Hsc82 in regulating life span in yeast and hunting for SMYD3 interacting proteins | en |
dc.type | Thesis | |
dc.date.schoolyear | 105-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 林敬哲(Jing-Jer Lin),劉雅雯(Ya-Wen Liu) | |
dc.subject.keyword | 老化,熱量限制,磷酸化,PKA,HSP90,SMYD3,SMYD3,甲基轉移?,癌化,酵母菌雙雜合系統,癌症, | zh_TW |
dc.subject.keyword | aging,calorie restriction,phosphorylation,PKA,HSP90,SMYD3,methyltransferase,oncogenic events,yeast two- hybrid,cancer, | en |
dc.relation.page | 93 | |
dc.identifier.doi | 10.6342/NTU201700056 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2017-01-12 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 微生物學研究所 | zh_TW |
顯示於系所單位: | 微生物學科所 |
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