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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 牟昀(Yun Mou) | |
| dc.contributor.author | Meng-Sen Huang | en |
| dc.contributor.author | 黃孟森 | zh_TW |
| dc.date.accessioned | 2021-07-10T21:42:37Z | - |
| dc.date.available | 2021-07-10T21:42:37Z | - |
| dc.date.copyright | 2020-09-10 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-07-30 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76989 | - |
| dc.description.abstract | 麩醯胺酸是身體產生能量的主要營養源之一並可作為其他物質生合成的前驅物來維持細胞生長所需，根據先前研究，麩醯胺酸在癌細胞增殖中扮演重要角色。兩種主要參與麩醯胺酸攝取的溶質轉運蛋白SLC1A5和SLC38A2可以調控細胞內麩醯胺酸的累積並藉由SLC7A5進一步交換必需氨基酸攝取，但是SLC1A5和SLC38A2之間的結構和功能關係尚未明瞭。在本研究中我們採用了鄰近標記蛋白質體學來剖析SLC1A5交互作用體。首先，我們發現APEX2與溶質轉運蛋白(SLCs)融合時會影響蛋白表達。我們利用基因工程製造出APEX2C32S並改善人類細胞中融合蛋白的表達，並且不會影響酵素催化癒創木酚氧化的能力。所有SLC-APEX2C32S在西方墨點法和免疫螢光實驗中均顯示出有效的鄰近標記活性同時融合蛋白正確地表達在細胞膜上。其次，蛋白質體學鄰近標記顯示SLC38A2可能與SLC1A5相互作用。SLC1A5和SLC38A2的相互作用透過雙分子螢光互補測定法證實。另外，我們利用核磁共振實驗研究了SLC1A5和SLC38A2在人類細胞中過量表達的影響。SLC38A2積累了細胞內必需胺基酸濃度而不是SLC1A5，我們進一步提出模型說明SLC1A5與SLC38A2具有協同作用來介導必需胺基酸的攝取。最後，5年生存率全癌分析表明SLC1A5、SLC38A2及SLC7A5基因共同高度表達的組別風險最高。總體而言，我們認為APEX2C32S在鄰近標記應用中優於APEX2。另外，在人類細胞中SLC1A5可以與SLC38A2相互作用並協同必需胺基酸的攝取。 | zh_TW |
| dc.description.abstract | Glutamine is the major resource for energy production and also acts as biosynthetic precursors to maintain cell growth. Thus, glutamine plays an important role in cancer cell proliferation. Two major glutamine transporters, SLC1A5 and SLC38A2, can mediate intracellular glutamine uptake and further exchange for essential amino acids (EAAs) through SLC7A5 as previously described. However, the structural and functional relations between SLC1A5 and SLC38A2 remain unclear. Here, we employed the proximity labeling-based proteomics to dissect SLC1A5 interactome. First, we found APEX2 affected the protein expression when it was fused to solute carriers (SLCs). We engineered APEX2C32S which improved the expression of fusion proteins in human cells without affecting enzyme activity to catalyze the oxidation of guaiacol. All SLC-APEX2C32S exhibited efficient proximity labeling and were correctly localized at the plasma membrane in western blot and immunofluorescence experiments. Second, proteomic proximity labeling revealed SLC38A2 may interact with SLC1A5. The interaction between SLC1A5 and SLC38A2 was confirmed by the bimolecular fluorescence complementation assay (BiFC). Next, we investigated the effects of SLC1A5 and SLC38A2 overexpression in human cells through nuclear magnetic resonance experiments. SLC38A2 accumulated intracellular EAAs rather than SLC1A5 and we proposed a model to illustrate that SLC1A5 synergized with SLC38A2 to mediate EAAs uptake. Finally, 5-year survival rates of the pan-cancer analysis showed high co-expression of SLC1A5/SLC38A2/SLC7A5 were at the highest risk. Overall, we believed that APEX2C32S is superior to APEX2 in proximity labeling applications and SLC1A5 can interact with SLC38A2 and synergize for the uptake of EAAs in human cells. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-10T21:42:37Z (GMT). No. of bitstreams: 1 U0001-2907202011300800.pdf: 5328680 bytes, checksum: 7477a00b996e00c61bc1d77742fa457a (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | PUBLICATIONS i 誌謝 ii 摘要 iii ABSTRACT iv CONTENTS vi LIST OF FIGURES ix LIST OF TABLES xi ABBREVIATION xii Chapter 1 Introduction 1 1.1 Solute carrier transporter 1 1.2 Biology of SLC transporters in this study 3 1.2.1 SLC1A5 3 1.2.2 SLC6A5 4 1.2.3 SLC6A14 4 1.2.4 SLC7A1 5 1.3 SLC1A5 acts as a target for cancer therapy 5 1.4 MYC oncogene promotes glutamine uptake 6 1.5 Amino acids signaling in the cellular process 7 1.5.1 mTOR signaling pathway 7 1.5.2 GCN2 pathway 8 1.6 Protein-protein interaction 9 1.6.1 Protein interactome mapping 10 1.6.2 Proximity labeling 10 1.7 Liquid chromatography mass spectrometry-based proteomics 12 1.8 Nuclear magnetic resonance-based metabolomics 14 1.9 Motivation 17 Chapter 2 Materials and Methods 20 2.1 Materials and Equipment 20 2.1.1 Cell line and cell culture 20 2.1.2 Chemicals and Reagents 20 2.1.3 Kits 24 2.1.4 Buffers and solutions 24 2.1.5 Oligonucleotides 28 2.1.6 Plasmid 31 2.1.7 Equipment 33 2.2 Methods 34 2.2.1 Cell line, transfection, and lentivirus transduction 34 2.2.2 Protein expression and purification 34 2.2.3 Construct cloning 35 2.2.4 Proximity labeling 35 2.2.5 Flow cytometry analysis 36 2.2.6 Immunofluorescence imaging 36 2.2.7 Western blot 36 2.2.8 Guaiacol steady-state kinetic assays 37 2.2.9 RNA extraction and Reverse Transcription-PCR 37 2.2.10 Quantitative Real-Time PCR 38 2.2.11 Proteomic sample preparations 38 2.2.12 LC-MS/MS analysis 39 2.2.13 Data analysis 39 2.2.14 Co-immunoprecipitation 40 2.2.15 Bimolecular fluorescence complementation assay 40 2.2.16 Protein docking 41 2.2.17 NMR sample preparations 41 2.2.18 NMR experiments 42 2.2.19 Pan-cancer analysis 42 Chapter 3 Results 44 3.1 APEX2 affected protein expressions when it was fused to solute carrier transporters 44 3.2 Crystal structures demonstrated that APEX2C32S mutant may not affect the enzyme activity in proximity labeling applications 46 3.3 APEX2 and APEX2C32S had the similar activity of guaiacol oxidation 48 3.4 APEX2C32S improved expressions of SLC fusion proteins in mammalian cells 49 3.5 APEX2C32S and BASU showed efficient proximity labeling in human cells 50 3.6 SLC38A2 may be a unique PPIs candidates in SLC1A5 interactome profile 53 3.7 SLC1A5 is not co-expressed with SLC38A2 57 3.8 SLC1A5 directly interacts with SLC38A2 at the plasma membrane 58 3.9 SLC1A5 synergizes with SLC38A2 to accumulate essential amino acids in human cells 66 3.10 High expression of SLC1A5/SLC38A2 or SLC1A5/SLC38A2/SLC7A5 resulted in poor prognosis across different cancers 69 Chapter 4 Discussion 74 Chapter 5 References 80 Chapter 6 Appendix 93 | |
| dc.language.iso | en | |
| dc.subject | 液相層析串聯質譜儀 | zh_TW |
| dc.subject | 溶質轉運蛋白 | zh_TW |
| dc.subject | 核磁共振代謝體學 | zh_TW |
| dc.subject | 鄰近標記 | zh_TW |
| dc.subject | 抗壞血酸過氧化酶 | zh_TW |
| dc.subject | 蛋白相互作用 | zh_TW |
| dc.subject | 生物素蛋白連接酶 | zh_TW |
| dc.subject | NMR metabolomics | en |
| dc.subject | Solute carriers | en |
| dc.subject | Ascorbate peroxidase | en |
| dc.subject | Biotin ligase | en |
| dc.subject | Proximity labeling | en |
| dc.subject | Protein-protein interactions | en |
| dc.subject | LC-MS/MS | en |
| dc.title | 利用鄰近標記蛋白質體學探討麩醯胺酸轉運蛋白SLC1A5的交互作用體及與癌症預後之關係 | zh_TW |
| dc.title | Investigation of the glutamine transporter SLC1A5 interactome using proximity labeling-based proteomics and its relevance to cancer prognosis | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 陳世淯(Shih-Yu Chen),林妙霞(Miao-Hsia Lin) | |
| dc.subject.keyword | 溶質轉運蛋白,抗壞血酸過氧化酶,生物素蛋白連接酶,鄰近標記,蛋白相互作用,液相層析串聯質譜儀,核磁共振代謝體學, | zh_TW |
| dc.subject.keyword | Solute carriers,Ascorbate peroxidase,Biotin ligase,Proximity labeling,Protein-protein interactions,LC-MS/MS,NMR metabolomics, | en |
| dc.relation.page | 100 | |
| dc.identifier.doi | 10.6342/NTU202002024 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2020-07-30 | |
| 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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