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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 劉雅雯(Ya-Wen, Liu) | |
| dc.contributor.author | Yu-Chen Chang | en |
| dc.contributor.author | 張瑜真 | zh_TW |
| dc.date.accessioned | 2021-06-17T08:22:57Z | - |
| dc.date.available | 2024-08-26 | |
| dc.date.copyright | 2019-08-26 | |
| dc.date.issued | 2019 | |
| dc.date.submitted | 2019-08-13 | |
| dc.identifier.citation | Adachi, Y., Itoh, K., Iijima, M. &Sesaki, H.(2017). Assay to Measure Interactions between Purified Drp1 and Synthetic Liposomes. Bio-Protocol 7.
Adachi, Y. et al. (2016).Coincident Phosphatidic Acid Interaction Restrains Drp1 in Mitochondrial Division. Mol. Cell 63, 1034–1043 Ban, T. et al. (2017). Molecular basis of selective mitochondrial fusion by heterotypic action between OPA1 and cardiolipin. Nat. Cell Biol. 19, 856–863 Barrera, G. et al.(2016) Mitochondrial Dysfunction in Cancer and Neurodegenerative Diseases: Spotlight on Fatty Acid Oxidation and Lipoperoxidation Products. Antioxidants 5, 7 Boissan, M., Schlattner, U. &Lacombe, M. L.(2018) The NDPK/NME superfamily: State of the art. Lab. Investig. 98, 164–174 Chakraborty, T. R., Vancura, A., Balija, V. S. &Haldar, D. (2002). Phosphatidic Acid Synthesis in Mitochondria. J. Biol. Chem. 274, 29786–29790 Chen, C.-W. et al. (2019). Two separate functions of NME3 critical for cell survival underlie a neurodegenerative disorder. Proc. Natl. Acad. Sci. 116, 566–574 Cipolat, S., deBrito, O. M., Dal Zilio, B. &Scorrano, L. (2004). OPA1 requires mitofusin 1 to promote mitochondrial fusion. Proc. Natl. Acad. Sci. 101, 15927–15932 Drin, G. &Antonny, B. Amphipathic helices and membrane curvature. (2010). FEBS Lett. 584, 1840–1847 ElKhoury, M. et al. (2017).Targeting Bacterial Cardiolipin Enriched Microdomains: An Antimicrobial Strategy Used by Amphiphilic Aminoglycoside Antibiotics. Sci. Rep. 7, 1–12 Flis, V.V &Daum, G. (2013).Lipid Transport between the Endoplasmic. Cold Spring Harb Perspect Biol 5, 1–22 González-Rubio, P., Gautier, R., Etchebest, C. &Fuchs, P. F. J. (2011).Amphipathic-Lipid-Packing-Sensor interactions with lipids assessed by atomistic molecular dynamics. Biochim. Biophys. Acta - Biomembr. 1808, 2119–2127 Horvath, S. E. &Daum, G. (2013).Lipids of mitochondria. Prog. Lipid Res. 52, 590–614 Lacombe, M.-L. et al. (2014). Nucleoside diphosphate kinases fuel dynamin superfamily proteins with GTP for membrane remodeling. Science (80-. ). 344, 1510–1515 Mishra, P. &Chan, D. C. (2014).Mitochondrial dynamics and inheritance during cell division, development and disease. Nat. Rev. Mol. Cell Biol. 15, 634–646 Mishra, P. &Chan, D. C. (2016).Metabolic regulation of mitochondrial dynamics. J. Cell Biol. 212, 379–387 Murphy, E. et al. (2015).The Ins and Outs of Mitochondrial Calcium. Circ. Res. 116, 1810–1819 Nelson, R. K. &Frohman, M. A. (2015)Physiological and pathophysiological roles for phospholipase D. J. Lipid Res. 56, 2229–2237. Okamoto, K., Sesaki, H., Adachi, Y., Iijima, M. &Kameoka, S. (2017). Phosphatidic Acid and Cardiolipin Coordinate Mitochondrial Dynamics. Trends in Cell Biology 28, 67–76 Praefcke, G. J. K. &McMahon, H. T. (2004) The dynamin superfamily: Universal membrane tubulation and fission molecules Nat. Rev. Mol. Cell Biol. 5, 133–147 Putta, P. et al. (2016). Phosphatidic acid binding proteins display differential binding as a function of membrane curvature stress and chemical properties. Biochim. Biophys. Acta - Biomembr. 1858, 2709–2716 Santel, A. &Fuller, M. T. (2001).Control of mitochondrial morphology by a human mitofusin. J. Cell Sci. 114, 867–74 Schrepfer, E. &Scorrano, L. (2016)Mitofusins, from Mitochondria to Metabolism. Mol. Cell 61, 683–694. Tait, S. W. G. &Green, D. R. (2012).Mitochondria and cell signalling. J. Cell Sci. 125, 807–815 Traut, T. W. (1994).Physiological concentrations of purines and pyrimidines. Mol. Cell. Biochem. 140, 1–22 van derBliek, A. M., Shen, Q. &Kawajiri, S. (2013).Mechanisms of mitochondrial fission and fusion. Cold Spring Harb. Perspect. Biol. 5, Vanni, S. et al. (2014).Polyunsaturated phospholipids facilitate membrane deformation and fission by endocytic proteins. Science (80-. ). 345, 693–697 Vanni, S. et al. (2013).Amphipathic lipid packing sensor motifs: Probing bilayer defects with hydrophobic residues. Biophys. J. 104, 575–584 Warnock, D. E., Hinshaw, J. E. &Schmid, S. L. (1996). Cell Biology and Metabolism : Dynamin Self-assembly Stimulates Its GTPase Activity Dynamin Self-assembly Stimulates Its GTPase Activity *. 271, 22310–22314 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/74174 | - |
| dc.description.abstract | 粒線體分裂、融合的動態平衡對於其功能極其重要且受到精準調控，神經性退化疾病、癌症、異常老化等與此動態失衡息息相關。然而在過去的研究中，即使負責控制粒線體型態改變的蛋白質已被充分研究，關於脂質如何影響粒線體形態的研究相對匱乏。演化上高度保守的核苷二磷酸激酶，功能為磷酸化核糖核苷二膦酸為核糖核苷三磷酸供給Dynamin蛋白進行膜重塑功能。本篇研究首先在骨骼肌細胞中發現核苷二磷酸激脢三的缺失會造成的造成粒線體碎裂。我們驚訝地透過基因下調-回復實驗發現，核苷二磷酸激脢三促進粒線體的融合，並非透過激酶作用，反而此酵素是否定位於粒線體為關鍵。核苷二膦酸激酶三N端形成特殊的兩親性螺旋結構，使其偏向結合粒線體外膜上的磷脂酸。突變N端氨基酸減低螺旋體兩親性大幅減低其粒線體定位功能。兩親性螺旋體偏好結合到脂質排列缺陷區域，透過製造多種不同成份的微脂體及浮選分析實驗，我們證實磷脂酸之錐狀結構易顯露疏水端吸引具有兩親性的純化核苷二磷酸激酶三結合。此外，利用不同構型的脂質與控制微脂體直徑，我們證實微脂體直徑顯著影響兩親性螺旋的結合，因為直徑越小的微脂體，表面弧度越大，易於顯露脂質排列缺陷區域，使脂質分子之鍊狀脂肪酸更容易被兩親性螺旋所辨認。透過螢光顯微鏡、動態光散射以及電子顯微鏡，我們觀察到核苷二磷酸激酶三透過此磷脂質結合特性以及其固有的六聚體結構，系鏈含有磷脂酸的微脂體形成較大的複微脂體聚合物，在生理上核苷二磷酸激酶三能牽引粒線體使其靠近彼此，促進粒線體融合。我的研究證明了核苷二磷酸激酶的新功能，也為脂質調控粒線體形態變化提供新的分子機制，並有利於發展純化粒線體的技術。 | zh_TW |
| dc.description.abstract | The fission and fusion dynamics of mitochondria are crucial for their role in physiological processes and are precisely regulated. As protein machineries governing mitochondrial dynamics have been fully discovered, how lipids affect mitohcondrial morphology remains comparatively unclear. Highly conserved nucleoside diphosphate kinases are previously discovered as GTP fueling enzymes for dynamin superfamily proteins during membrane remodeling processes. Here we demonstrated that NME3 is required for mitochondrial morphology maintenance that depletion of NME3 caused mitochondrial fragmentation in C2C12 myoblast as well as in myotube. Surprisingly, a kinase-dead NME3 could still rescued this phenotype, yet the N-terminus truncated NME3 could not. Utilizing biochemical reconstitution experiments, we found NME3 directly binds to phosphatidic acid (PA) and functions as a mitochondrial tethering protein through the amphipathic helix formation and oligomeric architecture. Together my study proposes a function of amphipathic helix in organelle integrity maintenance and provides a mechanistic explanation for the role of PA in mitofusin-mediated mitochondrial fusion. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T08:22:57Z (GMT). No. of bitstreams: 1 ntu-108-R06448013-1.pdf: 3218299 bytes, checksum: c368c847944e516ae299129d4d0d3e0e (MD5) Previous issue date: 2019 | en |
| dc.description.tableofcontents | Table of Contents
論文審定書 ………………………………………………………………i Acknowledgement………………………………………………………ii 中文摘要………………………………………………………………...iii ABSTRACT………………………………………………………….....iv Chapter 1 INTRODUCTION 1 1.1 Dynamin Related Proteins and mitochondrial dynamics 1 1.2 Nucleoside Diphosphate Kinase and Membrane Remodeling 2 1.3 Cardiolipin and Phosphatidic Acid Coordinate Mitochondrial Fusion and Fission 4 1.4 Role of Amphipathic helix in Membrane Biology 6 1.5 Our Hypothesis on the Function of NME3 in Mitochondrial Dynamics 7 Chapter 2 MATERIALS AND METHODS 8 2.1 Cell culture, transfection, and lentiviral infection 8 2.2 Immunofluorescent staining and imaging 10 2.3 Determination of the morphology of mitochondria 10 2.4 Liposome and SUV preparation 12 2.5 (His)6-NME3 Protein purification 12 2.6 Liposome flotation assay 14 2.7 Liposome tethering assay 15 2.8 Dynamic light scattering 15 2.9 Transmission electron microscopy 15 Chapter 3 RESULTS 17 3.1 NME3 depletion results in mitochondrial fragmentation in C2C12 myoblasts and myotubes 17 3.2 Direct mitochondrial binding of NME3 is required for the integrity of mitochondria while kinase activity is dispensable 18 3.3 The N-terminus of NME3 forms amphipathic helix thus favors association with phosphatidic acid 21 3.4 Oligomerization and PA-binding activity of NME3 facilitate mitochondrial tethering 25 Chapter 4 DISCUSSION 26 4.1 A new function of NME protein 27 4.2 Molecular basis of NME3 and PA interaction in mitochondrial fusion 28 4.3 Inhibitory effect of CL on liposome binding ability of NME3 29 4.4 A novel role of AH in membrane remodeling processes 30 Chapter 5 FIGURES 31 Figure 1. Mitochondrial remodel during C2C12 differentiation. 31 Figure 2. Effects of NME3 knockdown on mitochondrial morphology. 33 Figure 3. Mitochondrial localization of NME3 is required for the integrity of mitochondria. 35 Figure 4. Overexpression of NME3 with intact N-terminus causes mitochondrial hyperclustering. 37 Figure 5. NME3 localizes specifically to mitochondria. and have no effect on ER morphology. 39 Figure 6. The hyperclustering effect induced by NME3 overexpression is mitochondrial specific. 41 Figure 7. N-terminus of NME3 is predicted to form amphipathic helix. 43 Figure 8. N-terminal amphipathic helix formation is essential for NME3 mitochondrial localization. 45 Figure 9. schematic diagram of liposome flotation assay 47 Figure 10. Binding preference of NME3 towards phohphatidic acid (PA). 49 Figure 11. AH formation-defective mutant F9/13A has minor PA-binding competence. 51 Figure 12. Curvature-dependent binding of NME3 to 10% PA liposomes. 53 Figure 13. NME3 binds to 100% PC vesicles with extremely large curvature. 55 Figure 14. NME3 oligomerize into hexamers. 57 Figure 15. Through PA binding and oligomerization ability, NME3 tether different liposome compartments. 59 Figure 16. Dynamic light scattering analysis demonstrated the liposome tethering function of NME3 WT. 61 Figure 17. Multi-liposomes complex formed upon NME3 WT addition. 63 Figure 18. Model of NME3 tether mitochondria through PA binding ability. 65 Figure 19. Effect of NME3 in mitochondrial dynamics 67 Figure 20. Sequence alignment of human NME1/2 and NME3 69 Figure 21. Sequence alignment of NME3 in multiple species 71 Figure 22. Inhibitory effect of CL on NME3 PA binding 73 Figure 23. Role of amphipathic helix in supporting organelle integrity 75 Chapter 6 REFERENCES 77 | |
| dc.language.iso | en | |
| dc.subject | 粒線體 | zh_TW |
| dc.subject | 磷脂酸 | zh_TW |
| dc.subject | 蛋白兩親性螺旋 | zh_TW |
| dc.subject | 微脂體浮選 | zh_TW |
| dc.subject | amphipathic helix | en |
| dc.subject | phosphatidic acid | en |
| dc.subject | mitochondria | en |
| dc.subject | liposome flotation | en |
| dc.subject | membrane tethering | en |
| dc.title | 核苷二磷酸激酶三在粒線體形態變化之必要功能 | zh_TW |
| dc.title | Essential Role of Nucleoside Diphosphate Kinase NME3 on Mitochondrial Morphogenesis | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 107-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 詹迺立(Nei-Li Chan),曾秀如(Shiou-Ru Tzeng),張智芬(Zee-Fen Chang) | |
| dc.subject.keyword | 粒線體,磷脂酸,蛋白兩親性螺旋,微脂體浮選, | zh_TW |
| dc.subject.keyword | mitochondria,phosphatidic acid,amphipathic helix,membrane tethering,liposome flotation, | en |
| dc.relation.page | 79 | |
| dc.identifier.doi | 10.6342/NTU201903213 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2019-08-14 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 分子醫學研究所 | zh_TW |
| 顯示於系所單位： | 分子醫學研究所 | |
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