核苷二磷酸激酶三在粒線體形態變化之必要功能

Essential Role of Nucleoside Diphosphate Kinase NME3 on Mitochondrial Morphogenesis

張瑜真(Yu-Chen Chang)

指導教授 ： 劉雅雯

國立臺灣大學/醫學院/分子醫學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201903213

摘要

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。

關鍵字

粒線體 ； 磷脂酸 ； 蛋白兩親性螺旋 ； 微脂體浮選

並列摘要

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.

並列關鍵字

mitochondria ； phosphatidic acid ； amphipathic helix ； membrane tethering ； liposome flotation

參考文獻

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