Alpha-突触核蛋白与完整线粒体相互作用的NMR研究

doi:10.11938/cjmr20202841

摘要/Abstract

摘要：

天然无结构蛋白alpha-突触核蛋白（α-synuclein）能影响线粒体的形态、动力学及损害线粒体正常功能，被认为是帕金森症的致病机制之一.α-synuclein与线粒体模拟膜及分离提取的完整线粒体的相互作用研究是理解该蛋白如何影响线粒体的有效途径.文献报道α-synuclein能与磷脂模拟的线粒体内膜相互作用却不与模拟的外膜相互作用，且N端在与线粒体的结合中扮演重要角色，但具体的作用位点仍不十分清楚.本文利用核磁共振（NMR）方法研究了α-synuclein与大鼠肝脏中分离的完整线粒体的相互作用，发现α-synuclein能与线粒体外膜相互作用，且作用区域位于α-synuclein N端的前60个氨基酸残基.这一结果与文献报道并不相同，可能是因为α-synuclein与线粒体外膜的作用不仅依赖于膜组分，可能也和膜的曲率或膜上其他组分等相关，而这些是模拟膜所不易体现的.同时，我们的研究也证实NMR是研究蛋白质与分离完整线粒体相互作用的有效方法.

关键词: 核磁共振(NMR), alpha-突触核蛋白, 完整线粒体, 相互作用

Abstract:

The intrinsically disordered alpha-synuclein (α-synuclein) directly affects mitochondrial dynamics, morphology and function, and is closely related to Parkinson's disease. Studying the interactions between α-synuclein and mimic mitochondrial membranes/intact mitochondria is an effective way to understand how α-synuclein affects mitochondria. It has been reported that α-synuclein interacts with the mimic inner membrane of mitochondria (IMM), but not with the outer membrane of mitochondria (OMM), and the N-terminal plays an important role in binding with the mitochondria. However, little is known about the positions of interaction. Here, the interactions between α-synuclein and intact mitochondria isolated from rat liver were investigated by nuclear magnetic resonance (NMR). It was found that, different from the previous report, α-synuclein interacted with the OMM and the first 60 residues of N-terminus were crucial for the binding. It was postulated that the interaction between α-synuclein and OMM might depend not only on the lipid composition of the membrane, but also on other factors which were not reflected in the mimic membrane, such as the curvature of membrane and/or other components of the OMM. Our study also indicated that NMR is an effective method for studying the interaction of proteins with intact mitochondria.

Key words: nuclear magnetic resonance (NMR), α-synuclein, intact mitochondria, interaction

中图分类号:

O482.53

余锦波,张偲,张则婷,徐国华,李从刚. Alpha-突触核蛋白与完整线粒体相互作用的NMR研究[J]. 波谱学杂志, 2021, 38(2): 164-172.

Jin-bo YU,Cai ZHANG,Ze-ting ZHANG,Guo-hua XU,Cong-gang LI. Interactions Between α-synuclein and Intact Mitochondria Studied by NMR[J]. Chinese Journal of Magnetic Resonance, 2021, 38(2): 164-172.

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参考文献 34

1	POLYMEROPOULOS M H , LAVEDAN C , LEROY E , et al. Mutation in the alpha-synuclein gene identified in families with Parkinson's disease[J]. Science, 1997, 276 (5321): 2045- 2047. doi: 10.1126/science.276.5321.2045
2	SPILLANTINI M G , SCHMIDT M L , LEE V M Y , et al. Alpha-synuclein in Lewy bodies[J]. Nature, 1997, 388 (6645): 839- 840. doi: 10.1038/42166
3	MASLIAH E , ROCKENSTEIN E , VEINBERGS I , et al. Dopaminergic loss and inclusion body formation in alpha-synuclein mice: Implications for neurodegenerative disorders[J]. Science, 2000, 287 (5456): 1265- 1269. doi: 10.1126/science.287.5456.1265
4	FINK A L . The aggregation and fibrillation of alpha-synuclein[J]. Accounts Chem Res, 2006, 39 (9): 628- 634. doi: 10.1021/ar050073t
5	ZAMMIT V A , RAMSAY R R , BONOMINI M , et al. Carnitine, mitochondrial function and therapy[J]. Adv Drug Deliver Rev, 2009, 61 (14): 1353- 1362. doi: 10.1016/j.addr.2009.04.024
6	PALADE G E . The fine structure of mitochondria[J]. Anat Record, 1952, 114 (3): 427- 451. doi: 10.1002/ar.1091140304
7	FREY T G , MANNELLA C A . The internal structure of mitochondria[J]. Trends Biochem Sci, 2000, 25 (7): 319- 324. doi: 10.1016/S0968-0004(00)01609-1
8	LIN M T , CANTUTI-CASTELVETRI I , ZHENG K , et al. Somatic mitochondrial DNA mutations in early Parkinson and incidental Lewy body disease[J]. Ann Neurol, 2012, 71 (6): 850- 854. doi: 10.1002/ana.23568
9	SHAVALI S , BROWN-BORG H M , EBADI M , et al. Mitochondrial localization of alpha-synuclein protein in alpha-synuclein overexpressing cells[J]. Neurosci Lett, 2008, 439 (2): 125- 128. doi: 10.1016/j.neulet.2008.05.005
10	LI L , NADANACIVA S , BERGER Z , et al. Human A53T alpha-synuclein causes reversible deficits in mitochondrial function and dynamics in primary mouse cortical neurons[J]. PloS ONE, 2013, 8 (12): e85815. doi: 10.1371/journal.pone.0085815
11	PERFEITO R , LAZAR D F , OUTEIRO T F , et al. Linking alpha-synuclein phosphorylation to reactive oxygen species formation and mitochondrial dysfunction in SH-SY5Y cells[J]. Mol Cell Neurosci, 2014, 62, 51- 59. doi: 10.1016/j.mcn.2014.08.002
12	GANJAM G K , BOLTE K , MATSCHKE L A , et al. Mitochondrial damage by alpha-synuclein causes cell death in human dopaminergic neurons[J]. Cell Death Dis, 2019, 10 (11): 865- 879. doi: 10.1038/s41419-019-2091-2
13	SCHAPIRA A H V . Mitochondria in the aetiology and pathogenesis of Parkinson's disease[J]. Lancet Neurol, 2008, 7 (1): 97- 109. doi: 10.1016/S1474-4422(07)70327-7
14	MIZUNO Y , IKEBE S , HATTORI N , et al. Role of mitochondria in the etiology and pathogenesis of Parkinson's disease[J]. Biochim Biophys Acta, 1995, 1271 (1): 265- 274. doi: 10.1016/0925-4439(95)00038-6
15	ZIGONEANU I G , YANG Y J , KROIS A S , et al. Interaction of alpha-synuclein with vesicles that mimic mitochondrial membranes[J]. BBA-Biomembranes, 2012, 1818 (3): 512- 519. doi: 10.1016/j.bbamem.2011.11.024
16	ROBOTTA M , GERDING H R , VOGEL A , et al. Alpha-synuclein binds to the inner membrane of mitochondria in an alpha-helical conformation[J]. Chembiochem, 2014, 15 (17): 2499- 2502. doi: 10.1002/cbic.201402281
17	ROBOTTA M , HINTZE C , SCHILDKNECHT S , et al. Locally resolved membrane binding affinity of the N-terminus of alpha-synuclein[J]. Biochemistry, 2012, 51 (19): 3960- 3962. doi: 10.1021/bi300357a
18	GAO D L , SUN P , WANG Q W , et al. Interactions between albumin and fatty acids studied by NMR spectroscopy[J]. Chinese J Magn Reson, 2018, 35 (3): 338- 344.
	高东莉, 孙鹏, 王倩文, 等. 运用NMR研究白蛋白与脂肪酸的相互作用[J]. 波谱学杂志, 2018, 35 (3): 338- 344.
19	NING C F , MA M J , GUO C H , et al. Interactions between 5-fluorouracil and PAMAM dendrimers studies by NMR spectroscopy[J]. Chinese J Magn Reson, 2019, 36 (4): 555- 562.
	宁彩芳, 马敏珺, 郭朝晖, 等. PAMAM树状大分子与5-氟尿嘧啶相互作用的NMR研究[J]. 波谱学杂志, 2019, 36 (4): 555- 562.
20	THEILLET F X , BINOLFI A , BEKEI B , et al. Structural disorder of monomeric alpha-synuclein persists in mammalian cells[J]. Nature, 2016, 530 (7588): 45- 50. doi: 10.1038/nature16531
21	ZHENG W W , ZHANG Z T , YE Y S , et al. Phosphorylation dependent alpha-synuclein degradation monitored by in-cell NMR[J]. Chem Commun, 2019, 55 (75): 11215- 11218. doi: 10.1039/C9CC05662A
22	YE Y S , LIU X L , XU G H , et al. Direct observation of Ca²⁺-induced calmodulin conformational transitions in intact xenopus laevis oocytes by ¹⁹F NMR spectroscopy[J]. Angew Chem Int Edit, 2015, 54 (18): 5328- 5330. doi: 10.1002/anie.201500261
23	XU G H , YE Y S , LIU X L , et al. Strategies for protein NMR in escherichia coli[J]. Biochemistry, 2014, 53 (12): 1971- 1981. doi: 10.1021/bi500079u
24	YE Y S , LIU X L , CHEN Y H , et al. Labeling strategy and signal broadening mechanism of protein NMR spectroscopy in xenopus laevis oocytes[J]. Chemistry, 2015, 21 (24): 8686- 8690. doi: 10.1002/chem.201500279
25	YE Y S , LIU X L , ZHANG Z T , et al. ¹⁹F NMR spectroscopy as a probe of cytoplasmic viscosity and weak protein interactions in living cells[J]. Chem-Eur J, 2013, 19 (38): 12705- 12710. doi: 10.1002/chem.201301657
26	HOYER W , ANTONY T , CHERNY D , et al. Dependence of alpha-synuclein aggregate morphology on solution conditions[J]. J Mol Biol, 2002, 322 (2): 383- 393. doi: 10.1016/S0022-2836(02)00775-1
27	DAI C Y , LIU M L , LI C G . Salt content-dependent conformational changes of alpha-synuclein studied by ¹⁹F NMR[J]. Chinese J Magn Reson, 2015, 32 (1): 33- 44.
	戴晨晔, 刘买利, 李从刚. 低盐和高盐环境下α-synuclein构象的¹⁹F NMR研究[J]. 波谱学杂志, 2015, 32 (1): 33- 40.
28	COLLOMBET J M , WHEELER V C , VOGEL F , et al. Introduction of plasmid DNA into isolated mitochondria by electroporation[J]. J Biol Chem, 1997, 272 (8): 5342- 5347. doi: 10.1074/jbc.272.8.5342
29	PARIHAR M S , PARIHAR A , FUJITA M , et al. Mitochondrial association of alpha-synuclein causes oxidative stress[J]. Cell Mol Life Sci, 2008, 65 (7-8): 1272- 1284. doi: 10.1007/s00018-008-7589-1
30	SCHMITT S , SCHULZ S , SCHROPP E M , et al. Why to compare absolute numbers of mitochondria[J]. Mitochondrion, 2014, 19, 113- 123. doi: 10.1016/j.mito.2014.06.005
31	MIWA S , TREUMANN A , BELL A , et al. Carboxylesterase converts Amplex red to resorufin: Implications for mitochondrial H₂O₂ release assays[J]. Free Radical Biol Med, 2016, 90, 173- 183. doi: 10.1016/j.freeradbiomed.2015.11.011
32	ZHANG Z T , DAI C Y , BAI J , et al. Ca²⁺ modulating alpha-synuclein membrane transient interactions revealed by solution NMR spectroscopy[J]. BBA-Biomembranes, 2014, 1838 (3): 853- 858. doi: 10.1016/j.bbamem.2013.11.016
33	FUSCO G , DE SIMONE A , GOPINATH T , et al. Direct observation of the three regions in alpha-synuclein that determine its membrane-bound behaviour[J]. Nat Commun, 2014, 5, 3827- 3834. doi: 10.1038/ncomms4827
34	ZHANG Z T , JIANG X , XU D R , et al. Calcium accelerates SNARE-mediated lipid mixing through modulating alpha-synuclein membrane interaction[J]. BBA-Biomembranes, 2018, 1860 (9): 1848- 1853. doi: 10.1016/j.bbamem.2018.03.025

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