南京大学学报(自然科学版) ›› 2016, Vol. 52 ›› Issue (4): 609.
吕万胜1,2,朱伶俐1,2,吴先登1,王泽南1,郭文洁3,高 静3,华子春1,2*,郑伟娟1,2*
Lyu Wansheng1,2,Zhu Lingli1,2,Wu Xiandeng1,Wang Ze’nan1,Guo Wenjie3,Gao Jing3,Hua Zichun1,2*,Zheng Weijuan1,2*
摘要: 电压依赖性阴离子通道蛋白(VDAC)是线粒体外膜上最丰富的蛋白,在调节线粒体的物质代谢与能量代谢、以及线粒体介导的细胞凋亡中起重要作用,因而与肿瘤及神经退行性疾病的发生密切相关.VDAC1是三种VDAC变体中表达量最高、分布最广泛的一种.由于VDAC1的高度保守性,因此成为多种疾病治疗包括抗肿瘤治疗的理想靶点.获得足量的VDAC1蛋白是进一步研究其结构功能、筛选与VDAC1相互作用的药物分子的基础.从生物样品中直接分离纯化VDAC1,不仅条件苛刻,而且得率很低.因此利用DNA重组技术在大肠杆菌中重组表达VDAC1是获得足量蛋白、用于后续研究的重要途径.成功构建了C端带有Histag的hVDAC1的重组蛋白原核表达质粒pET28a/hVDAC1,在大肠杆菌中以包涵体形式表达,并通过盐酸胍变性、在变性条件下通过NTANi亲和层析柱纯化蛋白后进行复性的方法,获得了高纯度的hVDAC1.圆二色谱检测显示该蛋白二级结构以β-折叠为主、符合hVDAC1的二级结构特征.利用流式细胞仪检测结果显示:FITC荧光标记的hVDAC1可以与脂质体膜、细胞膜特异性结合.证明克隆表达纯化的hVDAC1具有特征的膜蛋白结构和功能,为体外筛选与hVDAC1相互作用的各类分子奠定了基础.
[1] Youle R J,Narendra D P.Mechanisms of mitophagy.Nature Reviews Molecular Cell Biology,2011,12(1):9-14. [2] Colombini M,Mannella C A.VDAC,the early days.Biochimica et Biophysica Acta,2012,1818(6):1438-1443. [3] ShoshanBarmatz V,De Pinto V,Zweckstetter M,et al.VDAC,a multifunctional mitochondrial protein regulating cell life and death.Molecular Aspects of Medicine,2010,31(3):227-285. [4] ShoshanBarmatz V,Golan M.Mitochondrial VDAC1:Function in cell life and death and a target for cancer therapy.Current Medicinal Chemistry,2012,19(5):714-735. [5] ShoshanBarmatz V,Mizrachi D.VDAC1:from structure to cancer therapy.Frontiers in Oncology,2012,2(164). [6] Reddy P H.Is the mitochondrial outermembrane protein VDAC1 therapeutic target for Alzheimer’s disease?Biochimica et Biophysica Acta,2013,1832(1):67-75. [7] Benz R.Permeation of hydrophilic solutes through mitochondrial outer membranes:Review on mitochondrial porins.Biochimica et Biophysica Acta,1994,1197(2):167-196. [8] Messina A,Reina S,Guarino F,et al.VDAC isoforms in mammals.Biochimica et Biophysica Acta,2012,1818(6):1466-1476. [9] Engelhardt H,Meins T,Poynor M,et al.Highlevel expression,refolding and probing the natural fold of the human voltagedependent anion channel isoforms Ⅰ and Ⅱ.The Journal of Membrane Biology,2007,216(2-3):93-105. [10] BlachlyDyson E,Zambronicz E B,Yu W H,et al.Cloning and functional expression in yeast of two human isoforms of the outer mitochondrial membrane channel,the voltagedependent anion channel.The Journal of Biological Chemistry,1993,268(3):1835-1841. [11] De Pinto V,Messina A,Lane D J,et al.Voltagedependent anionselective channel(VDAC)in the plasma membrane.FEBS Letters,2010,584(9):1793-1799. [12] ShoshanBarmatz V,Hadad N,et al.VDAC/porin is present in sarcoplasmic reticulum from skeletal muscle.FEBS Letters,1996,386(2-3):205-210. [13] ShoshanBarmatz V,Zalk R,Gincel D,et al.Subcellular localization of VDAC in mitochondria and ER in the cerebellum.Biochimica et Biophysica Acta,2004,1657(2-3):105-114. [14] Bayrhuber M,Meins T,Habeck M,et al.Structure of the human voltagedependent anion channel.Proceedings of the National Academy of Sciences of the United States of America,2008,105(40):15370-15375. [15] Hiller S,Garces R G,Malia T J,et al.Solution structure of the integral human membrane protein VDAC1 in detergent micelles.Science,2008,321(5893):1206-1210. [16] Ujwal R,Cascio D,Colletier J P,et al.The crystal structure of mouse VDAC1 at 2.3 A resolution reveals mechanistic insights into metabolite gating.Proceedings of the National Academy of Sciences of the United States of America,2008,105(46):17742-17747. [17] Hiller S,Abramson J,Mannella C,et al.The 3D structures of VDAC represent a native conformation.Trends in Biochemical Sciences,2010,35(9):514-521. [18] Geula S,BenHail D,ShoshanBarmatz V.Structurebased analysis of VDAC1:Nterminus location,translocation,channel gating and association with antiapoptotic proteins.Biochemical Journal,2012,444(3):475-485. [19] BrahimiHorn M C,BenHail D,Lie M,et al.Expression of a truncated active form of VDAC1 in lung cancer associates with hypoxic cell survival and correlates with progression to chemotherapy resistance.Cancer Research,2012,72(8):2140-2150. [20] Bai Z,Ye Y,Liang B,et al.Proteomicsbased identification of a group of apoptosisrelated proteins and biomarkers in gastric cancer.International Journal of Oncology,2011,38(2):375-383. [21] Simamura E,Shimada H,Ishigaki Y,et al.Bioreductive activation of quinone antitumor drugs by mitochondrial voltagedependent anion channel 1.Anatomical Science International,2008,83(4):261-266. [22] Yoo H J,Yun B R,Kwon J H,et al.Genetic and expression alterations in association with the sarcomatous change of cholangiocarcinoma cells.Experimental & Molecular Medicine,2009,41(2):102-115. [23] Bryson J M,Coy P E,Gottlob K,et al.Increased hexokinase activity,of either ectopic or endogenous origin,protects renal epithelial cells against acute oxidantinduced cell death.The Journal of Biological Chemistry,2002,277(13):11392-11400. [24] Pedersen P L,Mathupala S,Rempel A,et al.Mitochondrial bound type II hexokinase:Akey player in the growth and survival of many cancers and an ideal prospect for therapeutic intervention.Biochimica et Biophysica Acta,2002,1555(1-3):14-20. [25] Koppenol W H,Bounds P L,Dang C V.Otto Warburg’s contributions to current concepts of cancer metabolism.Nature Reviews Cancer,2011,11(5):325-337. [26] Shimizu S,Tsujimoto Y.Proapoptotic BH3only Bcl2 family members induce cytochrome c release,but not mitochondrial membrane potential loss,and do not directly modulate voltagedependent anion channel activity.Proceedings of the National Academy of Sciences,2000,97(2):577-582. [27] Arbel N,ShoshanBarmatz V.Voltagedependent anion channel 1based peptides interact with Bcl2 to prevent antiapoptotic activity.The Journal of Biological Chemistry,2010,285(9):6053-6062. [28] ShoshanBarmatz V,BenHail D,Admoni L,et al.The mitochondrial voltagedependent anion channel 1 in tumor cells.Biochimica et Biophysica Acta,2015,1848(10 Pt B):2547-2575. [29] ShoshanBarmatz V,BenHail D.VDAC,a multifunctional mitochondrial protein as a pharmacological target.Mitochondrion,2012,12(1):24-34. [30] Haridas V,Li X,Mizumachi T,et al.Avicins,a novel plantderived metabolite lowers energy metabolism in tumor cells by targeting the outer mitochondrial membrane.Mitochondrion,2007,7(3):234-240. [31] Yang Z,Schumaker L M,Egorin M J,et al.Cisplatin preferentially binds mitochondrial DNA and voltagedependent anion channel protein in the mitochondrial membrane of head and neck squamous cell carcinoma:Possible role in apoptosis.Clinical Cancer Research,2006,12(19):5817-5825. [32] Lai J C,Tan W,Benimetskaya L,et al.A pharmacologic target of G3139 in melanoma cells may be the mitochondrial VDAC.Proceedings of the National Academy of Sciences of the United States of America,2006,103(19):7494-7499. [33] Malia T J,Wagner G.NMR structural investigation of the mitochondrial outer membrane protein VDAC and its interaction with antiapoptotic BclxL.Biochemistry,2007,46(2):514-525. |
No related articles found! |
|