Materials and Methods Som Text Figs. S1 and S2 Table S1 References Abad Directly Links A␤ to Mitochondrial Toxicity in Alzheimer's Disease-猫眼课题宝

DOI：

发表时间：

期刊：

影响因子：

通讯作者：

中科院分区：

其他

文献类型：

作者：

J. Lustbader;M. Cirilli;Chang Lin;Hong Wei Xu;K. Takuma;Ning Wang;C. Caspersen;X. Chen;S. Pol

线粒体功能障碍是阿尔茨海默病（AD）中β-淀粉样蛋白（A β）诱导的神经元毒性的标志。在这里，我们证明了A β结合醇脱氢酶（ABAD）是从A β到线粒体毒性的直接分子联系。在AD患者和转基因小鼠的线粒体中，A β与ABAD相互作用。A β结合的ABAD的晶体结构显示阻止烟酰胺腺嘌呤二核苷酸（NAD）结合的活性位点的实质性变形。ABAD肽特异性抑制ABAD-A β相互作用并抑制神经元中A β诱导的细胞凋亡和自由基产生。在富含A β的环境中过表达ABAD的转基因小鼠表现出过度的神经元氧化应激和受损的记忆。这些数据表明，ABAD-A β相互作用可能是AD的治疗靶点。人ABAD（也称为ERAB和HSD-10）是在针对人脑和HeLa cDNA文库的酵母双杂交筛选中从结合A β的四个阳性克隆中鉴定的唯一蛋白质（1，2）。生物化学表征已经确定ABAD和A β之间的相互作用是高度特异性的，并且在纳摩尔浓度下开始发生。在微摩尔浓度下，可能以其寡聚体形式存在的A β抑制ABAD酶活性（1，3，4）。ABAD似乎在线粒体中具有重要的生理作用（1，3），果蝇ABAD（scully）的突变失活导致致死表型（5）。ABAD在AD中受影响的神经元中上调（1）（图S1），ABAD与突变型淀粉样前体蛋白（mAPP）的共表达加剧了AD诱导的细胞氧化应激和细胞死亡（1，3）。因此，我们推测，ABAD与A β的相互作用可能会导致线粒体功能障碍。然而，因为它还没有建立，细胞内的A β可以访问线粒体，它是必不可少的，以确定是否ABAD和A β在病理生理相关的设置相互作用。为了直接解决这一问题，我们通过用抗ABAD免疫球蛋白G（IgG）免疫沉淀皮质蛋白提取物，然后用抗ABAD免疫球蛋白G（IgG）免疫印迹，检测AD脑中的ABAD-A β复合物[图1A和支持性在线材料（SOM）文本S1]。匹配的非痴呆大脑显示很少的ABAD-A复合物。由于细胞和线粒体的完整性可能在死亡后不久就开始恶化，允许非生理性相互作用发生，我们从表达mAPP（6）、ABAD（7）或两者的12月龄小鼠的大脑皮层中分离线粒体，这些小鼠由血小板衍生生长因子B链启动子驱动（SOM文本S2和S3）。线粒体制备物的纯度通过细胞色素c氧化酶IV（考克斯IV）的富集以及溶酶体（组织蛋白酶D）和内质网（蛋白质二硫化物）的相对缺乏来证实。

Mitochondrial dysfunction is a hallmark of ␤-amyloid (A␤)–induced neuronal toxicity in Alzheimer's disease (AD). Here, we demonstrate that A␤-binding alcohol dehydrogenase (ABAD) is a direct molecular link from A␤ to mito-chondrial toxicity. A␤ interacts with ABAD in the mitochondria of AD patients and transgenic mice. The crystal structure of A␤-bound ABAD shows substantial deformation of the active site that prevents nicotinamide adenine dinucleotide (NAD) binding. An ABAD peptide specifically inhibits ABAD-A␤ interaction and suppresses A␤-induced apoptosis and free-radical generation in neurons. Trans-genic mice overexpressing ABAD in an A␤-rich environment manifest exaggerated neuronal oxidative stress and impaired memory. These data suggest that the ABAD-A␤ interaction may be a therapeutic target in AD. Human ABAD (also known as ERAB and HSD-10) was the only protein identified from four positive clones that bound A␤ in a yeast two-hybrid screen against human brain and HeLa cDNA libraries (1, 2). Biochemical characterization has established that the interaction between ABAD and A␤ is highly specific and starts to occur at nanomolar concentrations. At micromolar concentrations, A␤, likely in its oligomeric form, inhibits ABAD enzymatic activity (1, 3, 4). ABAD appears to have an essential physiological role in mitochondria (1, 3), and mutational inactiva-tion of Drosophila ABAD (scully) resulted in a lethal phenotype (5). ABAD is up-regulated in affected neurons in AD (1) (fig. S1), and coexpression of ABAD with mutant amyloid precursor protein (mAPP) exacerbates A␤-induced cellular oxidant stress and cell death (1, 3). Thus, we speculated that the interaction of A␤ with ABAD might induce mito-chondrial dysfunction. However, because it had not been established that intracellular A␤ can access mito-chondria, it was essential to determine whether ABAD and A␤ interact in pathophysiologically relevant settings. To address this directly, we detected ABAD-A␤ complex in AD brains by immunoprecipitating cortical protein extracts with anti-A␤ and then by immunoblot-ting with anti-ABAD immunoglobulin G (IgG) [Fig. 1A and supporting online material (SOM) text S1]. Age-matched nondemented brain displayed very little ABAD-A␤ complex. Because cellular and mitochondrial integrity may start to deteriorate soon after death, allowing nonphysiological interactions to occur, we isolated mitochondria from the cerebral cortex of 12-month-old mice expressing mAPP (6), ABAD (7), or both, driven by the platelet-derived growth factor B-chain promoter (SOM text S2 and S3). The purity of mitochondrial preparations was confirmed by the enrichment of cyto-chrome c oxidase IV (Cox IV), and the relative absence of lysosomal (cathepsin D) and endoplasmic reticulum [protein disul-fide …

Materials and Methods Som Text Figs. S1 and S2 Table S1 References Abad Directly Links A␤ to Mitochondrial Toxicity in Alzheimer's Disease

文献摘要

被引文献

相似文献