Kits to Quantitate and Analyze Mitochondrial Proteins

描述（由申请人提供）：线粒体在细胞能量代谢中发挥关键作用：它们是氧化磷酸化（OXPHOS）的所在地，其产生哺乳动物细胞所利用的所有ATP的98%。OXPHOS由5个大的膜结合蛋白复合物催化，这些蛋白复合物由90个多肽组成。底物进入OXPHOS系统是由第六个大的酶复合物，丙酮酸脱氢酶复合物控制的。这六种复合物中的遗传缺陷导致了一系列异质性的人类疾病，这些疾病被统称为线粒体疾病，每6000例活产中就有1例受到影响。线粒体疾病仍然难以诊断，这些疾病的基因型-表型关系仍然不清楚。有几种迟发性疾病，其中线粒体功能障碍被认为是因果关系和/或预测性的，包括帕金森病、阿尔茨海默病、早发性和迟发性糖尿病、精神分裂症和某些形式的心脏病。所有这些病症的共同之处在于它们是由氧化损伤的积累引起的，导致细胞死亡，所述细胞死亡是由特别是Escherion和OXPHOS组分引起的并涉及Escherion和OXPHOS组分。与线粒体遗传疾病一样，上述迟发性疾病的病因学仍然不清楚，并且由于缺乏足够的、简单的高通量方法来分析OXPHOS组分的组成、酶活性和翻译后（氧化）修饰，因此它们的诊断仍然困难。这项拨款将用于建立基于免疫学的分析方法。单克隆抗体（mAb）已从少量人类和其他哺乳动物组织中产生，其捕获功能活性完整形式的4种50 XPHOS复合物和丙酮酸脱氢酶。捕获复合物III的mAb的筛选正在进行中。这些将被制作成几个96孔的微量测定。将通过双染料荧光法比较实验/诊断样品与对照样品中每种复合物的水平。第二个将使用荧光和比色活性测定比较实验样品中每个复合物与对照的酶活性。第三组将以足以用于翻译后修饰的蛋白质组学分析的量免疫捕获各种复合物。这些试剂盒将是非常宝贵的，不仅对研究早发性和迟发性线粒体疾病的研究人员，而且对线粒体生理学的研究，以及对引起这些疾病的环境毒素和改善这些疾病的药物的高通量筛选。

DESCRIPTION (provided by applicant): Mitochondria play a key role in cellular energy metabolism: they are the seat of oxidative phosphorylation (OXPHOS), which produces 98 percent of all the ATP utilized by mammalian cells. OXPHOS is catalyzed by 5 large, membrane-bound, protein complexes together made up of 90 polypeptides. Substrate entry into the OXPHOS system is controlled by a sixth, large enzyme-complex, the pyruvate dehydrogenase complex. Genetic defects in these six complexes cause a heterogeneous set of human diseases collectively classified as mitochondrial disorders, which affect around 1 in 6000 live births. Mitochondrial diseases remain hard to diagnose and the genotype-phenotype relationships of these disorders is still obscure. There are several late-onset diseases where mitochondrial dysfunction is thought to be causal and/or predictive, including Parkinson's disease, Alzheimer's, early- and late-onset Diabetes, schizophrenia and some forms of heart disease. In common to all of these conditions is the idea that they result from accumulation of oxidative damage leading to cell death caused by and involving the mitochondrion and the OXPHOS components in particular. As with mitochondrial genetic diseases, the etiology of the late-onset diseases listed above remains obscure and their diagnosis is still difficult because of a lack of adequate, simple high-throughput methods for analyzing the composition, enzyme activities and post-translational (oxidative) modifications of the OXPHOS components. This grant is to establish immunologically-based methods for such analyses. Monoclonal antibodies (mAbs) have been generated from small amounts of human and other mammalian tissue, which capture functionally active intact forms of 4 of the 50XPHOS complexes and pyruvate dehydrogenase. Screening is in progress for a mAb to capture Complex III. These will be fabricated into several 96-well based microassays. One will compare the levels of each complex in an experimental/ diagnostic sample versus a control sample by a twodye fluorometric approach. A second will compare the enzymatic activities of each complex in an experimental sample with a control using fluorometric and colorimetric activity assays. A third set will immunocapture the various complexes in amount sufficient for proteomic analysis of post-translational modifications. These kits will be invaluable, not only for researchers studying early-onset and late-onset mitochondrial disorders, but also for studies of mitochondria physiology, and for high-throughput screening for environmental toxins that cause, and drugs that ameliorate, these conditions.