Quantification Assays for Total and Polyglutamine-Expanded Huntingtin Proteins
Quantification Assays for Total and Polyglutamine-Expanded Huntingtin Proteins
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DOI：
10.1371/journal.pone.0096854
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发表时间：
2014-05-09
期刊：
影响因子：
3.7
通讯作者：
Munoz-Sanjuan, Ignacio
中科院分区：
文献类型：
--
作者：
Macdonald, Douglas;Tessari, Michela A.;Munoz-Sanjuan, Ignacio
亨廷顿基因中CAG三核苷酸重复序列的扩增是亨廷顿病（HD）的原因，该扩增产生具有扩增的多聚谷氨酰胺束的亨廷顿蛋白。最近的研究报告称，在HD动物模型中对聚谷氨酰胺扩展的亨廷顿蛋白（突变HTT）的RNA干扰抑制可以改善疾病表型。旨在减少突变HTT暴露的此类临床前研究以及最终临床试验的关键要求是测量选定组织中HTT蛋白水平的稳健方法。我们已经开发了几种灵敏和选择性的检测方法，可以在电化学发光Meso Scale Discovery检测平台上测量总人HTT或聚谷氨酰胺扩增的人HTT蛋白，与其他方法相比，动态范围更大。此外，我们还开发了一种检测HD临床前模型中内源性小鼠和大鼠HTT蛋白的试验，以监测等位基因选择性和非选择性干预对野生型蛋白的影响。我们证明了这些检测方法的应用，以测量HTT蛋白在几个HD在体外细胞和体内动物模型系统，以及在HD患者的生物样品。此外，我们使用纯化的重组HTT蛋白作为标准品来定量这种生物样品中HTT蛋白的绝对量。
The expansion of a CAG trinucleotide repeat in the huntingtin gene, which produces huntingtin protein with an expanded polyglutamine tract, is the cause of Huntington's disease (HD). Recent studies have reported that RNAi suppression of polyglutamine-expanded huntingtin (mutant HTT) in HD animal models can ameliorate disease phenotypes. A key requirement for such preclinical studies, as well as eventual clinical trials, aimed to reduce mutant HTT exposure is a robust method to measure HTT protein levels in select tissues. We have developed several sensitive and selective assays that measure either total human HTT or polyglutamine-expanded human HTT proteins on the electrochemiluminescence Meso Scale Discovery detection platform with an increased dynamic range over other methods. In addition, we have developed an assay to detect endogenous mouse and rat HTT proteins in pre-clinical models of HD to monitor effects on the wild type protein of both allele selective and non-selective interventions. We demonstrate the application of these assays to measure HTT protein in several HD in vitro cellular and in vivo animal model systems as well as in HD patient biosamples. Furthermore, we used purified recombinant HTT proteins as standards to quantitate the absolute amount of HTT protein in such biosamples.