Rapid Profiling of Disease Alleles Using a Tunable Reporter of Protein Misfolding
Rapid Profiling of Disease Alleles Using a Tunable Reporter of Protein Misfolding
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DOI：
10.1534/genetics.112.143750
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发表时间：
2012-11-01
期刊：
影响因子：
3.3
通讯作者：
Tucker, Chandra L.
中科院分区：
文献类型：
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作者：
Pittman, Adrianne M. C.;Lage, Melissa D.;Tucker, Chandra L.
许多人类疾病是由降低蛋白质稳定性的基因突变引起的。此类突变可能不会具体影响活性位点，但可以改变蛋白质折叠、丰度或定位。在这里，我们描述了一种基于高通量细胞的稳定性测定，IDESA（DHFR 内酶稳定性测定），其中稳定性与模型酵母（酿酒酵母）中的细胞增殖相关。该测定不需要事先了解蛋白质的结构或活性，可以评估活性未知或难以表征的蛋白质的稳定性，并且我们证明了其可用于一系列疾病相关靶标，包括人丙氨酸：乙醛酸转氨酶 (AGT)、超氧化物歧化酶 (SOD-1)、DJ-1、p53 和 SMN1。该测定可以同时对数百个疾病等位基因进行，或用于鉴定不稳定等位基因的稳定小分子（药理学伴侣）。为了证明该测定的通用性，我们分析了 AGT 疾病等位基因的稳定性，AGT 的缺乏会导致肾结石疾病、I 型原发性高草酸尿症，并识别出专门影响蛋白质活性位点化学的突变。
Many human diseases are caused by genetic mutations that decrease protein stability. Such mutations may not specifically affect an active site, but can alter protein folding, abundance, or localization. Here we describe a high-throughput cell-based stability assay, IDESA (intra-DHFR enzyme stability assay), where stability is coupled to cell proliferation in the model yeast, Saccharomyces cerevisiae. The assay requires no prior knowledge of a protein's structure or activity, allowing the assessment of stability of proteins that have unknown or difficult to characterize activities, and we demonstrate use with a range of disease-relevant targets, including human alanine: glyoxylate aminotransferase (AGT), superoxide dismutase (SOD-1), DJ-1, p53, and SMN1. The assay can be carried out on hundreds of disease alleles in parallel or used to identify stabilizing small molecules (pharmacological chaperones) for unstable alleles. As demonstration of the general utility of this assay, we analyze stability of disease alleles of AGT, deficiency of which results in the kidney stone disease, primary hyperoxaluria type I, identifying mutations that specifically affect the protein-active site chemistry.