Department of Medicine, University of Wisconsin-Madison, Madison, WI, USA.
Cellular and Molecular Arrhythmias Research Program, University of Wisconsin-Madison, Madison, WI, USA.
Genet Med. 2020 Oct;22(10):1642-1652. doi: 10.1038/s41436-020-0842-1. Epub 2020 Jun 1.
DNA sequencing technology has unmasked a vast number of uncharacterized single-nucleotide variants in disease-associated genes, and efficient methods are needed to determine pathogenicity and enable clinical care.
We report an E. coli-based solubility assay for assessing the effects of variants on protein domain stability for three disease-associated proteins.
First, we examined variants in the Kv11.1 channel PAS domain (PASD) associated with inherited long QT syndrome type 2 and found that protein solubility correlated well with reported in vitro protein stabilities. A comprehensive solubility analysis of 56 Kv11.1 PASD variants revealed that disruption of membrane trafficking, the dominant loss-of-function disease mechanism, is largely determined by domain stability. We further validated this assay by using it to identify second-site suppressor PASD variants that improve domain stability and Kv11.1 protein trafficking. Finally, we applied this assay to several cancer-linked P53 tumor suppressor DNA-binding domain and myopathy-linked Lamin A/C Ig-like domain variants, which also correlated well with reported protein stabilities and functional analyses.
This simple solubility assay can aid in determining the likelihood of pathogenicity for sequence variants due to protein misfolding in structured domains of disease-associated genes as well as provide insights into the structural basis of disease.
DNA 测序技术揭示了大量与疾病相关基因中未被描述的单核苷酸变异，因此需要有效的方法来确定其致病性并为临床治疗提供依据。
我们报告了一种基于大肠杆菌的可溶性测定法，用于评估三种与疾病相关蛋白的变异对蛋白结构域稳定性的影响。
首先，我们研究了与遗传性长 QT 综合征 2 型相关的 Kv11.1 通道 PAS 结构域（PASD）中的变异，发现蛋白可溶性与体外报告的蛋白稳定性密切相关。对 56 个 Kv11.1 PASD 变异的全面可溶性分析表明，破坏膜运输是主要的失能疾病机制，这在很大程度上取决于结构域的稳定性。我们进一步通过使用该测定法鉴定改善 PASD 结构域稳定性和 Kv11.1 蛋白转运的第二部位抑制 PASD 变异来验证该测定法。最后，我们将该测定法应用于几种与癌症相关的 P53 肿瘤抑制 DNA 结合结构域和肌病相关的核纤层蛋白 A/C Ig 样结构域变异，这些变异与报告的蛋白稳定性和功能分析也很好地相关。
这种简单的可溶性测定法可用于确定由于疾病相关基因的结构域中蛋白质错误折叠而导致的序列变异的致病性可能性，同时为疾病的结构基础提供了深入了解。
