Purification and mutagenesis studies of TANC1 ankyrin repeats domain provide clues to understand mis-sense variants from diseases
Purification and mutagenesis studies of TANC1 ankyrin repeats domain provide clues to understand mis-sense variants from diseases
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TANC1锚蛋白重复结构域的纯化和诱变研究为理解疾病中的错义变异提供了线索
DOI：
10.1016/j.bbrc.2019.04.151
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发表时间：
2019-06-25
影响因子：
3.1
通讯作者：
Liu, Wei
中科院分区：
文献类型：
--
作者：
Yang, Qingqing;Liu, Haiyang;Liu, Wei
TANC 1及其近亲TANC 2是两种重要的突触支架蛋白，在调节突触棘密度和兴奋性突触强度方面起关键作用。最近的研究表明，TANC 1和TANC 2是多种神经发育障碍（NDD）的候选基因。到目前为止，TANC 1/2蛋白的生物化学性质仍然是未知的。本研究利用大肠杆菌表达并纯化了TANC 1的锚定重复序列（AR）结构域。（E.大肠杆菌）细胞，其在去除麦芽糖结合蛋白（MBP）标签后显示出低溶解度和稳定性。序列分析表明，TANC 1的AR结构域缺乏典型的N，C-帽单位。通过在C-帽单元中引入两个点突变并替换N-帽单元，单体和良好折叠的TANC 1 AR结构域被纯化，并通过尺寸排阻色谱结合多角度静态光散射（SEC-MALS）和圆二色性光谱（CD）进行表征。此外，来自智力残疾（ID）患者和癌症患者的突变被导入TANC 1 AR结构域。ID突变体在构象和蛋白质折叠稳定性变化方面表现出边际效应。相比之下，癌症突变体显着降低蛋白质溶解度。结合结构预测，我们推测本研究中测试的错义变体可能影响蛋白质折叠或破坏TANC 1/2 AR结构域与其结合伴侣之间的相互作用。(C)2019由Elsevier Inc.出版
TANC1 and its close relative TANC2 are two important synaptic scaffold proteins which play critical roles in regulating densities of synaptic spines and excitatory synapse strength. Recent studies indicated TANC1 and TANC2 are candidate genes of several neurodevelopmental disorders (NDD). So far, the biochemical properties of TANC1/2 proteins remain largely unknown. In this study, Ankyrin-repeats (AR) domain of TANC1 was expressed and purified using Escherichia coli. (E. coli.) cells, which showed low solubility and stability after removing the maltose binding protein (MBP) tag. Sequence analysis revealed that the TANC1 AR domain is lack of canonical N, C-capping units. By introducing two point mutations in the C-capping unit and replacing the N-capping unit, monomeric and well-folded TANC1 AR domain was purified and characterized by size exclusion chromatography coupled with multi-angle static light scattering (SEC-MALS) and circular dichroism spectroscopy (CD). In addition, mutations from intellectual disability (ID) patients and cancer patients were imported into the TANC1 AR domain. The ID mutant exhibited marginal effects in terms of conformation and protein folding stability changes. By contrast, the cancer mutants dramatically decreased protein solubility. Combined with structural prediction, we speculated that mis-sense variants tested in this study may either affect protein folding or disrupt the interaction between TANC1/2 AR domains and their binding partners. (C) 2019 Published by Elsevier Inc.