Identification of proteins that interact with TANK binding kinase 1 and testing for mutations associated with glaucoma.
Identification of proteins that interact with TANK binding kinase 1 and testing for mutations associated with glaucoma.
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DOI：
10.3109/02713683.2012.754047
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发表时间：
2013-02
影响因子：
2
通讯作者：
Fingert JH
中科院分区：
文献类型：
--
作者：
Seo S;Solivan-Timpe F;Roos BR;Robin AL;Stone EM;Kwon YH;Alward WL;Fingert JH
TANK结合激酶1（TBK 1）的拷贝数变异（重复）与正常眼压性青光眼（NTG）相关，NTG是全球常见的致盲原因。参与自噬的其他基因（TLR 4和OPTN）的突变与NTG有关。在这里，我们报告搜索其他蛋白质参与自噬，也可能有NTG的作用。转染HEK-293 T细胞以产生具有FLAG和S标签的合成TBK 1蛋白。使用串联亲和纯化（TAP）和聚丙烯酰胺凝胶电泳（PAGE）从HEK-293 T裂解物中分离与TBK 1缔合的蛋白质。用质谱法鉴定分离的蛋白质。来自爱荷华州的148名NTG患者和77名对照者的队列使用高分辨率熔解（HRM）分析和DNA测序检测编码与TBK 1相互作用的已鉴定蛋白质的基因中的致肉瘤突变。TAP研究表明，HEK-293 T细胞中表达的三种蛋白质（NAP 1，TANK和TBKBP 1）与TBK 1相互作用。对NTG和正常对照组的TANK致病突变进行测试，共鉴定出9种独特的变体，包括3种非同义变化、1种同义变化和5种内含子变化。当单独分析或作为一个组，非同义TBK 1编码序列的变化与NTG或原发性开角型青光眼无关。TAP表明，NAP 1，TANK和TBKBP 1与TBK 1相互作用，是促进NTG的良好候选者。TANK的突变筛查检测到三种非同义变体。尽管这些TANK突变中的一个或多个可能在NTG中起作用，但本报告中的数据并没有为TANK变体与NTG之间的关联提供统计学支持。
Copy number variations (duplications) of TANK binding kinase 1 (TBK1) have been associated with normal tension glaucoma (NTG), a common cause of blindness worldwide. Mutations in other genes involved in autophagy (TLR4 and OPTN) have been associated with NTG. Here we report searching for additional proteins involved in autophagy that may also have roles in NTG. HEK-293T cells were transfected to produce synthetic TBK1 protein with FLAG and S tags. Proteins that associate with TBK1 were isolated from HEK-293T lysates using tandem affinity purification (TAP) and polyacrylamide gel electrophoresis (PAGE). Isolated proteins were identified with mass spectrometry. A cohort of 148 NTG patients and 77 controls from Iowa were tested for glaucoma-causing mutations in genes that encode identified proteins that interact with TBK1 using high resolution melt (HRM) analysis and DNA sequencing. TAP studies show that three proteins expressed in HEK-293T cells (NAP1, TANK and TBKBP1) interact with TBK1. Testing cohorts of NTG and normal controls for disease-causing mutations in TANK, identified a total of nine unique variants including three non-synonymous changes, one synonymous changes and five intronic changes. When analyzed alone or as a group, the non-synonymous TBK1 coding sequence changes were not associated with either NTG or primary open angle glaucoma. TAP showed that NAP1, TANK and TBKBP1 interact with TBK1 and are good candidates for contributing to NTG. A mutation screen of TANK detected three non-synonymous variants. Although, it remains possible that one or more of these TANK mutations may have a role in NTG, the data in this report do not provide statistical support for an association between TANK variants and NTG.