In-depth Proteomic Characterization of Endogenous Nuclear Receptors in Mouse Liver
In-depth Proteomic Characterization of Endogenous Nuclear Receptors in Mouse Liver
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DOI：
10.1074/mcp.m112.022319
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发表时间：
2013-02-01
影响因子：
7
通讯作者：
Zhen, Bei
中科院分区：
文献类型：
--
作者：
Liu, Qiongming;Ding, Chen;Zhen, Bei
核受体（NR）是转录因子的超家族，其在与配体结合时结合特异性DNA序列并调节控制细胞增殖、分化和代谢的转录程序。在肝脏中，通过感知脂溶性激素和膳食脂质并控制关键肝脏代谢基因的表达，NR蛋白指导大量关键肝脏功能，包括脂质和葡萄糖代谢、胆汁分泌和胆汁酸稳态。虽然已经了解了很多关于NRs的生理学，但对它们在肝脏中的蛋白质表达和DNA结合活性知之甚少，因为它们的丰度低并且缺乏用于在蛋白质水平上检测的高通量方法。在这里，我们报告了一种分析小鼠肝脏NR转录因子超家族DNA结合活性的方法。我们使用DNA构建的激素反应元件（HREs）作为亲和试剂，以丰富NR蛋白质从小鼠肝细胞核提取物，然后使用质谱鉴定。我们评估了20个DNA构建体含有各种组合的HRE的能力，以丰富内源性NR蛋白，并发现，两种不同的HRE是足以实现几乎所有的内源性NR蛋白从一个小鼠肝脏的分离和鉴定。我们已经检测到蛋白质的NR家族的41个成员中的35个在小鼠肝脏中表达的mRNA水平。因此，该方法可以覆盖整个NR蛋白质组的大部分，并建立了一个实用的测定小鼠肝脏NR行动的调查。我们预计，这种方法将发现广泛的使用在未来的调查NR行动在肝脏生物学和病理学。此外，该工作流程对于NR生物学家来说是一个有用的工具，他们有兴趣在正常生理和病理条件下以及在药物干预期间测量NR表达，DNA结合，翻译后修饰，细胞定位和NR在器官中的其他功能方面。Molecular & Cellular Proteomics 12：10.1074/mcp. M112.022319，473-484，2013.
Nuclear receptors (NRs) are a superfamily of transcription factors that, upon binding to ligands, bind specific DNA sequences and regulate a transcriptional program governing cell proliferation, differentiation, and metabolism. In the liver, by sensing lipid-soluble hormones and dietary lipids and governing the expression of key liver metabolic genes, NR proteins direct a large array of key hepatic functions that include lipid and glucose metabolism, bile secretion, and bile acid homeostasis. Although much has been learned about the physiology of NRs, little is known about their protein expression and DNA binding activity in the liver because of their low abundance and the lack of high-throughput methods for detection at the protein level. Here we report a method for profiling the DNA binding activity of the NR transcription factor superfamily in mouse liver. We use DNA constructs of hormone response elements (HREs) as affinity reagents to enrich NR proteins from nuclear extracts of mouse liver and then identify them using mass spectrometry. We evaluated 20 DNA constructs containing various combinations of HREs for their ability to enrich endogenous NR proteins and found that two different HREs are sufficient to achieve isolation and identification of nearly all endogenous NR proteins from one mouse liver. We have detected proteins for 35 members of the NR family out of 41 that are expressed in mouse liver at mRNA level. Thus, this method allows coverage of most of the whole NR proteome and establishes a practical assay for the investigation of NR actions in mouse liver. We anticipate that this method will find widespread use in future investigations of NR actions in liver biology and pathology. Furthermore, this workflow is a useful tool for NR biologists interested in measuring NR expression, DNA binding, post-translational modifications, cellular localization, and other functional aspects of NRs in organs under normal physiological and pathological conditions, as well as during pharmacological intervention. Molecular & Cellular Proteomics 12: 10.1074/mcp.M112.022319, 473-484, 2013.