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Analysis of proteins which specifically binds to thyroid hormone receptor β

与甲状腺激素受体β特异性结合的蛋白质分析

基本信息

批准号：
18591018
负责人：
KANOU Yasuhiko
金额：
$ 2.35万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18591018/
关键词：
Resistance to thyroid hormone thyroid hormone receptors co-activator Yeast two-hybrid system cDNA cloning

项目摘要

There is a case where any mutations are not found in two types of thyroid hormone receptor(TR) in the resistance to thyroid hormone. We hypothesized that a specific coactivator for thyroid hormone receptor b existed. This coactivator may have any abnormality in the resistance to thyroid hormone. We tried to identify the cDNA for the specific protein. Yeast two-hybrid system was applied to screen a human liver cDNA library. We found two cDNA clones, No.60 and 93, as a candidate of TR-specific coactivator.1. A clone 60 was a fragment derived from 3 kinds of mRNA. The full-length cDNAs for these mRNAs were cloned. The deduced amino acid sequences showed that proteins had nuclear transport signals.2. In amino acid sequences of clone 60 proteins, there was a high homology sequence for another nuclear protein.3. To determine whether a clone 60 protein binds to TRb, pull-down assay was occurred. TRb and a clone 60 protein were expressed as a GST-fusion protein and His-tag fusion protein, respectively. Both fusion protein were mixed and precipitated with specific antibodies. Western blotting showed both fusion proteins bound in vitro.4. A clone 93 could not be expressed as His-tag fusion protein in vitro. We are advancing the analysis of the clone 60 proten further continuously.

在对甲状腺激素的抵抗中，有两种类型的甲状腺激素受体（TR）中未发现任何突变的情况。我们假设存在甲状腺激素受体B的特异性共激活因子。这种辅激活因子可能具有对甲状腺激素抵抗的任何异常。我们试图鉴定特定蛋白质的cDNA。应用酵母双杂交系统筛选人肝cDNA文库。我们发现了两个cDNA克隆，No.60和93，作为TR特异性共激活因子的候选者。克隆60是来自3种mRNA的片段。克隆这些mRNA的全长cDNA。推导的氨基酸序列表明蛋白质具有核转运信号.在克隆60蛋白的氨基酸序列中，与另一种核蛋白有高度同源性.为了确定克隆60蛋白是否与TRb结合，进行下拉测定。TRb和克隆60蛋白分别表达为GST-融合蛋白和His-标签融合蛋白。将两种融合蛋白混合并用特异性抗体沉淀。Western blotting显示两种融合蛋白在体外结合.克隆93不能在体外表达为His标签融合蛋白。我们正在继续进一步推进克隆60蛋白的分析。