Waardenburg综合征致病蛋白MITF核定位信号的鉴定及其功能研究  

Analysis of nuclear localization and signal function of MITF protein predisposing to Warrdenburg syndrome

作　　者：张华[1,2] 冯娟[1] 陈红胜[3] 李家大[4] 罗浑金[4] 冯永[3] 

机构地区：[1]新疆医科大学第一附属医院耳鼻咽喉科,830011 [2]上海交通大学医学院附属仁济医院耳鼻咽喉科 [3]中南大学湘雅医院耳鼻咽喉科 [4]医学遗传学国家重点实验室

出　　处：《中华医学遗传学杂志》2015年第6期805-809,共5页Chinese Journal of Medical Genetics

基　　金：国家自然科学基金（81280160）

摘　　要：目的对MITF蛋白核定位信号（nuclear localization signals，NLS）进行鉴定并探讨其功能异常在Waardenburg综合征（Waardenburg syndrome，WS）发病中的作用。方法通过分子克隆技术设计突变引物，以野生型真核细胞表达质粒pcMv_MITF_Flag（MITF）为模板构建突变型真核细胞表达质粒pCMV-MITFANLS-Flag（MITFANLS），并将二者瞬时单转或共转染UACC903细胞进行荧光素酶活性检测，观察其对靶基因酪氨酸酶（tyrosinase，TYR）转录活性的影响。将MITFNLS核苷酸序列5f_GAAcGAAGAAGAAGATTT＇3’克隆到真核细胞表达质粒pEGFP-N1上，构建重组真核细胞表达质粒pEGFP-N1-MITF-NLS（’pEGFP-N1-NLS）。将MITF、MITF／kNLS、pEGFP-N1和pEGFP-N1-NLS分别瞬时转染NIH3T3细胞后，利用免疫荧光检测对其亚细胞定位进行观察。结果成功构建了MITF／kNLS和pEGFP-N1-NLS的真核细胞表达质粒，前者缺失MITF的NLS核心序列213ERRRRF218，后者则偶联了MITFNLS。与野生型MITF相比，MITF△NLS完全失去了激活TYR启动子作用，并且不影响野生型MITF的正常功能。与野生型MITF仅在细胞核分布相比，MITF△NLS仅在细胞质分布。pEGFP—N1在细胞核与细胞质中均有分布，而pEGFP—N1-NLS主要在细胞核分布。结论验证并明确了213ERRRRF218在MITF蛋白中的NLS核心序列，缺失NLS的MITF突变蛋白出现异常亚细胞定位而失去调控靶基因TYR转录活性功能可导致WS临床表型。Objective To study the role of dysfunction of nuclear localization signals （NLS） of MITF protein in the pathogenesis of Waardenburg syndrome. Methods Eukaryotic expression plasmid pCMV- MITF-Flag was used as a template to generate mutant plasmid pCMV-MITF△NLS-Flag by molecular cloning technique in order to design the mutagenic primers. The UACC903 cells were transfected transiently with MITF and MITFANLS plasmids, and the luciferase activity assays were performed to determine their impact on the transcriptional activities of target gene tyrosinase （TYR）. The oligonucleotide 5＇- GAACGAAGAAGAAGATTT-3＇ was subcloned into pEGFP-N1 to generate recombinant eukaryotic expression plasmid pEGFP-N1-MITF-NLS. The NIH3T3 cells were transfeeted separately with MITF, MITF△NLS, pEGFP-N1 and pEGFP-N1-NLS plasmids, and their subeellular distribution was observed by immunoflorescence assays. Results Expression plasmids for the mutant MITF△NLS with loss of core NLS sequence and pEGFP-N1-NLS coupled with MITFANLS were successfully generated. Compared with the wild-type MITF, MITFANLS was not able to transactivate the transcriptional activities of promoter TYR and did not affect the normal function of MITF. MITF△NLS was only localized in the cytoplasm and pEGFP-N1 was found in both the cytoplasm and nucleus, whereas pEGFP-N1-NLS was mainly located in the nucleus. Conclusion This study has confirmed the localization function of NLS sequence 213ERRRRF218 within the MITF protein. Mutant MITF with loss of NLS has failed to transactivate the transcriptional activities of target gene TYR, which can result in melanocyte defects and cause WS.

关 键 词：MITF蛋白 WAARDENBURG综合征 核定位信号 

分 类 号：R596.1[医药卫生—内科学]