镉胁迫下MAPKs对通道蛋白的调控机理

Cadmium ions are toxic to human health. Cd enters cells through proteins involved in the uptake of essential cations, however regulatory mechanism of the transporters under cadmium stress condition have not been previously reported. Our lab has identified two mitogen-activate protein (MAP) kinases, Hog1 and Slt2, involved in the response to cadmium stress, and revealed the molecular mechanisms of which two MAPKs regulated manganese transporter. Regulatory mechanisms involved in Cd transported, and the relationship between transporter and MAPKs will involve further analysis of the study. Through gene expression profile assay, Cd2+ fluorescent probes analysis, electrophoretic mobility shift assay and chromatin immunoprecipitation, we will identify and sort mechanism of transporters regulated by Hog1 and Slt2. Transport proteins mediated by MAPKs and their response pathway under cadmium stress can be systematic studied and elucidated at the cellular and molecular levels, which will be revealing cadmium toxicity in mammals.

镉污染对人类健康造成严重威胁。有研究报道镉通过必需金属的通道蛋白进入细胞，但胁迫条件下通道蛋白的调控机制研究尚不明确。申请人前期工作中发现，酿酒酵母对外界刺激做出应答的核心组份MAPKs-Hog1和Slt2参与镉胁迫条件下的应答，并调控锰通道蛋白，在镉离子进入细胞中发挥功能，揭示其分子机制。本研究将在酵母细胞中进一步分析参与镉运输相关通道蛋白的调控机理，及其与MAPKs之间的关系。利用基因表达谱筛选、结合Cd2+荧光探针PDI-DIDPA、EMSA和ChIP等遗传学实验和理化特性研究，分析镉胁迫条件下Hog1和Slt2调控离子通道的种类和机制，建立MAPKs调控通道蛋白的信号转导通路，从细胞和分子水平阐明酵母细胞对镉离子胁迫的应答规律，为揭示镉离子在哺乳动物中的毒性奠定理论基础。

镉作为人体的非必需金属会对生物体造成不可逆的毒性损伤，因而对镉致毒机理和脱毒机制的研究显得尤为重要。已知镉进入后会破坏细胞内ROS和Ca2+的平衡，引发包括MAPKs在内的信号通路激活。但对于MAPKs的认识，目前仅局限在镉激活包括Hog1和Slt2在内的MAPKs磷酸化，对于这条经典信号通路的调控机制研究相关报道鲜见。本项目采用RNA测序的生物信息学方法，分析并预测出镉胁迫下MAPKs的潜在效应物。以野生型菌株BY4741为对照，分析hog1Δ和slt2Δ菌株在镉处理后的差异基因表达谱。发现野生型菌株在镉处理前后共有1545个差异表达基因，韦恩图分析得出与氧化损伤、DNA复制和脱毒相关的Hog1调控的31个基因和Slt2调控的2个基因。其中高亲和力半胱氨酸通道蛋白Yct1编码基因的缺失可以部分恢复hog1对镉的敏感性表型，Yct1的转录水平、蛋白表达水平以及原子吸收检测实验，初步证实Hog1位于上游正调控Yct1的表达。经过三年的实验验证，我们成功构建了MAPKs-Hog1和Slt2响应镉胁迫应答的效应基因表达谱。在本课题的支持下，三年内在酵母学领域高水平期刊发表了两篇SCI论文，两篇会议论文，尚有一篇文章刚刚收到修改意见，另一篇正在修改准备重新投稿。本项目的实施为揭示镉离子对真核细胞的毒性提供重要科学依据。