NTE相关酯酶在小鼠能量代谢中的作用研究

项目名称： NTE相关酯酶在小鼠能量代谢中的作用研究

项目编号： No.30870298

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 轻工业、手工业

项目作者： 常平安

作者单位： 重庆邮电大学

项目金额： 30万元

中文摘要： 在克隆小鼠NRE基因及其2个剪接变异体的基础上，通过本项目研究发现：（1）mNRE具有一个N-端跨膜结构域（TM）、一个调节域(R)和催化结构域(C)。酯酶分析显示TM和R结构域并不是其催化活性所必需的，然而TM对其活力影响较大。mNRE通过其TM镶嵌于内质网膜上， mNRE大部分结构暴露在内质网膜上的细胞质侧面。（2）mNRE通过自噬和蛋白酶体途径被降解，其TM和R结构域分别在2条降解途径中其一定作用。（3）mNRE剪接变异体1和2(mNREV1和mNREV2)定位于内质网膜上。mNREV1具有mNRE相同的蛋白结构域和催化活性，mNREV2缺乏patatin结构域，没有催化活性。（4）饥饿增强具有催化活性的mNRE剪接变异体1的mRNA水平，而降低没有催化活性的mNRE剪接变异体2的表达，因此转录剪接是mNRE在小鼠能量稳态中的表达调节方式。（5）降低mNRE的表达抑制前脂肪细胞的分化和胰岛素诱导的脂肪细胞中甘油三酯的水平，表明NRE在脂肪细胞脂代谢中起一定作用。这些结果不仅是对NRE特定功能和表达调节的深刻认识，而且是对动物脂代谢和能量平衡新机理的揭示。

中文关键词： NTE相关酯酶；能量代谢；表达调节；脂肪细胞；小鼠

英文摘要： As a novel lysophospholipase, NRE (neuropathy target esterase-related esterase) was proposed to play a role in energy metabolism. On the basis of cloning of mouse NRE (mNRE) and two splice variants, protein domains, catalytic activity, subcellular location and degradation pathway of mNRE, the regulation and role of mNRE in mouse energy homeostasis, preadipocyte differentiation and adipocyte triacylglycerol biosynthesis were investigated. These results showed that: (1) mNRE had a predicted amino-terminal transmembrane region (TM), a putative regulatory (R) domain, and a hydrophobic catalytic (C) domain. Esterase assays indicated that neither the TM nor R-domain was essential for mNRE esterase activity, but the TM significantly contributed to its activity. Subcellular distribution showed that mNRE was anchored in ER via its TM domain and most of mNRE molecule was exposed on the cytoplasmic face of ER membranes. (2) mNRE was degraded by macroautophagy and the proteasome pathways and the TM and R-domain played a role in autophagy and proteasome pathways respectively. (3) Green fluorescent protein- mNREV1 or mNREV2 fusion proteins localized to the endoplasmic reticulum as mNRE. mNREV1 had the same protein domains with mNRE, while mNREV2 lacked the patatin domain in the C-terminal catalytic region. mNREV1 and mNRE exhibited equal hydrolytic activity to the substrate phenyl valerate, whereas mNREV2 did not have any catalytic activity. (4) mNRE was regulated by nutrition status. Induction and down-regulation of the mRNA levels of mNREV1 with catalytic activity andmNREV2 without enzyme activity by fasting respectively, indicated that the major isoform of mNRE mRNA generated switched from mNREV2 to mNREV1 during fasting and suggested a nutritional regulation of the enzyme expression by transcription splicing. (5) Knockdown of mNRE suppressed preadipocyte differentiation and decreased triacylglycerol accumulation in adipocyte induced by insulin, which showed that mNRE played a role in lipid metabolism in adipocyte. In summary, these results were not only the findings of the specific function and regulation of mNRE, but also the revealing of the mechanism of animal lipid metabolism and energy balance.

英文关键词： NTE-related esterase; energy metabolism; expression regulation; adipocyte; mouse