去泛素化酶USP37和USP46调控脂质代谢的机制研究

Mechanism of deubiquitinating enzymes USP37 and USP46 in regulating lipid metabolism

目的探索去泛素化酶USP37和USP46在脂质代谢中的作用。方法构建固醇调控元件(SRE)荧光素酶报告基因质粒pGL4-SRE-luc,利用去泛素化酶基因表达库筛选去泛素化酶,采用荧光素报告基因检测系统观察去泛素化酶活性对固醇调节元件结合蛋白(SREBP)-1a转录活性的调控作用,免疫共沉淀法检测去泛素化酶和SREBP-1a的相互作用。结果成功构建了pGL4-SRE-luc,可以响应细胞内低脂应激以及SREBP介导的基因转录。经筛选发现去泛素化酶USP37和USP46能显著上调SRE报告基因荧光素酶活性。与空载体相比,在低脂浓度下USP37能显著上调SRE报告基因荧光素酶活性(P<0.05),并显著上调硬脂酰CoA去饱和酶(SCD)基因和脂肪酸合酶(FASN)基因的表达(P<0.05),而其酶活突变克隆USP37 C350S上调作用不显著(P<0.05),USP37与SREBP-1a存在相互作用。USP46及其酶活突变克隆USP46-C44S均上调SRE报告基因荧光素酶活性和SREBP下游靶基因SCD的表达(P<0.05),而USP46与SREBP-1a不存在相互作用。结论USP37可通过去泛素化酶活性影响SREBP下游靶基因调控脂质代谢,而USP46上调部分SREBP下游靶基因的过程不依赖于其去泛素化酶活性。

AIM To investigate the role of deubiquitinating enzymes USP37 and USP46 in lipid metabolism.METHODS The sterol regulatory element(SRE)luciferase reporter gene plasmid pGL4-SRE-luc was constructed,the deubiquitinating enzymes were screened by the deubiquitinase gene expression library.The regulation activity of deubiquitinating enzymes on the transcription activity of sterol regulatory element binding protein(SREBP)-1a was observed by fluorescein reporter gene detection system.The interaction between deubiquitinating enzymes and SREBP-1a was detected by immunoprecipitation.RESULTS The pGL4-SRE-luc was successfully constructed to respond to low lipid stress and SREBP-mediated gene transcription.Ubiquitination enzymes USP37 and USP46 were shown to significantly up-regulate luciferase activity of SRE reporter gene.Compared with empty vector,USP37 significantly up-regulated the luciferase activity of SRE reporter gene(P<0.05)and the expression of stearoyl coenzyme A desaturase(SCD)gene and fatty acid synthase(FASN)gene(P<0.05),but its mutant clone USP37 C350S had no significant up-regulation(P<0.05).There was interaction between USP37 and SREBP-1a.Both USP46 and its mutant clone USP46 C44S up-regulated the luciferase activity of SRE reporter gene and the expression of SCD gene(P<0.05),but there was no interaction between USP46 and SREBP-1a.CONCLUSION USP37 affects the regulation of lipid metabolism by the downstream target gene of SREBP through its deubiquitinating enzyme activity,while USP46 up-regulates downstream target genes of SREBP partly independent of its deubiquitinating enzyme activity.