新型泛素蛋白连接酶MARCH2降解低密度脂蛋白受体的机制研究  

A mechanistic investigation into the degradation of low-density lipoprotein receptor by the novel ubiquitin ligase MARCH2

作　　者：王跃峰 倪东升 满永[1] 李国平[1] Wang Yuefeng;Ni Dongsheng;Man Yong;Li Guoping(The Key Laboratory of Geriatrics,Beijing Institute of Geriatrics,Institute of Geriatric Medicine,Chinese Academy of Medical Sciences,Beijing Hospital/National Center of Gerontology of National Health Commission,Beijing 100730,China;Graduate School of Peking Union Medical College,Beijing 100730,China)

机构地区：[1]北京医院、国家老年医学中心、国家卫生健康委北京老年医学研究所、国家卫生健康委老年医学重点实验室、中国医学科学院老年医学研究院,北京100730 [2]北京协和医学院研究生院,北京100730

出　　处：《中华老年医学杂志》2024年第12期1599-1606,共8页Chinese Journal of Geriatrics

基　　金：中国医学科学院医学与健康科技创新工程项目(2021-I2M-1-008);国家自然科学基金面上项目(82370877, 81970739);中央高水平医院临床科研业务费(BJ-2023-237)

摘　　要：目的探讨E3泛素蛋白连接酶MARCH2对肝细胞中低密度脂蛋白受体(LDLR)降解的影响及其生物学作用。方法在数据库中预测降解LDLR的MARCH家族的泛素连接酶,通过分子对接预测其结合位点,并通过文献检索筛选出MARCH2与胆固醇水平相关。在肝细胞系中进行实时荧光定量PCR、免疫印迹、环己酰亚胺追踪实验、免疫共沉淀等实验研究MARCH2对LDLR的泛素化降解的影响。DiⅠ标记的低密度脂蛋白(DiⅠ-LDL)摄取实验用于研究过表达MARCH2对肝细胞摄取低密度脂蛋白(LDL)的影响。结果实时荧光定量PCR、免疫印迹、环己酰亚胺追踪实验表明,MARCH2在蛋白水平而不是转录水平促进LDLR的降解,24 h内MARCH2降解了13%~33%的LDLR蛋白。血清饥饿后再恢复血清,其降解水平显著上调(t=2.280,P=0.046)。该降解过程不能被蛋白酶体途径抑制剂MG132阻断,能被溶酶体抑制剂氯喹阻断。免疫共沉淀实验证明MARCH2与LDLR之间存在相互作用。DiⅠ-LDL摄取实验表明过表达MARCH2在3 h时,肝细胞对LDL的摄取从4039.8±16.2降低到2630.3±185.9,降低了34.9%(t=16.89,P<0.001)。结论MARCH2通过泛素依赖的溶酶体途径,在翻译后水平,结合并降解LDLR,减少肝细胞对LDL的摄取。ObjectiveTo explore the impact and biological relevance of E3 ubiquitin ligase MARCH2 on the degradation of low-density lipoprotein receptor(LDLR)in hepatic cell lines.MethodsThe ubiquitin ligase of the MARCH family involved in LDLR degradation was predicted using databases,and their binding sites were predicted through molecular docking.The association between MARCH2 and cholesterol levels was screened through a literature search.Various experiments including real-time quantitative PCR,immunoblotting,cycloheximide tracing assay,immunoprecipitation,were conducted in hepatic cell lines to investigate the impact of MARCH2 on the ubiquitination degradation of LDLR.Additionally,DiⅠ-labeled low-density lipoprotein(DiⅠ-LDL)uptake assays were performed to examine the effect of MARCH2 overexpression on LDL uptake in hepatocytes.ResultsReal-time quantitative PCR,immunoblotting,and cycloheximide tracing experiments illustrated that MARCH2 facilitates the degradation of LDLR at the protein level rather than the transcriptional level.MARCH2 degraded 13%to 33%of LDLR protein within 24 hours,Following serum starvation and subsqnent serum refeeding,the degradation level significantly increased(t=2.280,P=0.046).This degradation process was unaffected by the proteasome pathway inhibitor MG132 but was hindered by the lysosome inhibitor chloroquine.Immunoprecipitation experiments validated the interaction between MARCH2 and LDLR.DiⅠ-LDL uptake experiments showed that MARCH2 overexpression decreased LDL uptake by hepatocytes from 4039.8±16.2 to 2630.3±185.9 at 3 hours,indicating a 34.9%reduction(t=16.89,P<0.001).ConclusionsMARCH2 facilitates the degradation of LDLR via the ubiquitin-dependent lysosome pathway,thereby decreasing the uptake of LDL by hepatic cells.

关 键 词：E3泛素蛋白连接酶MARCH2 低密度脂蛋白受体 溶酶体途径 蛋白酶体途径 

分 类 号：R341[医药卫生—基础医学]