A mechanistic investigation into the degradation of low-density lipoprotein receptor by the novel ubiquitin ligase MARCH2
10.3760/cma.j.issn.0254-9026.2024.12.015
- VernacularTitle: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
- Author:
Yuefeng WANG
1
;
Dongsheng NI
;
Yong MAN
;
Guoping LI
Author Information
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
- Publication Type:Journal Article
- Keywords:
Ubiquitin ligase MARCH2;
Low density lipoprotein receptor;
Lysosome pathway;
Proteasome pathway
- From:
Chinese Journal of Geriatrics
2024;43(12):1599-1606
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To explore the impact and biological relevance of E3 ubiquitin ligase MARCH2 on the degradation of low-density lipoprotein receptor(LDLR)in hepatic cell lines.Methods:The 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.Results:Real-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 4 039.8±16.2 to 2 630.3±185.9 at 3 hours, indicating a 34.9% reduction( t=16.89, P<0.001). Conclusions:MARCH2 facilitates the degradation of LDLR via the ubiquitin-dependent lysosome pathway, thereby decreasing the uptake of LDL by hepatic cells.
