A mechanistic investigation into the degradation of low-density lipoprotein receptor by the novel ubiquitin ligase MARCH2
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,Di Ⅰ-labeled low-density lipoprotein(Di Ⅰ-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.Di Ⅰ-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.
Ubiquitin ligase MARCH2Low density lipoprotein receptorLysosome pathwayProteasome pathway
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