二甲双胍通过抑制泛素-蛋白酶体系统上调ABCA1表达

Metformin upregulates ABCA1 expression via inhibiting ubiquitin-proteasome system

摘要:

目的]探讨二甲双胍调控ATP结合盒转运体A1(ABCA1)表达的分子机制。 [方法]使用二甲双胍和放线菌酮共同或分别刺激J774A.1巨噬细胞,采用蛋白免疫印迹法检测ABCA1的表达；将His-tagged ABCA1和HA-tagged Ub质粒共同转染至HEK293细胞中,采用二甲双胍刺激细胞,免疫共沉淀检测ABCA1与泛素的结合能力；采用基于免疫共沉淀的蛋白质组学,寻找二甲双胍调控ABCA1表达的候选E3泛素蛋白连接酶；构建MIB1过表达质粒并转染至HEK293细胞,采用蛋白免疫印迹法验证二甲双胍能否通过MIB1调控ABCA1的表达。 [结果]二甲双胍可增加J774A.1巨噬细胞中ABCA1的表达(P＜0.01),并抑制ABCA1的降解(P＜0.05)；二甲双胍可抑制ABCA1与泛素的结合(P＜0.05)；二甲双胍调控ABCA1表达的蛋白主要富集于细胞发育、炎症和免疫防御等通路；二甲双胍可通过MIB1上调ABCA1的表达(P＜0.05)。 [结论]二甲双胍可通过阻断泛素-蛋白酶体系统抑制ABCA1的降解,MIB1是上调ABCA1表达的候选E3泛素蛋白连接酶之一。

Abstract:

Aim To explore the potential mechanism of metformin on ATP-binding cassette transport A1 (ABCA1) expression. Methods J774A.1 macrophages were treated with metformin and cycloheximide, and ABCA1 expression was determined by Western blot. His-tagged ABCA1 and HA-tagged Ub plasmids were co-transferred into HEK293 cells and stimulated with metformin. Co-immunoprecipitation (Co-IP) was used to test the binding ability of ABCA1 and ubiquitin. Candidate E3 ubiquitin-protein ligases (CE3) of ABCA1 were identified through Co-IP-based proteomics. The MIB1 plasmid was constructed and transferred into HEK293 cells, and Western blot was used to determine the effect of metformin and MIB1 on ABCA1 expression. Results Metformin increased the expression of ABCA1 in J774A.1 cells (P＜0.01), and inhibited ABCA1 degradation (P＜0.05). Metformin disrupted the binding of ABCA1 to ubiquitin (P＜0.05). The proteins regulated by metformin in ABCA1 expression were primarily enriched in pathways related to cell development, inflammation and immune defense. Metformin may upregulate ABCA1 protein expression via MIB1 (P＜0.05). Conclusion Metformin inhibits the degradation of ABCA1 by blocking the ubiquitin-proteasome system (UPS), and MIB1 might act as a candidate E3 ubiquitin-protein ligase (CE3) for ABCA1.