AGE-albumin enhances ABCA1 degradation by ubiquitin-proteasome and lysosomal pathways in macrophages
AGE-albumin enhances ABCA1 degradation by ubiquitin-proteasome and lysosomal pathways in macrophages
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DOI：
10.1016/j.jdiacomp.2017.09.012
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发表时间：
2018-01-01
影响因子：
3
通讯作者：
Passarelli, Marisa
中科院分区：
文献类型：
--
作者：
Iborra, Rodrigo Tallada;Machado-Lima, Adriana;Passarelli, Marisa
背景和目的：晚期糖基化终产物（AGEs）诱导细胞氧化/内质网应激和炎症反应。我们研究了其动脉粥样硬化形成的潜在机制，重点是调节巨噬细胞中ABCA 1蛋白的衰变。在用LXR激动剂处理后，并通过与对照（C）或AGE-白蛋白一起孵育或不与放线菌酮孵育，评估巨噬细胞中ABCA 1衰减速率，MG-132、氯化铵和钙蛋白酶抑制剂分别用于抑制蛋白酶体，溶酶体和ABCA 1蛋白水解。通过免疫印迹测定ABCA 1，并通过线性回归的斜率计算沿着时间的蛋白质衰减速率。在从全细胞裂解物或散装细胞膜免疫沉淀的ABCA 1中测定泛素化水平。在用siRNA-siRNA转染的THP-1细胞中也分析了AGE的作用。结果：AGE-白蛋白组CML和PYR-白蛋白含量均高于C组（P < 0. 05）; AGE-白蛋白减少了J774和THP-1巨噬细胞中的ABCA 1（20-30%），并诱导了更高的ABCA 1泛素化和更快的蛋白质衰变速率，这取决于在放线菌酮存在下测量动力学期间AGE的存在。在用AGE-白蛋白处理的细胞中，蛋白酶体抑制恢复，溶酶体抑制部分恢复ABCA 1。钙蛋白酶抑制不能拯救ABCA 1。结论：AGE-白蛋白通过蛋白酶体和溶酶体系统加速ABCA 1的降解，从而减少ABCA 1。这可能通过减少胆固醇流出而增加巨噬细胞中的脂质积聚，胆固醇流出通过促进糖尿病中的动脉粥样硬化的信号传导。(C)2017爱思唯尔公司All rights reserved.
Background and aims: Advanced glycation end products (AGEs) induce cellular oxidative/endoplasmic reticulum stress and inflammation. We investigated its underlying mechanisms for atherogenesis focusing on regulation of ABCA1 protein decay in macrophages.Methods: The ABCA1 decay rate was evaluated in macrophages after treatment with LXR agonist and by incubation with control (C) or AGE-albumin concomitant or not with cycloheximide, MG-132, ammonium chloride and calpain inhibitors were utilized to inhibit, respectively, proteasome, lysosome and ABCA1 proteolysis at cell surface. ABCA1 was determined by immunoblot and the protein decay rate calculated along time by the slope of the linear regression. Ubiquitination level was determined in ABCA1 immunoprecipitated from whole cell lysate or bulk cell membrane. AGE effect was also analyzed in THP-1 cells transfected with siRNA-RAGE. Carboxymethyllysine (CML) and pyrraline (PYR) were determined by LC/MS. One-way ANOVA and Student t test were utilized to compare results.Results: CML and PYR-albumin were higher in AGE-albumin as compared to C. AGE-albumin reduced ABCA1 in J774 and THP-1 macrophages (20-30%) and induced a higher ABCA1 ubiquitination and a faster protein decay rate that was dependent on the presence of AGE during the kinetics of measurement in the presence of cycloheximide. Proteasomal inhibition restored and lysosomal inhibition partially recovered ABCA1 in cells treated with AGE-albumin. Calpain inhibition was not able to rescue ABCA1. RAGE knockdown prevented the reduction in ABCA1 elicited by AGE.Conclusions: AGE-albumin.diminishes ABCA1 by accelerating its degradation through the proteasomal and lysosomal systems. This may increase lipid accumulation in macrophages by diminishing cholesterol efflux via RAGE signaling contributing to atherosclerosis in diabetes mellitus. (C) 2017 Elsevier Inc. All rights reserved.