Wenner C, Lorkowski S, Engel T, Cullen P
Institute of Arteriosclerosis Research, University of Münster, Germany.
Biochem Biophys Res Commun. 2001 Mar 30;282(2):608-14. doi: 10.1006/bbrc.2001.4611.
Macrophage-derived apolipoprotein E (apoE) influences the susceptibility of the arterial wall to atherosclerosis. Previous studies have shown that production of apoE in these cells is regulated at a posttranscriptional level and is increased by inhibitors of proteasomal degradation. To further investigate this mechanism, we stably transfected RAW 264.7 macrophages and HepG2 cells with a construct overexpressing ubiquitin, the peptide targeting proteins to the proteasome, fused to an influenza virus hemagglutinin epitope tag. Ubiquitination of apoE was investigated by immunoprecipitation and Western blot analysis. In both cell types, apoE was ubiquitinated, and inhibition of proteasome function by lactacystin led to accumulation of ubiquitinated apoE. These studies provide strong evidence for proteasomal degradation of apoE in the two main cell types responsible for its production and indicate a possible new level of regulation of this important protein.
巨噬细胞衍生的载脂蛋白E（apoE）会影响动脉壁对动脉粥样硬化的易感性。先前的研究表明，这些细胞中apoE的产生在转录后水平受到调控，并且蛋白酶体降解抑制剂可使其增加。为了进一步研究这一机制，我们用一个过表达泛素（将蛋白质靶向蛋白酶体的肽段，并与流感病毒血凝素表位标签融合）的构建体稳定转染了RAW 264.7巨噬细胞和HepG2细胞。通过免疫沉淀和蛋白质印迹分析研究了apoE的泛素化情况。在这两种细胞类型中，apoE均被泛素化，而乳胞素对蛋白酶体功能的抑制导致泛素化apoE的积累。这些研究为负责产生apoE的两种主要细胞类型中apoE的蛋白酶体降解提供了有力证据，并表明了对这一重要蛋白质进行调控的一个可能的新层面。
