N-terminal mutation of apoA-I and interaction with ABCA1 reveal mechanisms of nascent HDL biogenesis
N-terminal mutation of apoA-I and interaction with ABCA1 reveal mechanisms of nascent HDL biogenesis
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DOI：
10.1194/jlr.m084376
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发表时间：
2019-01-01
影响因子：
6.5
通讯作者：
Atkinson, David
中科院分区：
文献类型：
--
作者：
Liu, Minjing;Mei, Xiaohu;Atkinson, David
apoa - 1和ABCA1在新生高密度脂蛋白（nHDL）的生物发生中起重要作用，这是逆向胆固醇运输途径的第一步，可以预防心血管疾病。根据我们实验室确定的c端截断形式的apoA-I[Delta(185-243)]的晶体结构，我们假设打开n端螺旋束会促进脂质结合。为此，我们在结构上设计了一个突变体（L38G/K40G）来破坏第一个铰链区域的n端螺旋束的稳定性。该突变体在溶液中的构象特征显示，螺旋含量最低限度地降低，整体结构不致密，脂质结合能力增强。在溶液结合研究中，apoA-I和纯化的ABCA1也表现出直接结合。在abca1转染的HEK293细胞中，L38G/K40G形成nHDL的能力显著增强，这表明不稳定的n端束促进了nHDL的形成。然而，abca1转染的HEK293细胞的总胆固醇外排在突变体与WT apoA-I相比没有变化，这表明胆固醇外排和nHDL颗粒形成可能是不耦合的事件。对流出介质中颗粒的分析显示，一群无apoa - i脂质颗粒与nHDL一起存在。该模型为未来的研究提高了对nHDL形成的认识。
ApoA-I and ABCA1 play important roles in nascent HDL (nHDL) biogenesis, the first step in the pathway of reverse cholesterol transport that protects against cardiovascular disease. On the basis of the crystal structure of a C-terminally truncated form of apoA-I[Delta(185-243)] determined in our laboratory, we hypothesized that opening the N-terminal helix bundle would facilitate lipid binding. To that end, we structurally designed a mutant (L38G/K40G) to destabilize the N-terminal helical bundle at the first hinge region. Conformational characterization of this mutant in solution revealed minimally reduced -helical content, a less-compact overall structure, and increased lipid-binding ability. In solution-binding studies, apoA-I and purified ABCA1 also showed direct binding between them. In ABCA1-transfected HEK293 cells, L38G/K40G had a significantly enhanced ability to form nHDL, which suggests that a destabilized N-terminal bundle facilitates nHDL formation. The total cholesterol efflux from ABCA1-transfected HEK293 cells was unchanged in mutant versus WT apoA-I, though, which suggests that cholesterol efflux and nHDL particle formation might be uncoupled events. Analysis of the particles in the efflux media revealed a population of apoA-I-free lipid particles along with nHDL. This model improves knowledge of nHDL formation for future research.