Oram John F, Wolfbauer Gertrud, Tang Chongren, Davidson W Sean, Albers John J
Division of Metabolism, Endocrinology and Nutrition, Department of Medicine, Box 356426, University of Washington, Seattle, Washington 98195, USA.
J Biol Chem. 2008 Apr 25;283(17):11541-9. doi: 10.1074/jbc.M800117200. Epub 2008 Feb 19.
Phospholipid lipid transfer protein (PLTP) mimics high-density lipoprotein apolipoproteins in removing cholesterol and phospholipids from cells through the ATP-binding cassette transporter A1 (ABCA1). Because amphipathic alpha-helices are the structural determinants for ABCA1 interactions, we examined the ability of synthetic peptides corresponding to helices in PLTP to remove cellular cholesterol by the ABCA1 pathway. Of the seven helices tested, only one containing PLTP residues 144-163 (p144), located at the tip of the N-terminal barrel, promoted ABCA1-dependent cholesterol efflux and stabilized ABCA1 protein. Mutating methionine 159 (Met-159) in this helix in PLTP to aspartate (M159D) or glutamate (M159E) nearly abolished the ability of PLTP to remove cellular cholesterol and dramatically reduced PLTP binding to phospholipid vesicles and its phospholipid transfer activity. These mutations impaired PLTP binding to ABCA1-generated lipid domains and PLTP-mediated stabilization of ABCA1 but increased PLTP binding to ABCA1. PLTP interactions with ABCA1 also mimicked apolipoproteins in activating Janus kinase 2; however, the M159D/E mutants were also able to activate this kinase. Structural analyses showed that the M159D/E mutations had only minor effects on PLTP conformation. These findings indicate that PLTP helix 144-163 is critical for removing lipid domains formed by ABCA1, stabilizing ABCA1 protein, interacting with phospholipids, and promoting phospholipid transfer. Direct interactions with ABCA1 and activation of signaling pathways likely involve other structural determinants of PLTP.
磷脂转运蛋白（PLTP）通过ATP结合盒转运体A1（ABCA1）从细胞中清除胆固醇和磷脂，其功能类似于高密度脂蛋白载脂蛋白。由于两亲性α螺旋是ABCA1相互作用的结构决定因素，我们研究了与PLTP中螺旋相对应的合成肽通过ABCA1途径清除细胞胆固醇的能力。在所测试的七个螺旋中，只有一个包含位于N端桶状结构顶端的PLTP第144 - 163位残基（p144），它能促进ABCA1依赖的胆固醇流出并稳定ABCA1蛋白。将PLTP中该螺旋的甲硫氨酸159（Met - 159）突变为天冬氨酸（M159D）或谷氨酸（M159E），几乎消除了PLTP清除细胞胆固醇的能力，并显著降低了PLTP与磷脂囊泡的结合及其磷脂转运活性。这些突变损害了PLTP与ABCA1产生的脂质结构域的结合以及PLTP介导的ABCA1稳定，但增加了PLTP与ABCA1的结合。PLTP与ABCA1的相互作用在激活Janus激酶2方面也类似于载脂蛋白；然而，M159D/E突变体也能够激活该激酶。结构分析表明，M159D/E突变对PLTP构象只有轻微影响。这些发现表明，PLTP的144 - 163螺旋对于清除由ABCA1形成的脂质结构域、稳定ABCA1蛋白、与磷脂相互作用以及促进磷脂转运至关重要。与ABCA1的直接相互作用和信号通路的激活可能涉及PLTP的其他结构决定因素。
