Chowdhury Saiful M, Zhu Xuewei, Aloor Jim J, Azzam Kathleen M, Gabor Kristin A, Ge William, Addo Kezia A, Tomer Kenneth B, Parks John S, Fessler Michael B
From the ‡Laboratory of Respiratory Biology and.
¶Section on Molecular Medicine, Department of Internal Medicine, Wake Forest School of Medicine, Winston-Salem, NC 27157.
Mol Cell Proteomics. 2015 Jul;14(7):1859-70. doi: 10.1074/mcp.M114.045179. Epub 2015 Apr 24.
Lipid raft membrane microdomains organize signaling by many prototypical receptors, including the Toll-like receptors (TLRs) of the innate immune system. Raft-localization of proteins is widely thought to be regulated by raft cholesterol levels, but this is largely on the basis of studies that have manipulated cell cholesterol using crude and poorly specific chemical tools, such as β-cyclodextrins. To date, there has been no proteome-scale investigation of whether endogenous regulators of intracellular cholesterol trafficking, such as the ATP binding cassette (ABC)A1 lipid efflux transporter, regulate targeting of proteins to rafts. Abca1(-/-) macrophages have cholesterol-laden rafts that have been reported to contain increased levels of select proteins, including TLR4, the lipopolysaccharide receptor. Here, using quantitative proteomic profiling, we identified 383 proteins in raft isolates from Abca1(+/+) and Abca1(-/-) macrophages. ABCA1 deletion induced wide-ranging changes to the raft proteome. Remarkably, many of these changes were similar to those seen in Abca1(+/+) macrophages after lipopolysaccharide exposure. Stomatin-like protein (SLP)-2, a member of the stomatin-prohibitin-flotillin-HflK/C family of membrane scaffolding proteins, was robustly and specifically increased in Abca1(-/-) rafts. Pursuing SLP-2 function, we found that rafts of SLP-2-silenced macrophages had markedly abnormal composition. SLP-2 silencing did not compromise ABCA1-dependent cholesterol efflux but reduced macrophage responsiveness to multiple TLR ligands. This was associated with reduced raft levels of the TLR co-receptor, CD14, and defective lipopolysaccharide-induced recruitment of the common TLR adaptor, MyD88, to rafts. Taken together, we show that the lipid transporter ABCA1 regulates the protein repertoire of rafts and identify SLP-2 as an ABCA1-dependent regulator of raft composition and of the innate immune response.
脂筏膜微区通过许多典型受体来组织信号传导，包括先天免疫系统的Toll样受体（TLR）。蛋白质在脂筏中的定位普遍被认为受脂筏胆固醇水平的调节，但这在很大程度上是基于使用如β - 环糊精等粗糙且特异性差的化学工具来操纵细胞胆固醇的研究。迄今为止，尚未有关于细胞内胆固醇转运的内源性调节因子，如ATP结合盒（ABC）A1脂质外流转运蛋白，是否调节蛋白质靶向脂筏的蛋白质组规模的研究。据报道，Abca1(-/-)巨噬细胞具有富含胆固醇的脂筏，其中某些蛋白质的水平有所增加，包括脂多糖受体TLR4。在这里，我们使用定量蛋白质组分析，在来自Abca1(+/+)和Abca1(-/-)巨噬细胞的脂筏分离物中鉴定出383种蛋白质。ABCA1的缺失引起了脂筏蛋白质组的广泛变化。值得注意的是，其中许多变化与脂多糖暴露后Abca1(+/+)巨噬细胞中观察到的变化相似。类stomatin蛋白（SLP）-2是膜支架蛋白的stomatin - prohibitin - flotillin - HflK/C家族的成员，在Abca1(-/-)脂筏中显著且特异性地增加。在研究SLP - 2的功能时，我们发现SLP - 2沉默的巨噬细胞的脂筏具有明显异常的组成。SLP - 2沉默并不损害ABCA1依赖性胆固醇外流，但降低了巨噬细胞对多种TLR配体的反应性。这与TLR共受体CD14的脂筏水平降低以及脂多糖诱导的常见TLR衔接蛋白MyD88向脂筏的募集缺陷有关。综上所述，我们表明脂质转运蛋白ABCA1调节脂筏的蛋白质组成，并确定SLP - 2是一种依赖ABCA1的脂筏组成和先天免疫反应的调节因子。
