Tamehiro Norimasa, Zhou Suiping, Okuhira Keiichiro, Benita Yair, Brown Cari E, Zhuang Debbie Z, Latz Eicke, Hornemann Thorsten, von Eckardstein Arnold, Xavier Ramnik J, Freeman Mason W, Fitzgerald Michael L
Lipid Metabolism Unit and Center for Computational and Integrative Biology, Massachusetts General Hospital, Harvard Medical School, 185 Cambridge Street, Boston, Massachusetts 02114, USA.
Biochemistry. 2008 Jun 10;47(23):6138-47. doi: 10.1021/bi800182t. Epub 2008 May 17.
ABCA1 transport of cholesterol and phospholipids to nascent HDL particles plays a central role in lipoprotein metabolism and macrophage cholesterol homeostasis. ABCA1 activity is regulated both at the transcriptional level and at the post-translational level. To explore mechanisms involved in the post-translational regulation of the transporter, we have used affinity purification and mass spectrometry to identify proteins that bind ABCA1 and influence its activity. Previously, we demonstrated that an interaction between beta1-syntrophin stimulated ABCA1 activity, at least in part, be slowing the degradation of the transporter. This work demonstrates that one subunit of the serine palmitoyltransferase enzyme, SPTLC1, but not subunit 2 (SPTLC2), is copurified with ABCA1 and negatively regulates its function. In human THP-I macrophages and in mouse liver, the ABCA1-SPTLC1 complex was detected by co-immunoprecipitation, demonstrating that the interaction occurs in cellular settings where ABCA1 activity is critical for HDL genesis. Pharmacologic inhibition of SPTLC1 with myriocin, which resulted in the disruption of the SPTLC1-ABCA1 complex, and siRNA knockdown of SPTLC1 expression both stimulated ABCA1 efflux by nearly 60% ( p < 0.05). In contrast, dominant-negative mutants of SPTLC1 inhibited ABCA1 efflux, indicating that a reduced level of sphingomyelin synthesis could not explain the effect of myriocin on ABCA1 activity. In 293 cells, the SPTLC1 inhibition of ABCA1 activity led to the blockade of the exit of ABCA1 from the endoplasmic reticulum. In contrast, myriocin treatment of macrophages increased the level of cell surface ABCA1. In composite, these results indicate that the physical interaction of ABCA1 and SPTLC1 results in reduction of ABCA1 activity and that inhibition of this interaction produces enhanced cholesterol efflux.
ABCA1将胆固醇和磷脂转运至新生高密度脂蛋白（HDL）颗粒的过程在脂蛋白代谢和巨噬细胞胆固醇稳态中起着核心作用。ABCA1的活性在转录水平和翻译后水平均受到调控。为探究该转运蛋白翻译后调控所涉及的机制，我们利用亲和纯化和质谱技术来鉴定与ABCA1结合并影响其活性的蛋白质。此前，我们证明β1 - 肌养蛋白之间的相互作用至少部分地通过减缓转运蛋白的降解来刺激ABCA1活性。这项工作表明，丝氨酸棕榈酰转移酶的一个亚基SPTLC1，而非亚基2（SPTLC2），与ABCA1共纯化并负向调节其功能。在人THP - 1巨噬细胞和小鼠肝脏中，通过免疫共沉淀检测到ABCA1 - SPTLC1复合物，表明这种相互作用发生在ABCA1活性对HDL生成至关重要的细胞环境中。用鞘氨醇霉素对SPTLC1进行药理学抑制，导致SPTLC1 - ABCA1复合物的破坏，以及对SPTLC1表达进行小干扰RNA（siRNA）敲低，均使ABCA1流出增加近60%（p < 0.05）。相反，SPTLC1的显性负性突变体抑制ABCA1流出，表明鞘磷脂合成水平降低无法解释鞘氨醇霉素对ABCA1活性的影响。在293细胞中，SPTLC1对ABCA1活性的抑制导致ABCA1从内质网的出口受阻。相反，用鞘氨醇霉素处理巨噬细胞会增加细胞表面ABCA1的水平。综合来看，这些结果表明ABCA1与SPTLC1的物理相互作用导致ABCA1活性降低，而抑制这种相互作用会增强胆固醇流出。
