Parr Rebecca D, Martin Gregory G, Hostetler Heather A, Schroeder Megan E, Mir Kiran D, Kier Ann B, Ball Judith M, Schroeder Friedhelm
Department of Pathobiology, Texas A&M University, TVMC, College Station, Texas 77843-4467, USA.
Biochemistry. 2007 Jul 17;46(28):8301-14. doi: 10.1021/bi7002636. Epub 2007 Jun 20.
Although plasma membrane domains, such as caveolae, provide an organizing principle for signaling pathways and cholesterol homeostasis in the cell, relatively little is known regarding specific mechanisms, whereby intracellular lipid-binding proteins are targeted to caveolae. Therefore, the interaction between caveolin-1 and sterol carrier protein-2 (SCP-2), a protein that binds and transfers both cholesterol and signaling lipids (e.g., phosphatidylinositides and sphingolipids), was examined by yeast two-hybrid, in vitro binding and fluorescence resonance energy transfer (FRET) analyses. Results of the in vivo and in vitro assays identified for the first time the N-terminal amino acids (aa) 1-32 amphipathic alpha helix of SCP-2 functionally interacted with caveolin-1. This interaction was independent of the classic caveolin-1 scaffolding domain, in which many signaling proteins interact. Instead, SCP-2 bound caveolin-1 through a new domain identified in the N-terminal domain of caveolin-1 between aa 34-40. Modeling studies suggested that electrostatic interactions between the SCP-2 N-terminal aa 1-32 amphipathic alpha-helical domain (cationic, positively charged face) and the caveolin-1 N-terminal aa 33-59 alpha helix (anionic, negatively charged face) may significantly contribute to this interaction. These findings provide new insights on how SCP-2 enhances cholesterol retention within the cell as well as regulates the distribution of signaling lipids, such as phosphoinositides and sphingolipids, at plasma membrane caveolae.
尽管质膜结构域，如小窝，为细胞内信号通路和胆固醇稳态提供了一种组织原则，但对于细胞内脂质结合蛋白靶向小窝的具体机制却知之甚少。因此，通过酵母双杂交、体外结合和荧光共振能量转移（FRET）分析，研究了小窝蛋白-1与固醇载体蛋白-2（SCP-2）之间的相互作用，SCP-2是一种结合并转运胆固醇和信号脂质（如磷脂酰肌醇和鞘脂）的蛋白。体内和体外实验结果首次确定，SCP-2的N端1-32个氨基酸的两亲性α螺旋与小窝蛋白-1发生功能相互作用。这种相互作用独立于许多信号蛋白相互作用的经典小窝蛋白-1支架结构域。相反，SCP-2通过在小窝蛋白-1 N端结构域中34-40位氨基酸之间鉴定出的一个新结构域与小窝蛋白-1结合。模型研究表明，SCP-2的N端1-32个氨基酸的两亲性α螺旋结构域（阳离子、带正电荷的面）与小窝蛋白-1的N端33-59位氨基酸α螺旋（阴离子、带负电荷的面）之间的静电相互作用可能对这种相互作用有显著贡献。这些发现为SCP-2如何增强细胞内胆固醇保留以及调节质膜小窝处信号脂质（如磷酸肌醇和鞘脂）的分布提供了新的见解。
