Department of Biochemistry and Molecular Biology, Faculty of Biology, Complutense University, Madrid, Spain.
Research Institute "Hospital 12 de Octubre (imas12)", Madrid, Spain.
Protein Sci. 2024 Jan;33(1):e4835. doi: 10.1002/pro.4835.
Pulmonary surfactant (PS) is a lipid-protein complex that forms films reducing surface tension at the alveolar air-liquid interface. Surfactant protein C (SP-C) plays a key role in rearranging the lipids at the PS surface layers during breathing. The N-terminal segment of SP-C, a lipopeptide of 35 amino acids, contains two palmitoylated cysteines, which affect the stability and structure of the molecule. The C-terminal region comprises a transmembrane α-helix that contains a ALLMG motif, supposedly analogous to a well-studied dimerization motif in glycophorin A. Previous studies have demonstrated the potential interaction between SP-C molecules using approaches such as Bimolecular Complementation assays or computational simulations. In this work, the oligomerization state of SP-C in membrane systems has been studied using fluorescence spectroscopy techniques. We have performed self-quenching and FRET assays to analyze dimerization of native palmitoylated SP-C and a non-palmitoylated recombinant version of SP-C (rSP-C) using fluorescently labeled versions of either protein reconstituted in different lipid systems mimicking pulmonary surfactant environments. Our results reveal that doubly palmitoylated native SP-C remains primarily monomeric. In contrast, non-palmitoylated recombinant SP-C exhibits dimerization, potentiated at high concentrations, especially in membranes with lipid phase separation. Therefore, palmitoylation could play a crucial role in stabilizing the monomeric α-helical conformation of SP-C. Depalmitoylation, high protein densities as a consequence of membrane compartmentalization, and other factors may all lead to the formation of protein dimers and higher-order oligomers, which could have functional implications under certain pathological conditions and contribute to membrane transformations associated with surfactant metabolism and alveolar homeostasis.
肺表面活性剂（PS）是一种脂质-蛋白复合物，可在肺泡气-液界面形成薄膜以降低表面张力。表面活性蛋白 C（SP-C）在呼吸过程中对 PS 表面层的脂质重排起着关键作用。SP-C 的 N 端片段是一个由 35 个氨基酸组成的脂肽，其中包含两个棕榈酰化半胱氨酸，它们影响分子的稳定性和结构。C 端区域包含一个跨膜α-螺旋，其中包含一个 ALLMG 基序，推测类似于糖蛋白 A 中研究充分的二聚化基序。先前的研究已经使用双分子互补测定或计算模拟等方法证明了 SP-C 分子之间的潜在相互作用。在这项工作中，使用荧光光谱技术研究了 SP-C 在膜系统中的寡聚状态。我们使用荧光标记的天然棕榈酰化 SP-C 和非棕榈酰化重组 SP-C（rSP-C）进行了自猝灭和 FRET 测定，这些蛋白版本在不同的脂质系统中进行了重建，模拟了肺表面活性剂环境。我们的结果表明，双棕榈酰化天然 SP-C 主要保持单体状态。相比之下，非棕榈酰化重组 SP-C 表现出二聚化，在高浓度下增强，尤其是在具有脂质相分离的膜中。因此，棕榈酰化可能在稳定 SP-C 的单体α-螺旋构象中起关键作用。去棕榈酰化、由于膜分隔而导致的高蛋白密度以及其他因素都可能导致蛋白二聚体和更高阶的寡聚体形成，这可能在某些病理条件下具有功能意义，并有助于与表面活性剂代谢和肺泡稳态相关的膜转化。
