跨膜蛋白16F在免疫细胞信号传导中的作用及其机制研究

The plasma membrane (PM) is important not only for structural integrity of the cell, but also for many cell functions such as adhesion, migration and signaling. The PM is made of two asymmetric layers of lipids that include phospholipids (the most abundant), cholesterol, sphingomyelin and glycosphingolipids. Phosphatidylserine (PS) is the most important acidic phospholipid and is primarily located in the inner leaflet of the PM. Several other lipids are also distributed asymmetrically across the PM. This asymmetry is key for normal cell functions and is maintained by enzymes named flippases, floppases and scramblases. Flippases maintain PS in the inner leaflet of the plasma, while floppases carry out the opposite function. In contrast, scramblases randomize lipids, resulting in loss of lipid asymmetry and increased exposure of PS on the outer leaflet of the PM. Scramblases are activated by intracellular Ca2+. . Acute exposure of PS on the outer leaflet of the PM is critical for platelet aggregation and phagocytosis of apoptotic cells by macrophages. In addition, the sustained presence of PS and other acidic phospholipids in the inner aspect of the PM may be important. Indeed, acidic phospholipids are implicated in the recruitment, at the PM, of intracellular signaling molecules such as Src family protein tyrosine kinases (PTKs) and G proteins like Ras. Thus, loss of acidic phospholipids in the inner leaflet could compromise membrane recruitment of these signaling molecules and alter cell functions. . Until recently, the identity of the scramblases was largely unknown. However, recently the transmembrane protein TMEM16F (or ANO6) was identified as a Ca2+-dependent scramblase. Loss-of-function mutations of TMEM16F were identified in patients with Scott syndrome, a bleeding disorder in which platelets fail to expose PS on the outer membrane. Conversely, expression of a point mutant in TMEM16F, that is hypersensitive to Ca2+ ionophores such as ionomycin, resulted in augmented exposure of PS on the outer membrane.. Considering these findings, I postulated that, in addition to their role in platelet aggregation, scramblases might control the functions of intracellular signaling molecules, such as Src kinases and Ras proteins, which require inner leaflet phospholipids. Such a mechanism, possibly regulated by intracellular Ca2+, could be a very rapid way to down-regulate cell functions. This may be especially important for immune cells, which express the high amounts of TMEM16F.

细胞膜脂非对称的分布在细胞膜内外层上。然而细胞膜脂为什么非对称的分布在细胞膜上，是长期困扰学界的重要问题。近年来，关于细胞膜脂在免疫细胞信号传导中的作用，有了很多突破性的进展。细胞膜的非对称结构可以在许多生理过程中打破，其中，以细胞膜搅乱酶为代表的膜蛋白，能够快速的、可逆的、随机的重分布细胞膜脂。本项目推测，细胞膜搅乱酶活化导致的细胞膜脂的重排，不但改变细胞膜非对称性，而且将影响免疫细胞跨膜信号的传导，进而影响免疫细胞的功能。本项目将以细胞膜搅乱酶为切入点，利用免疫学方法，以基因敲除小鼠为动物模型，深入研究其在免疫细胞跨膜信号传导中的作用，致力于揭示细胞膜非对称结构对于细胞信号传导的作用。全面了解细胞膜搅乱酶在免疫细胞中的作用及其工作机制，不但具有重要理论意义，更能够指导开发调节细胞膜搅乱酶活性的分子药物，用于调节免疫细胞功能，具有重要应用潜力。

基于细胞膜脂非对称性结构对于免疫细胞跨膜信号传导影响的科学假说，我们以细胞膜脂搅乱酶TMEM16F为切入点，对淋巴细胞，尤其是T细胞和NK细胞进行了细胞膜受体信号跨膜传导和相关功能的研究，并取得了丰硕的成果和极其意外的收获。主要结果如下：.一、.TMEM16F参与细胞膜完整性维持.该项目最初的设想认为在TCR信号导致的钙内流可以通过活化TMEM16F这个钙离子依赖的细胞膜脂搅乱酶，来影响和调节TCR信号及T细胞活化。然而，我们在TMEM16F过表达和敲除的细胞模型中都未观察到T细胞信号和功能的异常（结果未展示）。我们仍然坚信TMEM16F在T细胞中扮演着重要角色，相关课题仍然在其他的场景下继续进行探索。意外的是，我们发现在细胞膜破损中，TMEM16F发挥重要的作用，即通过搅乱细胞膜脂，增强细胞膜脂流动性，抵抗细胞膜损伤，维持细胞膜完整性。这个全新的分子功能不仅仅在T细胞中存在，在大量的免疫细胞中都存在。该成果发表在Cell Reports上（2020）。该意外收获在具有重要科学意义，揭示了TMEM16F类细胞膜脂搅乱酶的进化保守意义，即在从低等到高等生物中维持细胞膜流动性。..二、.以TMEM16F为分子工具，证明细胞膜脂的非对称性维持在免疫细胞跨膜信号传导中的重要作用.在NK细胞中，TMEM16F介导的细胞膜脂重分布可以强烈的影响NK细胞膜受体2B4的跨膜信号传导。这种重分布膜脂是严格16F依赖的，瞬时可逆的效应。进一步研究发现TMEM16F活化后，细胞膜的生物化学和生物物理特性发生了多个层面的改变。2B4的跨膜信号传导异常可能是由于其本身构象改变、Src家族激酶定位改变、细胞膜理化特性（如电荷分布、脂质分子的空间分布、流动性、膜蛋白的分布）改变综合导致。就我们了解，这是目前该领域首次系统性的通过实验直接证明细胞膜脂非对称性分布对于免疫细胞信号传导的作用，并影响免疫细胞功能，且从分子水平提出了机制解释。相关成果以共同通讯身份发表在Cell Mol Immunol （2021）。该成果提出了有力的证据，全面支持细胞膜非对称性除了在结构上是生命体的特征，还对生命体信号传导具有至关重要的作用。