内质网–质膜互作在凋亡细胞磷脂酰丝氨酸外翻过程中的作用机制研究

Phosphatidylserine (PS) exposure, act as a “eat me” signal released by apoptotic cells, plays a crucial role in the process of engulfment recognition and clearance of apoptotic cells, and its molecular regulation mechanism is a research hotspot in the field of apoptotic cell clearance. Many scientific questions remain to be solved in the non-vesicular transport of PS via the lipid transporter at Endoplasmic reticulum- Plasma membrane contact site. Our preliminary data suggest homologous gene of ORP5/8 in drosophila melanogaster CG1513, and Sac1 involved in the non-vesicular transport of PS at the Endoplasmic reticulum- Plasma membrane contact site were both involved in PS exposure of apoptotic cells. Endoplasmic reticulum- Plasma Membrane contact site is very important for the physiological function of mammalian cells, including excitation-contraction coupling, lipid non-vesicular transport and so on. However, the relationship between apoptotic PS exposure and non-vesicular transport of PS at the Endoplasmic reticulum - Plasma Membrane contact site has not been reported. This project proposed to use simple model organism drosophila melanogaster, and the integrated use of genetics, biochemistry and cell biology research methods, to in depth study the molecular regulation mechanism of non-vesicular transport of PS at the Endoplasmic reticulum - Plasma Membrane contact site in the process of PS exposure during apoptosis. In completion of this research project, we anticipate that our work may provide new insights into current mechanism of apoptotic cells clearance and will lead to the design of new strategies to fight autoimmune and inflammation disease by defects in apoptotic cells clearance.

磷脂酰丝氨酸（PS）外翻作为凋亡细胞释放的“食我”信号，在吞噬细胞识别、清除凋亡细胞的过程中起着至关重要的作用，其分子调控机制是凋亡细胞清除领域的研究热点。PS在内质网-细胞质膜接触位点通过脂质转运蛋白的非囊泡转运仍有许多科学问题尚待解决。内质网-细胞质膜接触位点是亚细胞位点，两者之间的距离约为30nm, 对哺乳动物细胞的生理功能非常重要，包括兴奋-收缩耦合，脂质非囊泡运输等。我们的前期研究基础发现，参与PS在内质网-细胞质膜接触位点的非囊泡转运的黑腹果蝇ORP5/8的同源基因CG1513和Sac1均参与了凋亡细胞的PS外翻。本项目拟以简单模式生物黑腹果蝇为载体，综合运用遗传学、生物化学和细胞生物学等综合研究手段，对内质网-细胞质膜接触位点PS的非囊泡运输与细胞凋亡PS外翻的分子调控机制进行深入的解析和研究，以期为治疗因凋亡细胞清除障碍而引发的严重炎症或致命性自体免疫疾病提供理论基础。

磷脂酰丝氨酸（PS）外翻作为凋亡细胞释放的“食我”信号，在吞噬细胞识别、清除凋亡细胞的过程中起着至关重要的作用，其分子调控机制是凋亡细胞清除领域的研究热点。PS在内质网-细胞质膜接触位点通过脂质转运蛋白的非囊泡转运仍有许多科学问题尚待解决。因此本项目拟借助黑腹果蝇这一优异的模式动物，拟深入探讨内质网–质膜互作（内质网-细胞质膜接触位点 PS 的非囊泡运输）是否参与了细胞凋亡的 PS外翻，是否由 TM9SF4、Sac1 和 dCED-8 三者之间形成蛋白复合体所介导，其分子机制如何。.目前我们已证实：1）黑腹果蝇Xkr8同源蛋白dCED-8具有磷脂翻转酶的活性，调控细胞凋亡期间的PS外翻，并影响吞噬细胞对其识别；2）跨膜蛋白TM9SF4和磷脂酶Sac1和dCED-8形成蛋白复合体，能够影响细胞膜上PS的分布，进而介导了S2细胞凋亡期间PS外翻事件；3） PS的非囊泡运输对细胞凋亡的PS外翻非常重要，OSBP蛋白CG3860、CG1513、CG9205及内质网锚定蛋白Vap33都能通过介导PS的非囊泡运输调控凋亡细胞的PS外翻，其中CG3860和CG1513均能与dCED-8直接互作参与细胞凋亡早期的PS外翻进程。进一步研究发现只有跨膜蛋白TM9SF4与OSBP蛋白CG3860能在果蝇体内影响吞噬细胞对凋亡细胞的识别、清除，说明内质网-细胞膜接触位点的PS非囊泡运输是凋亡早期PS翻转的重要途径之一，且参与了后期PS作为“食我”信号对吞噬细胞的招募。.本项目以简单模式生物黑腹果蝇为载体，综合运用遗传学、生物化学和细胞生物学等综合研究手段，对内质网-细胞质膜接触位点PS的非囊泡运输与细胞凋亡PS外翻的分子调控机制进行深入的解析和研究，以期为治疗因凋亡细胞清除障碍而引发的严重炎症或致命性自体免疫疾病提供理论基础。