细胞器膜接触介导胆固醇转运的分子机制与功能研究

Cholesterol is the most abundant sterol in mammalian cells and unevenly distributed in cellular membranes. The intracellular transport of cholesterol among organelles is essential for multiple cellular functions. Our recent study has shown for the first time the existence of lysosomal-peroxisome membrane contacts essential for the cellular trafficking of cholesterol. The results explain the key step of how cholesterol leaves the lysosomal membrane. However, there are still important questions unanswered. For example, how does the hydrophobic cholesterol cross the water phase between the cell membranes? How does the cholesterol pass from peroxisomes to other organelles? In our preliminary experiment, we found LPCTP protein can mediate the transport of cholesterol between lysosomes and peroxisomes and we observed the membrane contacts between peroxisomes and endoplasmic reticulum. In this proposal, we will use the organelle in vitro reconstruction, artificial liposome and other methods to elucidate the LPCTP function in the cholesterol transport between lysosomal-peroxisome membrane contacts. We will reveal the molecular mechanism of peroxisomes and endoplasmic reticulum contact. In addition, we will explore the physiological and pathological functions of lysosome-peroxisome-ER cholesterol transport system. The results will deepen our understanding of organelle membrane contacts and intracellular cholesterol transport mechanism.

胆固醇是哺乳动物细胞含量最丰富的甾醇类分子，它在各细胞器间动态运输并呈不均匀分布，胆固醇的正常运输对维持细胞的功能至关重要。我们发现过氧化物酶体可通过和溶酶体形成膜接触而实现胆固醇的转运，揭示了胆固醇离开溶酶体膜这一关键步骤的分子机制。但是仍然有许多重要问题没有回答，如疏水的胆固醇是如何跨水相在细胞器膜之间进行转运？胆固醇到达过氧化物酶体后再如何转运到其他细胞器？我们前期实验筛选到LPCTP蛋白可介导溶酶体与过氧化物酶体之间的胆固醇转运，同时也观察到过氧化物酶体和内质网存在膜接触。本项目将利用细胞器体外重构、人工脂质体等方法阐明LPCTP在胆固醇细胞器膜之间进行跨水相转运的分子机制，揭示过氧化物酶体与内质网形成膜接触的分子基础，最后在动物水平探索溶酶体-过氧化物酶体-内质网胆固醇转运的生理病理功能。研究结果将揭示细胞器膜接触介导胆固醇转运机制, 开辟胆固醇代谢研究的新方向。

胆固醇是哺乳动物细胞内重要的而且也是含量最高的甾醇类分子，血液胆固醇以低密度脂蛋白形式被细胞膜表面的低密度脂蛋白受体识别并内吞，LDL 继而被分选入溶酶体、水解释放出游离的胆固醇。在我们的早期工作中鉴定出溶酶体-过氧化物酶体之间的膜接触可介导胆固醇从溶酶体中运出。在本研究中，我们观察到过氧化物酶体和内质网有紧密的膜接触，并且使用高度纯化的过氧化物酶体和内质网在体外成功重构了这种细胞器间的膜接触。无论是降低过氧化物酶体上PI(4,5)P2含量还是敲低E-Syts 都会减少过氧化物酶体与内质网之间的膜接触，并导致大量胆固醇在溶酶体中积累。通过使用体外重构系统，更进一步证明了胆固醇可从过氧化物酶体或含有PI(4,5)P2的脂质体向内质网膜的转运。综上，过氧化物酶体和内质网通过PI(4,5)P2 和 E-Syts 形成膜接触，并且胆固醇可以通过这种膜接触由过氧化物酶体向内质网转运。结合前期已经发现的溶酶体-过氧化物酶体膜接触，我们阐述了低密度脂蛋白来源胆固醇沿溶酶体-过氧化物酶体-内质网轴运输这样一条新的胆固醇转运途径。