VPS53调控细胞内胆固醇转运的机制与生理功能

Intracellular cholesterol transport is a vital process required for natural cellular activities. The deficient low-density lipoprotein (LDL)-derived cholesterol egress from lysosomes could induce Niemann–Pick C disease (NPC disease), a severe neurodegenerative disease. By employing an Amphotericin B treatment, we screened out two mutant cell lines with lysosomal cholesterol accumulation. A mutation in vps53 gene was identified to be responsible for the defective cholesterol trafficking in the mutant cells. It is known that mutations of vps53 caused another neurodegenerative disease, progressive cerebello-cerebral atrophy type 2 (PCCA2), which is similar to the NPC disease. However, the mechanism through which VPS53 protein regulates intracellular cholesterol trafficking and how the vps53 mutations lead to PCCA2 disease is poorly understood. This proposal will first investigate the mechanism whereby the deficiency of VPS53 protein impairs the cholesterol trafficking at cellular level. To elucidate the mechanism underlying PCCA2 disease development, we will investigate the regulatory functions of VPS53 in body cholesterol metabolism using vps53-deficient mice as well as fibroblast from patients with PCCA2. Furthermore, the therapeutic effect on PCCA2 disease after restoring the cellular cholesterol trafficking of vps53-deficient mice by Hydroxypropyl-β-Cyclodextrin (HPCD) administration will be assessed. This project aims to elucidate the working mechanism of VPS53 protein that was identified to be implicated in the intracellular cholesterol trafficking, and reveal the defective lysosomal cholesterol egress caused by vps53 mutations contribute to the development of PCCA2 disease.

细胞内胆固醇的运输是维持细胞正常活动的重要过程，其中低密度脂蛋白（LDL）来源的胆固醇向溶酶体外的转运发生障碍会诱发神经退行性疾病——尼曼匹克C症。前期我们利用两性霉素筛选到胆固醇转运异常的变异细胞，通过转录组测序鉴定出突变基因为vps53。已知vps53突变会导致另一种神经系统疾病进行性小脑萎缩（PCCA2），但VPS53蛋白如何调控细胞内胆固醇转运及其变异引发PCCA2的机制仍不清楚。本项目将在细胞水平深入揭示VPS53缺陷引起胆固醇转运障碍的机理。同时，为探寻vps53基因突变导致PCCA2的机制，我们将利用vps53基因缺陷小鼠和PCCA2病人细胞分析VPS53蛋白对机体胆固醇代谢的调控功能；进一步采用HPCD注射等方法恢复胆固醇转运，探索对PCCA2疾病的治疗效果。本课题旨在揭示新鉴定的胆固醇转运关键蛋白VPS53的作用机制及其突变引起的胆固醇转运障碍在PCCA2疾病发生中的贡献

细胞内胆固醇的正常转运对于生命活动是至关重要的，其中低密度脂蛋白来源的胆固醇向溶酶体外转运障碍会诱发神经退行性疾病。已知两个溶酶体蛋白NPC1和NPC2介导胆固醇向溶酶体外的转运，然而目前对调控这一过程的其余因子还所知甚少。通过基于两性霉素的筛选，我们得到胆固醇转运缺陷的细胞株。转录组测序分析发现缺陷细胞株携带 Vps53基因突变。抑制包括VPS53蛋白在内的GARP 复合物亚基的表达会导致NPC2蛋白向溶酶体分选异常并引起胆固醇堆积。机制研究表明，GARP复合物缺陷会阻碍CI-MPR向反面高尔基网的运输。Vps54基因突变小鼠出现细胞内NPC2减少和大量胆固醇堆积的表型，表明GARP复合物在机体胆固醇代谢过程中发挥重要功能。综上，本项研究发现GARP复合物通过促进CI-MPR依赖的NPC2蛋白向溶酶体的分选运输参与细胞内胆固醇转运。已知NF-κB2蛋白是一个转录调控因子，参与免疫和炎症反应过程。我们鉴定发现包括NF-κB2在内的非经典NF-κB信号通路控制NPC2的表达，从而调节胆固醇向溶酶体外的运输过程。进一步研究发现NF-κB2能直接结合到NPC2基因的启动子上调控其转录。小鼠模型研究显示NFKB2突变小鼠的脾脏、淋巴结和胸腺等免疫器官均表现出明显的胆固醇累积。该项研究揭示了NF-κB信号对细胞内胆固醇运输的调控功能，提示免疫系统与胆固醇运输之间的紧密联系。