The Role of Phosphatidylinositides in Lipid Metabolism

摘要 作为细胞内转运的关键因子，磷脂酰肌肽(PI)是一类低丰度脂类， 几乎影响到真核细胞内的每一个过程。低密度脂蛋白受体(LDLR)是一种主要的 血低密度脂蛋白水平的决定因素，是预防心血管疾病最有效的治疗靶点 疾病。当低密度脂蛋白结合时，细胞表面的低密度脂蛋白受体被内化，在那里它释放结合的低密度脂蛋白，然后 回收回细胞表面或直接进入溶酶体进行降解。最近，我们发现敲门- 一种磷脂酰肌醇(4，5)二磷酸[PI(4，5)P]的跨膜蛋白55B 磷酸酶可促进细胞内LDLR蛋白的降解，并使小鼠血浆LDLC升高。由于PI(4，5)P是 参与溶酶体的形成，我们推测PI(4，5)P磷酸酶(如TMEM55B)可能会改变 低密度脂蛋白通过调节低密度脂蛋白受体的胞内转运。我们还发现PI(4，5)P磷酸酶的减少导致了 严重损害甘油三酯向血浆的分泌。值得注意的是，这种情况发生时没有肝脏积聚。 有趣的是，脂肪只在雄性小鼠身上观察到，对雌性小鼠没有影响。肝脏脂肪缺乏 蓄积可以归因于许多不同的机制，包括吞脂性增加(一个过程 选择性自噬，其中脂滴是溶酶体腐烂的靶标)。PI(4，5)P是一种重要的调节剂 自噬小体融合后的溶酶体重塑，我们发现TMEM55B的敲除增加 溶酶体。这些发现提示PI(4，5)P磷酸酶可能调节肝脏TG的分泌和储存 通过溶酶体依赖的机制。因此，这项提案的总体目标是评价 PI(4，5)P参与LDLR循环和甘油三酯分泌。在目标1中，我们将i)测试PI(4，5)P代谢酶是否影响 利用转基因小鼠模型在缺乏低密度脂蛋白受体的情况下血浆低密度脂蛋白；ii)评估修饰的效果 PI(4，5)P对LDLR细胞内水平和小鼠肝细胞定位的影响；III)酶促表达 不活跃的Tem55b挽救在Tem55b基因敲除小鼠中观察到的表型；以及iv)评估是否 PI(4，5)P磷酸酶对低密度脂蛋白受体循环的调节依赖于已知的低密度脂蛋白受体衰变调节因子。在AIM 2 我们将比较全身和肝脏特异的Tem55b基因敲除雄性小鼠的甘油三酯分泌，以证实 缺陷是肝脏的机制；ii)测试是否缺乏肝脏脂肪堆积，这将是预期的结果 Tem55b基因敲除小鼠肝细胞甘油三酯分泌受损是由于脂肪酸的变化 氧化、合成或摄取，或TG合成；III)取决于溶酶体决定PI(4，5)P磷酸酶 和/或自噬小体以损害甘油三酯的分泌；以及iv)评估PI(4，5)P调节是否具有性别特异性 甘油三酯的分泌是由性染色体组成(XX对XY)和/或性腺激素引起的。调查 研究PI(4，5)P在脂类和脂蛋白代谢中的作用可能导致新的治疗方案的开发 降低心脏代谢性疾病的风险。

SUMMARY As key directors of intracellular trafficking, phosphotidylinsotides (PIs) are a family of low abundance lipids that impact almost every process within the eukaryotic cell. The low-density lipoprotein receptor (LDLR), a major determinant of blood LDL levels, is the most efficacious therapeutic target for the prevention of cardiovascular disease. Upon LDL binding, LDLR at the cell surface is internalized, where it releases the bound LDL and then recycles back to the cell surface or is directed to the lysosome for degradation. Recently, we found that knock- down of transmembrane protein 55B (TMEM55B), a phosphatidylinositol (4,5) bisphosphate [PI(4,5)P] phosphatase, stimulated decay of LDLR protein in cells and raised plasma LDLC in mice. Since PI(4,5)P is involved in lysosome formation, we hypothesize that PI(4,5)P phosphatases (such as TMEM55B) may alter LDLC by regulating LDLR intracellular trafficking. We also found that reduction of PI(4,5)P phosphatases caused severely impaired secretion of triglycerides into plasma. Notably, this occurred without the accumulation of liver fat, and interestingly, was observed only in male mice, with no effect in females. The lack of hepatic fat accumulation may be attributed to a number of different mechanisms including increased lipophagy (a process of selective autophagy in which lipid droplets are targeted for lysosomal decay). PI(4,5)P is an important regulator of lysosome reformation after autophagosome fusion, and we found that TMEM55B knock-down increased lysosomes. These findings suggest that PI(4,5)P phosphatases may modulate hepatic TG secretion and storage through a lysosome-dependent mechanism. Thus, the overall objective of this proposal is to evaluate the role of PI(4,5)P in LDLR recycling and TG secretion. In Aim 1 we will i) Test if PI(4,5)P metabolizing enzymes impact plasma LDL in the absence of LDLR using genetically modified mouse models; ii) Evaluate the effect of modifying PI(4,5)P on Ldlr intracellular levels and localization in mouse hepatocytes; iii) Test if expression of enzymatically inactive Tmem55b rescues the phenotypes observed in the Tmem55b knockout mouse; and iv) Evaluate if PI(4,5)P phosphatase regulation of LDLR recycling is dependent on known regulators of LDLR decay. In Aim 2 we will i) Compare TG secretion in whole body versus liver-specific Tmem55b knockout male mice to confirm that the defect is a hepatic mechanism; ii) Test if lack of hepatic fat accumulation, as would be expected to result from impaired TG secretion, in hepatocytes from Tmem55b knockout mice is due to changes in fatty acid oxidation, synthesis, or uptake, or TG synthesis; iii) Determine PI(4,5)P phosphatases depend on lysosome and/or autophagosomes to impair TG secretion; and iv) Evaluate if the sex-specific effect of PI(4,5)P modulation of TG secretion is due to the sex chromosome complement (XX vs. XY) and/or gonadal hormones. Investigation of the role of PI(4,5)P in lipid and lipoprotein metabolism may lead to the development of new therapeutic options for reducing cardiometabolic disease risk.