Regulation of VLDL Transport and Secretion

肝脏极低密度脂蛋白(VLDL)的分泌受到其复杂的细胞内转运事件的严格控制。极低密度脂蛋白的异常分泌导致脂质平衡失衡，与许多代谢性疾病如高脂血症、肝脏脂肪变性等有关。极低密度脂蛋白的生物发生发生在肝脏内质网(ER)，但它们的成熟发生在高尔基体。我们已经确定了肝脏特有的内质网到高尔基体极低密度脂蛋白的转运过程，并确定了这一步骤的关键调控因素；然而，成熟的极低密度脂蛋白从TGN到PM的运输及其分泌仍有待分子水平的研究。在目标1中，我们扩展了我们正在进行的确定内质网到高尔基体转运的调控方面的研究。我们已经证明，SVIP是VTV从肝细胞分泌ER和VLDL生物合成所必需的。我们的初步研究表明，SVIP在VTV生物发生过程中被磷酸化；然而，使SVIP磷酸化的特定蛋白激酶尚不清楚。我们建议在AIM 1中确定使SVIP磷酸化的特定蛋白激酶。由于SVIP的磷酸化与VTV的生物发生以及VLDL的分泌特定相关，因此鉴定特定的蛋白激酶将为控制VLDL的分泌提供一个新的选择性靶点。在目标2和目标3中，我们建议描述调控TGN后成熟极低密度脂蛋白运输的分子机制。我们的初步数据表明，成熟的VLDL是通过不同的运输小泡从TGN运输到PM的，即高尔基体后VLDL运输小泡(PG-VTV)，这些小泡在形态和生化上不同于其他TGN衍生的蛋白质运输小泡(PTV)。由于PG-VTV在其大小、载量、浮力密度和蛋白质组成上都不同，我们假设蛋白质在TGN到PM的运输中并不重要，它介导了VLDL的选择和PG-VTV的形成。推测这些蛋白在货物选择、囊泡生物发生和PG-VTV与PM的融合中具有重要作用在目标2中，我们建议确定在PG-VTV中有效包装成熟的极低密度脂蛋白所需的货物选择蛋白。我们最近报道L-FABP是极低密度脂蛋白正常分泌所必需的；然而，其潜在的机制尚不清楚。在目标3中，我们将确定L-FABP和RBP4在肝脏极低密度脂蛋白分泌中的作用。拟议的研究将对鲜为人知的过程提供新的分子见解，即成熟的极低密度脂蛋白如何被选择进入PG-VTV，并被输送到PM，最终在血液中分泌，并将这些过程联系起来，以确定控制VLDL分泌的潜在靶点。

Secretion of very-low-density lipoprotein (VLDL) from the liver is tightly controlled by its complex intracellular transport events. Aberrant VLDL secretion causes imbalance in lipid homeostasis which is associated with many metabolic diseases such as hyperlipidemia, hepatic steatosis etc. Biogenesis of VLDLs occurs in hepatic endoplasmic reticulum (ER); however, their maturation occurs in the Golgi. We have characterized the ER-to- Golgi VLDL transport process that is unique to the liver and have identified key regulators of this step; however, the transport of mature VLDL from the TGN to the PM for its secretion remains to be studied at the molecular level. In Aim 1, we extend our ongoing studies of determining the regulatory aspects of ER-to-Golgi transport. We have shown that SVIP is required for VTV biogenesis from the ER and VLDL secretion from hepatocytes. Our preliminary studies suggest that SVIP gets phosphorylated during VTV biogenesis; however, a specific protein kinase that phosphorylates SVIP is not known. We propose to identify the specific protein kinase that phosphorylates SVIP in Aim 1. Because phosphorylation of SVIP is specifically associated with VTV biogenesis and thus VLDL secretion, identification of a specific protein kinase will provide a novel selective target in controlling VLDL secretion. In Aim 2 and Aim 3, we propose to delineate the molecular machinery that regulates the post-TGN trafficking of mature VLDL. Our preliminary data suggest that mature VLDLs are transported from the TGN to the PM in distinct transport vesicles, the post-Golgi VLDL transport vesicle (PG-VTV), which are morphologically and biochemically different from other TGN-derived protein transport vesicles (PTV). Since the PG-VTV is distinct in its size, cargo, buoyant density and protein composition, we hypothesize that proteins, not previously considered important in the TGN-to-PM transport, mediate VLDL-selection and PG-VTV formation. These proteins are postulated to be important for cargo-selection, vesicle biogenesis and PG-VTV fusion with PM. In Aim 2, we propose to identify cargo-selecting protein that is required for effective packaging of mature VLDL in PG-VTV. We recently reported that L-FABP is required for normal VLDL secretion; however, the underlying mechanism is not known. In Aim 3, we will define the role of L-FABP and RBP4 in VLDL secretion from the liver. The proposed studies will shed new molecular insights into the poorly understood process of how mature VLDL is selected into the PG-VTV and is being transported to the PM for its eventual secretion in the blood and link these processes to identify potential targets in controlling VLDL secretion.