Regulation of VLDL Transport and Secretion

肝脏中极低密度脂蛋白（VLDL）的分泌受到其复杂的细胞内转运事件的严格控制。 VLDL分泌异常会导致脂质稳态失衡，这与许多代谢性疾病如高脂血症、肝脂肪变性等有关。VLDL的生物合成发生在肝内质网（ER）中；然而，它们的成熟发生在高尔基体中。我们已经描述了肝脏特有的 ER 至高尔基体 VLDL 转运过程，并确定了该步骤的关键调节因子；然而，成熟VLDL从TGN转运至PM并进行分泌的过程仍有待在分子水平上进行研究。在目标 1 中，我们扩展了正在进行的确定内质网到高尔基体运输的监管方面的研究。我们已经证明，SVIP 是 ER 和肝细胞分泌的 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，磷酸化 SVIP 的特定蛋白激酶尚不清楚。我们建议在目标 1 中鉴定磷酸化 SVIP 的特定蛋白激酶。由于 SVIP 磷酸化与 VTV 生物发生以及 VLDL 分泌特异性相关，因此特定蛋白激酶的鉴定将为控制 VLDL 分泌提供新的选择性靶标。在目标 2 和目标 3 中，我们建议描述调节 TGN 后成熟 VLDL 运输的分子机制。我们的初步数据表明，成熟的 VLDL 在不同的转运囊泡中从 TGN 转运到 PM，即高尔基后 VLDL 转运囊泡 (PG-VTV)，其在形态和生化上与其他 TGN 衍生的蛋白质转运囊泡 (PTV) 不同。由于 PG-VTV 的大小、货物、浮力密度和蛋白质组成不同，我们假设以前认为在 TGN 到 PM 运输中不重要的蛋白质介导了 VLDL 选择和 PG-VTV 形成。这些蛋白质被认为对于货物选择、囊泡生物发生以及 PG-VTV 与 PM 融合很重要。在目标 2 中，我们建议鉴定 PG-VTV 中有效包装成熟 VLDL 所需的货物选择蛋白。我们最近报道，正常 VLDL 分泌需要 L-FABP；然而，根本机制尚不清楚。在目标 3 中，我们将定义 L-FABP 和 RBP4 在肝脏分泌 VLDL 中的作用。拟议的研究将对成熟的 VLDL 如何被选入 PG-VTV 并被转运至 PM 以最终在血液中分泌的过程提供新的分子见解，并将这些过程联系起来以确定控制 VLDL 分泌的潜在目标。

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.