Regulation of VLDL Transport and Secretion

极低密度脂蛋白（VLDL）从肝脏的分泌受到其复杂的细胞内转运事件的严格控制。异常VLDL分泌导致脂质稳态失衡，这与许多代谢疾病如高脂血症、肝脂肪变性等相关。VLDL的生物发生发生在肝内质网（ER）中;然而，它们的成熟发生在高尔基体中。我们已经表征了ER到高尔基体的VLDL转运过程，这是肝脏所特有的，并且已经确定了这一步骤的关键调节剂;然而，成熟VLDL从TGN到PM的转运以用于其分泌仍有待于在分子水平上研究。在目标1中，我们扩展了我们正在进行的研究，确定ER到高尔基体运输的监管方面。我们已经证明SVIP是VTV从肝细胞分泌的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。我们建议确定特定的蛋白激酶，磷酸化SVIP的目标1。由于SVIP的磷酸化与VTV生物发生和VLDL分泌特异性相关，因此鉴定特异性蛋白激酶将为控制VLDL分泌提供新的选择性靶点。在目标2和目标3中，我们提出描绘的分子机制，调节后TGN贩运成熟VLDL。我们的初步数据表明，成熟的VLDL从TGN运输到PM在不同的运输囊泡，后高尔基体VLDL运输囊泡（PG-VTV），这是形态学和生物化学上不同于其他TGN衍生的蛋白质运输囊泡（PTV）。由于PG-VTV在其大小、货物、浮力密度和蛋白质组成方面是不同的，因此我们假设蛋白质，先前在TGN至PM运输中不被认为是重要的，介导VLDL选择和PG-VTV形成。这些蛋白质被认为是重要的货物选择，囊泡的生物合成和PG-VTV与PM融合。在目的2中，我们提出鉴定PG-VTV中成熟VLDL有效包装所需的货物选择蛋白。我们最近报道，L-FABP是正常VLDL分泌所必需的，然而，其潜在机制尚不清楚。在目的3中，我们将确定L-FABP和RBP 4在肝脏分泌VLDL中的作用。拟议的研究将揭示新的分子见解，了解成熟VLDL如何被选择进入PG-VTV并被转运到PM以最终分泌到血液中的过程，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.