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运输事件的严格控制。VLDL分泌异常导致脂质稳态失衡，与高脂血症、肝脂肪变性等代谢疾病有关。肝内质网（ER）是vldl的生物生成途径；然而，它们的成熟发生在高尔基体中。我们描述了ER-to-高尔基VLDL运输过程，这是肝脏独有的，并确定了这一步骤的关键调节因子；然而，成熟VLDL从TGN转运到PM分泌的过程仍有待于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er到高尔基转运的调控方面的研究。我们已经证明SVIP是内质网VTV生物生成和肝细胞VLDL分泌所必需的。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Aim 1中鉴定特异性磷酸化SVIP的蛋白激酶。由于SVIP的磷酸化与VTV的生物发生和VLDL的分泌特异性相关，因此鉴定特定的蛋白激酶将为控制VLDL分泌提供新的选择性靶点。在Aim 2和Aim 3中，我们建议描述调节成熟VLDL在tgn后转运的分子机制。我们的初步数据表明，成熟的VLDL通过不同的转运囊泡从TGN转运到PM，即高尔基后VLDL转运囊泡（PG-VTV），它在形态和生化上与其他TGN来源的蛋白转运囊泡（PTV）不同。由于PG-VTV在其大小、载重量、浮力密度和蛋白质组成方面都是不同的，我们假设蛋白质在tgn到pm的运输中并不重要，但介导了vldl选择和PG-VTV的形成。这些蛋白被认为是重要的货物选择，囊泡生物发生和PG-VTV与PM融合。在目标2中，我们建议确定在PG-VTV中有效包装成熟VLDL所需的货物选择蛋白。我们最近报道了L-FABP是正常VLDL分泌所必需的；然而，潜在的机制尚不清楚。在Aim 3中，我们将定义L-FABP和RBP4在肝脏VLDL分泌中的作用。提出的研究将为成熟VLDL如何被选择进入PG-VTV并被运输到PM最终在血液中分泌的鲜为人知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.