Molecular Mechanisms of Proglucagon Sorting to the Regulated Secretory Pathway

背景：多肽激素在内质网上合成为较大的前体或前体激素，通过高尔基体运输，并分类到调节分泌途径的分泌颗粒中。在这些颗粒中，前荷尔蒙被加工成它们的组成多肽荷尔蒙，并被储存起来，直到刺激触发它们的释放。胰高血糖素原是胰腺激素胰高血糖素和肠道激素胰高血糖素样肽(GLP)-1和-2的前体。这些多肽必须储存在分泌颗粒中，才能对营养做出反应而分泌。胰高血糖素是一种主要的葡萄糖逆向调节激素，在低血糖时分泌，以维持正常血糖。GLP-1和GLP-2都是在营养摄入时分泌的；GLP-1刺激葡萄糖依赖的胰岛素分泌，GLP-2增加肠道血流量和营养吸收。我的研究重点是确定原高血糖素被导向分泌颗粒的分子机制。 进展：我们已经证明，胰高血糖素原可能与一种分选受体CPE相互作用，该受体针对胰腺α细胞中的颗粒，而肠道L细胞中可能需要另一种受体。我们还在胰高血糖素和GLP-1的结构中发现了特定的分选信号，这些信号将原胰高血糖素引导成颗粒。我们将继续利用蛋白质组学和定量超分辨显微镜来表征控制原胰高血糖素分选的分子机制。 具体目标： 1.鉴定作为α细胞中胰高血糖素原分选受体的候选先导。我们的工作表明，CPE可能是α细胞的分选受体，而不是L细胞的分选受体。我们有更多的数据表明GLP-1分选信号可以与CGA结合。我们将确定CPE和/或CGA是否直接与胰高血糖素原相互作用。 2.鉴定与胰高血糖素原分选信号相互作用的蛋白质簇。我们将使用亲和纯化-质谱法来鉴定在将原胰高血糖素分选为α细胞和L细胞颗粒的过程中的新角色。我们将使用超分辨率显微镜和siRNA功能丧失实验来验证我们的发现。 3.鉴定分泌颗粒中调节前高血糖素分选的蛋白质网络。对胰腺α细胞分泌颗粒的蛋白质组学分析将揭示新的网络参与原胰高血糖素的翻译后加工和分选，并将提供颗粒组成、结构和生物发生的信息。 意义：胰高血糖素、GLP-1和GLP-2是营养动态平衡的关键调节因子。为了阿尔法细胞和L细胞对营养物质的反应，需要将这些多肽正确地分类为分泌颗粒。我们将使用最先进的定量技术来研究控制前胰高血糖素在细胞内运输的分子机制。

Background: Peptide hormones are synthesized in the endoplasmic reticulum as larger precursors or prohormones, transported through the Golgi, and sorted to the secretory granules of the regulated secretory pathway. Within these granules, prohormones are processed to their constituent peptide hormones and stored until a stimulus triggers their release. Proglucagon is the precursor of the pancreatic hormone glucagon and the intestinal hormones, glucagon-like peptide (GLP)-1 and -2. These peptides must be stored in secretory granules in order to be secreted in response to nutrients. Glucagon, the major glucose counter-regulatory hormone, is secreted in response to low blood glucose levels in order to maintain euglycemia. Both GLP-1 and GLP-2 are secreted in response to nutrient ingestion; GLP-1 stimulates glucose-dependent insulin secretion, and GLP-2 increases intestinal blood flow and nutrient absorption. My research program focuses on identifying the molecular mechanisms by which proglucagon is directed to secretory granules. Progress: We have shown that proglucagon may interact with a sorting receptor, CPE, to be targeted to granules in pancreatic alpha cells, and that another receptor may be required in intestinal L cells. We have also identified specific sorting signals within the structures of glucagon and GLP-1 that direct proglucagon into granules. We will continue to characterize the molecular mechanisms that govern the sorting of proglucagon by using proteomics and quantitative super-resolution microscopy. Specific Aims: 1. To characterize the lead candidates as sorting receptors for proglucagon in alpha cells. Our work indicates that CPE may be a sorting receptor in alpha cells but not L cells. We have additional data suggesting that the GLP-1 sorting signal can bind to CgA. We will determine if CPE and/or CgA directly interact with proglucagon. 2. To identify clusters of proteins that interact with proglucagon sorting signals. We will use affinity purification-mass spectrometry to identify novel players in the sorting of proglucagon to granules in alpha and L cells. We will validate our findings using super-resolution microscopy and siRNA loss-of-function experiments. 3. To identify protein networks within secretory granules that regulate proglucagon sorting. Proteomic analysis of pancreatic alpha cell secretory granules will reveal novel networks involved in the post-translational processing and sorting of proglucagon, and will yield information on granule composition, architecture and biogenesis. Significance: Glucagon, GLP-1 and GLP-2 are key regulators of nutrient homeostasis. Correct sorting of these peptides to secretory granules is required for the alpha and L cells' response to nutrients. We will be using state-of-the-art quantitative techniques to investigate the molecular mechanisms that govern the intracellular trafficking of proglucagon.