INTEGRATED MODELING OF PROTEIN AND MEMBRANE TRAFFIC IN THE SECRETORY PATHWAY

描述（由申请人提供）：真核生物分泌途径由内质网、高尔基体、分泌囊泡、核内体、溶酶体和质膜组成。这条通路负责合成、加工和分泌所有由特定细胞类型产生的蛋白质、激素和神经递质。它还通过向细胞表面传递所有受体、酶、离子通道和转运体来独特地定义每个细胞的功能和反应。随着绿色荧光蛋白（GFP）标签的出现，我们和其他人已经在活细胞中使用延时共聚焦显微镜测量了分泌途径中蛋白质运输的动力学。这些测量使我们能够测试当前的高尔基函数理论，并提出一个新的理论来解释当前范式无法解释的数据。虽然这些信息是有用的，但它不能解释膜脂和胆固醇在驱动和调节分泌途径中的突出作用。本研究的长期目标是制定和测试分泌途径的机械动力学模型，该模型整合了五个关键子系统或模块：货物蛋白、甘油磷脂（GPL）、鞘脂、胆固醇和常驻高尔基蛋白。为实现这一目标，我们提出三个具体目标：1)建立一个结合货物、甘油磷脂、鞘脂和常驻蛋白模块的动力学模型，并根据经典观察结果和我们关于gfp标记的货物蛋白水泡性口炎病毒G蛋白（VSVG-GFP）高尔基出口的新数据对其和两个竞争模型进行测试；2)构建细胞内胆固醇运输及其对货物出口的假设调节动力学模型；将其与Aim 1中的模型结合，并在用降低、增加或化学修饰细胞胆固醇含量的药物处理的细胞中测试其分泌货物和固醇动力学数据。3)修改Aim 2中的模型，以包括假设的蛋白质-胆固醇相互作用，并测试结果模型对过表达货物蛋白或常驻内质网或高尔基蛋白的细胞中的货物和固醇动力学。方法：采用ProcessDB和Berkeley Madonna软件进行动力学建模，GFP融合蛋白，延时荧光显微镜，活细胞成像。细胞胆固醇处理的紊乱是包括心脏病和糖尿病在内的主要人类疾病的核心方面。由于分泌途径对正常细胞功能至关重要，我们需要知道胆固醇是如何控制它的。

DESCRIPTION (provided by applicant): The eukaryotic secretory pathway consists of the endoplasmic reticulum, Golgi apparatus, secretory vesicles, endosomes, lysosomes, and the plasma membrane. This pathway is responsible for synthesis, processing, and secretion of all proteins, hormones, and neurotransmitters produced by a given cell type. It also uniquely defines the functions and responses of each cell by delivering to the cell surface all receptors, enzymes, ion channels and transporters.With the advent of green fluorescent protein (GFP) tags we and others have measured, using time-lapse confocal microscopy in living cells, the dynamics of protein transport n the secretory pathway. These measurements have allowed us to test current theories of Golgi function and to propose a new theory that accounts for data that are unexplained by current paradigms. While this information is useful, it fails to account for the prominent role of membrane lipids and cholesterol in powering and regulating the secretory pathway. The long term goal of this research is formulation and testing of a mechanistic kinetic model of the secretory pathway that integrates five key subsystems or modules: cargo protein, glycerophospholipids (GPL), sphingolipids, cholesterol, and resident Golgi protein. To advance this goal we propose three specific aims: 1) Develop a kinetic model that combines cargo, glycerophospholipid, sphingolipid and resident protein modules and test it and two competing models against both classical observations and our new data on Golgi exit of a GFP-tagged cargo protein, vesicular stomatitis virus G protein (VSVG-GFP), 2) Construct a kinetic model of intracellular cholesterol trafficking and its hypothesized regulation of cargo export, combine it with the model from Aim 1 and test it against secretory cargo and sterol kinetic data in cells treated with agents that decrease, increase, or chemically modify cellular cholesterol content, 3) Modify the model from Aim 2 to include hypothesized protein-cholesterol interactions and test the resulting model against cargo and sterol kinetics in cells overexpressing either cargo proteins or resident ER or Golgi proteins. Methods: Kinetic modeling using ProcessDB and Berkeley Madonna software, GFP fusion proteins, time-lapse fluorescence microscopy, live cell imaging. Derangements of cellular cholesterol processing are central aspects of major human diseases including heart disease and diabetes. Since the secretory pathway is vital to normal cellular function we need to know how cholesterol controls it.