INTEGRATED MODELING OF PROTEIN AND MEMBRANE TRAFFIC IN THE SECRETORY PATHWAY

描述（由申请人提供）：真核分泌途径由内质网、高尔基体、分泌囊泡、核内体、溶酶体和质膜组成。该途径负责合成、加工和分泌由给定细胞类型产生的所有蛋白质、激素和神经递质。它还通过将所有受体、酶、离子通道和转运蛋白递送到细胞表面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，我们和其他人已经测量了，使用活细胞中的延时共聚焦显微镜，蛋白质运输的动态分泌途径。这些测量使我们能够测试当前的高尔基体功能理论，并提出一个新的理论，解释目前的范式无法解释的数据。虽然这一信息是有用的，但它未能解释膜脂质和胆固醇在驱动和调节分泌途径中的突出作用。这项研究的长期目标是制定和测试的分泌途径的机械动力学模型，该模型集成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体蛋白。为了实现这一目标，我们提出了三个具体目标：1）开发一个动力学模型，该模型结合了货物、甘油磷脂、鞘脂和常驻蛋白模块，并针对经典观察和我们关于GFP标记的货物蛋白、水泡性口炎病毒G蛋白的高尔基体退出的新数据，测试该模型和两个竞争模型（VSVG-GFP），2）构建细胞内胆固醇运输的动力学模型及其对货物输出的假设调节，将其与来自目标1的模型联合收割机组合，并针对用降低、增加或化学修饰细胞胆固醇含量的试剂处理的细胞中的分泌性货物和甾醇动力学数据测试其，3）修改来自目标2的模型以包括假设的蛋白质-胆固醇相互作用，并在过表达货物蛋白或驻留ER或高尔基体蛋白的细胞中针对货物和固醇动力学测试所得模型。方法：动力学建模采用ProcessDB和Berkeley Madonna软件，GFP融合蛋白，延时荧光显微镜，活细胞成像。细胞胆固醇加工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如何控制它。

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.