MAL2 regulation of hepatic protein trafficking: mechanisms and binding partners

项目摘要 肝脏是人体的代谢中心，将食物转化为生命所需的化学物质，并合成 无数的化合物被释放到循环中供其他器官使用。肝脏也是主要的 解毒中心，清除体内的外源性物质和内源性废物。其中大部分功能 在肝细胞中进行，肝细胞是肝脏的主要上皮细胞。这些细胞形成了一个屏障， 内部和外部环境通过紧密连接的形成将自身粘合在一起， 将不同的活性限制在特定的质膜（PM）区域：基底外侧和顶端。的 基底外侧表面面向血液（内部环境），而顶表面面向胆汁（内部环境）。 外部环境），复杂的分子“肥皂”，有助于吸收膳食脂肪和废物 的拔除. PM蛋白的不对称分布反映了功能的不对称性（或极性）; 每个结构域的特征在于不同的蛋白质子集。因为正常的肝功能取决于 肝细胞极性，这一提议询问极性是如何建立和维持的？答案，在某种程度上， 来自于对极化膜运输的理解我们的重点是确定肝顶细胞的调节因子， 蛋白质递送。与直接靶向来自TGN的新合成蛋白质的简单上皮细胞不同， 在顶端PM，肝细胞主要使用间接途径，其中顶端蛋白首先被递送到 基底外侧PM，通过内吞作用回收，然后转胞吞至顶面。我们确定 肝胞吞转运分选需要胆固醇和鞘糖脂。因为MAL 2（髓磷脂和 淋巴细胞蛋白2）被鉴定为转胞吞作用的调节因子，因为其活性需要胆固醇 和鞘糖脂，我们一直在研究MAL 2如何在顶端递送中发挥作用。对于这些研究， 将使用WIF-B细胞。该细胞系是一种极好的极化肝模型系统。目标1旨在 鉴定间接途径中的哪些步骤受MAL 2的调节。目标2检查什么 MAL 2的结构特征是重要的，并询问细胞质N-和C-末端是否是必需的， 功能目的3寻求鉴定新的和表征已知的MAL 2结合蛋白，其在以下方面协作： 调节顶端蛋白运输。在目标3A和B中，建议开展研究，以表征相互作用 MAL 2和一种已知的相互作用物肿瘤蛋白D52之间的联系。在目标3C中，描述了将 使用分裂泛素酵母双杂交系统鉴定新的结合配偶体。因为我们的WIF-B细胞 伟大的极化，肝脏模型系统，因为我们有许多重要的工具和试剂，我们很好- 准备进行这些实验，并希望为我们对根尖细胞的理解提供根本性的进展， 蛋白质靶向

Project Summary The liver is the body's metabolic center that converts food into chemicals required for life and synthesizes countless compounds that are released into the circulation for use by other organs. The liver is also the major detoxifying center, ridding the body of xenobiotics and endogenous waster products. Most of these functions are carried out in hepatocytes, the major epithelial cell of the liver. These cells form a barrier between the internal and external environments by cementing themselves together by the formation of tight junctions that restrict distinct activities to specific plasma membrane (PM) domains: the basolateral and apical. The basolateral surface faces the blood (the internal environment) whereas the apical surface faces the bile (the external environment), the complex molecular "soap" that helps in absorption of dietary fats and waste removal. The functional asymmetry (or polarity) is mirrored by the asymmetrical distribution of PM proteins; each domain is characterized by distinct subsets of proteins. Because proper liver function depends on hepatocyte polarity, this proposal asks how is polarity established and maintained? The answer, in part, comes from understanding polarized membrane trafficking. Our focus is to identify regulators of hepatic apical protein delivery. Unlike simple epithelial cells that directly target newly synthesized proteins from the TGN to the apical PM, hepatocytes mainly use an indirect pathway where apical proteins are first delivered to the basolateral PM, retrieved by endocytosis and then transcytosed to the apical surface. We determined that hepatic transcytotic sorting requires cholesterol and glycosphingolipids. Because MAL2 (myelin and lymphocyte protein 2) was identified as a regulator of transcytosis and because its activity requires cholesterol and glycosphingolipids, we have been examining how MAL2 functions in apical delivery. For these studies, WIF-B cells will be used. This cell line is an excellent polarized, hepatic model system. Aim 1 is aimed at identifying what steps in the indirect pathway are under the regulation of MAL2. Aim 2 examines what structural features of MAL2 are important and asks whether the cytoplasmic N- and C-termini are required for function. Aim 3 seeks to identify new and characterize known MAL2 binding proteins that collaborate in regulating apical protein trafficking. In Aims 3A and B, studies are proposed to characterize interactions between MAL2 and a known interactor, tumor protein D52. In Aim 3C, experiments are described that will identify new binding partners using the split-ubiquitin yeast two-hybrid system. Because our WIF-B cells are a great polarized, hepatic model system and because we have many important tools and reagents, we are well- poised to perform these experiments and hope to provide fundamental advances in our understanding of apical protein targeting.