MAL2 regulation of hepatic protein trafficking: mechanisms and binding partners

项目摘要 肝脏是人体的代谢中心，它将食物转化为生命所需的化学物质，并合成 不计其数的化合物被释放到循环中供其他器官使用。肝也是主治 排毒中心，清除体内的外来物质和内源性废物。其中的大多数函数 是在肝细胞中进行的，肝细胞是肝脏的主要上皮细胞。这些细胞形成了一道屏障， 内部和外部环境通过形成紧密的连接将自己粘合在一起， 将不同的活性限制在特定的质膜(PM)区域：基侧和顶端。这个 基侧表面面向血液(内环境)，而心尖表面面向胆汁( 外部环境)，这是一种复杂的分子“肥皂”，有助于吸收饮食脂肪和废物 移走。这种功能的不对称性(或极性)反映在PM蛋白的不对称分布上； 每个结构域都有不同的蛋白质亚集。因为正常的肝功能取决于 肝细胞的极性，这项建议询问极性是如何建立和维持的？在某种程度上，答案是 来自于对极化膜运输的理解。我们的重点是确定肝尖部的调节因子。 蛋白质递送。不同于简单的上皮细胞直接靶向TGN到TGN的新合成的蛋白质 在顶端PM，肝细胞主要使用间接途径，顶端蛋白首先被输送到 基底侧PM，通过内吞作用取回，然后转胞到根尖表面。我们决定 肝脏跨细胞分选需要胆固醇和神经鞘糖脂。因为MAL2(髓鞘和 淋巴细胞蛋白2)被确定为跨细胞作用的调节器，因为它的活性需要胆固醇 和鞘糖脂，我们一直在研究MAL2在根尖交付中的作用。在这些研究中， 将使用WiF-B细胞。该细胞系是一个极好的极化肝脏模型系统。目标1的目标是 确定间接途径中哪些步骤受MAL2的调控。《目标2》考察了 MAL2的结构特征很重要，它询问细胞质的N-末端和C-末端是否需要 功能。AIM 3试图识别新的并表征已知的MAL2结合蛋白，这些蛋白在 调节顶端蛋白的运输。在AIMS 3A和B中，提出了描述相互作用的研究 在MAL2和已知的相互作用因子肿瘤蛋白D52之间。在Aim 3C中，描述了将 使用裂解泛素酵母双杂交系统确定新的结合伙伴。因为我们的WIF-B细胞是 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，并希望为我们对心尖的理解提供根本性的进步 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.