MAL2 regulation of hepatic protein trafficking: mechanisms and binding partners

MAL2 对肝蛋白运输的调节：机制和结合伙伴

基本信息

批准号：
8281689
负责人：
PAMELA L. TUMA
金额：
$ 17.56万
依托单位：
CATHOLIC UNIVERSITY OF AMERICA
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-01 至 2015-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8281689
关键词：
Accounting Adenoviruses Apical B-Lymphocytes Bile fluid Binding Binding Proteins Biological Models Blood Blood Circulation C-terminal Cancerous Cell Line Cell Polarity Cell membrane Cell physiology Cells Chemicals Chimera organism Chimeric Proteins Cholesterol Collaborations Complex Cytoplasmic Tail Dietary Fats Endocytosis Environment Epithelial Epithelial Cells Excision Face Family member Food Glycosphingolipids Goals Hepatic Hepatocyte Human Immunoglobulin A Investigation Life Lipids Liver Location Lymphocyte Malignant Neoplasms Mediating Membrane Protein Traffic Membrane Proteins Metabolic Modeling Molecular Myelin Organ Pathway interactions Phosphorylation Protein Family Proteins Reagent Recombinants Recruitment Activity Regulation Science Soaps Sorting - Cell Movement Specificity Surface System TPD52 gene Testing Tight Junctions Tissues Ubiquitin Vesicle Waste Products Xenobiotics Yeasts absorption base basolateral membrane experience liver function member protein protein interaction protein transport receptor research study tool transcytosis tumor wasting yeast two hybrid system

项目摘要

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.

项目概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系统中供其他器官使用。肝脏也是主要的解毒中心，清除体内的外源性废物和内源性废物。大多数这些功能是在肝细胞（肝脏的主要上皮细胞）中进行的。这些细胞在细胞之间形成屏障通过形成紧密连接将内部和外部环境粘合在一起将不同的活动限制在特定的质膜（PM）区域：基底外侧和顶端。这基底外侧表面面向血液（内部环境），而顶端表面面向胆汁（内环境）外部环境），复杂的分子“肥皂”，有助于吸收膳食脂肪和废物移动。 PM 蛋白的不对称分布反映了功能的不对称性（或极性）；每个结构域都有不同的蛋白质子集。因为正常的肝功能取决于肝细胞极性，该提案询问极性是如何建立和维持的？答案部分是，来自对极化膜运输的理解。我们的重点是确定肝尖的调节因子蛋白质输送。与简单的上皮细胞不同，简单的上皮细胞直接靶向从 TGN 到新合成的蛋白质在顶端 PM，肝细胞主要使用间接途径，其中顶端蛋白首先被递送至基底外侧 PM，通过内吞作用回收，然后转胞吞至顶端表面。我们确定肝转细胞分选需要胆固醇和鞘糖脂。因为MAL2（髓磷脂和淋巴细胞蛋白 2) 被确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 MAL2 在心尖递送中的功能。对于这些研究，将使用 WIF-B 细胞。该细胞系是一种优秀的极化肝脏模型系统。目标1旨在确定间接途径中的哪些步骤受到 MAL2 的调节。目标 2 检查什么 MAL2 的结构特征很重要，并询问细胞质 N 端和 C 端是否是必需的功能。目标 3 寻求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质运输。在目标 3A 和 B 中，建议进行研究来描述相互作用 MAL2 和已知的相互作用蛋白 D52 之间的关系。在目标 3C 中，描述的实验将使用分裂泛素酵母双杂交系统识别新的结合伴侣。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，所以我们很好- 准备进行这些实验，并希望为我们对顶端的理解提供根本性的进展蛋白质靶向。