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释放到循环中的无数化合物供其他器官使用。肝脏也是主要的排毒中心，摆脱异种生物和内源性浪潮产物的身体。这些功能大多是在肝细胞中进行的，肝细胞是肝脏的主要上皮细胞。这些单元在内部和外部环境通过形成紧密连接的形成来巩固自己将不同的活性限制在特定的质膜（PM）域：基底外侧和顶端。这基底外侧表面面对血液（内部环境），而顶部表面面对胆汁（外部环境），复杂的分子“肥皂”，有助于吸收饮食脂肪和废物移动。 PM蛋白的不对称分布反映了功能不对称性（或极性）。每个结构域的特征是蛋白质的不同子集。因为适当的肝功能取决于肝细胞极性，该提议问如何建立和维护极性？答案部分是来自了解两极分化的膜贩运。我们的重点是确定肝根尖的调节剂蛋白质递送。与简单的上皮细胞不同，该细胞直接靶向新合成的蛋白质从TGN到顶端PM，肝细胞主要使用间接途径，首先将根尖蛋白传递到该途径基底外侧PM，通过内吞作用检索，然后转到顶端表面。我们确定了这一点肝胞单胞菌分选需要胆固醇和糖磷脂。因为mal2（髓鞘和淋巴细胞蛋白2）被鉴定为跨胞菌病的调节剂，因为其活性需要胆固醇和糖磷脂，我们一直在研究MAL2在顶端递送中的功能。对于这些研究，将使用Wif-B细胞。该细胞系是一种极好的极化肝模型系统。目标1针对在MAL2的调节下，确定间接途径中的哪些步骤。 AIM 2检查什么 MAL2的结构特征很重要，并询问是否需要细胞质N和C末端功能。 AIM 3试图确定合作的新的和特征已知的MAL2结合蛋白调节根尖蛋白运输。在AIMS 3A和B中，提出了研究以表征相互作用 MAL2和已知相互作用的肿瘤蛋白D52之间。在AIM 3C中，描述了将会使用分裂泛素酵母两杂交系统确定新的结合伴侣。因为我们的Wif-B细胞是一个伟大的两极化，肝模型系统，因为我们有许多重要的工具和试剂，所以我们很擅长准备进行这些实验，并希望在我们对顶的理解中提供基本进步蛋白质靶向。