Cholesterol and Sphingolipid Perturbation of Membrane Traffic in Human Disease

人类疾病中膜运输的胆固醇和鞘脂扰动

• 批准号: 7577211
• 负责人: RICHARD E PAGANO
• 金额: $ 30.22万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-12-22 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7577211
• 关键词: Affect; Bacteria; Cell Line; Cell membrane; Cell surface; Cells; Chloride Channels; Chloride Ion; Chlorides; Cholesterol; Chronic; Cilia; Collection; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Data; Development; Disease; Distal; Dominant-Negative Mutation; Effectiveness; Endocytosis; Environment; Epithelial Cells; Exhibits; Golgi Apparatus; Grant; Guanosine Triphosphate Phosphohydrolases; In Vitro; Infection; Inherited; Lead; Learning; Lipids; Lung; Membrane Protein Traffic; Membrane Proteins; Methods; Missense Mutation; Mucous body substance; Mutation; Pathway interactions; Phenotype; Phenylalanine; Process; Proteins; Recycling; Regulation; Respiratory Failure; Role; Signal Pathway; Sodium; Sphingolipidoses; Sphingolipids; Symptoms; Techniques; Testing; Therapeutic Intervention; Thick; Transmembrane Transport; Transport Process; absorption; base; caveolin 1; cell type; cholesterol-binding protein; cilium biogenesis; cystic fibrosis patients; disease phenotype; disease-causing mutation; human disease; improved; in vivo; insight; knock-down; multicatalytic endopeptidase complex; mutant; novel therapeutic intervention; premature; protein misfolding; public health relevance; research study; tool; trafficking

DESCRIPTION (provided by applicant): Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cAMP-dependent chloride channel CF transmembrane conductance regulator (CFTR). Some missense mutations, including deletion of phenylalanine 508 (?F508), the most common mutation in CF disease, cause retention of the protein in the ER and premature degradation. As a result there is a significant reduction in functional CFTR in the plasma membrane of airway epithelial cells leading to defective chloride secretion, hyper-absorption of sodium, and other changes that reduce the capacity of cilia to clear bacteria from the airways. Recently several groups, including our own, have shown that cholesterol and sphingolipids (SLs) accumulate in CF cells, similar to that seen in various sphingolipid storage diseases. These findings and key preliminary data led to our central hypothesis that increased levels of cholesterol and SLs exacerbate the symptoms of CF by interfering with normal membrane transport processes and CFTR function. This will be tested by pursuing three Specific Aims. Under Aim 1 we will focus on over-expression of Rab1 and Rab2, two GTPases involved in ER to Golgi transport, as a tool for enhancing the delivery of F508 to the plasma membrane. These studies will provide a new method for increasing ?F508-CFTR at the plasma membrane and allow us to carry out studies on the underlying mechanism for this enhanced delivery as well as the function of the mutant protein at the cell surface. Under Aim 2, we will study the effects of SLs and cholesterol on endocytosis, recycling, and function of WT- and ?F508-CFTR since these lipids can significantly affect membrane transport as well as the microenvironment of other membrane proteins. Based on preliminary data showing reduced cilia in ?F508-CFTR cells, we will also study the effect of alterations in cholesterol on (i) the distribution of cilia in cells expressing wild type or mutant CFTR, (ii) modulation of RabGTPases involved in ciliogenesis, and (iii) the distribution of cholesterol binding proteins that are normally associated with cilia. Under Aim 3, we will first study the mechanism underlying elevation of SLs and cholesterol in mutant CFTR cells to learn whether the absence of CFTR function or the presence of a misprocessed mutant CFTR in the distal secretory pathway is responsible for lipid storage. We will also investigate the causal relationships between mutant CFTR expression, elevated lysosomal pH, and lipid storage. Second, we will use methods developed in the previous grant period and in preliminary studies (e.g., Caveolin-1 knock-down) to deplete elevated cholesterol and SLs from cells, in an attempt to reverse various aspects of the ?F508 phenotype. Together these proposed experiments should increase our understanding of the connection between mutant CFTR and lipid storage and may also provide additional approaches for treatment of CF. PUBLIC HEALTH RELEVANCE Cystic fibrosis is an inherited disease caused by mutations in a protein that results in thick mucus secretions that are not properly cleared from the lung and can lead to chronic infection and respiratory failure. Recent studies have shown that cholesterol and other lipids accumulate in airway cells from Cystic fibrosis patients. The following studies will examine the role of these lipids in exacerbating the symptoms of Cystic fibrosis and will be used in developing new therapeutic approaches for this disease.

描述（由申请人提供）：囊性纤维化（CF）是由依赖CAMP依赖性氯化物通道CF跨膜电导调节剂（CFTR）突变引起的常染色体隐性疾病。一些错义突变，包括苯丙氨酸508（？f508）的缺失，这是CF疾病中最常见的突变，会导致蛋白质保留在ER和过早降解中。结果，气道上皮细胞的质膜中功能性CFTR显着降低，导致氯化物分泌有缺陷，钠的高吸收以及其他变化，从而降低了纤毛清除呼吸道细菌的能力。最近，包括我们自己的几个组表明胆固醇和鞘脂（SLS）积聚在CF细胞中，类似于在各种鞘脂储存疾病中看到的胆固醇和胆固醇（SLS）。这些发现和关键的初步数据导致了我们的中心假设，即通过干扰正常的膜运输过程和CFTR功能，增加了胆固醇和SL的水平加剧CF的症状。这将通过追求三个具体目标来测试。在AIM 1下，我们将重点放在Rab1和Rab2的过表达上，这两个GTPases涉及ER到Golgi Transpers，作为增强F508向质膜递送的工具。这些研究将为质膜上增加f508-CFTR的新方法，并让我们对这种增强的递送以及突变蛋白在细胞表面的基础机制进行研究。在AIM 2下，我们将研究SLS和胆固醇对WT-和？F508-CFTR的内吞作用，回收和功能的影响，因为这些脂质会显着影响膜转运以及其他膜蛋白的微环境。基于初步数据显示了f508-CFTR细胞中纤毛的减少，我们还将研究胆固醇改变对（i）表达野生型或突变体CFTR的细胞中纤毛的分布，（ii）与纤毛生成有关的RabGTPase的调节，以及（iii）与（III）相关的蛋白质蛋白酶的分布，该蛋白酶与ciL的分布相关。在AIM 3下，我们将首先研究突变CFTR细胞中SLS和胆固醇升高的基础机制，以了解是否缺乏CFTR功能或在远端分泌途径中存在错误的突变CFTR的存在是负责脂质储存的。我们还将研究突变体CFTR表达，溶酶体pH升高和脂质储存之间的因果关系。其次，我们将使用在上一个赠款期间开发的方法，并在初步研究（例如，小窝蛋白-1敲击）中耗尽细胞升高胆固醇和SLS，以试图扭转？F508表型的各个方面。这些提出的实验共同加在一起，应增强我们对突变体CFTR和脂质储存之间的联系的理解，还可以提供其他CF治疗的方法。公共卫生相关性囊性纤维化是一种由蛋白质中突变引起的遗传疾病，导致浓厚的粘液分泌物无法从肺部清除，可能导致慢性感染和呼吸衰竭。最近的研究表明，囊性纤维化患者的气道细胞中积累胆固醇和其他脂质。以下研究将研究这些脂质在加剧囊性纤维化症状中的作用，并将用于开发该疾病的新治疗方法。