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: 7741247
• 负责人: ANDREW H LIMPER
• 金额: $ 29.92万
• 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 2011-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7741247
• 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）是一种由cAMP依赖性氯离子通道CF跨膜传导调节因子（CFTR）突变引起的常染色体隐性遗传疾病。一些错义突变，包括苯丙氨酸508（？F508），CF疾病中最常见的突变，导致蛋白质在ER中的滞留和过早降解。因此，气道上皮细胞的质膜中的功能性CFTR显著降低，导致氯分泌缺陷、钠的过度吸收和降低纤毛从气道清除细菌的能力的其他变化。最近，包括我们自己的研究小组在内的几个研究小组已经表明，胆固醇和鞘脂（SL）在CF细胞中积累，类似于在各种鞘脂储存疾病中看到的情况。这些发现和关键的初步数据导致了我们的中心假设，即胆固醇和SL水平的增加通过干扰正常的膜转运过程和CFTR功能而加剧了CF的症状。这将通过追求三个具体目标来检验。在目标1下，我们将专注于Rab 1和Rab 2的过表达，这两种GTP酶参与ER到高尔基体的转运，作为增强F508向质膜递送的工具。这些研究将提供一种新的方法来提高？F508-CFTR在质膜上的表达，使我们能够对这种增强的递送的潜在机制以及突变蛋白在细胞表面的功能进行研究。根据目标2，我们将研究SL和胆固醇对WT-和？F508-CFTR，因为这些脂质可以显著影响膜转运以及其他膜蛋白的微环境。根据初步数据显示减少纤毛在？F508-CFTR细胞，我们还将研究胆固醇改变对（i）表达野生型或突变型CFTR的细胞中纤毛分布的影响，（ii）参与纤毛发生的RabGTP酶的调节，以及（iii）通常与纤毛相关的胆固醇结合蛋白的分布。在目标3下，我们将首先研究突变CFTR细胞中SL和胆固醇升高的潜在机制，以了解远端分泌途径中CFTR功能的缺失或错误加工的突变CFTR的存在是否是脂质储存的原因。我们还将研究突变CFTR表达，溶酶体pH值升高和脂质储存之间的因果关系。第二，我们将使用上一个资助期和初步研究中开发的方法（例如，小窝蛋白-1敲低）消耗升高的胆固醇和SL细胞，试图扭转的各个方面？F508表型。总之，这些拟议的实验应该增加我们的理解突变CFTR和脂质储存之间的联系，也可能提供额外的方法治疗CF。囊性纤维化是一种遗传性疾病，由一种蛋白质突变引起，导致粘稠的粘液分泌物不能从肺部正确清除，并可导致慢性感染和呼吸衰竭。最近的研究表明，胆固醇和其他脂质积聚在囊性纤维化患者的气道细胞中。以下研究将检查这些脂质在加重囊性纤维化症状中的作用，并将用于开发这种疾病的新治疗方法。