Cholesterol and Sphingolipid Perturbation of Membrane Traffic in Human Disease

描述（由申请人提供）：囊性纤维化（CF）是一种常染色体隐性遗传病，由camp依赖性氯离子通道CF跨膜电导调节因子（CFTR）突变引起。一些错义突变，包括苯丙氨酸508 (？F508)是CF疾病中最常见的突变，导致内质网中蛋白质的滞留和过早降解。因此，气道上皮细胞质膜上功能性CFTR显著减少，导致氯化物分泌缺陷、钠的高吸收以及其他变化，降低了纤毛清除气道细菌的能力。最近，包括我们自己在内的几个研究小组已经表明，胆固醇和鞘脂（SLs）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支持了我们的中心假设，即胆固醇和SLs水平的升高通过干扰正常的膜运输过程和CFTR功能而加剧CF的症状。这将通过追求三个具体目标来检验。在Aim 1中，我们将重点关注Rab1和Rab2的过表达，这两种gtpase参与内质网到高尔基转运，作为增强F508向质膜递送的工具。这些研究将为增加？F508-CFTR在质膜上的表达，使我们能够对这种增强传递的潜在机制以及突变蛋白在细胞表面的功能进行研究。在Aim 2下，我们将研究SLs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。初步数据显示？在F508-CFTR细胞中，我们还将研究胆固醇变化对(i)表达野生型或突变型CFTR细胞中纤毛分布的影响，（ii）参与纤毛发生的rabgtpase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Aim 3中，我们将首先研究突变CFTR细胞中SLs和胆固醇升高的机制，以了解CFTR功能缺失或远端分泌途径中存在错误加工的突变CFTR是导致脂质储存的原因。我们还将研究突变CFTR表达、溶酶体pH升高和脂质储存之间的因果关系。其次，我们将使用在之前的资助期和初步研究中开发的方法（例如，Caveolin-1敲除）来消耗细胞中升高的胆固醇和SLs，试图逆转这种疾病的各个方面。F508表现型。总之，这些拟议的实验应该增加我们对突变CFTR和脂质储存之间联系的理解，也可能为CF的治疗提供额外的方法。公共卫生相关性囊性纤维化是一种遗传性疾病，由一种蛋白质突变引起的粘稠粘液分泌物不能正确地从肺部清除，并可能导致慢性感染和呼吸衰竭。最近的研究表明，胆固醇和其他脂质在囊性纤维化患者的气道细胞中积聚。接下来的研究将探讨这些脂质在加剧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。

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.