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: 7996545
• 负责人: ANDREW H LIMPER
• 金额: $ 29.62万
• 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/7996545
• 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; Health; 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; 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）是一种常染色体隐性遗传疾病，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) 的缺失（CF 疾病中最常见的突变），会导致蛋白质滞留在 ER 中并过早降解。结果，气道上皮细胞质膜中的功能性 CFTR 显着减少，导致氯离子分泌缺陷、钠吸收过度以及其他降低纤毛清除气道细菌能力的变化。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 (SL) 在 CF 细胞中积聚，类似于各种鞘脂贮积病中所见的情况。这些发现和关键的初步数据得出了我们的中心假设：胆固醇和 SL 水平升高会干扰正常的膜转运过程和 CFTR 功能，从而加剧 CF 的症状。这将通过追求三个具体目标来检验。在目标 1 下，我们将重点关注 Rab1 和 Rab2 的过度表达，这两种参与 ER 至高尔基体转运的 GTP 酶，作为增强 F508 向质膜递送的工具。这些研究将为增加质膜上的?F508-CFTR提供一种新方法，并使我们能够对这种增强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。在目标 2 下，我们将研究 SL 和胆固醇对 WT- 和 ?F508-CFTR 的内吞作用、再循环和功能的影响，因为这些脂质可以显着影响膜运输以及其他膜蛋白的微环境。基于显示 ?F508-CFTR 细胞中纤毛减少的初步数据，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 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。在目标 3 下，我们将首先研究突变型 CFTR 细胞中 SL 和胆固醇升高的机制，以了解 CFTR 功能的缺失或远端分泌途径中存在错误处理的突变型 CFTR 是否是脂质储存的原因。我们还将研究突变型 CFTR 表达、溶酶体 pH 值升高和脂质储存之间的因果关系。其次，我们将使用先前资助期和初步研究中开发的方法（例如 Caveolin-1 敲除）来消除细胞中升高的胆固醇和 SL，试图逆转 ?F508 表型的各个方面。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，并且还可能为 CF 的治疗提供其他方法。公共卫生相关性囊性纤维化是一种由蛋白质突变引起的遗传性疾病，会导致浓稠的粘液分泌物无法从肺部正确清除，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。以下研究将检查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。

项目成果

Role of protein kinase d in Golgi exit and lysosomal targeting of the transmembrane protein, Mcoln1.

蛋白激酶 d 在高尔基体出口和跨膜蛋白 Mcoln1 的溶酶体靶向中的作用。

• DOI: 10.1111/j.1600-0854.2012.01331.x
• 发表时间: 2012
• 期刊: Traffic (Copenhagen, Denmark)
• 作者: Marks,DavidL;Holicky,EileenL;Wheatley,ChristineL;Frumkin,Ayala;Bach,Gideon;Pagano,RichardE
• 通讯作者: Pagano,RichardE

Use of Bodipy-labeled sphingolipid and cholesterol analogs to examine membrane microdomains in cells.

• DOI: 10.1007/s00418-008-0509-5
• 发表时间: 2008-11
• 期刊: HISTOCHEMISTRY AND CELL BIOLOGY
• 影响因子: 2.3
• 作者: Marks, David L.;Bittman, Robert;Pagano, Richard E.
• 通讯作者: Pagano, Richard E.

Rab proteins mediate Golgi transport of caveola-internalized glycosphingolipids and correct lipid trafficking in Niemann-Pick C cells.

• DOI: 10.1172/jci15420
• 发表时间: 2002-06
• 期刊: The Journal of clinical investigation
• 作者: A. Choudhury;M. Dominguez;V. Puri;D. Sharma;K. Narita;C. Wheatley;D. Marks;R. Pagano

Clathrin-dependent and -independent internalization of plasma membrane sphingolipids initiates two Golgi targeting pathways.

• DOI: 10.1083/jcb.200102084
• 发表时间: 2001-08-06
• 期刊: The Journal of cell biology
• 作者: Puri V;Watanabe R;Singh RD;Dominguez M;Brown JC;Wheatley CL;Marks DL;Pagano RE
• 通讯作者: Pagano RE