Membrane Traffic in Sphingolipid Storage Diseases

鞘脂贮积病中的膜运输

• 批准号: 6865170
• 负责人: RICHARD E PAGANO
• 金额: $ 26.99万
• 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: 2000-04-01 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6865170
• 关键词: CHO cells; Golgi apparatus; N acylation; Niemann Pick disease; cell component structure /function; cell line; cholesterol; cholesterol ester storage disease; endocytosis; fibroblasts; fluorescent dye /probe; gene mutation; guanosinetriphosphatases; immunofluorescence technique; intracellular transport; lipid metabolism; lipid transport; lysosomes; membrane activity; membrane lipids; membrane structure; membrane transport proteins; molecular pathology; protein localization; sphingolipidosis

DESCRIPTION (provided by applicant): We previously demonstrated the occurrence of defective lipid transport and sorting along the endosome/lysosome pathway in a broad collection of sphingolipid storage disease (SLSD) fibroblasts, and in preliminary studies have found that plasma membrane lipid and protein recycling are also significantly perturbed as a result of lipid storage. During the previous grant period we made the exciting finding that over-expression of selected Rab GTPases (rabs 4, 7, or 9) corrects defective membrane traffic in several SLSDs and dramatically reduces accumulation of cholesterol, and perhaps other stored lipids. We hypothesize that the function of selected rab proteins is impaired in SLSDs due to accumulation of SLs and cholesterol. The overall purpose of this grant is to understand how the function of some rab proteins (but not others) is modulated by stored lipids and to develop strategies for overcoming this impaired function. To do so, we will (i) test the hypothesis that stored SLs and cholesterol perturb the mosaic organization of selected rab proteins on endosomes in situ. This will be accomplished by systematically altering cellular lipid composition, by studying lipid microdomains on endosomes of living cells, and by studying the behavior of chimeric rab proteins in response to lipid storage; (ii) perform in vitro studies to determine the mechanism by which stored lipids modulate function of selected rab proteins. These studies will utilize an enriched endosome fraction prepared from normal or SLSD fibroblasts, and will focus on the extraction of prenylated rabs from target membranes by the GDP-dissociation inhibitor. We will determine whether the sensitivity of some (but not other) rabs to stored lipids results from a direct interaction of the lipids with motifs within rab proteins using chimeras of lipid-sensitive and -insensitive rabs; (iii) study the mechanism by which rab protein transduction corrects lipid storage in various SLSD fibroblasts, by quantifying lipid secretion, esterification, or degradation in the treated cells; and (iv) generate a transgenic mouse strain that inducibly over-expresses rab9 in the brain, and cross these mice with an SLSD mouse model to learn whether there is delayed symptom onset and/or increased life span. Together, these studies will provide novel insights into the mechanisms by which lipids and cholesterol regulate membrane trafficking, and may also provide "proof of principle" that modulation of membrane trafficking is a useful approach for treatment of SLSDs.

描述(申请人提供)：我们先前证明，在广泛的鞘脂沉积病(SLSD)成纤维细胞中，沿着内体/溶酶体途径发生了有缺陷的脂类运输和分选，并且在初步研究中发现，由于脂类的储存，质膜的脂类和蛋白质循环也受到了显著的干扰。在之前的授权期内，我们做出了令人兴奋的发现，即过表达选定的Rab GTP酶(RAB 4、7或9)可以纠正几个SLSD中有缺陷的膜交通，并显著减少胆固醇的积累，可能还有其他储存的脂类。我们推测，在SLSD中，由于SLS和胆固醇的积累，选定的Rab蛋白的功能受到了损害。这项资助的总体目的是了解一些Rab蛋白质(但不是其他蛋白质)的功能是如何受到储存的脂类调节的，并开发克服这种受损功能的策略。为了做到这一点，我们将(I)检验假设，即储存SLS和胆固醇扰乱了选定的Rab蛋白在原位内吞体内的马赛克组织。这将通过系统地改变细胞脂质组成，通过研究活细胞内体上的脂质微域，以及通过研究嵌合Rab蛋白对脂质储存的响应来实现；(Ii)进行体外研究，以确定储存的脂质调节选定的Rab蛋白的功能的机制。这些研究将利用从正常或SLSD成纤维细胞中制备的丰富的内体部分，并将重点放在通过GDP解离抑制剂从靶膜中提取预烯基化的RAB。我们将确定一些(但不是其他)RAB对储存的脂类的敏感性是否源于利用脂类敏感和不敏感的RAB嵌合体使Rab蛋白中的脂类与Rab蛋白中的基序直接相互作用；(Iii)通过量化处理细胞中的脂类分泌、酯化或降解，研究Rab蛋白转导纠正各种SLSD成纤维细胞中的脂类储存的机制；(Iv)建立一个可诱导在大脑中过表达Rab9的转基因小鼠株，并将这些小鼠与SLSD小鼠模型杂交，以了解是否有延迟的症状出现和/或延长寿命。总之，这些研究将为脂类和胆固醇调节膜转运的机制提供新的见解，并可能为调节膜转运是治疗SLSD的有用方法提供“原则证据”。