Membrane Traffic in Sphingolipid Storage Diseases

鞘脂贮积病中的膜运输

基本信息

批准号：
6986822
负责人：
RICHARD E PAGANO
金额：
$ 26.36万
依托单位：
MAYO CLINIC ROCHESTER
依托单位国家：
美国
项目类别：
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

项目摘要

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（由申请人提供）：我们先前证明了在广泛的鞘脂储存疾病（SLSD）的成纤维细胞中发生有缺陷的脂质传输和沿内体/溶酶体途径的出现，并且在初步研究中，Plipids corese coresse coresse coresse coresse coresse coresse coresse coresse coresse coresse coresse coresse coresse coresse corection。在上一个赠款期间，我们提出了令人兴奋的发现，所选RAB GTPases（RABS 4、7或9）的过度表达纠正了几种SLSD中有缺陷的膜流量，并且大大减少了胆固醇的积累，也许还有其他储存的脂质。我们假设，由于SLS和胆固醇的积累，SLSD中选定的RAB蛋白的功能受损。这笔赠款的总体目的是了解某些RAB蛋白（但没有其他Rab蛋白）的功能是如何通过储存的脂质调节的，并制定了克服这种受损功能的策略。为此，我们将（i）检验以下假设，该假设储存了SLS和胆固醇的摩西蛋白在原位内体上的镶嵌组织。这将通过系统地改变细胞脂质组成，通过研究活细胞内体的脂质微域以及研究嵌合Rab蛋白对脂质储存的行为来实现。（ii）进行体外研究，以确定储存的脂质调节所选RAB蛋白功能的机制。这些研究将利用由正常或SLSD成纤维细胞制备的富含内体分数，并将集中于通过GDP分解抑制剂从靶膜中提取靶膜的trenylaty Rab。我们将确定使用脂质对脂质蛋白质中的脂质直接相互作用（使用脂质敏感性和不敏感的Rabs）在Rab蛋白中的直接相互作用而引起的某些（但没有其他）Rab对储存脂质的敏感性是否引起。（iii）通过量化处理细胞中的脂质分泌，酯化或降解，研究了Rab蛋白转导矫正脂质储存的机制；（iv）产生一种转基因小鼠菌株，可诱导大脑中的Rab9过表达，并使用SLSD小鼠模型跨这些小鼠，以了解是否存在延迟的症状发作和/或增加寿命。总之，这些研究将为脂质和胆固醇调节膜运输的机制提供新的见解，并可能提供“原理证明”，即膜运输的调节是治疗SLSD的有用方法。