Membrane Traffic in Sphingolipid Storage Diseases

鞘脂贮积病中的膜运输

• 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
• 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/6986822
• 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（由申请人提供）：我们先前证明了在广泛收集的鞘脂贮积病（SLSD）成纤维细胞中沿着内体/溶酶体途径发生缺陷性脂质转运和分选沿着，并且在初步研究中发现，由于脂质贮积，质膜脂质和蛋白质再循环也受到显著干扰。在先前的资助期间，我们取得了令人兴奋的发现，即所选Rab GTP酶（rab 4、7或9）的过表达纠正了几种SLSD中有缺陷的膜运输，并显著降低了胆固醇的积累，也许还有其他储存的脂质。我们推测，由于SL和胆固醇的积累，选定的rab蛋白的功能在SLSD中受损。这项资助的总体目的是了解一些rab蛋白（而不是其他蛋白）的功能是如何被储存的脂质调节的，并开发克服这种受损功能的策略。为此，我们将（i）检验储存的SL和胆固醇原位扰乱内体上选定rab蛋白的镶嵌组织的假设。这将通过系统地改变细胞脂质组成，通过研究活细胞的内体上的脂质微结构域，并通过研究嵌合rab蛋白质的行为响应于脂质储存来实现;（ii）进行体外研究以确定储存的脂质调节所选rab蛋白质的功能的机制。这些研究将利用从正常或SLSD成纤维细胞制备的富集的内体部分，并且将集中于通过GDP解离抑制剂从靶膜提取异戊二烯化的rab。 我们将确定是否敏感的一些（而不是其它）rab与储存的脂质的直接相互作用是使用脂质敏感性和非敏感性rab的嵌合体由脂质与rab蛋白内的基序的直接相互作用引起的;（iii）通过定量处理的细胞中的脂质分泌、酯化或降解，研究rab蛋白转导校正各种SLSD成纤维细胞中的脂质储存的机制;和（iv）产生在脑中诱导性过表达rab 9的转基因小鼠品系，并将这些小鼠与SLSD小鼠模型杂交以了解是否存在延迟的症状发作和/或延长的寿命。 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，并且还可以提供“原理证明”，即膜运输的调节是治疗SLSD的有用方法。