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Membrane Traffic in Sphingolipid Storage Diseases

鞘脂贮积病中的膜运输

基本信息

批准号：
7149161
负责人：
RICHARD E PAGANO
金额：
$ 25.59万
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

来源：
https://reporter.nih.gov/project-details/7149161
关键词：
Animals Ataxic Gait BODIPY Behavior Boron Bos taurus Bovine Serum Albumin Brain Cattle Cell membrane Cell model Cell physiology Cells Chimera organism Cholera Toxin Protomer B Cholesterol Collection Cyclodextrins Cytosol Defect Detection Disease Disease model Dominant-Negative Mutation Early Endosome Endosomes Environment Esterification Fibroblasts Fluorescence GDP dissociation inhibitor Ganglioside GM1 Ganglioside Sialidase Deficiency Disease Glycosphingolipids Golgi Apparatus Grant Green Fluorescent Proteins Guanine Nucleotide Dissociation Inhibitors Human Imagery In Situ In Vitro Intracellular Membranes Lactosylceramides Learning Life Link Lipids Lipoproteins Liposomes Longevity Low Density Lipoprotein Receptor Low-Density Lipoproteins Lysosomes Measures Mediating Membrane Membrane Lipids Membrane Protein Traffic Monitor Mouse Strains Mus Mutate Neurons Organelles Pathway interactions Play Proteins Publishing Purpose Rab escort protein RabGDI Recycling Relative (related person)Research Personnel Role SRE-1 binding protein Serum Skin Sorting - Cell Movement Sphingolipidoses Sphingolipids Sphingomyelins Symptoms Testing Therapeutic Transferrin Transgenic Organisms Transmembrane Transport Transport Vesicles Tremor Vesicle analog bis(monoacylglyceryl)phosphate cell type disease phenotype fetal in vivo insight late endosome lipid transport monomer mouse model neuron loss novel preference programs protein function rab GTP-Binding Proteins research study response

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）：我们之前证明了在广泛收集的鞘脂储存病（SLSD）成纤维细胞中沿内核/溶酶体途径发生缺陷的脂质运输和分选，并且在初步研究中发现质膜脂质和蛋白质循环也因脂质储存而显着受到干扰。在之前的资助期间，我们取得了令人兴奋的发现，选定的Rab gtpase （rabs 4、7或9）的过表达纠正了几种slsd中有缺陷的膜运输，并显著减少了胆固醇的积累，可能还有其他储存的脂质。我们假设，在slsd中，由于SLs和胆固醇的积累，选定的兔蛋白的功能受损。这项资助的总体目的是了解一些兔蛋白（而不是其他）的功能是如何被储存的脂质调节的，并制定克服这种受损功能的策略。为此，我们将(i)验证存储的SLs和胆固醇会原位扰乱选定的兔蛋白在核内体上的马赛克组织的假设。这将通过系统地改变细胞脂质组成，通过研究活细胞内体上的脂质微域，以及通过研究嵌合兔蛋白对脂质储存的反应行为来实现；（ii）进行体外研究，以确定储存的脂质调节选定兔蛋白功能的机制。这些研究将利用从正常或SLSD成纤维细胞制备的富集内体部分，并将重点放在用gdp -解离抑制剂从靶膜上提取戊烯基化的rabs上。我们将使用脂敏感兔和不敏感兔的嵌合体来确定某些（但不是其他）兔对储存的脂质的敏感性是否源于脂质与兔蛋白内基序的直接相互作用；（iii）通过定量处理细胞中的脂质分泌、酯化或降解，研究兔蛋白转导纠正各种SLSD成纤维细胞中脂质储存的机制；（iv）产生在大脑中诱导过表达rab9的转基因小鼠品系，并将这些小鼠与SLSD小鼠模型杂交，以了解是否有延迟症状发作和/或延长寿命。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，也可能提供“原理证明”，即调节膜运输是治疗slsd的有用方法。