MEMBRANE TRAFFIC IN SPHINGOLIPID STORAGE DISEASES

鞘脂储存疾病中的膜运输

• 批准号: 6685236
• 负责人: RICHARD E PAGANO
• 金额: $ 23.64万
• 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 2004-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6685236
• 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 (Verbatim from the applicant's abstract): The investigator will study membrane lipid transport and sphingolipid/cholesterol interactions in sphingolipid storage disease (SLSD) fibroblasts. Recently he found that several fluorescent SL analogs were internalized from the plasma membrane (PM) predominantly to the Golgi complex of normal cells, while in 10 different SLSD cell types, these lipids accumulated in endosomes and lysosomes. This accumulation was in general unrelated to the extent of SL analog degradation in the different cell types. He also showed that cholesterol bomeostasis is perturbed in multiple SLSDs secondary to SL accumulation and that mistargeting of SL analogs was regulated by cholesterol. Based on these results, they hypothesize that endogenous lipids which accumulate in SLSD cells due to primary defects in lipid catabolism result in an altered intracellular distribution of cholesterol, and that this alteration in membrane composition then results in defective sorting and transport of SLs. Four broad projects pertaining to this hypothesis will.be pursued. They will (i) examine the itinerary of fluorescent internalized recycled from the PM to various intracellular compartments over time: Dominant negative constructs of several different Rab proteins will be used to help define the compartment(s) where defective SL sorting occurs; (ii) study the intracellular transport of endogenous (e.g., using fluorescent SL binding toxins, anti-SL antibodies, or resialylation of SLs) to confirm their preliminary observations that the perturbation in PM to Golgi traffic seen in SLSD fibroblasts with fluorescent SLs is mirrored by endogenous SLs; (iii) evaluate potential mechanisms responsible for the perturbation of cholesterol homeostasis in SLSD cells. In particular, they will determine whether the ability of exogenous SLs to perturb cholesterol homeostasis in cells is related to the strength of specific SL/cholesterol interactions in vitro. They will also evaluate the potential roles of several sterol-sensing proteins in modulating cholesterol homeostasis in SLSD cells; and (iv) evaluate mechanistic hypotheses modulating Sl transport targeting in SLSD cells. The role of "membrane fluidity" will be studied by modifying endogenous membrane lipids and by systematically varying the chain length and hydrophobicity of the analogs used to observe SL sorting in SLSD cells. They will also examine the function of the sterol transport protein, NPC 1, suggested by their preliminary data to play a critical role in SL targeting. These studies will test a novel concept concerning the involvement of SL/cholesterol interactions in the regulation of lipid trafficking in normal and SLSD cells.

描述（来自申请人摘要的逐字记录）：研究者将 研究膜脂质转运和鞘脂/胆固醇相互作用， 鞘脂贮积病（SLSD）成纤维细胞。最近他发现， 荧光SL类似物从质膜（PM）内化 主要是正常细胞的高尔基复合体，而在10种不同的SLSD中， 细胞类型，这些脂质积累在内体和溶酶体中。这 积累一般与SL类似物降解的程度无关， 不同的细胞类型。他还表明，胆固醇代谢停滞是 在多个SLSD中扰动，继发于SL积累和错误启动 SL类似物的合成受胆固醇的调节。根据这些结果，他们 假设内源性脂质在SLSD细胞中积累是由于 脂质代谢的主要缺陷导致细胞内 胆固醇的分布，这种膜组成的改变 则导致SL的有缺陷的分拣和运输。四大项目 关于这一假设will.be所追求的。他们将（一）审查 行程荧光内化回收从PM到各种 随时间推移的细胞内区室：几种主要的负性结构 将使用不同的Rab蛋白来帮助定义隔室， 缺陷SL分选发生;（ii）研究细胞内转运 内源性（例如，使用荧光SL结合毒素、抗SL抗体，或 SL的再唾液酸化），以证实他们的初步观察结果， 在SLSD成纤维细胞中观察到的PM到高尔基体交通的扰动， SL由内源性SL反映;（iii）评估潜在机制 负责SLSD细胞中胆固醇稳态的扰动。在 特别是，它们将确定外源SL干扰 细胞中胆固醇的稳态与特异性 体外SL/胆固醇相互作用。他们还将评估 几种甾醇敏感蛋白在调节胆固醇稳态中的作用 在SLSD细胞中;和（iv）评估调节S1转运的机制假说 靶向SLSD细胞。“膜流动性”的作用将通过以下方法进行研究： 修饰内源性膜脂，并通过系统地改变链 用于观察SLSD中SL分选的类似物的长度和疏水性 细胞他们还将研究固醇转运蛋白NPC的功能 1，他们的初步数据表明，在SL靶向中发挥关键作用。 这些研究将测试一个新的概念， SL/胆固醇相互作用在正常人脂质运输调节中的作用 SLSD细胞。