MEMBRANE TRAFFIC IN SPHINGOLIPID STORAGE DISEASES

鞘脂储存疾病中的膜运输

• 批准号: 6256427
• 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/6256427
• 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（逐字研究申请人的摘要）：调查员将 研究膜脂质转运和鞘脂/胆固醇相互作用 鞘脂储存疾病（SLSD）成纤维细胞。最近他发现有几个 荧光SL类似物从质膜内部内部化（PM） 主要是正常细胞的高尔基体配合物，而在10种不同的SLSD中 细胞类型，这些脂质积聚在内体和溶酶体中。这 总体上，积累与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bomeostasis是 在SL累积的多个SLSD中扰动 SL类似物的胆固醇调节。基于这些结果，他们 假设由于 脂质分解代谢中的主要缺陷导致细胞内改变 胆固醇的分布以及膜组成的这种改变 然后导致SLS的分类和运输有缺陷。四个广泛的项目 与这一假设有关。他们将（i）检查 从PM到各种的荧光内部回收的行程 随着时间的流逝，细胞内室：几个的主要负面结构 不同的Rab蛋白将用于帮助定义隔室 SL分类有缺陷； （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 重置SLS的重新养老），以确认他们的初步观察 PM的扰动与荧光的SLSD成纤维细胞中看到的高尔基交通 SLS由内源性SL镜像； （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，他们将确定外源SLS扰动的能力是否 细胞中的胆固醇稳态与特定的强度有关 体外SL/胆固醇相互作用。他们还将评估潜力 几种固醇激素蛋白在调节胆固醇稳态中的作用 在SLSD细胞中； （iv）评估机械假设调节SL传输 靶向SLSD细胞。 “膜流动性”的作用将由 修饰内源性膜脂质，并系统地改变链 用于观察SLSD中SL排序的类似物的长度和疏水性 细胞。他们还将检查固醇转运蛋白NPC的功能 1，由他们的初步数据提出，在SL靶向中发挥关键作用。 这些研究将测试有关参与的新颖概念 正常脂质运输调节中的SL/胆固醇相互作用 和SLSD细胞。