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（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 鞘脂贮积症（SLSD）成纤维细胞。最近他发现有几个 荧光 SL 类似物从质膜 (PM) 内化 主要针对正常细胞的高尔基复合体，而在 10 种不同的 SLSD 中 对于不同的细胞类型，这些脂质在内体和溶酶体中积累。这 积累通常与 SL 类似物降解程度无关 不同的细胞类型。他还表明胆固醇平衡是 继发于 SL 积累和误定位的多个 SLSD 中受到干扰 SL 类似物的含量受胆固醇调节。根据这些结果，他们 假设 SLSD 细胞中积累的内源性脂质是由于 脂质分解代谢的主要缺陷导致细胞内的改变 胆固醇的分布，以及膜成分的改变 然后导致 SL 的分拣和运输出现缺陷。四大项目 与该假设相关的研究将被追究。他们将 (i) 检查 从 PM 到各个地方的荧光内化循环行程 随着时间的推移，细胞内区室：几个显性负结构 不同的 Rab 蛋白将用于帮助定义区室 发生 SL 分选不良； (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 SLs 的再唾液酸化）以证实他们的初步观察结果 荧光 SLSD 成纤维细胞中 PM 到高尔基体交通的扰动 SL 是内生 SL 的镜像； (iii) 评估潜在机制 负责扰乱 SLSD 细胞中的胆固醇稳态。在 特别是，他们将确定外源 SL 是否有能力扰乱 细胞内胆固醇稳态与特定胆固醇的强度有关 SL/胆固醇体外相互作用。他们还将评估潜力 几种甾醇感应蛋白在调节胆固醇稳态中的作用 在 SLSD 细胞中； (iv) 评估调节 Sl 转运的机制假设 靶向 SLSD 细胞。 “膜流动性”的作用将通过以下方式研究 修饰内源性膜脂质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。他们还将检查甾醇转运蛋白 NPC 的功能 1，他们的初步数据表明在 SL 瞄准中发挥关键作用。 这些研究将测试一个关于参与的新概念 SL/胆固醇相互作用在正常脂质运输调节中的作用 和 SLSD 细胞。