MEMBRANE TRAFFIC IN SPHINGOLIPID STORAGE DISEASES

鞘脂储存疾病中的膜运输

• 批准号: 6625111
• 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/6625111
• 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(逐字摘自申请人的摘要)：调查员将 膜脂转运及鞘磷脂/胆固醇相互作用的研究 鞘脂沉积病(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 主要为正常细胞的高尔基复合体，而在10种不同的SLSD中 细胞类型，这些脂类聚集在内质体和溶酶体中。这 累积总体上与SL类似物退化的程度无关 不同的细胞类型。他还表明，胆固醇平衡是 在多个SLSD中受到干扰，继发于SL积累和错误定位 的SL类似物受胆固醇调节。基于这些结果，他们 假设SLSD细胞中积累的内源性脂质是由于 脂类分解代谢的原发缺陷导致细胞内改变 胆固醇的分布，以及膜成分的这种变化 从而导致SLS的有缺陷的分类和运输。四大工程 与这一假说有关的问题将继续下去。他们将(I)审查 从PM回收到各种类型的荧光内在化的行程 随时间推移的细胞内隔间：几种主要的否定结构 不同的Rab蛋白将被用来帮助定义间隔(S) SL分选出现缺陷；(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 重新分析SLS)，以确认他们的初步观察结果 用荧光观察SLSD成纤维细胞中PM对高尔基体交通的扰动 系统性红斑狼疮由内源性系统性红斑狼疮反映；(Iii)评估潜在的机制 负责SLSD细胞中胆固醇动态平衡的扰动。在……里面 特别是，他们将决定外源SLS是否具有干扰能力 细胞内胆固醇的稳态与特异性的强度有关 SL/胆固醇在体外的相互作用。他们还将评估潜在的 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；以及(Iv)评估调节S1运输的机制假说 靶向SLSD细胞。“膜流动性”的作用将通过 通过系统地改变链来修饰内源性膜脂 用于观察SLSD中SL分类的类似物的长度和疏水性 细胞。他们还将研究固醇运输蛋白npc的功能。 1，根据他们的初步数据，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，涉及到 SL/胆固醇相互作用在调节正常人体脂质转运中的作用 和SLSD细胞。