Intracellular Cholesterol Transport

细胞内胆固醇转运

• 批准号: 10059259
• 负责人: Frederick R. Maxfield
• 金额: $ 33.9万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-01-01 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10059259
• 关键词: Affect; Atherosclerosis; Autophagosome; Binding; Biological; Biological Assay; Bone Marrow; Cell Line; Cell membrane; Cell physiology; Cells; Cholesterol; Cholesterol Homeostasis; Complex; Computational Biology; Computer Analysis; Computer Models; Crystallization; Data; Development; Feedback; HepG2; Hepatocyte; Hereditary Disease; Human; In Vitro; Lipids; Liposomes; Maintenance; Mediating; Membrane; Methods; Modeling; Molecular; Mus; Mutation; NMR Spectroscopy; Organelles; Phosphatidylinositol Phosphates; Physiological; Play; Property; Protein Dynamics; Proteins; Protocols documentation; Recycling; Reporting; Role; Site; Specificity; Sterols; Structural Protein; Structure; Testing; Work; X-Ray Crystallography; cell type; cholesterol control; computer studies; experimental analysis; experimental study; knock-down; late endosome; macrophage; molecular dynamics; mutant; novel; structural biology; uptake; vector

Cholesterol plays an essential role in determining the properties of biological membranes. Control of cholesterol levels in organelles depends on sterol transport mechanisms that are poorly understood. Novel methods to study sterol transport among organelles quantitatively have been developed, and this is leading to precise models for transport among organelles. Preliminary data show that a widely expressed, sterol- regulated, soluble sterol transporter, STARD4, plays an important role in nonvesicular transport of cholesterol, and its role and mechanism of transport will be studied. STARD4 is an important component of a complex sterol regulatory network. Recent work showed that STARD4 is responsible for about 25% of sterol transport between two major pools – the plasma membrane and the endocytic recycling compartment - in U2OS cells. The role of STARD4 will also be assessed in macrophages, a cell type in which cholesterol metabolism plays a major role in development of atherosclerosis, and in HepG2 cells, a model for hepatocytes. In preliminary studies it has been found that STARD4 has unique interactions with phosphatidylinositol phosphates (PIPs). STARD4 extraction of sterol from membranes is accelerated nearly 10-fold when the membranes contain PI(4,5)P2, but there is no effect of PI(4,5)P2 in membranes accepting sterol from STARD4. Conversely, PI(3,5)P2 or PI5P in acceptor membranes accelerates transfer ∼10-fold with no effect when these PIPs are in donor membranes. PI3P has a similar but smaller effect. The role of STARD4 in sterol transport to late endosomes, lipid droplets, and autophagosomes (i.e., organelles with good acceptor PIPs) will be analyzed. Molecular dynamics simulations of interactions of STARD4 with membranes is identifying potential sites of interaction with PIPs. The effects of mutations of these sites will be assayed in a liposome to liposome sterol transport assay. Mutant STARD4 molecules with altered PIP sensitivity will be expressed in cells to determine effects on sterol transport and distribution. In preliminary studies, mutants identified by molecular dynamics were confirmed by tests in the liposome assay, and it was found that they do affect transport of sterol among organelles. X-ray crystallography has identified differences between human STARD4 with and without bound sterol. NMR spectroscopy is being used to analyze STARD4 protein dynamics and to determine the sites of interaction with lipids containing various PIPs.

胆固醇在决定生物膜的特性方面发挥着重要作用。 细胞器中的胆固醇水平取决于甾醇转运机制，但人们对此知之甚少。 定量研究细胞器之间甾醇转运的方法已经开发出来，这导致 细胞器之间运输的精确模型 初步数据表明，广泛表达的甾醇- 受调节的可溶性甾醇转运蛋白 STARD4 在胆固醇的非囊泡转运中发挥重要作用， STARD4是一个复合体的重要组成部分，其作用和运输机制将得到研究。 最近的研究表明 STARD4 负责大约 25% 的甾醇运输。 U2OS 细胞中两个主要池（质膜和内吞回收室）之间。 STARD4 的作用也将在巨噬细胞中进行评估，巨噬细胞是一种胆固醇代谢在其中发挥重要作用的细胞类型。 在动脉粥样硬化的发展以及肝细胞模型 HepG2 中发挥着重要作用。 研究发现 STARD4 与磷酸磷脂酰肌醇 (PIP) 具有独特的相互作用。 当膜含有时，STARD4 从膜中提取甾醇的速度加快了近 10 倍 PI(4,5)P2，但 PI(4,5)P2 在接受来自 STARD4 的甾醇的膜中没有影响， 受体膜中的 PI(3,5)P2 或 PI5P 可将转移加速约 10 倍，但当这些 PIP 存在时没有任何影响 PI3P 在甾醇转运中具有类似但较小的作用。 将分析核内体、脂滴和自噬体（即具有良好受体 PIP 的细胞器）。 STARD4 与膜相互作用的分子动力学模拟正在识别潜在的位点 与 PIP 的相互作用将在脂质体到脂质体甾醇中进行测定。 具有改变的 PIP 敏感性的突变 STARD4 分子将在细胞中表达以确定 在初步研究中，通过分子动力学鉴定了突变体。 经脂质体试验证实，发现它们确实影响甾醇在体内的转运 X 射线晶体学鉴定了有结合和无结合的人类 STARD4 之间的差异。 核磁共振波谱法用于分析 STARD4 蛋白质动力学并确定其位点。 与含有各种 PIP 的脂质相互作用。

项目成果

Membrane order in the plasma membrane and endocytic recycling compartment.

• DOI: 10.1371/journal.pone.0188041
• 发表时间: 2017
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Iaea DB;Maxfield FR
• 通讯作者: Maxfield FR

Role of STARD4 in sterol transport between the endocytic recycling compartment and the plasma membrane.

• DOI: 10.1091/mbc.e16-07-0499
• 发表时间: 2017-04-15
• 期刊: Molecular biology of the cell
• 影响因子: 3.3
• 作者: Iaea DB;Mao S;Lund FW;Maxfield FR
• 通讯作者: Maxfield FR

Phosphatidylinositol phosphates modulate interactions between the StarD4 sterol trafficking protein and lipid membranes.

• DOI: 10.1016/j.jbc.2022.102058
• 发表时间: 2022-07
• 期刊: JOURNAL OF BIOLOGICAL CHEMISTRY
• 影响因子: 4.8
• 作者: Zhang, Xiaoxue;Xie, Hengyi;Iaea, David;Khelashvili, George;Weinstein, Harel;Max, Frederick R.
• 通讯作者: Max, Frederick R.

Comment on 'Orthogonal lipid sensors identify transbilayer asymmetry of plasma membrane cholesterol'.

• DOI: 10.7554/elife.38493
• 发表时间: 2018-11-13
• 期刊: eLife
• 影响因子: 7.7
• 作者: Courtney KC;Fung KY;Maxfield FR;Fairn GD;Zha X
• 通讯作者: Zha X