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.

胆固醇在确定生物膜的特性中起着至关重要的作用。控制 细胞器中的胆固醇水平取决于固醇转运机制，这些机制知之甚少。小说 已经开发了研究细胞器之间立体传输的方法，这导致 初步数据表明，广泛表达的固醇 受调节的，固体固醇转运蛋白，Stard4在胆固醇的非维化运输中起重要作用， 其运输的作用和机制将研究。 stard4是复合物的重要组成部分 固醇调节网络。最近的工作表明Stard4负责约25％的固醇运输 U2OS细胞中的两个主要池（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，巨噬细胞是胆固醇代谢的细胞类型 在动脉粥样硬化的发育以及HEPG2细胞中的主要作用，这是肝细胞的模型。在初步 研究已经发现，Stard4与磷脂酰肌醇磷酸盐（PIPS）具有独特的相互作用。 当膜中，从膜上提取立体声的stard4将几乎加速10倍 Pi（4,5）P2，但膜中Pi（4,5）P2没有影响Stard4的立体声。反过来， 受体膜中的Pi（3,5）P2或PI5P加速转移约10倍，当这些PIP在 供体膜。 PI3P具有相似但较小的效果。 stard4在立体声传输到迟到的角色 将分析内体，脂质液滴和自噬体（即具有良好受体PIPS的细胞器）。 Stard4与膜的相互作用的分子动力学模拟正在识别 与PIP的相互作用。这些位点突变的影响将以脂质体为脂质体固醇分配 运输测定法。在细胞中将表达具有改变PIP敏感性的突变体星分子，以确定 对立体传输和分布的影响。在初步研究中，通过分子动力学鉴定的突变体 通过脂质体测定中的测试证实，发现它们确实会影响固醇的运输 细胞器。 X射线晶体学已经确定了具有和没有绑定的人类Stard4之间的差异 固醇。 NMR光谱法被用于分析Stard4蛋白动力学并确定 与包含各种PIP的脂质的相互作用。

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