Cellular cholesterol movement in cardiovascular disease

项目3：心血管疾病中的类固醇转运途径 摘要 项目3的目标是定义调节细胞脂质流量的基本机制，并阐明 对全身新陈代谢的影响。剖析控制细胞存储、运输和传输的信号通路 代谢脂有望为代谢性疾病的治疗干预发现新的机会。 尽管长期以来，非囊泡胆固醇转运一直被认为是脂类稳态的关键。 在哺乳动物细胞中，其潜在的机制仍不清楚。我们发现了一种新的运输机 它被称为Aster-B，似乎填补了我们对类固醇运输理解上的这一重要空白。Aster-B是一种 促进胆固醇从质膜直接运输的一种以前未确定的蛋白质 (下午)到急诊室。我们提出了一系列的分子、细胞生物学和小鼠研究，以探讨 Aster-B胆固醇在生理和疾病中的转运途径。目的1是阐明Aster-B在体内的作用。 细胞内胆固醇的运输、外排和酯化。我们发现Asterb是一种新型的胆固醇敏感剂 LXR靶基因。Aster-B的得失改变大鼠的胆固醇分布和胆固醇酯合成 对胆固醇负荷的反应。使用生化方法和互补成像方式 包括电子显微镜和活细胞显微镜，我们将定义Aster-B的作用机制及其在 巨噬细胞的甾醇流量。目的2确定Aster-B对体内固醇转运的影响。初步数据 表明AsterB在巨噬细胞、肾上腺和性腺中的表达最高。我们将决定 Aster-B表达缺失对全身和组织特异性脂质稳态的影响。我们假设 Aster-B是高密度脂蛋白受体SR-BI下游细胞胆固醇运输的关键介质。目标3 目的是明确巨噬细胞Aster通路在动脉粥样硬化中的作用。LXR途径是 动脉粥样硬化病变发展的最强已知决定因素。我们观察到Aster-B的表达 提示Aster依赖的胆固醇转运可能影响巨噬细胞泡沫细胞 动脉粥样硬化的形成和发展。我们将测试Aster函数的得失对 巨噬细胞胆固醇摄取和外流。我们将对LDLR缺陷患者进行骨髓移植研究 检测Aster-B缺乏对小鼠病变形成的影响。目标4是确定其他组件 Aster途径。我们将使用生物素邻近标记策略进行蛋白质相互作用筛选。我们会 使用装载有胆固醇模拟探针的高密度脂蛋白颗粒进行化学蛋白质组筛选，该探针可以 与蛋白质交叉连接，并使用点击化学处理检索。此应用程序利用独特的 我们PPG的每个成员优势互补，汇集了一系列方法和 任何一名调查员都无法获得的技术能力。理解分子 控制巨噬细胞中胆固醇运动的途径是这篇PPG的总体主题的中心 应用并将促进我们对生理学和病理生理学的理解。

Project 3: Sterol Transport Pathways in Cardiovascular Disease ABSTRACT The objective of Project 3 is to define fundamental mechanisms that regulate cellular lipid flux and to elucidate their impact on systemic metabolism. Dissecting signaling pathways that govern how cells store, transport, and metabolize lipids is expected to uncover new opportunities for therapeutic intervention in metabolic disease. Although nonvesicular cholesterol transport has long been hypothesized to be critical for lipid homeostasis in mammalian cells, the underlying mechanisms have remained obscure. We have discovered a novel transporter called Aster-B that appears to fill this important gap in our understanding of sterol transport. Aster-B is a previously uncharacterized protein that facilitates the direct transport of cholesterol from the plasma membrane (PM) to the ER. We propose a series of molecular, cell biological, and mouse studies to investigate the roles of the Aster-B cholesterol transport pathway in physiology and disease. Aim 1 is to elucidate the role of Aster-B in cellular cholesterol transport, efflux, and esterification. We identified Asterb as a novel cholesterol-responsive LXR target gene. Gain or loss of Aster-B alters cholesterol distribution and impairs cholesterol ester synthesis in response to cholesterol loading. Using biochemical approaches and complementary imaging modalities including electron and live-cell microscopy, we will define the mechanism of action of Aster-B and its role in macrophage sterol flux. Aim 2 is to determine the impact of Aster-B on sterol transport in vivo. Preliminary data indicate that Asterb is most highly expressed in macrophages, adrenal gland, and gonads. We will determine the effect of loss of Aster-B expression on whole-body and tissue-specific lipid homeostasis. We hypothesize that Aster-B is a critical mediator of cellular cholesterol transport downstream of the HDL receptor SR-BI. Aim 3 is to define the contribution of the macrophage Aster pathway to atherosclerosis. The LXR pathway is one of the strongest known determinants of atherosclerotic lesion development. Our observation that Aster-B expression is regulated by LXRs suggests that Aster-dependent cholesterol transport may impact macrophage foam cell formation and the development of atherosclerosis. We will test the impact of gain or loss of Aster function on macrophage cholesterol uptake and efflux. We will perform bone marrow transplant studies into LDLR-deficient mice to test the impact of Aster-B deficiency on lesion formation. Aim 4 is to identify additional components of the Aster pathway. We will perform protein-interaction screens using a biotin proximity labeling strategy. We will perform a chemoproteomic screen using HDL particles loaded with a cholesterol-mimetic probe that can be crosslinked to proteins and retrieved using a click-chemistry handle. This application leverages the unique and complementary strengths of each member of our PPG, bringing together a range of approaches and technological capabilities that would be unavailable to any single investigator. Understanding the molecular pathways that control cholesterol movement in macrophages is central to the overall theme of this PPG application and will advance our understanding of both physiology and pathophysiology.