Biophysical Mechanisms of Cholesterol Homeostasis

胆固醇稳态的生物物理机制

• 批准号: 10624260
• 负责人: FREDRIC S COHEN
• 金额: $ 34.7万
• 依托单位: RUSH UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10624260
• 关键词: Back; Binding; Biological Assay; Biophysical Process; Caveolae; Caveolins; Cell Signaling Process; Cell membrane; Cell physiology; Cells; Cellular biology; Chemicals; Cholesterol; Cholesterol Homeostasis; Closure by clamp; Communication; Cues; Data; Devices; Disease; Exposure to; FRAP1 gene; Feeds; Fluorescence; Fluorescence Resonance Energy Transfer; Growth Factor; Lead; Link; Measurement; Measures; Membrane; Methods; PIK3CG gene; Pathology; Perfusion; Phosphorylation; Physiological; Plasma Cells; Positioning Attribute; Regulation; Resolution; Rest; Scaffolding Protein; Signal Pathway; Signal Transduction; Signaling Molecule; Signaling Protein; Site; Techniques; Tertiary Protein Structure; Testing; Time; caveolin 1; cholesterol control; dehydroergosterol; experimental study; extracellular; flotillin; inhibitor; mTOR Signaling Pathway; membrane activity; novel; optogenetics; restoration; scaffold; sensor

Abstract Understanding mechanisms cells use to maintain cholesterol homeostasis are critical in cell biology and many diseases. To achieve this, the chemical activity of cholesterol in cell plasma membranes must be measured because activity controls cholesterol’s effects on cellular processes. To date, plasma membrane cholesterol concentration has been used to quantify cholesterol activity. But the activity of cholesterol is determined by its chemical potential; concentration contributes to, but does not accurately reflect membrane activity. Because a method to measure cholesterol chemical potential had not been available, it was not possible to properly evaluate many of cholesterol’s effects, including those on cellular signaling. We have now developed methods to do so. These methods and a new perfusion fluorimetry apparatus we have devised allow us to follow the chemical potential of cholesterol of plasma membranes in real time. We have discovered that cells quickly respond to changes in extracellular cholesterol by adjusting the cholesterol chemical potential of their plasma membranes without changing the total content of cellular cholesterol. This finding reveals a previously unknown mechanism to maintain cholesterol homeostasis: quick adjustment of plasma membrane chemical potentials to control cholesterol influx and efflux. We have identified protein scaffolded domains, as typified by caveolae, as sites at which cells sense and rapidly respond to external cholesterol. The abundance and total amount of cholesterol that resides in caveolae are determined by the extent of phosphorylation at position Ser80 of caveolin-1, the foundational protein of the domain. The shuttling of cholesterol between scaffolded domains and the surround which must result upon Ser80 phosphorylation alters cholesterol chemical potential. We therefore hypothesize that signaling cascades initiated within scaffolded domains are responsible for maintaining cholesterol homeostasis when cells are subjected to changes in external cholesterol and to growth factors. We further posit that these activated signaling cascades feed back to the plasma membrane to maintain chemical potentials. Cells will be stimulated with growth factors and relevant signaling cascades will be identified. The abundance of caveolae will be assessed by measuring the FRET (fluorescence resonance energy transfer) signals between caveolins. Our preliminary evidence strongly implicates that growth factors and/or changes in the level of external cholesterol stimulate the PI3K/Akt/mTOR signaling pathway that feeds back to achieve cholesterol homeostasis. Optogenetic techniques will be used to determine whether it and/or others are indeed responsible for control of cholesterol. Parallel experiments using the same strategies will determine if flotillins, analogous to caveolin, also serve as sensors/regulators of cholesterol chemical potentials.

抽象的 了解细胞用于维持胆固醇稳态的机制在细胞生物学中至关重要，许多 疾病。为此，必须测量胆固醇在细胞质膜中的化学活性 因为活性控制胆固醇对细胞过程的影响。迄今为止，质膜胆固醇 浓度已用于量化胆固醇活性。但是胆固醇的活性取决于 化学势；浓度有助于，但不能准确反映膜活性。因为 测量胆固醇化学势的方法尚不可用，无法正确地 评估许多胆固醇的作用，包括对细胞信号传导的作用。我们现在已经开发了方法 这样做。这些方法和一种新的灌注荧光学设备使我们能够遵循 质膜的胆固醇的化学潜力。我们发现细胞很快 通过调节血浆的胆固醇化学潜力来应对细胞外胆固醇的变化 膜而不改变细胞胆固醇的总含量。这一发现揭示了以前未知的 维持胆固醇稳态的机制：快速调整质膜化学势 控制胆固醇的影响和外排。我们已经确定了蛋白质支架的结构域，如小窝的特征 作为细胞感知并迅速反应外部胆固醇的部位。丰度和总数 居住在小窝中的胆固醇取决于位置在Ser80的位置的磷酸化程度 Caveolin-1，域的基础蛋白。脚手架域之间的胆固醇穿梭 Ser80磷酸化必须导致的周围环境改变胆固醇的化学潜力。我们 因此，假设在脚手架域中启动的信号级联反应是负责的 当细胞发生外部胆固醇的变化并生长时，保持胆固醇稳态 因素。我们进一步确认这些激活的信号级联反馈回到质膜以维持 化学势。细胞将被生长因子刺激，相关信号级联将是 确定。将通过测量FRET（荧光共振能量）来评估大小的丰度 转移）信号在小窝之间。我们的初步证据强烈暗示增长因素和/或 外部胆固醇水平的变化刺激PI3K/AKT/MTOR信号传导途径，该途径回到 达到胆固醇稳态。光遗传技术将用于确定它和/或其他技术是否是 确实负责控制胆固醇。使用相同策略的并行实验将确定是否 类似于小窝蛋白的植物素也是胆固醇化学势的传感器/调节剂。