Biophysical Mechanisms of Cholesterol Homeostasis

胆固醇稳态的生物物理机制

• 批准号: 10117604
• 负责人: FREDRIC S COHEN
• 金额: $ 35.4万
• 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/10117604
• 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; Foundations; Growth Factor; Lead; Link; Measurement; Measures; Membrane; Methods; 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/antagonist; 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对于细胞生物学和许多领域至关重要 为了实现这一目标，必须测量细胞质膜中胆固醇的化学活性。 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。 浓度已被用来量化胆固醇活性，但胆固醇的活性是由其决定的。 化学势；浓度有助于但不能准确反映膜活性。 测量胆固醇化学势的方法尚未可用，不可能正确地 评估胆固醇的许多影响，包括对细胞信号传导的影响，我们现在已经开发出方法。 为此，我们设计了这些方法和新的灌注荧光测定装置。 我们发现细胞质膜的实时化学势很快。 通过调整血浆胆固醇化学势来响应细胞外胆固醇的变化 膜不改变细胞胆固醇的总含量这一发现揭示了一个以前未知的事实。 维持胆固醇稳态的机制：质膜化学势的快速调节 控制胆固醇的流入和流出，我们已经确定了以小凹为代表的蛋白质支架结构域。 作为细胞感知外部胆固醇并快速做出反应的部位。 驻留在小窝中的胆固醇由小窝中 Ser80 位点的磷酸化程度决定 Caveolin-1，该结构域的基础蛋白 胆固醇在支架结构域之间的穿梭。 Ser80 磷酸化所引起的周围环境会改变胆固醇化学势。 因此捕获了支架结构域内启动的信号级联负责 当细胞受到外部胆固醇变化和生长时维持胆固醇稳态 我们进一步认为这些激活的信号级联反馈到质膜以维持。 细胞将受到生长因子的刺激，并且相关的信号级联反应将被激活。 通过测量 FRET（荧光共振能量）来评估小凹的丰度。 我们的初步证据强烈暗示生长因子和/或小窝蛋白之间的信号。 外部胆固醇水平的变化刺激 PI3K/Akt/mTOR 信号通路，该通路反馈 实现胆固醇稳态。将使用光遗传学技术来确定它和/或其他是否是。 使用相同策略的平行实验将确定是否确实负责控制胆固醇。 与 Caveolin 类似，flotillins 也可作为胆固醇化学势的传感器/调节器。