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.

摘要 了解细胞用来维持胆固醇动态平衡的机制在细胞生物学和许多 疾病。为此，必须测量细胞质膜中胆固醇的化学活性。 因为活动控制着胆固醇对细胞过程的影响。到目前为止，质膜胆固醇 浓度已被用来量化胆固醇的活性。但胆固醇的活性是由它的 化学势；浓度对膜活性有贡献，但不能准确反映膜的活性。因为一个 目前还没有测量胆固醇化学势的方法，不可能正确地 评估胆固醇的许多影响，包括对细胞信号的影响。我们现在已经开发出了方法 这样做。这些方法和我们设计的一种新的灌注荧光测定仪使我们能够遵循 实时测定质膜胆固醇的化学势。我们很快就发现细胞 通过调节血浆胆固醇化学势来应对细胞外胆固醇的变化 在不改变细胞总胆固醇含量的情况下，膜。这一发现揭示了一个以前未知的 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。我们已经确定了蛋白质支架结构域，以小窝为代表， 作为细胞感知和快速反应外界胆固醇的场所。的丰度和总量 小窝中的胆固醇是由SER80位的磷酸化程度决定的 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 而必须导致Ser80磷酸化的周围环境改变了胆固醇的化学势。我们 因此，假设在脚手架域内启动的信令级联负责 当细胞受到外部胆固醇变化和生长的影响时，维持胆固醇稳态 各种因素。我们进一步假设，这些激活的信号级联反馈到质膜以维持 化学势。细胞将受到生长因子的刺激，相关的信号级联将被 确认身份。小窝的丰度将通过测量FRET(荧光共振能量)来评估 在洞穴之间传递)信号。我们的初步证据有力地表明，生长因子和/或 外部胆固醇水平的变化刺激PI3K/Akt/mTOR信号通路反馈到 实现胆固醇动态平衡。光遗传技术将被用来确定它和/或其他人是否 确实负责控制胆固醇。使用相同策略的平行实验将确定 Flotillins类似于小窝蛋白，也是胆固醇化学势的感应器/调节器。