Biophysical Mechanisms of Cholesterol Homeostasis

胆固醇稳态的生物物理机制

基本信息

批准号：
10454109
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
项目类别：
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/10454109
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 mTOR Signaling Pathway membrane activity novel optogenetics restoration scaffold sensor

项目摘要

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，许多为了实现这一目标，必须测量胆固醇的化学活性由于活性控制胆固醇对细胞过程的影响。浓度已用于量化胆固醇活性。化学潜力; 测量胆固醇化学潜力潜力的方法尚无可用，因此无法正确评估许多胆固醇的作用，包括我们现在开发的方法。这样做。质膜的化学潜力实时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改变了细胞胆固醇的总含量。维持胆固醇稳态的机制：快速调整质量惊奇电势为了控制胆固醇的流入和外排。如细胞感知外部胆固醇的位置。居住在口腔中的胆固醇取决于phosporition在Ser80的phosporition的程度 Caveolin-1，结构域的基础蛋白。必须在Ser80磷酸化时导致的周围改变Chesterol化学势因此，假设胸肌施舍胸肌施舍施舍施舍施施施施施施施施施施施施刺脚脚架脚手架脚轮造成的领域是负责的当细胞受到外部胆固醇的变化并生长时，保持胆固醇稳态我们进一步认为，激活的信号级联反馈到质膜化学电位将被生长因子刺激，相关信号级联鉴定出来的小窝的丰度将通过测量fret（荧光共振能量）进行评估 Caveolins之间的转移信号。外部胆固醇刺激水平的变化PI3K/AKT/MTOR信号传导途径，该途径回到实现胆固醇稳态。确实可以使用相同的策略来控制胆固醇。类似于小窝蛋白的植物素也是胆固醇化学势的传感器/常规者。