Investigating protein supersaturation as a driver of aging

研究蛋白质过饱和作为衰老的驱动因素

• 批准号: 10605634
• 负责人: Alex Von Schulze
• 金额: $ 6.95万
• 依托单位: STOWERS INSTITUTE FOR MEDICAL RESEARCH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10605634
• 关键词: Acceleration; Age; Age of Onset; Aging; Amyloid; Biological; Biological Process; Cell Aging; Cell Nucleus; Cell physiology; Cells; Characteristics; Clinical; Cytoplasm; Cytosol; DNA Methylation; Data; Deceleration; Deposition; Development; Disease; Epigenetic Process; Event; Feedback; Fibroblasts; Fluorescence Resonance Energy Transfer; Foundations; Genetic Transcription; Goals; Human; Intrinsic drive; Investigation; Kinetics; Libraries; Light; Mass Spectrum Analysis; Measures; Molecular; Molecular Conformation; Nuclear; Nuclear Export; Nuclear Proteins; Phase; Physiological; Prevention; Process; Proteins; Proteome; Proteomics; Reporter; Reporting; Research; Research Personnel; Role; Solubility; Testing; Thermodynamics; Time; age related; aged; cell age; conformational conversion; design; driving force; experimental study; insight; novel; novel therapeutics; polypeptide; prediction algorithm; protein aggregation; proteostasis; self assembly; transcriptome sequencing

PROJECT SUMMARY Protein aggregation is a hallmark of aging and age-associated disease, however a causal relationship has not been demonstrated. Elucidating whether there is a predestined, irreversible driving force in cell aging would enable the development of novel therapies to decelerate aging. Therefore, my long-term goal is to understand this relationship at a fundamental level. My central hypothesis is that probabilistic, irreversible conformational transitions in physiologically supersaturated proteins not only initiate the process of cellular aging, but drive it!. The accumulation of amyloids following such transitions will compromise kinetic proteostasis, or kinetic barriers for supersaturated proteins to remain soluble, and this ultimately compromises thermodynamic proteostasis -- or the processes that maintain the concentrations and stabilities of soluble proteins. I will utilize the following Specific Aims and synergistic approaches, distributed amphifluoric FRET (DAmFRET), Epigenetic Clocks, and RNA-Seq, to distinguish the kinetic from thermodynamic determinants of protein solubility as a function of cell age. In Aim 1, I will compare thermodynamic and kinetic proteostasis as a function of biological age. To do so, I will first obtain primary human fibroblasts (PHFs) from differentially aged donors, and validate their epigenetic age using DNA methylation signatures (DNAm) referenced against previously developed DNAm age prediction algorithms, as well as RNA-Seq. I will then perform DAmFRET experiments in each of the PHFs using a panel of inducible constructs that reliably aggregate in a nucleation- and/or concentration-limited manner. These data will reveal the degree to which kinetic proteostasis and/or thermodynamic proteostasis are impacted by biological age. In Aim 2, I will test if conformational nuclei accelerate the aging of PHFs. I will generate generic light-activated optoSeeds from our reporter library in Aim 1 to elicit a conformational transition, or cross-seeding event, in PHFs of young age. I will then use multiple mass spectrometry approaches to evaluate whether the nucleation event precipitated endogenous proteins, and determine their identities. I will then determine if the treatment accelerates the progression of cell age via DNAmAge and RNA-Seq. In Aim 3, I will test if perturbing kinetic proteostasis in the nucleus enhances the rate of aging as compared to the cytosol. Using our optoSeeds, I will elicit a conformational transition in the nuclear and cytoplasmic compartments. I will again use DNAm age prediction and RNA-Seq to determine whether age is accelerated via conformational transitioning in the nucleus versus the cytosol. Completion of these aims will provide fundamental insights into the thermodynamic reasons for why we age. In addition, completion of the proposed studies will provide me with a strong foundation to continue my research as an independent investigator.

项目概要 蛋白质聚集是衰老和年龄相关疾病的标志，但因果关系并不存在 已被证明。阐明细胞衰老是否存在预定的、不可逆转的驱动力 促进新疗法的开发以延缓衰老。因此，我的长期目标是了解 这种关系在根本层面上。我的中心假设是概率性的、不可逆的 生理上过饱和蛋白质的构象转变不仅启动了 细胞老化，但驱动它！这种转变后淀粉样蛋白的积累将损害动力学 蛋白质稳态，或过饱和蛋白质保持可溶性的动力学障碍，这最终会损害 热力学蛋白质稳态——或维持可溶性物质浓度和稳定性的过程 蛋白质。我将利用以下具体目标和协同方法，分布式双氟 FRET (DAmFRET)、表观遗传时钟和 RNA-Seq，以区分动力学和热力学决定因素 蛋白质溶解度与细胞年龄的关系。在目标 1 中，我将比较热力学和动力学蛋白质稳态 生物年龄的函数。为此，我将首先从不同年龄的人中获取原代人成纤维细胞（PHF） 捐赠者，并使用参考的 DNA 甲基化特征 (DNAm) 验证他们的表观遗传年龄 之前开发的 DNAm 年龄预测算法以及 RNA-Seq。然后我将执行 DAmFRET 在每个 PHF 中进行实验，使用一组可诱导的构建体，这些构建体可靠地聚集在成核- 和/或浓度限制的方式。这些数据将揭示动力学蛋白质稳态和/或 热力学蛋白质稳态受生物年龄的影响。在目标 2 中，我将测试构象核是否 加速PHF的老化。我将从 Aim 的记者库中生成通用的光激活 optoSeeds 1 在年轻的 PHF 中引发构象转变或交叉播种事件。然后我将使用多个 质谱方法评估成核事件是否沉淀内源蛋白质， 并确定他们的身份。然后我将通过以下方式确定治疗是否加速细胞年龄的进展 DNAmAge 和 RNA 测序。在目标 3 中，我将测试扰动细胞核中的动力学蛋白质稳态是否会提高速率 与细胞质相比，衰老的发生。使用我们的 optoSeeds，我将引发核构象转变 和细胞质区室。我将再次使用 DNAm 年龄预测和 RNA-Seq 来确定年龄是否 通过细胞核相对于细胞质的构象转变加速。完成这些目标将 为我们衰老的热力学原因提供基本见解。此外，完成 拟议的研究将为我作为独立的人继续我的研究提供坚实的基础 研究者。