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.

项目总结 蛋白质聚集是衰老和年龄相关疾病的标志，但因果关系不是 已经被证明了。阐明细胞衰老是否存在注定的、不可逆转的驱动力 能够开发新的治疗方法来减缓衰老。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的，不可逆转的 生理过饱和蛋白质中的构象转变不仅启动了 细胞老化，但驱使它！在这种转变之后淀粉样蛋白的积累将损害动力学。 蛋白质稳定，或过饱和蛋白质保持可溶的动力学障碍，这最终会妥协 热力学蛋白质平衡--或维持可溶性蛋白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，分布式两氟FRET (DAmFRET)、表观遗传学时钟和RNA-Seq，以区分动力学和热力学决定因素 蛋白质的溶解度是细胞年龄的函数。在目标1中，我将比较热力学和动力学蛋白质平衡 生物年龄的作用。为此，我将首先从不同年龄的原代人成纤维细胞(PHF)中获得 供者，并使用参照的DNA甲基化签名(DNaM)验证他们的表观遗传年龄 以前开发的dNaM年龄预测算法，以及RNA-Seq.然后我将执行DAMFRET 在每个PHF中使用一组可诱导的结构进行实验，这些结构可靠地聚集在成核过程中- 和/或浓度受限方式。这些数据将揭示运动蛋白平衡和/或 热力学蛋白质平衡受到生物年龄的影响。在目标2中，我将测试构象核是否 加速PHF的老化。我将从我们在AIM的报告文库中生成通用的光激活光种子 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 质谱学方法来评估成核事件是否沉淀了内源蛋白质， 并确定他们的身份。然后我将确定治疗是否通过以下途径加速细胞年龄的进展 DNAmAge和RNA-Seq.在目标3中，我将测试干扰细胞核内的动力学蛋白平衡是否会提高这一速度 与细胞溶质相比，老化的可能性更大。使用我们的光种子，我将在原子核中引发构象转变 和细胞质隔间。我将再次使用dNaM年龄预测和RNA-Seq来确定年龄 是通过细胞核相对于胞浆的构象转变而加速的。完成这些目标将 提供对我们衰老的热力学原因的基本见解。此外，完成 拟议的研究将为我作为独立研究人员继续我的研究提供坚实的基础 调查员。