Investigating protein supersaturation as a driver of aging

项目摘要 蛋白质聚集是衰老和年龄相关疾病的标志，然而， 被证明。阐明细胞衰老是否有一种注定的、不可逆转的驱动力， 使新疗法的发展能够减缓衰老。因此，我的长期目标是了解 这种关系在一个基本的水平。我的核心假设是概率性的，不可逆转的 生理上过饱和的蛋白质中的构象转变不仅启动了 细胞老化，但驱动它！.淀粉样蛋白在这种转变后的积累将损害动力学 蛋白质稳态，或动力学障碍，过饱和蛋白质保持可溶性，这最终损害 热力学蛋白质稳态-或维持可溶性蛋白质的浓度和稳定性的过程 proteins.我将利用以下具体目标和协同的方法，分布式双功能FRET （DAmFRET）、表观遗传时钟和RNA-Seq，以区分基因的动力学和热力学决定因素。 蛋白质溶解度作为细胞年龄的函数。在目标1中，我将比较热力学和动力学蛋白质稳态作为一种 生理年龄的功能。为了做到这一点，我将首先从不同年龄的人中获得原代人成纤维细胞（PHFs）。 供体，并使用DNA甲基化特征（DNAm）验证其表观遗传年龄， 先前开发的DNAm年龄预测算法，以及RNA-Seq。然后我将执行DamFRET 使用一组诱导型构建体在每个PHF中进行实验，所述诱导型构建体可靠地聚集成核- 和/或浓度限制方式。这些数据将揭示动力学蛋白质稳态和/或代谢的程度。 热力学蛋白质稳态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Aim的报告库中生成通用的光激活optoSeeds 1引起构象转变，或交叉播种事件，在年轻的PHF。然后我将使用多个 质谱分析方法来评价成核事件是否沉淀了内源性蛋白， 并确定他们的身份然后，我将确定治疗是否通过以下方式加速细胞年龄的进展： DNAmAge和RNA-Seq.在目标3中，我将测试是否在细胞核中扰乱动力学蛋白质稳态会提高 与胞质溶胶相比，利用我们的optoSeeds，我将在核中引发构象转变， 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 通过细胞核相对于胞质溶胶的构象转变而加速。实现这些目标将 为我们衰老的热力学原因提供了基本的见解。此外， 拟议的研究将为我提供一个坚实的基础，继续我的研究作为一个独立的 调查员

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.