Reverse Engineering of Cell Senescence

摘要 细胞衰老是一种难以捉摸的细胞状态-它在胚胎发生、伤口愈合和衰老中被识别， 它不仅是一种症状，而且是衰老病理学的主要贡献者。这些引人注目的实验 小鼠，消除衰老细胞赋予广泛的健康益处，甚至逆转衰老 这一过程，驱使我们对理解并最终控制正常细胞向 衰老如果我们了解了细胞衰老是如何发生的，我们就有机会找到抑制衰老的方法。 它或逆转它，以及开发从我们的身体清除这些细胞的实用方法。这项建议是为了 在显微镜和分子水平上使用新的工具来观察细胞从正常的 衰老状态并揭示其分子电路的变化。在细胞进行的转变中， 是细胞大小的急剧增加。已知肥大由mTOR驱动，并由药物抑制 雷帕霉素，它与几个遗传多样性物种的寿命延长有关。我们专注于肥大 帮助破解衰老，因为作为一种表型，它完全违反了正常的细胞经济， 其中跨细胞类型的大小被精确地保持。不幸的是，细胞的大小一直是最困难的 表型进行研究。随着新形式显微镜的发展，这种情况发生了根本性的变化 直接测量细胞干质量，甚至直接分别测量蛋白质和脂质质量。 使用这样的仪器，我们建议以前所未有的时间和质量分辨率研究细胞， 提示压力或老化，成为肥大，以及肥大是如何连接到其他表型， 衰老我们将沿着这些相同的轨迹进行深度定量质谱分析， 蛋白质表达和磷酸化与大小和其它衰老标志物的关系。我们交叉对比了 我们的发现之间的体外研究细胞培养和体内研究在年轻和老年小鼠。一旦我们建成 衰老过程的基线描述，与诱导-成熟-死亡的生命周期相同， 衰老的细胞在药物的干扰下会重复。我们可以从描述发展到因果 使用几种衰老清除药物是激酶抑制剂的知识进行分析。当我们扰乱衰老 细胞通过一个小的最佳信息，预先选择的多特异性激酶抑制剂，其抑制 活动平铺整个激酶组，我们可以确定调节衰老的关键激酶。进一步将此 磷酸质谱法将使我们能够追踪信号级联到特定的蛋白质底物， 磷酸盐这些观察结果和药理学扰动可以提出新的senolytic策略 甚至建议使用特定的抗衰老药物。

Abstract Cellular senescence is an elusive cell state - it is recognized in embryogenesis, wound healing and aging, where it is not only a symptom, but a major contributor to aging pathology. The striking experiments in the mouse, where eliminating senescent cells bestows broad health benefits and even a reversal of the aging process, drives our interest in understanding and ultimately controlling the transition of normal cells towards senescence. If we understood how cell senescence arises, we would have a chance to find ways to suppress it or reverse it, as well as to develop practical ways of clearing such cells from our bodies. This proposal is to use new tools on the microscopic and on the molecular level to observe the passage of cells from a normal to senescent state and reveal the changes in their molecular circuitry. Among the transitions that cells make is a dramatic increase in cell size. Hypertrophy is known to be driven by mTOR, and inhibited by the drug rapamycin, which is tied to life extension in several phylogenetically diverse species. We focus on hypertrophy to help crack senescence because, as a phenotype, it is in such stark violation of the normal cellular economy, where across cell types size is precisely maintained. Unfortunately, cell size has been one of the hardest phenotypes to study. This situation has changed radically with the development of new forms of microscopy by our group, that directly measure cell dry mass or even directly measure protein and lipid mass separately. Using such instruments, we propose to study with unprecedented temporal and mass resolution how cells, prompted by stress or aging, become hypertrophic and how hypertrophy is connected to other phenotypes of senescence. We will follow these same trajectories with deep quantitative mass spectrometry to correlate protein expression and phosphorylation with size and other senescent markers. We cross-reference some of our findings between in-vitro studies in cell culture and in-vivo studies in young and aged mice. Once we build a baseline description of the process of senescence, the same induction-maturation-death lifecycle of senescent cells will be repeated under perturbation by drugs. We can progress from description to causal analysis using the knowledge that several senolytic drugs are kinase inhibitors. When we perturb senescing cells by a small optimally informative, pre-selected set of poly-specific kinase inhibitors, whose inhibitory activities tile the whole kinome, we can identify key kinases that regulate senescence. Further coupling this to phospho-mass spectrometry will allow us to trace the signaling cascades to specific protein substrates and phosphosites. These observations and pharmacological perturbations can suggest new senolytic strategies and even suggest specific senolytic drugs.