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、伤口愈合和衰老过程中被认识到， 在那里，它不仅是一种症状，而且是衰老病理的主要因素。世界上最引人注目的实验 小鼠，消除衰老细胞可以带来广泛的健康益处，甚至可以逆转衰老 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 或者逆转它，以及开发从我们的身体中清除这种细胞的实际方法。这项建议是为了 使用显微镜和分子水平上的新工具来观察正常细胞的传代 以达到衰老状态，并揭示它们分子电路的变化。在细胞进行的转变中 是细胞大小的急剧增加。已知肥大是由mTOR驱动的，并被药物抑制 雷帕霉素，它在几个系统发育不同的物种中与延长生命有关。我们专注于肥大 来帮助破解衰老，因为作为一种表型，它是如此明显地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，单元格大小一直是最难处理的问题之一 要研究的表型。随着新型显微镜的发展，这种情况发生了根本性的变化 由我们小组直接测量细胞干质量，甚至直接分别测量蛋白质和脂肪质量。 利用这些仪器，我们计划以前所未有的时间和质量分辨率研究细胞是如何， 在压力或衰老的刺激下，变得肥大，以及肥大与其他表型 衰老。我们将用深度定量质谱学跟踪这些相同的轨迹来关联 蛋白质表达和磷酸化与大小和其他衰老标志物。我们交叉参考了一些 我们在细胞培养的体外研究和对年轻和老年小鼠的体内研究之间的发现。一旦我们建造了 衰老过程的基线描述，相同的诱导-成熟-死亡生命周期 衰老的细胞会在药物的干扰下重复。我们可以从描述到因果关系 利用几种能分解衰老的药物是激酶抑制剂的知识进行分析。当我们扰乱衰老的时候 通过一小组最佳信息量的、预先选择的多聚特异性激酶抑制剂，其抑制作用 活动覆盖整个基因组，我们可以确定关键的蛋白激酶调节衰老。进一步结合这一点 将使我们能够追踪信号级联到特定的蛋白质底物和 亚磷酸盐。这些观察和药理学上的干扰可以提出新的感觉神经分解策略。 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.