Combining Genomics and Phosphoproteomics to Identify Kinase-Substrate Networks that Promote Mitochondrial

线粒体是细胞内发现的专门的隔间，可以将燃料转化为能量。在发育等能量需求很高的过程中，胰岛素会激活多个生物开关，最终产生新的线粒体。线粒体数量和功能的减少和/或受损线粒体的积累加速了衰老过程。此外，线粒体功能障碍可导致慢性炎症状态的发生，而慢性炎症状态是2型糖尿病、癌症和神经退行性疾病发病机制的基础。胰岛素抵抗是这些与年龄相关的疾病的一个标志，强烈表明抑制胰岛素介导的线粒体生产是导致衰老和疾病的常见途径。然而，将胰岛素刺激与线粒体健康联系起来的生化反应在很大程度上还没有被描绘出来。从系统水平上了解将胰岛素刺激与线粒体产生联系起来的信号网络是至关重要的。我们的目标是使用遗传和磷蛋白质组技术的组合来绘制对胰岛素介导的线粒体动态平衡至关重要的关键生化反应图。我们乐观地认为，对这些反应的全面了解将使我们最终能够开发出操纵胰岛素信号网络的方法，以改善衰老过程中的福祉。使用我们最近开发的一种新的基因筛查，我们首先旨在全面识别线粒体健康所需的所有基因。这一具有成本效益的筛查使用计算算法在基因组中的每个基因被抑制后，一次一个地自动测量数百万单个细胞的线粒体形状。如果特定基因的抑制导致线粒体形状异常，这强烈表明该基因在促进线粒体自我稳定方面发挥了作用。我们最近开发了一种基于尖端质谱学的技术来监测细胞中所有蛋白质的蛋白质磷酸化水平。激酶蛋白将不同的蛋白质磷酸化，使不同的蛋白质“开”“开”“关”。通过监测胰岛素刺激后细胞中发生的所有磷酸化事件，我们可以确定哪些蛋白质可能受到胰岛素的调节。通过完成这两个目标，我们将识别出同时参与线粒体健康(通过目标1中完成的基因筛选)和受胰岛素调节(通过完成目标2)的蛋白质组。为了确定负责AIMS 1和2中确定的底物磷酸化的激酶，我们将结合遗传学和质谱学来监测胰岛素处理细胞中所有激酶被系统抑制后的蛋白质磷酸化。

Mitochondria are specialized compartments found within cells that convert fuel into energy. During processes such as development, where energy demands are high, insulin activates multiple biological switches that ultimately generates new mitochondria. Decreases in mitochondrial number and function, and/or the accumulation of damaged mitochondria accelerate the ageing process. Moreover dysfunctional mitochondria can drive the onset of a chronic inflammatory state that underpins to the pathogenesis of type 2 diabetes, cancer, and neurodegenerative disorders. Insulin-resistance is a hallmark of such age-associated diseases strongly suggesting that suppression of insulin-mediated mitochondrial production is a common route to ageing and disease. However, the biochemical reactions that link insulin stimulation to mitochondrial health are largely unmapped. Gaining a systems-level view into the signaling networks that link insulin stimulation to mitochondrial production is essential.We aim to use a combination of genetic and phosphoproteomic technologies to map key biochemical reactions that are critical for insulin-mediated mitochondrial homeostasis. We are optimistic that a comprehensive understanding of these reactions will allow us to ultimately develop means to manipulate insulin signalling networks to improve well-being during the ageing process. Using a novel genetic screen that we have recently developed, we first aim to comprehensively identify all genes required for mitochondrial health. This cost-effective screen that use computational algorithms to automatically measure mitochondrial shape in millions of single cells following inhibition of each gene in the genome one at a time. If inhibition of a particular gene leads to abnormally shaped mitochondria this strongly suggests a role for this gene in promoting mitochondrial homeostasis.We have recently developed a cutting-edge mass-spectrometry based technology to monitor levels of protein phosphorylation on all proteins in a cell. Kinase proteins phosphorylate different proteins as a means to turn different proteins "on" on "off". By monitoring all phosphorylation events that occur in cells following stimulation of insulin, we can then determine which proteins are likely to be regulated by insulin.By completing these two aims we will thus identify sets of proteins that are both involved in mitochondrial health (through genetic screens completed in Aim 1), and that are regulated by insulin (through completion of Aim 2). In order to determine the kinases that are responsible for phosphorylating substrates identified in Aims 1 and 2, we will then combine genetics and mass spectrometry to monitor protein phosphorylation following systematic inhibition of all kinases in insulin-treated cells.