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

线粒体是在细胞内发现的将燃料转化为能量的专门隔室。在发育过程中，能量需求很高，胰岛素激活多个生物开关，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衰老过程。此外，功能障碍的线粒体可以驱动慢性炎症状态的发作，这是2型糖尿病、癌症和神经退行性疾病发病机制的基础。胰岛素抵抗是这种年龄相关疾病的标志，强烈表明胰岛素介导的线粒体产生的抑制是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是未映射的。获得一个系统水平的观点到信号网络，连接胰岛素刺激线粒体生产是必不可少的。我们的目标是使用遗传和磷酸蛋白质组学技术的组合，映射关键的生化反应，胰岛素介导的线粒体稳态是至关重要的。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健康状况。使用我们最近开发的一种新的遗传筛选，我们的目标首先是全面识别线粒体健康所需的所有基因。这种具有成本效益的筛选使用计算算法自动测量数百万个单细胞中的线粒体形状，然后一次一个地抑制基因组中的每个基因。如果抑制一个特定的基因导致异常形状的线粒体，这强烈表明该基因在促进线粒体稳态中的作用。我们最近开发了一种基于质谱的尖端技术来监测细胞中所有蛋白质的蛋白质磷酸化水平。激酶蛋白质磷酸化不同的蛋白质，作为一种手段，打开不同的蛋白质“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，我们将识别出既参与线粒体健康（通过目标1中完成的遗传筛选），又受胰岛素调节（通过完成目标2）的蛋白质组。为了确定负责磷酸化目的1和2中鉴定的底物的激酶，我们将结合联合收割机遗传学和质谱法，在胰岛素处理的细胞中系统性抑制所有激酶后监测蛋白磷酸化。

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.