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

结合基因组学和磷酸蛋白质组学来识别促进线粒体的激酶底物网络

• 批准号: BB/J017450/1
• 负责人: Chris Bakal
• 金额: $ 83.56万
• 依托单位: Institute of Cancer Research
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2012
• 资助国家: 英国
• 起止时间: 2012 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FJ017450%2F1
• 关键词: Combining; Genomics; Phosphoproteomics; Identify; Kinase

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.

线粒体是细胞内发现的将燃料转化为能量的特殊隔室。在发育等能量需求较高的过程中，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会加速衰老过程。此外，功能失调的线粒体会引发慢性炎症状态，从而导致 2 型糖尿病、癌症和神经退行性疾病的发病机制。胰岛素抵抗是此类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产生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尚未被绘制出来。从系统层面了解将胰岛素刺激与线粒体产生联系起来的信号网络至关重要。我们的目标是结合遗传和磷酸蛋白质组技术来绘制对胰岛素介导的线粒体稳态至关重要的关键生化反应。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。使用我们最近开发的新型遗传筛选，我们的首要目标是全面鉴定线粒体健康所需的所有基因。这种经济高效的屏幕使用计算算法，在一次抑制基因组中的每个基因后，自动测量数百万个单细胞中的线粒体形状。如果抑制特定基因会导致线粒体形状异常，则强烈表明该基因在促进线粒体稳态中发挥作用。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。激酶蛋白磷酸化不同的蛋白质，作为将不同蛋白质“打开”或“关闭”的手段。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胰岛素调节。通过完成这两个目标，我们将识别出一组既参与线粒体健康（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。为了确定负责目标 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法来监测胰岛素处理细胞中所有激酶的系统抑制后的蛋白质磷酸化。

项目成果

Cross-talk between Rho and Rac GTPases drives deterministic exploration of cellular shape space and morphological heterogeneity.

• DOI: 10.1098/rsob.130132
• 发表时间: 2014-01-22
• 期刊: Open biology
• 影响因子: 5.8
• 作者: Sailem H;Bousgouni V;Cooper S;Bakal C
• 通讯作者: Bakal C

Reproducible automated phosphopeptide enrichment using magnetic TiO2 and Ti-IMAC.

• DOI: 10.1021/ac5025842
• 发表时间: 2014-10-21
• 期刊: ANALYTICAL CHEMISTRY
• 影响因子: 7.4
• 作者: Tape, Christopher J.;Worboys, Jonathan D.;Sinclair, John;Gourlay, Robert;Vogt, Janis;McMahon, Kelly M.;Trost, Matthias;Lauffenburger, Douglas A.;Lamont, Douglas J.;Jorgensen, Claus
• 通讯作者: Jorgensen, Claus

Phenotypic Characterization of Mitochondria in Breast Cancer Cells using Morphology and Texture Properties

利用形态学和纹理特性对乳腺癌细胞线粒体进行表型表征

• 作者: Chris Bakal (Author)

Systematic evaluation of quantotypic peptides for targeted analysis of the human kinome.

• DOI: 10.1038/nmeth.3072
• 发表时间: 2014-10
• 期刊: NATURE METHODS
• 影响因子: 48
• 作者: Worboys, Jonathan D.;Sinclair, John;Yuan, Yinyin;Jorgensen, Claus
• 通讯作者: Jorgensen, Claus

Cell cycle progression is an essential regulatory component of phospholipid metabolism and membrane homeostasis.

• DOI: 10.1098/rsob.150093
• 发表时间: 2015-09
• 期刊: Open biology
• 影响因子: 5.8
• 作者: Sanchez-Alvarez M;Zhang Q;Finger F;Wakelam MJ;Bakal C
• 通讯作者: Bakal C