Revealing Secrets of Cellular Phosphorylation Systems Using Biological Mass Spectrometry

利用生物质谱揭示细胞磷酸化系统的秘密

• 批准号: RGPIN-2020-05099
• 负责人: Vacratsis, Panayiotis
• 金额: $ 2.33万
• 依托单位: University of Windsor
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=716673
• 关键词: Revealing; Secrets; Cellular; Phosphorylation; Systems

All organisms contain proteins (e.g. enzymes) that perform the necessary chemical reactions that allow cellular life to exist. In order to restrict protein function to when it is needed by the cell, nature has developed molecular systems that can change the three-dimensional shape of proteins in a reversible fashion, and thus turn protein function “on” and “off”. Protein phosphorylation is a common chemical modification utilized by all living organisms to alter the shape and function of proteins. The enzymes that mediate the addition and removal of the phosphate molecules are called kinases and phosphatases respectively. Since a delicate balance of reversible phosphorylation is essential for nearly all cellular processes to function properly, the activities of all kinases/phosphatases are under tight control. One of the most powerful analytical instruments to study phosphorylation systems is a mass spectrometer. Mass spectrometry-based proteome sequencing techniques are the protein equivalent to genome sequencing techniques, and thus have the potential to discover fundamental aspects of phosphorylation systems. Accordingly, the long-term goal of our research program is to integrate mass spectrometry, biochemistry, and cell biology techniques to better understand the cellular role of poorly studied kinases and phosphatases predicted to regulate important cellular processes. We propose to utilize advanced mass spectrometry techniques towards the study of human YVH1 (hYVH1), a scantily studied member of the atypical dual specificity phosphatase (DUSP) subfamily. Also known as DUSP12, YVH1 orthologues are widely conserved in the animal and plant kingdom, and recent studies from our laboratory and others have provided evidence that hYVH1 is a regulator of protein synthesis, cell survival, and cellular DNA content. Moreover, we recently reported the results of a hYVH1 mass spectrometry study that discovered hYVH1 associates with various ribonucleoprotein particles (RNP) and functions as a novel stress granule disassembly factor. This initial mass spectrometry analysis also provided preliminary evidence that a well characterized tyrosine kinase known as Src, may control the activities of hYVH1. We have confirmed that Src can phosphorylate hYVH1 in human cells and have sequenced a novel phospho-tyrosine site using mass spectrometry. We are now interested in thoroughly characterizing the functional significance of Src-mediated phosphorylation of hYVH1. Specifically, we are interested in determining how this phosphorylation modification changes the shape of hYVH1 in order to alter the function of hYVH1 in the cell. The methods employed within our proposal will be transferrable to the study of other poorly understood phosphatases while the findings will deepen our understanding of the role of hYVH1 in cellular regulation.

所有生物体都含有蛋白质（例如酶），这些蛋白质可以进行必要的化学反应，使细胞生命得以存在。为了将蛋白质功能限制在细胞需要的时候，大自然开发了分子系统，可以以可逆的方式改变蛋白质的三维形状，从而“打开”和“关闭”蛋白质功能。蛋白质磷酸化是所有生物体用来改变蛋白质的形状和功能的常见化学修饰。介导磷酸盐分子添加和去除的酶分别称为激酶和磷酸酶。 由于可逆磷酸化的微妙平衡对于几乎所有细胞过程的正常运行至关重要，因此所有激酶/磷酸酶的活性都受到严格控制。研究磷酸化系统最强大的分析仪器之一是质谱仪。基于质谱的蛋白质组测序技术相当于基因组测序技术的蛋白质，因此有可能发现磷酸化系统的基本方面。因此，我们研究计划的长期目标是整合质谱、生物化学和细胞生物学技术，以更好地了解研究不足的激酶和磷酸酶的细胞作用，这些激酶和磷酸酶预计可调节重要的细胞过程。 我们建议利用先进的质谱技术来研究人类 YVH1 (hYVH1)，这是非典型双特异性磷酸酶 (DUSP) 亚家族的一个研究很少的成员。 YVH1 直向同源物也称为 DUSP12，在动植物界广泛保守，我们实验室和其他实验室的最新研究提供了证据，表明 hYVH1 是蛋白质合成、细胞存活和细胞 DNA 含量的调节因子。 此外，我们最近报告了 hYVH1 质谱研究的结果，发现 hYVH1 与各种核糖核蛋白颗粒 (RNP) 相关，并作为一种新型的应激颗粒分解因子发挥作用。该初始质谱分析还提供了初步证据，表明一种被称为 Src 的特征明确的酪氨酸激酶可能控制 hYVH1 的活性。我们已经证实 Src 可以磷酸化人类细胞中的 hYVH1，并使用质谱法对一个新的磷酸酪氨酸位点进行了测序。我们现在有兴趣彻底表征 Src 介导的 hYVH1 磷酸化的功能意义。具体来说，我们感兴趣的是确定这种磷酸化修饰如何改变 hYVH1 的形状，从而改变 hYVH1 在细胞中的功能。 我们的提案中采用的方法将可转移到其他知之甚少的磷酸酶的研究中，而这些发现将加深我们对 hYVH1 在细胞调节中的作用的理解。