Revealing Secrets of Cellular Phosphorylation Systems Using Mass Spectrometry

使用质谱揭示细胞磷酸化系统的秘密

• 批准号: RGPIN-2015-06000
• 负责人: Vacratsis, Panayiotis
• 金额: $ 2.77万
• 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: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=667427
• 关键词: Revealing; Secrets; Cellular; Phosphorylation; Systems

Protein phosphorylation is a chemical modification utilized by all living organisms to alter the function of proteins and controls processes such as cellular communication, growth, metabolism, differentiation, and survival. The enzymes that add and remove phosphate molecules to proteins are called kinases and phosphatases respectively.  Since a delicate balance of reversible phosphorylation is essential for cellular homeostasis, the actions of all kinases/phosphatases are under tight surveillance. One of the most powerful analytical tools to study phosphorylation systems is biological mass spectrometry. Mass spectrometry based methods can rapidly identify functional multiprotein complexes containing kinases and phosphatases as well as directly map the location of phosphate addition to better study the consequence of the protein modification. Accordingly, the long term goal of our research program is to integrate mass spectrometry, biochemistry, and cell biology techniques to decipher the mechanistic details regulating poorly understood kinases and phosphatases predicted to regulate fundamental cellular processes. ***     Currently we are directing much of our focus on a poorly understood phosphatase identified as YVH1 (also known as DUSP12). Human YVH1 (hYVH1) is a so called dual specificity phosphatase (DSP) widely expressed in human tissues. The YVH1 genes are found throughout the animal and plant kingdom and all gene products possess a characteristic zinc binding domain in addition to their phosphatase domain. Remarkably, although YVH1 was the first eukaryotic DSP identified over 22 years ago, relatively little is known about its specific biological function in human cells. ***    The recent acquisition in 2014 of a new multiplexed mass spectrometry system capable of biological applications such as discovery/targeted proteomics, mapping of post translational modifications, structural protein dynamics, and interactome analysis has fundamentally altered the depth of analysis for our research program. Utilizing this system, we have recently isolated novel hYVH1 associating proteins from human cell lines using classical mass spectrometry based affinity chromatography techniques. Our preliminary findings suggest that hYVH1 is active at cellular components that control the rate at which genes are transformed into functional proteins (mRNA regulation). We are now interested in employing next generation affinity capture methods and magnetic bead technology to thoroughly delineate and functionally validate the hYVH1 interactome. Follow-up experiments will include examining how the cell controls the access of hYVH1 to these macromolecular workstations.  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 DSPs play in cellular regulation.

蛋白质磷酸化是所有生物体利用的一种化学修饰，用于改变蛋白质的功能并控制细胞通讯、生长、代谢、分化和生存等过程。 在蛋白质上添加和去除磷酸盐分子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细胞稳态至关重要，因此所有激酶/磷酸酶的作用都受到严格监视。 研究磷酸化系统最强大的分析工具之一是生物质谱法。 基于质谱的方法可以快速识别含有激酶和磷酸酶的功能性多蛋白复合物，并直接绘制磷酸盐添加的位置，以更好地研究蛋白质修饰的结果。 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，破译人们对激酶和磷酸酶的调控机制细节，而人们对这些激酶和磷酸酶的调控却知之甚少，而这些激酶和磷酸酶预计可以调控基本的细胞过程。 ***    目前，我们将大部分注意力集中在一种人们知之甚少的磷酸酶上，该酶被称为 YVH1（也称为 DUSP12）。人类 YVH1 (hYVH1) 是一种广泛表达于人体组织中的双特异性磷酸酶 (DSP)。 YVH1 基因遍布于动植物界，所有基因产物除了磷酸酶结构域外，还具有特征性锌结合结构域。 值得注意的是，尽管 YVH1 是 22 年前发现的第一个真核 DSP，但人们对其在人类细胞中的具体生物学功能知之甚少。 ***    最近于 2014 年收购了一种新型多重质谱系统，该系统能够进行生物应用，例如发现/靶向蛋白质组学、翻译后修饰图谱、结构蛋白动力学和相互作用组分析，从根本上改变了我们研究项目的分析深度。 利用该系统，我们最近使用基于经典质谱的亲和层析技术从人类细胞系中分离出新型 hYVH1 关联蛋白。我们的初步研究结果表明，hYVH1 对控制基因转化为功能蛋白（mRNA 调节）速率的细胞成分具有活性。我们现在有兴趣采用下一代亲和捕获方法和磁珠技术来彻底描述和功能验证 hYVH1 相互作用组。后续实验将包括检查细胞如何控制 hYVH1 对这些大分子工作站的访问。  我们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，而研究结果将加深我们对 DSP 在细胞调节中所发挥作用的理解。