Structure and function of PTP like inositol polyphosphatases

PTP 类似肌醇多磷酸酶的结构和功能

• 批准号: 238738-2010
• 负责人: Mosimann, Steven
• 金额: $ 2.4万
• 依托单位: University of Lethbridge
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2013
• 资助国家: 加拿大
• 起止时间: 2013-01-01 至 2014-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=524429
• 关键词: Structure; function; PTP; like; inositol

The overall objective of my research program is understanding the molecular structure and function of a recently discovered family of enzymes (bacterial inositol polyphosphatases or PTP-IPPs) that are distantly related to protein tyrosine phosphatases. These enzymes remove one or more phosphates from many different inositol polyphosphates, an important class of signal transduction molecule that has known roles in many essential cellular processes including DNA repair, mRNA export, plant development, apotosis and bacterial pathogenicity. We utilize a variety of biochemical and molecular biology techniques to study the substrate specificity, kinetic and molecular mechanism of action of these enzymes. In particular, this research utilizes X-ray crystallographic techniques to determine atomic resolutions structures of PTP-IPPs alone and in complex with naturally occuring substrates. These studies allow us to visualize the enzyme and its interaction with substrate. Site-directed mutagenesis, fluorescense-based binding assays and steady-state kinetics are then utilized toextend and refine our understanding of the structure and function of these enzymes. Specific, interelated questions of interest that can be addressed using these techniques include: (1) How do these enzymes discriminate between the many naturally occuring InsPs? (2) What is their detailedkinetic mechanism and how does it differ from the well studied protein tyrosine phosphatases? And (3) Can these enzymes be utlized to cheaply produce biologically important InsPs? In turn, the answers to these questions will shed light upon their role in pathogenesis and the role of InsPs in eucaryotes.

我的研究计划的总体目标是了解最近发现的酶家族（细菌肌醇多磷酸酶或PTP-IPP）的分子结构和功能，这些酶与蛋白酪氨酸磷酸酶有很大关系。这些酶从许多不同的肌醇多磷酸中去除一个或多个磷酸，肌醇多磷酸是一类重要的信号转导分子，其在许多基本细胞过程中具有已知的作用，包括DNA修复、mRNA输出、植物发育、细胞凋亡和细菌致病性。我们利用各种生物化学和分子生物学技术来研究这些酶的底物特异性、动力学和分子作用机制。特别是，本研究利用X射线晶体学技术来确定PTP-IPP单独和与自然发生的基板的复杂的原子分辨率结构。这些研究使我们能够可视化酶及其与底物的相互作用。定点诱变，荧光结合试验和稳态动力学，然后利用textending和完善我们的理解这些酶的结构和功能。使用这些技术可以解决的具体的、相互关联的感兴趣的问题包括：（1）这些酶如何区分许多天然存在的InsPs？(2)它们的详细动力学机制是什么？它与研究得很好的蛋白酪氨酸磷酸酶有什么不同？（3）这些酶是否可以廉价地生产具有重要生物学意义的InsPs？反过来，这些问题的答案将揭示其在发病机制中的作用和InsPs在真核生物中的作用。