Molecular Properties of Diacylglycerol Kinase Epsilon

二酰甘油激酶 Epsilon 的分子特性

• 批准号: RGPIN-2018-05585
• 负责人: Epand, Richard
• 金额: $ 3.06万
• 依托单位: McMaster University
• 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=713741
• 关键词: Molecular; Properties; Diacylglycerol; Kinase; Epsilon

The proposal is focused on a unique member of the family of mammalian diacylglycerol kinases, i.e. DGK. DGK plays a key role in signal transduction and lipid synthesis. It is the only known member of this group that appears to have specificity for one particular species of substrate, i.e. 1-stearoyl-2-arachidonoyl glycerol. This is the same acyl chain composition as is found in lipid intermediates of the phosphatidylinositol (PI) cycle, the sole pathway for the synthesis of PI and its phosporylated products that play a major role in metabolic regulation and cancer. Interconversion of these phosphorylated forms of PI is highly dependent on the nature of the acyl chains. DGK contributes to the specific acyl chain composition of several lipids involved in cell signaling. The product of the reaction catalyzed by DGK, phosphatidic acid, is also an important signaling agent as well as being a precursor for several other lipids. We have successfully purified catalytically active DGK. This provides an opportunity to understand the molecular structure of this protein. It would be the first mammalian DGK isoform to have its structure determined and would provide a model for the analysis of other isoforms that have some homologous segments and for understanding the functioning of this important family of signaling proteins. We will also study how this protein interacts with membranes and with its substrate. It will be an important example of a peripheral membrane protein that interacts with lipid segments buried in the membrane. In cells DGK functions when bound to a bilayer membrane. Before our success in purifying this enzyme, all activity studies were done in assays using detergent micelles. However, we are now able to use the purified enzyme to assay the activity in liposomes made of bilayer membranes. We found a marked difference between the micellar-based and the liposome-based assays and a dependence of both the activity and specificity on the chemical and physical properties of the lipids used to make the liposomes. This will enable us to gain an understanding of how the functioning of DGK is dependent on the nature of the membrane to which it is bound. This research program will determine how peripheral membrane proteins can bind to membranes and recognize lipid substrates. The example of DGK is of particular importance because of its involvement in determining the acyl chain composition of PI. There is currently no structural information on this protein and no reported studies of its activity on liposomes. Our work will provide important novel structural and functional information. It will contribute to our general understanding of the mechanism of acyl chain recognition by interfacial enzymes and in particular as applied to DGK. This information will provide a more accurate description of an aspect of the regulation of biological systems that eventually will be used for their control.

该提案的重点是哺乳动物二酰甘油激酶家族中的一种独特成员，即DGK。DGK在信号转导和脂质合成中起关键作用。它是这一组中唯一对一种特定底物具有特异性的已知成员，即1-硬脂酰基-2-花生四烯基甘油。这与磷脂酰肌醇(PI)循环的脂类中间体中发现的酰链组成相同，PI循环是合成PI及其磷孢子化产物的唯一途径，后者在代谢调节和癌症中发挥主要作用。这些磷酸化形式的PI的相互转化高度依赖于酰基链的性质。DGK参与参与细胞信号转导的几种脂类的特定酰链组成。DGK催化的反应产物，磷脂酸，也是一个重要的信号转导分子，也是其他几种脂质的前体。我们成功地提纯了具有催化活性的DGK。这为了解这种蛋白质的分子结构提供了机会。这将是第一个确定其结构的哺乳动物DGK亚型，并将为分析其他具有一些同源片段的亚型以及了解这一重要信号蛋白家族的功能提供一个模型。我们还将研究这种蛋白质如何与膜及其底物相互作用。这将是外周膜蛋白与埋藏在膜中的脂段相互作用的一个重要例子。在细胞中，DGK在结合到双层膜上时起作用。在我们成功地提纯这种酶之前，所有的活性研究都是在洗涤剂胶束中进行的。然而，我们现在能够使用纯化的酶来检测由双层膜制成的脂质体的活性。我们发现胶束为基础的检测和脂质体为基础的检测有显著的不同，并且其活性和特异性都依赖于用于制造脂质体的脂类的化学和物理性质。这将使我们能够了解DGK的功能如何依赖于它所结合的膜的性质。这项研究计划将确定外周膜蛋白如何与膜结合并识别脂质底物。DGK的例子特别重要，因为它参与了确定PI的酰基链组成。目前还没有关于这种蛋白质的结构信息，也没有关于其在脂质体上的活性的报道。我们的工作将提供重要的新颖的结构和功能信息。这将有助于我们全面理解界面酶识别酰基链的机理，特别是在DGK中的应用。这些信息将更准确地描述生物系统调控的一个方面，最终将用于对其进行控制。