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: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=688515
• 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基甘油激酶的独特成员，即DGK。 DGK在信号转导和脂质合成中起关键作用。它是该组中唯一已知的成员，它似乎对一种特定的底物具有特异性，即1 stearoyl-2-芳烃甘油。这是与磷脂酰肌醇（PI）周期的脂质中间体相同的酰基链组成，这是合成PI及其在代谢调节和癌症中起主要作用的PI的唯一途径。这些磷酸化形式的PI的相互转换高度取决于酰基链的性质。 DGK有助于几种参与细胞信号的脂质的特异性酰基链组成。由DGK（磷脂酸）催化的反应的乘积也是重要的信号剂，也是其他几种脂质的前体。我们已经成功纯化了催化活性的DGK。这提供了了解该蛋白质的分子结构的机会。它将是第一个确定其结构的哺乳动物DGK同工型，并将为分析具有某些同源段的其他同工型分析并理解这种重要信号蛋白家族的功能。我们还将研究该蛋白质如何与膜及其底物相互作用。这将是与埋在膜中的脂质片段相互作用的周围膜蛋白的重要例子。在细胞中，当与双层膜结合时，DGK起作用。在我们成功纯化这种酶之前，所有活性研究均在使用洗涤剂胶束的测定中进行。但是，我们现在能够使用纯化的酶来测定由双层膜制成的脂质体的活性。我们发现基于胶束的基于胶束和基于脂质体的测定以及对脂质体用于制造脂质体的化学和物理性质的活性和特异性的依赖性之间有明显的差异。这将使我们能够了解DGK的功能如何取决于其绑定的膜的性质。该研究计划将确定外围膜蛋白如何与膜结合并识别脂质底物。 DGK的示例特别重要，因为它参与了确定PI的酰基链组成。目前尚无有关该蛋白质的结构信息，也没有报道其对脂质体的活性的研究。我们的工作将提供重要的新型结构和功能信息。它将有助于我们对界面酶识别酰基链识别机制的一般理解，尤其是应用于DGK。该信息将对生物系统调节的一个方面提供更准确的描述，这些方面最终将用于其控制。