Biochemistry and Physiological Role of Diacylglycerol Kinase Theta

描述(由申请人提供)：本修订方案中的研究侧重于了解二酰基甘油酸激酶(DGK)的调节和生理作用。拟议研究的完成将填补我们目前对这种DGK的调节和生理作用的几个重要空白。这一点尤为重要 DGK-Q主要定位于神经元，其调节和生理作用尚不清楚。最重要的假设是，DGK-theta受一个含有多碱区(PBR)的神经元蛋白的调节，这种调节介导了中枢神经系统神经元谷氨酸释放的调节。我们进一步假设，谷氨酸释放的调节涉及突触小泡周期的调节。这一假说基于以下观察：(A)DGK-theta受调控，(B)这种调节涉及一种含有PBR的辅助蛋白，(C)该酶改变已知参与突触囊泡释放的脂质，(D)该酶参与调节乙酰胆碱的释放，(E)抑制DGK-theta可减弱诱导的神经元谷氨酸的释放，以及(F)DGK-theta的抑制导致突触囊泡循环明显放缓。这项提案中概述的研究将检验我们的假设，并检验参与调节DGK-theta活性、调节谷氨酸释放和突触小泡循环的分子机制。这将通过三个具体目标来实现。在特定的目标I中，我们将通过(A)在激活剂存在和不存在的情况下定量DGK-，theta的动力学参数，以及(B)特定的磷脂对这些参数的影响来研究DGK-theta的调节。然后，我们将(C)通过确定激活剂是否影响酶的固有活性、膜结合或两者兼而有之，进一步深入研究磷脂和蛋白质激活剂对酶活性的影响。我们还将识别与之相互作用的神经元蛋白 和激活DGK-theta在特定目的II中，我们将通过体外和原位研究DGK-theta‘S在调节皮层和海马神经元谷氨酸释放中的作用，采用原代培养的神经元和来自野生型和DGK-theta基因敲除小鼠的脑片进行研究。我们还将研究DGK-theta对突触小泡周期的影响。在特定目标III中，我们将 将通过评估DGK-theta消融对参与突触囊泡循环和钙稳态的脂质水平的影响来研究DGK theta调节谷氨酸的机制。这些研究将在神经元DGK-theta的调节和功能之间建立新的机制和生理联系。

DESCRIPTION (provided by applicant): The studies in this revised proposal focus on understanding the regulation and physiological role of a diacylglycerol kinase (DGK), DGK-theta. The completion of the proposed studies will fill several important gaps in our current knowledge about the regulation and physiological role of this DGK. This is particularly significant for DGK-q as it is predominately localized to neurons and its regulation and physiological role are unknown. The overarching hypothesis is that DGK- theta is regulated by a polybasic region (PBR) - containing neuronal protein and this regulation mediates the modulation of glutamate release from neurons in the central nervous system. We further hypothesize that the regulation of glutamate release involves modulation of the synaptic vesicle cycle. This hypothesis is based on the following observations: (a) DGK- theta is regulated, (b) this regulation involves an accessory protein that contains a PBR, (c) the enzyme alters lipids known to be involved in synaptic vesicle release, (d) the enzyme has been implicated in modulating acetylcholine release, (e) suppression of DGK- theta attenuates induced release of neuronal glutamate, and (f) DGK- theta suppression results in an apparent slowing of synaptic vesicle cycling. The studies outlined in this proposal will test our hypothesis and examine the molecular mechanisms involved in the regulation of DGK- theta activity, and the modulation of glutamate release and synaptic vesicle cycling. This will be approached in three specific aims. In specific aim I, we wil examine the regulation of DGK- theta by (a) quantifying the kinetic parameters of DGK-, theta in the presence and absence of activators, as well as (b) the effect of specific phospholipids on these parameters. We will then (c) delve further into the effect of phospholipids and protein activators on enzyme activity by determining whether activators affect the enzyme's intrinsic activity, membrane association, or both. We will also identify neuronal proteins that interact with and activate DGK- theta in specific aim II, we will determine DGK theta -'s role in modulating glutamate release from cortical and hippocampal neurons using in vitro and in situ studies with primary cultured neurons and brain slices from wild-type and DGK- theta knockout mice. We will also examine the effect of DGK- theta ablation on synaptic vesicle cycling. In specific aim III, we will examine the mechanism by which DGK theta - modulates glutamate by evaluating the effect of DGK- theta ablation on the levels of lipids involved in synaptic vesicle cycling and calcium homeostasis. These studies will forge new mechanistic and physiological links between the regulation and function of neuronal DGK- theta.