Biochemistry and Physiological Role of Diacylglycerol Kinase Theta

描述（由申请人提供）：本修订提案中的研究重点是了解二酰甘油激酶（DGK）DGK-θ的调节和生理作用。拟议研究的完成将填补我们目前对该DGK的调节和生理作用的认识中的几个重要空白。这一点特别重要 对于DGK-q，因为它主要定位于神经元，并且其调节和生理作用是未知的。总体假设是DGK-θ受含多元区（PBR）的神经元蛋白调节，并且这种调节介导中枢神经系统中神经元的谷氨酸释放的调节。我们进一步假设谷氨酸释放的调节涉及突触囊泡周期的调节。这一假设基于以下观察：（a）DGK-θ受到调节，（B）这种调节涉及含有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）该酶涉及调节乙酰胆碱释放，（e）DGK-θ的抑制减弱神经元谷氨酸的诱导释放，和（f）DGK-θ抑制导致突触囊泡循环明显减慢。本提案中概述的研究将检验我们的假设，并检查参与DGK-θ活性调节的分子机制，以及谷氨酸释放和突触囊泡循环的调节。将从三个具体目标着手。在具体的目的I中，我们将通过（a）在存在和不存在活化剂的情况下定量DGK-θ的动力学参数，以及（B）特定磷脂对这些参数的影响来检查DGK-θ的调节。然后，我们将（c）通过确定激活剂是否影响酶的内在活性、膜结合或两者，进一步深入研究磷脂和蛋白质激活剂对酶活性的影响。我们还将识别与神经元相互作用的神经元蛋白质。 并激活DGK-θ在特定的目标II，我们将确定DGK-θ的作用，在调节谷氨酸释放皮质和海马神经元使用在体外和原位研究与原代培养的神经元和脑切片野生型和DGK-θ敲除小鼠。我们还将研究DGK-θ消融对突触囊泡循环的影响。在具体目标三中，我们 将通过评估DGK-θ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-θ调节谷氨酸的机制。这些研究将在神经元DGK-θ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.