Biochemistry and Physiological Role of Diacylglycerol Kinase Theta

二酰甘油激酶 Theta 的生物化学和生理作用

基本信息

批准号：
9084674
负责人：
Daniel M. Raben
金额：
$ 35.44万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-06-01 至 2018-05-31
项目状态：
已结题

项目摘要

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.

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-theta 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。这一点尤为重要对于 DGK-q 来说，它主要定位于神经元，其调节和生理作用尚不清楚。总体假设是 DGK-theta 受到含有神经元蛋白的多碱基区 (PBR) 的调节，并且这种调节介导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。该假设基于以下观察：（a）DGK-theta 受到调节，（b）这种调节涉及包含 PBR 的辅助蛋白，（c）该酶改变已知参与突触小泡释放的脂质，（d） ) 该酶与调节乙酰胆碱释放有关，(e) DGK-theta 的抑制会减弱神经元谷氨酸的诱导释放，(f) DGK-theta 的抑制会导致突触小泡循环明显减慢。本提案中概述的研究将检验我们的假设，并检查参与 DGK-theta 活性调节、谷氨酸释放和突触小泡循环调节的分子机制。这将通过三个具体目标来实现。在具体目标 I 中，我们将通过 (a) 量化存在和不存在激活剂的情况下 DGK-theta 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-theta 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、膜缔合或两者，进一步深入研究磷脂和蛋白质激活剂对酶活性的影响。我们还将鉴定与相互作用的神经元蛋白并在特定目标 II 中激活 DGK-theta，我们将使用原代培养神经元以及来自野生型和 DGK-theta 的脑切片进行体外和原位研究，确定 DGK-theta 在调节皮质和海马神经元谷氨酸释放中的作用基因敲除小鼠。我们还将检查 DGK-theta 消融对突触小泡循环的影响。在具体目标 III 中，我们将通过评估 DGK-theta 消融对参与突触小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。