Defining the role of phosphatidic acid as an allosteric regulator of mitochondrial glutaminase

项目摘要/摘要 线粒体谷氨酰胺酶(GLS和GLS2)催化谷氨酰胺水解为谷氨酸和铵， 对哺乳动物生理的许多方面都至关重要的一种代谢反应。鉴于GLS2的表达 GLS主要局限于肝脏，普遍表达，尤其在肾脏、消化器官中含量较高 脑部和大脑。它的作用包括调节全身酸碱平衡和生物合成 兴奋性和抑制性神经递质。因此，涉及突变的人类先天新陈代谢错误 的GLS基因具有严重的表型，包括神经兴奋性毒性和致死性新生儿脑病。在……里面 除了在机体健康方面的功能外，GLS活性失调还与一系列 人类的病理，从神经退化到癌症。因此，进行了密集的努力。 开发GLS的选择性变构抑制剂，其中一种目前正在进行临床试验 实体和血液系统恶性肿瘤的治疗。然而，到目前为止的结果表明， 耐药性的存在限制了这种治疗策略的有效性。值得注意的是，尽管GLS研究有很长的历史， 目前还不清楚这种酶的活性是如何在细胞中调节的。纯化的重组GLS具有最低限度的 催化活性和大量超生理浓度的无机磷酸盐(100-150 mM)是必需的 体外激活GLS。我们最近发现，生物活性磷脂磷脂酸(PA) 是一种非常有效的GLS激活剂，大约是无机磷酸盐的107倍。 重要的是，我们已经发现，即使在临床上存在GLS抑制剂的情况下，PA也可以激活GLS，因此 使这些药物失效。在这个项目中，我们建议定义PA的分子机制 激活GLS，然后探索线粒体PA信号的上游调控，最终目的是 干扰这一过程以克服对GLS抑制剂的耐药性。在这些研究过程中，我们会 开发新的化学探针用于研究PA-GLS相互作用，包括磷基共价 捕获探针以确定PA磷酸单酯头部基团的结合位置。然后我们将进行一次 筛选以确定其他潜在的代谢物-GLS相互作用，并从功能上验证所获得的任何“命中”。 在第二个目标中，我们将建立在初步数据的基础上，这些数据强烈地涉及PA生成酶 磷脂酶D2(PLD2)以及未知蛋白PLD5在线粒体调节中的作用 PA-GLS轴。我们将尝试确定PLD5的功能，重点是它是否报告的相互作用 与PLD2一起调节后者的活性或亚细胞定位。最后，由于我们的初步数据 提示PLD2/5介导对临床GLS抑制剂的耐药性，我们将测试是否药理学或遗传学 阻断PLD2或PLD5可以克服这种耐药机制。因此，我们建议的研究将界定 调节GLS活动的生理过程，然后将应用这一知识来识别机会 以提高针对谷氨酰胺代谢的治疗策略的有效性。

PROJECT SUMMARY/ABSTRACT Mitochondrial glutaminases (GLS and GLS2) catalyze the hydrolysis of glutamine to glutamate and ammonium, a metabolic reaction that is critical for numerous aspects of mammalian physiology. Whereas GLS2 expression is largely restricted to the liver, GLS is ubiquitously expressed, with particularly high levels in the kidney, digestive tract, and brain. Its roles include the regulation of systemic acid-base homeostasis and the biosynthesis of excitatory and inhibitory neurotransmitters. Accordingly, human inborn errors of metabolism involving mutation of the GLS gene have severe phenotypes, including neural excitotoxicity and lethal neonatal encephalopathy. In addition to its functions in organismal health, dysregulated GLS activity has been implicated in a spectrum of human pathologies, ranging from neurodegeneration to cancer. Consequently, there have been intensive efforts to develop selective allosteric inhibitors of GLS, one of which is now being evaluated in clinical trials for the treatment of solid and hematological malignancies. However, results to date indicate that the rapid development of resistance limits the efficacy of this therapeutic strategy. Remarkably, despite the long history of GLS research, it is not understood how the activity of this enzyme is regulated in cells. Purified recombinant GLS has minimal catalytic activity, and vastly supraphysiological concentrations of inorganic phosphate (100-150 mM) are required to activate GLS in vitro. We have recently discovered that that the bioactive phospholipid phosphatidic acid (PA) is an extremely potent activator of GLS, approximately 107-fold more potent than inorganic phosphate. Importantly, we have found that PA can activate GLS even in the presence of clinical GLS inhibitors, thus rendering these drugs ineffective. In this project, we propose to define the molecular mechanism by which PA activates GLS, and then to probe the upstream regulation of mitochondrial PA signaling, with the ultimate goal of perturbing this process to overcome resistance to GLS inhibitors. In the course of these studies, we will develop new chemical probes for investigating the PA-GLS interaction, including phosphorus-based covalent capture probes to determine the binding site of the PA phosphomonoester head group. We will then conduct a screen to identify additional potential metabolite-GLS interactions, and functionally validate any ‘hits’ obtained. In the second Aim, we will build on preliminary data that strongly implicate the PA-generating enzyme phospholipase D2 (PLD2), as well as the uncharacterized protein PLD5, in the regulation of the mitochondrial PA-GLS axis. We will attempt to determine the function of PLD5, focusing on whether its reported interaction with PLD2 regulates either the activity or subcellular localization of the latter. Finally, since our preliminary data indicate that PLD2/5 mediate resistance to clinical GLS inhibitors, we will test whether pharmacological or genetic blockade of PLD2 or PLD5 can overcome this resistance mechanism. Thus, our proposed studies will define the physiological processes that regulate GLS activity, and will then apply this knowledge to identify opportunities for enhancing the efficacy of therapeutic strategies targeting glutamine metabolism.