Dynamic Control of Tryptophan Hydroxylase 2:Regulating Brain Serotonin Synthesis

描述（由申请人提供）：血清素能功能障碍与许多神经精神疾病有关。治疗这些疾病的药物旨在稳定突触间隙中的血清素水平。虽然目前的治疗方法为数百万患者提供了缓解，但它们存在耐受性和疗效问题。因此，需要能够安全地治疗高比例患者的概念上新颖的疗法。我们的长期目标是了解色氨酸羟化酶2（TPH 2）。TPH 2催化色氨酸在大脑中转化为5-羟色胺的第一步和限速步骤。已发现TPH 2被磷酸化;据报道，这种修饰导致TPH 2稳定性增加和活性增强。我们推测，翻译后修饰（PTM）的TPH 2在体内调控这一关键酶发挥重要作用。为了证实这一假设，我计划通过基于质谱（MS）的蛋白质组学在TPH 2上绘制体内PTM。此外，通过使用亲和纯化结合MS，我的目标是表征TPH 2的结合伴侣。这项工作是创新的，因为它利用现代技术来解决传统生物化学实验无法解决的问题。这项研究的结果可以提供有关TPH 2生理控制的重要信息，并可以为新一代更有选择性的抗抑郁药打开大门，副作用更少，能够通过增强大脑特异性TPH 2活性来增加5-羟色胺合成。我们将在两个具体目标下进行这些研究：具体目标#1：通过基于MS的蛋白质组学探索TPH 2的体内翻译后修饰。我们的目的是通过质谱（MS）蛋白质组学研究TPH 2在生理相关环境中的翻译后调控。为此，我将稳定地将6XHis标记的TPH 2转化到哺乳动物（PC 12）细胞中。或者，我计划从大鼠脑缝中提取TPH 2。我将通过基于MS的蛋白质组学分析这两个来源的TPH 2的PTM。具体目标#2：通过亲和纯化结合MS鉴定涉及TPH 2的蛋白质-蛋白质相互作用。我们的目标是鉴定新的TPH 2结合伴侣。相互作用蛋白可以调节TPH 2的功能。迄今为止，14-3-3蛋白是唯一已知的TPH 2结合伴侣。在这些实验中，我将从PC 12细胞中提取标记的TPH 2，并通过MS鉴定共纯化蛋白。同样，共纯化（相互作用）蛋白将通过MS鉴定。 公共卫生相关性：低水平的神经递质血清素与各种精神疾病有关，如抑郁症、强迫症、精神分裂症和自闭症等。在大脑中，色氨酸羟化酶2（TPH 2）催化色氨酸转化为5-羟色胺的第一步和限速步骤。对这种蛋白质的调控知之甚少;我们的目标是了解它是如何控制的。这项研究的结果最终可以为新一代更有选择性的抗抑郁药打开大门，副作用更少。

DESCRIPTION (provided by applicant): Serotonergic dysfunctions have been linked to many neuropsychiatric illnesses. Medications to treat these disorders aim to stabilize the levels of serotonin in the synaptic cleft. While current treatments have provided relief to millions of patients, they present tolerance and efficacy problems. Owing to this, there is a need for conceptually novel therapies capable of safely treat a high proportion of patients. Our long-term objective is to understand Tryptophan Hydroxylase 2 (TPH2). TPH2 catalyzes the first and rate-limiting step in the transformation of tryptophan into serotonin in the brain. TPH2 has been found to be phosphorylated; this modification has been reported to result in increased TPH2 stability and enhanced activity. We hypothesize that post-translational modification (PTM) of TPH2 to play an important role in the in vivo regulation of this key enzyme. To corroborate this hypothesis, I plan to map in vivo PTMs on TPH2 through mass spectrometry (MS) based proteomics. Furthermore, through the use of affinity purification in combination with MS, I aim to characterize binding partners for TPH2. The proposed work is innovative, because it utilizes modern techniques to solve questions inaccessible through conventional biochemical experiments. The results of this study can provide important information about the physiological control of TPH2 and can open the door to a new, more selective generation of antidepressants with fewer side effects, able to increase serotonin synthesis through the enhancement of brain-specific TPH2 activity. We will pursue these studies in two specific aims: Specific Aim #1: Explore the in vivo post-translational modification of TPH2 through MS -based proteomics. We aim to explore the post-translational regulation of TPH2 in physiologically relevant settings through mass spectrometry (MS) proteomics. To do this, I will stably transform 6XHis-tagged TPH2 into mammalian (PC12) cells. Alternatively, I plan to extract TPH2 from rat brain raphe. I will analyze PTMs of TPH2 from these two sources through MS based proteomics. Specific Aim #2: Identify protein-protein interactions involving TPH2 through affinity purification in combination with MS. We aim to identify novel TPH2 binding partners. Interacting proteins can regulate TPH2's function. To date, 14-3-3 proteins are the only known TPH2 binding partner. In these experiments, I will extract tagged TPH2 from PC12 cells, and identify co-purifying proteins through MS. Again, co-purifying (interacting) proteins will be identified through MS. PUBLIC HEALTH RELEVANCE: Low levels of the neurotransmitter serotonin are linked to various psychiatric disorders such as depression, obsessive compulsive disorder, schizophrenia and autism among many others. In the brain, Tryptophan hydroxylase 2 (TPH2) catalyzes the first and rate-limiting step in the transformation of tryptophan into serotonin. The regulation of this protein is poorly understood; we aim to comprehend how it is controlled. The results of this study can ultimately open the door to a new, more selective generation of antidepressants with fewer side effects.