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血清素的水平。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，需要一种概念新颖的疗法，能够安全地治疗高比例的患者。我们的长期目标是了解色氨酸羟化酶2 （TPH2）。TPH2催化大脑中色氨酸转化为血清素的第一步和限速步骤。发现TPH2被磷酸化；据报道，这种修饰可提高TPH2的稳定性和活性。我们假设TPH2的翻译后修饰（PTM）在该关键酶的体内调控中发挥重要作用。为了证实这一假设，我计划通过基于质谱（MS）的蛋白质组学来绘制体内TPH2上的ptm。此外，通过结合MS使用亲和纯化，我的目标是表征TPH2的结合伙伴。这项工作是创新的，因为它利用现代技术来解决传统生化实验无法解决的问题。这项研究的结果可以提供关于TPH2生理控制的重要信息，并可以打开一扇门，新的，更有选择性的一代抗抑郁药，副作用更少，能够通过增强脑特异性TPH2活性来增加血清素合成。我们将在两个特定的目标下进行这些研究：特定目标#1：通过基于MS的蛋白质组学探索TPH2的体内翻译后修饰。我们的目标是通过质谱（MS）蛋白质组学来探索TPH2在生理相关环境下的翻译后调控。为此，我将稳定地将6xhis标记的TPH2转化为哺乳动物（PC12）细胞。或者，我计划从大鼠脑raphe中提取TPH2。我将通过基于MS的蛋白质组学分析这两个来源的TPH2的PTMs。特异性目标#2：通过与ms结合的亲和纯化鉴定涉及TPH2的蛋白质-蛋白质相互作用。我们的目标是鉴定新的TPH2结合伙伴。相互作用的蛋白质可以调节TPH2的功能。到目前为止，14-3-3蛋白是唯一已知的TPH2结合伙伴。在这些实验中，我将从PC12细胞中提取标记的TPH2，并通过质谱鉴定共纯化蛋白。再次，将通过质谱鉴定共纯化（相互作用）蛋白。

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.