Dynamic Control of Tryptophan Hydroxylase 2:Regulating Brain Serotonin Synthesis

色氨酸羟化酶2的动态控制：调节脑血清素合成

• 批准号: 8125534
• 负责人: Mariana Plazas Torrente
• 金额: $ 4.84万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8125534
• 关键词: 14-3-3 Proteins; Adverse effects; Affinity; Affinity Chromatography; Anabolism; Antidepressive Agents; Autistic Disorder; Binding; Biochemical; Brain; Cultured Cells; Development; Disease; Enzyme Stability; Enzymes; Escherichia coli; Functional disorder; Generations; Goals; Immunoprecipitation; Incubated; Link; Maps; Mass Spectrum Analysis; Mental Depression; Mental disorders; Methods; Modification; Neurotransmitters; Obsessive-Compulsive Disorder; PC12 Cells; Patients; Pharmaceutical Preparations; Phosphorylation Site; Physiological; Play; Post-Translational Protein Processing; Post-Translational Regulation; Protein Binding; Proteins; Proteomics; Rattus; Regulation; Reporting; Research; Role; Schizophrenia; Serotonin; Site; Source; Symptoms; Synaptic Cleft; Techniques; Tryptophan; Tryptophan 5-monooxygenase; Work; animal tissue; base; enzyme activity; in vivo; innovation; neuropsychiatry; novel; novel therapeutics; protein protein interaction; raphe nuclei; research study; uptake

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.

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已经为数百万患者带来了缓解，但它们也存在耐受性和疗效问题。因此，需要能够安全治疗大部分患者的概念性新颖疗法。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，也是限速步骤。已发现TPH2被磷酸化；据报道，这种修饰可提高 TPH2 稳定性并增强活性。我们假设 TPH2 的翻译后修饰 (PTM) 在该关键酶的体内调节中发挥重要作用。为了证实这一假设，我计划通过基于蛋白质组学的质谱 (MS) 来绘制 TPH2 上的体内 PTM 图谱。此外，通过结合使用亲和纯化和 MS，我的目标是表征 TPH2 的结合配偶体。这项工作具有创新性，因为它利用现代技术解决了传统生化实验无法解决的问题。这项研究的结果可以提供有关 TPH2 生理控制的重要信息，并可以为新一代、选择性更强、副作用更少的抗抑郁药打开大门，能够通过增强大脑特异性 TPH2 活性来增加血清素合成。我们将围绕两个具体目标开展这些研究： 具体目标#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) 蛋白质组学探索 TPH2 在生理相关环境中的翻译后调控。为此，我将把 6XHis 标记的 TPH2 稳定地转化到哺乳动物 (PC12) 细胞中。或者，我计划从大鼠脑中缝中提取 TPH2。我将通过基于 MS 的蛋白质组学分析这两个来源的 TPH2 PTM。具体目标#2：通过亲和纯化结合 MS 鉴定涉及 TPH2 的蛋白质-蛋白质相互作用。我们的目标是识别新型 TPH2 结合伙伴。相互作用的蛋白质可以调节 TPH2 的功能。迄今为止，14-3-3 蛋白是唯一已知的 TPH2 结合伴侣。在这些实验中，我将从 PC12 细胞中提取标记的 TPH2，并通过 MS 鉴定共纯化蛋白。同样，共纯化（相互作用）蛋白质将通过 MS 进行鉴定。 公共健康相关性：神经递质血清素水平低与各种精神疾病有关，例如抑郁症、强迫症、精神分裂症和自闭症等。在大脑中，色氨酸羟化酶 2 (TPH2) 催化色氨酸转化为血清素的第一步，也是限速步骤。人们对这种蛋白质的调节知之甚少。我们的目标是了解它是如何控制的。这项研究的结果最终将为开发新一代、选择性更强、副作用更少的抗抑郁药物打开大门。