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: 8263768
• 负责人: Mariana Plazas Torrente
• 金额: $ 2.24万
• 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 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8263768
• 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; public health relevance; 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裂中的5-羟色胺水平。尽管目前的治疗方法可以缓解数百万患者，但他们带来了耐受性和功效问题。因此，需要能够安全治疗大量患者的概念新疗法。我们的长期目标是了解色氨酸羟化酶2（TPH2）。 TPH2催化色氨酸转化为大脑中5-羟色胺的第一个限制步骤。已经发现TPH2被磷酸化；据报道，这种修饰会导致TPH2稳定性和活动增强。我们假设TPH2​​的翻译后修饰（PTM）在该关键酶的体内调节中起重要作用。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TPH2上绘制体内PTM。此外，通过将亲和力纯化与MS结合使用，我旨在表征TPH2的结合伙伴。拟议的工作具有创新性，因为它利用现代技术来通过常规的生化实验来解决无法访问的问题。这项研究的结果可以提供有关TPH2生理控制的重要信息，并可以为具有较少副作用的新的，更具选择性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5-羟色胺合成。我们将以两个具体的目的进行这些研究：特定目的＃1：通过基于MS的蛋白质组学探索TPH2的体内翻译后修饰。我们旨在通过质谱（MS）蛋白质组学探索在生理相关环境中TPH2的翻译后调节。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的蛋白质组学从这两个来源分析TPH2的PTM。特定目标＃2：通过与MS结合使用亲和力纯化，鉴定涉及TPH2的蛋白质蛋白质相互作用。我们旨在确定新颖的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鉴定共纯化的蛋白质。同样，将通过MS鉴定共纯化（相互作用）蛋白质。 公共卫生相关性：少量神经递质5-羟色胺与抑郁症，强迫症，精神分裂症和自闭症等各种精神疾病有关。在大脑中，色氨酸羟化酶2（TPH2）催化了色氨酸转化为5-羟色胺的第一个和速率限制步骤。该蛋白的调节知之甚少。我们旨在理解它的控制方式。这项研究的结果最终可以打开新的，更具选择性的抗抑郁药具有更少副作用的抗抑郁药。