THE TRANSCRIPTIONAL REGULATION OF MONOAMINE OXIDASE A

单胺氧化酶 A 的转录调控

• 批准号: 6924638
• 负责人: Jean Chen Shih
• 金额: $ 36.16万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6924638
• 关键词: RNA interference; amine oxidase (flavin); apoptosis; chromatin immunoprecipitation; enzyme activity; gel mobility shift assay; gene expression; glucocorticoids; green fluorescent proteins; immunocytochemistry; in situ hybridization; laboratory mouse; liquid chromatography mass spectrometry; luciferin monooxygenase; mental disorders; molecular pathology; neoplastic cell culture for noncancer research; neuroblastoma; northern blottings; nucleic acid repetitive sequence; prostate neoplasms; serotonin; site directed mutagenesis; transcription factor; western blottings

DESCRIPTION (provided by applicant): The long-term objective of this project is to understand the role of monoamine oxidase A (MAO A) in mental disorders. MAO A is the key enzyme, which degrades serotonin (5-HT). This application focuses on the transcriptional regulation of MAO A. The new information obtained will provide new insights on the molecular basis of diseases associated with abnormal levels of 5-HT. It will also help develop a new series of MAO A inhibitors targeted at the transcriptional level. We have recently cloned a novel Repressor R1 specific for MAO A promoter. We have also found that steroids (androgen and glucocorticoid) activate the MAO A gene expression. With these new findings we hypothesize that the interactions among R1, Spl, a functional polymorphism, 30 bp variable number of tandem repeat (VNTR), steroid receptors and other associated proteins play important roles in the transcriptional regulation of MAO A. Specific aims are: 1. To investigate if Repressor R1 binds to Spl site or the GC rich region by site-directed mutagenesis, luciferase functional assay and gel shift assay. Human neuroblastoma (SK-N-BE (2)-C) and androgen dependent prostate carcinoma (LNCaP) cell lines will be used. The role of Cysteines on R1 function will be studied. The interaction between endogenous R1 and native MAO A promoter will be studied by chromatin immunoprecipitation (CHIP) assay. The biological function of R1 will be studied by RNA interference (RNAi) and serum starvation induced apoptosis. 2. To investigate the dynamic intracellular location of R1 protein in living cells by fusion R1 with enhanced Green Fluorescence Protein (eGFP). The brain regional distribution of R1 protein and mRNA will be studied by immunohistochemistry and in situ hybridization, respectively. They will be correlated with that of MAO A and B. The embryonic and postnatal development of R1 will be studied. 3. The effect of VNTR on R1 function and MAO A promoter activity will be investigated. Repressor R1 interacting proteins in SK-N-BE (2)-C cells will be co-immuno-precipitated using specific polyclonal R1 antibody developed in this laboratory.The proteins associated with R1 will be identified by their partial amino acid sequences by LC/MS/MS mass spectrometry. The validity and the function of these R1 interacting proteins will be investigated. 4. To identify the functional glucocorticoid (androgen) response element and to study the direct effect of AR/GR on MAO A promoter activity. The effect of VNTR on AR/GR activation of MAO A promoter will be studied. 5. To investigate if AR/GR indirectly activates MAC) A gene expression by interacting with Spl and other coactivators. The interactions among AR, GR, Spl and R1 on MAO A gene expression will be studied by luciferase assay (promoter activity), Northern blot (mRNA), Western blot (protein) and catalytic activity. AR (PC-3) and GR (Cos 7) negative cell lines will also be used. Co-regulators interacting with APJGR during agonist stimulated or basal state will be co-immuno-precipitated by AR or GR specific antibody. The validity and the function of identified protein(s) will be investigated.

描述（由申请人提供）：本项目的长期目标是了解单胺氧化酶A（MAO A）在精神障碍中的作用。单胺氧化酶A是降解5-羟色胺（5-HT）的关键酶。本申请集中于MAO A的转录调控。所获得的新信息将为与5-HT水平异常相关的疾病的分子基础提供新的见解。这也将有助于开发一系列新的针对转录水平的单胺氧化酶A抑制剂。我们最近克隆了一个新的抑制子R1特异性的单胺氧化酶A启动子。我们还发现类固醇（雄激素和糖皮质激素）激活MAOA基因表达。根据这些新发现，我们推测R1、Spl、30 bp可变数目串联重复序列（VNTR）、类固醇受体和其他相关蛋白之间的相互作用在MAOA的转录调控中起重要作用。具体目标是：1。通过定点突变、荧光素酶功能分析和凝胶迁移分析研究阻遏蛋白R1是否与Spl位点或GC富集区结合。将使用人神经母细胞瘤（SK-N-BE（2）-C）和雄激素依赖性前列腺癌（LNCaP）细胞系。将研究半胱氨酸对R1功能的作用。将通过染色质免疫沉淀（CHIP）测定来研究内源R1和天然MAO A启动子之间的相互作用。R1的生物学功能将通过RNA干扰（RNAi）和血清饥饿诱导细胞凋亡来研究。2.目的研究R1蛋白与增强型绿色荧光蛋白（eGFP）融合后在活细胞内的动态定位。分别采用免疫组织化学和原位杂交技术研究R1蛋白和mRNA的脑区分布。它们将与MAO A和B相关。将研究R1的胚胎和出生后发育。3.将研究VNTR对R1功能和MAO A启动子活性的影响。本实验室研制的R1特异性多克隆抗体将SK-N-BE（2）-C细胞中与R1相互作用的蛋白质免疫共沉淀，并通过LC/MS/MS质谱分析鉴定与R1相互作用的蛋白质。将研究这些R1相互作用蛋白的有效性和功能。4.目的鉴定功能性糖皮质激素（雄激素）反应元件，研究AR/GR对MAOA启动子活性的直接影响。将研究VNTR对MAOA启动子的AR/GR激活的影响。5.研究AR/GR是否通过与Spl等共激活因子相互作用而间接激活MAC A基因表达。AR、GR、Spl和R1之间对MAO A基因表达的相互作用将通过荧光素酶测定（启动子活性）、北方印迹（mRNA）、蛋白质印迹（蛋白质）和催化活性来研究。还将使用AR（PC-3）和GR（Cos 7）阴性细胞系。在激动剂刺激或基础状态期间与APJGR相互作用的共调节剂将被AR或GR特异性抗体共免疫沉淀。将研究已鉴定蛋白质的有效性和功能。