THE TRANSCRIPTIONAL REGULATION OF MONOAMINE OXIDASE A

单胺氧化酶 A 的转录调控

• 批准号: 7085373
• 负责人: Jean Chen Shih
• 金额: $ 35.42万
• 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/7085373
• 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）：该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。 MAO A 是降解血清素 (5-HT) 的关键酶。该应用重点关注 MAO A 的转录调控。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。它还将有助于开发一系列针对转录水平的新 MAO A 抑制剂。我们最近克隆了一种针对 MAO A 启动子的新型阻遏蛋白 R1。我们还发现类固醇（雄激素和糖皮质激素）可激活 MAO A 基因表达。根据这些新发现，我们假设 R1、Spl、功能多态性、30 bp 可变串联重复序列数 (VNTR)、类固醇受体和其他相关蛋白之间的相互作用在 MAO A 的转录调节中发挥重要作用。 具体目标是： 1. 通过定点诱变、荧光素酶功能测定和凝胶研究阻遏物 R1 是否与 Spl 位点或 GC 丰富区结合。 位移测定。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。内源性 R1 和天然 MAO A 启动子之间的相互作用将通过染色质免疫沉淀 (CHIP) 测定进行研究。将通过 RNA 干扰 (RNAi) 和血清饥饿诱导的细胞凋亡来研究 R1 的生物学功能。 2. 通过将R1与增强型绿色荧光蛋白（eGFP）融合，研究R1蛋白在活细胞中的动态定位。将分别通过免疫组织化学和原位杂交研究R1蛋白和mRNA的脑区域分布。它们将与 MAO A 和 B 相关。将研究 R1 的胚胎和出生后发育。 3.研究VNTR对R1功能和MAO A启动子活性的影响。 SK-N-BE (2)-C 细胞中的阻遏物 R1 相互作用蛋白将使用本实验室开发的特异性多克隆 R1 抗体进行共免疫沉淀。与 R1 相关的蛋白将通过 LC/MS/MS 质谱法通过其部分氨基酸序列进行鉴定。这些 R1 相互作用蛋白的有效性和功能将得到研究。 4. 鉴定功能性糖皮质激素（雄激素）反应元件并研究AR/GR对MAO A启动子活性的直接影响。将研究VNTR对MAO A启动子的AR/GR激活的影响。 5.研究AR/GR是否通过与Spl和其他共激活因子相互作用间接激活MAC)A基因表达。通过荧光素酶测定（启动子活性）、Northern印迹（mRNA）、Western印迹（蛋白质）和催化活性来研究AR、GR、Spl和R1之间对MAO A基因表达的相互作用。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。