THE TRANSCRIPTIONAL REGULATION OF MONOAMINE OXIDASE A
单胺氧化酶 A 的转录调控
基本信息
- 批准号:6825855
- 负责人:
- 金额:$ 36.16万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:RNA interferenceamine oxidase (flavin)apoptosischromatin immunoprecipitationenzyme activitygel mobility shift assaygene expressionglucocorticoidsgreen fluorescent proteinsimmunocytochemistryin situ hybridizationlaboratory mouseliquid chromatography mass spectrometryluciferin monooxygenasemental disordersmolecular pathologyneoplastic cell culture for noncancer researchneuroblastomanorthern blottingsnucleic acid repetitive sequenceprostate neoplasmsserotoninsite directed mutagenesistranscription factorwestern blottings
项目摘要
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(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。MAO A是降解5-羟色胺(5-HT)的关键酶。这一应用的重点是MAO A的转录调控,所获得的新信息将为研究与5-羟色胺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新的针对转录水平的MAO A抑制剂。我们最近克隆了一个新的针对MAO A启动子的抑制因子R1。我们还发现类固醇(雄激素和糖皮质激素)激活了MAO A基因的表达。根据这些新的发现,我们推测,在MAO A的转录调控中,R1、SPL、功能多态、30BP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起着重要作用。具体目的是:1.通过定点突变、荧光素酶功能分析和凝胶移位分析,研究抑制子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)和雄激素依赖型前列腺癌(LNCaP)细胞系。我们将研究半胱氨酸对R1功能的影响。内源R1与天然MAO A启动子之间的相互作用将用染色质免疫沉淀(ChIP)方法进行研究。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R1与增强型绿色荧光蛋白(EGFP)的融合,研究R1蛋白在活细胞内的动态定位。用免疫组织化学和原位杂交法分别研究R1蛋白和mRNA在脑内的区域分布。它们将与MAO A和MAO B相关,并将研究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。用本实验室研制的特异性多克隆抗体免疫共沉淀SK-N-BE(2)-C细胞中与抑制子R1相互作用的蛋白,并通过LC/MS/MS的部分氨基酸序列鉴定与R1相关的蛋白。这些R1相互作用蛋白的有效性和功能将被研究。4.鉴定功能性糖皮质激素(雄激素)反应元件,研究AR/GR对MAO A启动子活性的直接影响。我们将研究VNTR对MAO A启动子AR/GR激活的影响。5.探讨AR/GR是否通过与SPL等共激活因子相互作用间接激活MACA基因的表达。通过荧光素酶活性(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对MAO A基因表达的相互作用。也将使用AR(PC-3)和GR(CoS 7)阴性细胞系。在激动剂刺激或基础状态下与APJGR相互作用的协同调节因子将被AR或GR特异性抗体共同免疫沉淀。将对鉴定出的蛋白(S)的有效性和功能进行研究。
项目成果
期刊论文数量(0)
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Jean Chen Shih其他文献
Introduction to the special issue on monoamine oxidase A and B: eternally enigmatic isoenzymes
- DOI:
10.1007/s00702-018-1920-2 - 发表时间:
2018-09-26 - 期刊:
- 影响因子:4.000
- 作者:
Jean Chen Shih;Peter Riederer;Wakako Maruyama;Makoto Naoi - 通讯作者:
Makoto Naoi
Jean Chen Shih的其他文献
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{{ truncateString('Jean Chen Shih', 18)}}的其他基金
THE TRANSCRIPTIONAL REGULATION OF MONOAMINE OXIDASE A
单胺氧化酶 A 的转录调控
- 批准号:
6924638 - 财政年份:2004
- 资助金额:
$ 36.16万 - 项目类别:
THE TRANSCRIPTIONAL REGULATION OF MONOAMINE OXIDASE A
单胺氧化酶 A 的转录调控
- 批准号:
7085373 - 财政年份:2004
- 资助金额:
$ 36.16万 - 项目类别:
THE TRANSCRIPTIONAL REGULATION OF MONOAMINE OXIDASE A
单胺氧化酶 A 的转录调控
- 批准号:
7252478 - 财政年份:2004
- 资助金额:
$ 36.16万 - 项目类别:
THE TRANSCRIPTIONAL REGULATION OF MONOAMINE OXIDASE A
单胺氧化酶 A 的转录调控
- 批准号:
7456338 - 财政年份:2004
- 资助金额:
$ 36.16万 - 项目类别: