THE TRANSCRIPTIONAL REGULATION OF MONOAMINE OXIDASE A

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。本研究的重点是MAO a的转录调控，获得的新信息将为5-HT异常相关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靶向转录水平的MAO a抑制剂。我们最近克隆了一个新的抑制子R1特异性MAO a启动子。我们还发现类固醇（雄激素和糖皮质激素）激活MAO A基因表达。基于这些新发现，我们推测R1、Spl、一个功能多态性、30bp可变数目的串串重复序列（VNTR）、类固醇受体和其他相关蛋白之间的相互作用在MAO a的转录调控中发挥重要作用。通过定点诱变、荧光素酶功能试验和凝胶移位试验研究阻遏因子R1是否与Spl位点或GC富区结合。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。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）试验进行研究。R1的生物学功能将通过RNA干扰（RNAi）和血清饥饿诱导凋亡来研究。2. 通过增强的绿色荧光蛋白（eGFP）与R1融合，研究R1蛋白在活细胞内的动态定位。采用免疫组织化学和原位杂交技术分别研究R1蛋白和mRNA的脑区分布。它们可能与MAO A和MAO b基因相关。我们将对R1的胚胎和出生后发育进行研究。3. 研究VNTR对R1功能和MAO A启动子活性的影响。在SK-N-BE (2)-C细胞中，抑制因子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。通过LC/MS/MS质谱法对R1相关蛋白的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被研究。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MAO A启动子活性的直接影响。研究VNTR对MAO A启动子AR/GR活化的影响。5. 目的探讨AR/GR是否通过与Spl及其他共激活因子的相互作用间接激活MAC) A基因的表达。通过荧光素酶（启动子活性）、Northern blot （mRNA）、Western blot（蛋白）和催化活性研究AR、GR、Spl和R1对MAO A基因表达的相互作用。AR （PC-3）和GR （Cos 7）阴性细胞系也将被使用。在激动剂刺激或基础状态下与APJGR相互作用的共调节因子将被AR或GR特异性抗体共同免疫沉淀。将对鉴定的蛋白质的有效性和功能进行研究。

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.