PPP2R2B：外遺傳研究暨藥物篩檢模式的建立
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2011
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Protein phosphatase 2A (簡稱PP2A)是細胞內重要的蛋白去磷酸化酵素，其次單元B調控PP2A在細胞內作用的位置及催化的受質種類。PPP2R2B是表現在腦神經細胞中的PP2A調控次單元B。腦部表現的PP2A調節tau蛋白的磷酸化。Tau蛋白過度磷酸化和PP2A活性下降，皆和阿茲海默氏症(AD)相關。本實驗室的研究亦發現PPP2R2B的低啟動子活性，和國人的阿茲海默氏症顯著相關。本研究第一部份是以PPP2R2B啟動子接上EGFP報導基因，來建立人類胚胎腎HEK-293及神經腫瘤SK-N-SH細胞，轉染入轉錄因子SP1及CREB1後，分析綠螢光量變化，並未呈現預期的調控情形。第二部分為探討PPP2R2B DNA甲基化的外遺傳調控，與阿茲海默氏症的相關性。選取五組年齡及性別配對的病人及正常人DNA樣品，經Bisulfite定序，結果發現AD病人的PPP2R2B的5'端甲基化程度有高於正常人的趨勢，尤其是-311及-310的位置，雖然並沒有到達顯著差異。進一步的神經(SK-N-SH、SH-SY5Y)及非神經(HEK-293)腫瘤細胞PPP2R2B啟動子定序，顯示HEK-293細胞PPP2R2B啟動子上的甲基化，但以去甲基化藥物5-aza-dC處理HEK-293後，PPP2R2B表現量並未顯著上升，MeCP2的抗體的染色質免疫沈澱亦未看到MeCP2蛋白結合到PPP2R2B啟動子上。
Protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase expressed in all eukaryotic cells. The regulatory B subunit confers substrate specificity and sub-cellular localization of the PP2A holoenzyme. PPP2R2B is a regulatory B subunit expressed throughout the brain. The brain specific PPP2R2B regulates PP2A dephosphorylation activity for microtubule-associated protein tau. The association of pathological hyperphosphorylated tau and reduced PP2A activity with Alzheimer's disease (AD) has been established. Our case-control study and reporter assay have also revealed that the rare low transcriptional activity alleles of PPP2R2B are associated with AD. In the first part of the study, PPP2R2B-driven EGFP construct was used to generate human embryonic kidney (HEK)-293 and neuroblastoma SK-N-SH cell lines. SP1 and CREB1 co-transfection did not enhance PPP2R2B expression. In the second part of the study, epigenetic control of DNA methylation affecting AD susceptivity was explored. Bisulfite sequencing was performed to assess the DNA methylation using lymphocyte DNA from five AD patients and age- and gender-matched controls. The results of increased DNA methylation (although not significantly) in the PPP2R2B gene 5' region, especially -311 and -310, suggest that the epigenetic change may alter the PPP2R2B expression in AD patients. Further direct bisulfite-sequencing of HEK-293 cells revealed increased DNA methylation in the PPP2R2B gene 5' region. However, real-time PCR quantitation of 5-aza-dC treated HEK-293 cells did not show increased PPP2R2B expression. Chromatin IP with MeCP2 antibody and PCR also did not support MeCP2 binding to PPP2R2B promoter.
Protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase expressed in all eukaryotic cells. The regulatory B subunit confers substrate specificity and sub-cellular localization of the PP2A holoenzyme. PPP2R2B is a regulatory B subunit expressed throughout the brain. The brain specific PPP2R2B regulates PP2A dephosphorylation activity for microtubule-associated protein tau. The association of pathological hyperphosphorylated tau and reduced PP2A activity with Alzheimer's disease (AD) has been established. Our case-control study and reporter assay have also revealed that the rare low transcriptional activity alleles of PPP2R2B are associated with AD. In the first part of the study, PPP2R2B-driven EGFP construct was used to generate human embryonic kidney (HEK)-293 and neuroblastoma SK-N-SH cell lines. SP1 and CREB1 co-transfection did not enhance PPP2R2B expression. In the second part of the study, epigenetic control of DNA methylation affecting AD susceptivity was explored. Bisulfite sequencing was performed to assess the DNA methylation using lymphocyte DNA from five AD patients and age- and gender-matched controls. The results of increased DNA methylation (although not significantly) in the PPP2R2B gene 5' region, especially -311 and -310, suggest that the epigenetic change may alter the PPP2R2B expression in AD patients. Further direct bisulfite-sequencing of HEK-293 cells revealed increased DNA methylation in the PPP2R2B gene 5' region. However, real-time PCR quantitation of 5-aza-dC treated HEK-293 cells did not show increased PPP2R2B expression. Chromatin IP with MeCP2 antibody and PCR also did not support MeCP2 binding to PPP2R2B promoter.
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阿茲海默氏症, 小腦萎縮症第十二型, 外遺傳調控, 甲基化, AD, SCA12, epigenetic regulation, methylation