泛素特異性胜酶24對組蛋白的影響

The Effect of Ubiquitin Specific Peptidase 24 on Histones

莊縕沛(Yun-Pei Chuang)

指導教授 ： 洪建中

共同指導教授 ： 張文昌(Wen-Chang Chang)

國立成功大學/醫學院/藥理學研究所/碩士(2012年)

摘要

真核細胞的DNA由核心組蛋白 (core histones) 及連結組蛋白 (linker histone) 纏繞而成核小體 (nucleosomes)。組蛋白之N端尾 (N-terminal tail) 的轉譯後修飾 (posttranslational modifications, PTMs) 如甲基化 (methylation)、乙醯化 (acetylation)、磷酸化 (phosphorylation) 以及泛素化 (ubiquitination) 所形成之組蛋白碼 (histone code) 可進一步調控細胞的基因轉錄作用 (gene transcription)，進而影響 DNA 修復、重組、複製以及染色質結構。在將 DNA 包裹成染色質 (chromatin) 時，細胞必須嚴謹地維持組蛋白和 DNA 合成之間的平衡。目前已有研究指出，過多的組蛋白堆積會造成染色質缺失並增加細胞對 DNA 損害及細胞毒性的敏感度，由此可知，組蛋白表現對於維持基因穩定性是非常重要的。但是，如何維持組蛋白的穩定表現仍然是未知的。在此項研究中，我們發現一種去泛素化蛋白 ─ 泛素特異性胜肽酶 24 (ubiquitin specific peptidase 24, USP24) 能夠調控組蛋白表現。在人類肺腺癌細胞株 A549 中靜默 (knockdown) USP24 表現後，大部分的組蛋白包括 H1、H2A、H2B 和 H4 表現量都有顯著增加。此外，經由細胞計數 (cell counting) 和細胞存活率分析 (MTT assay) 得知，靜默 USP24 表現顯著增強細胞增生和存活率。相較於蛋白質表現量， USP24 對於組蛋白 mRNA 所造成的影響較為有限，因此，為了進一步提出 USP24 是透過何種機制影響組蛋白表現，我們接著分析蛋白質穩定性，結果發現在抑制 USP24 之後，細胞中的組蛋白質穩定性有顯著上升，因此證明 USP24 是透過維持組蛋白的穩定而調節其表現。反之，從免疫螢光染色 (immunofluorescence assay) 結果得知，在過度表現 USP24 的細胞中，組蛋白表現訊號明顯減弱，最終促使細胞走向死亡。最後，根據免疫螢光染色結果，我們大膽推測 USP24 可能與細胞核中的泛素蛋白酶體系統 (nucleus ubiquitin proteasome system, nUPS) 存在共位現象 (colocalization)，暗示了 USP24 也許在細胞核中負責進行蛋白質的降解作用。總結此項研究結果，我們證實 USP24 能夠調控組蛋白表現並進而提高細胞的存活率且維持基因組的穩定性。

關鍵字

組蛋白 ； 基因穩定性 ； 泛素特異性胜肽酶 24

並列摘要

Core histones involves in DNA packaging hence regulate gene expression in interphase and divide genome into daughter cells in mitosis. The variety of post-translational modifications, including acetylation, phosphorylation, methylation and ubiquitination, play important roles in the epigenetic control of gene expression. However, how to maintain histones level has yet to be addressed. In this study, we found that a deubiquitin protein, ubiquitin specific peptidase 24 (USP24) could affect histone level. Under transient knock-down of USP24 in A549 cells, most histones including H1, H2A, H2B and H4 were increased dramatically, leading an increase in cell number and viability. To further address the mechanism of how USP24 affect the histone level, only little contribution from modulating the mRNA level of histones by USP24. The stability of most histones was increased significantly, indicating that the increase in histones level under silence of USP24 was due to the enhancement of histones protein stability. On the other hand, as GFP-USP24 was overexpressed inside A549 cells, the signal of histones was decreased and chromatin was looser, finally resulting in cell death. Furthermore, localization study found that USP24 stayed mainly in cytoplasm, but some of which still in nucleus. Our recent preliminary results in Immunofluorescence assay indicated that USP24 might colocalized with nucleus ubiquitin proteasome system (nUPS), suggesting that it might work with nUPS to perform the protein degradation inside nucleus. Therefore, unraveling the detailed mechanism of how USP24 affect the stability of histones is ongoing in our laboratory. In conclusion, it is important to clarify the role of USP24 in protein degradation and might be advantage to the study of abnormal protein aggregation such as Parkinson’s disease and tumorignensis.

並列關鍵字

histones ； genome stability ； USP 24

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