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National Chung Hsing University Institutional Repository - NCHUIR > 生命科學院 > 分子生物學研究所 > 依資料類型分類 > 碩博士論文 >  1. 一新穎類泛素 SUMO 變異體 SUMO5 之功能性探討。 2. 聚腺苷二磷酸核醣聚合酶-2 在轉錄調控上之功能性探討。

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/151715

標題: 1. 一新穎類泛素 SUMO 變異體 SUMO5 之功能性探討。 2. 聚腺苷二磷酸核醣聚合酶-2 在轉錄調控上之功能性探討。
1. The Functional Studies of a Novel SUMO Variant, SUMO5. 2. The Functional Studies of Poly(ADP-ribosyl)ase-2 in Transcriptional Regulation.
作者: 梁雅珍
Liang, Ya-Chen
Contributors: 楊文明
Wen-Ming Yang
分子生物學研究所
關鍵字: 類泛素修飾;急性前骨髓性白血病;聚腺苷二磷酸核醣聚合作用
SUMO5;SUMOylation;PML;APL;arsenic trioxide;ATO;PARP-2
日期: 2013
Issue Date: 2013-11-07 12:18:23 (UTC+8)
Publisher: 分子生物學研究所
摘要: 一、一新穎類泛素 SUMO 變異體 SUMO5 之功能性探討

  類泛素修飾 (SUMOylation) 是由類泛素蛋白 (small ubiquitin-related modifier, SUMO) 所介導之後轉譯修飾作用,參與多種細胞生理過程,其中包含協助細胞核次結構 PML 核小體 (PML nuclear body, PML-NB) 之形成。先前研究指出,PML 核小體形成缺失將導致疾病如急性前骨髓性白血病 (acute promyelocytic leukemia, APL)。在實驗室先前的研究中發現ㄧ新類泛素蛋白,為人類第五號類泛素蛋白 (SUMO5),此蛋白可增加內生性 PML 核小體之體積與數量,但 SUMO5 之特性與其調控 PML 核小體之分子機制仍不明。在此研究中,我們闡述 SUMO5 之分子特性,與其在正常狀態、致癌機制與抗癌療法中調控 PML 核小體生成與降解之機制。首先,我們證實 SUMO5 為一具有高度序列保留性及類泛素酵素活性之類泛素蛋白家族成員。在正常狀態下,SUMO5 可藉由直接修飾並穩定 PML 蛋白來促使 PML 核小體形成,而當 SUMO5 在 PML 蛋白上之修飾被其他類泛素蛋白成員 SUMO2 及 SUMO3 置換後, PML 核小體會藉由 RNF4 蛋白所介導之泛素修飾作用 (ubiquitination) 而瓦解。在急性前骨髓性白血病的致癌機制中,致癌蛋白 PML-PARα 會與 PML 蛋白競爭 SUMO5 的修飾作用,並使 PML 蛋白轉分布至異常之細胞質區,最終導致 PML 核小體結構瓦解。在三氧化二砷 (arsenic trioxide, As2O3) 抗急性前骨髓性白血病的療法中,三氧化二砷會促使 SUMO5 大量修飾 PML,並將其轉分布至泛細胞核基質區 (detergent-insoluble fraction) 以形成功能性 PML 核小體,而三氧化二砷無法促使致癌蛋白 PML-PARα 之 類泛素修飾作用與轉分布,最終導致致癌蛋白 PML-PARα 之降解,因而達成抗癌功效。此研究鑑定一新穎類泛素 SUMO 變異體 SUMO5 之特性與其調控 PML 核小體之功能,並進一步闡釋其參與癌症之致癌機轉與抗癌療法之機制。


二、 聚腺苷二磷酸核醣聚合酶-2 在轉錄調控上之功能性探討

  聚腺苷二磷酸核醣聚合酶-2 (Poly(ADP-ribose) polymerase-2, PARP-2) 會催化ㄧ後轉譯修飾作用,稱為聚腺苷二磷酸核醣聚合反應 (poly(ADP-ribosyl)ation, PARylation) ,並參與如基因轉錄調控等諸多細胞核生理功能。先前研究指出,喪失 PARP-2 會降低轉錄因子之轉錄活性並改變細胞內總體基因表現,但 PARP-2 如何調控啓動子 (promoter) 之分子機制仍不明。在此研究中,我們闡釋 PARP-2 具有與其酵素活性無關之轉錄抑制活性。PARP-2 藉由與組蛋白去乙醯化酵素五與七 (histone deacetylase-5/-7, HDAC5/7) 以及組蛋白甲基化酵素 (histone methyltransferase) G9a 交互作用,而將此些組蛋白修飾蛋白招募至細胞週期相關基因之啓動子區域,產生抑制型染色質標記 (repressive chromatin signatures),進而抑制細胞週期相關基因之表現。此研究提供ㄧ後轉譯修飾蛋白 PARP-2 不藉由本身酵素作用而以蛋白質交互作用來抑制基因轉錄之新機制,並突顯 PARP-2 於調控細胞週期之重要性。
1. The Functional Studies of A Novel SUMO Variant, SUMO5

SUMOylation is conducted by small ubiquitin-related modifiers (SUMO) and involves in numerous cellular processes including the formation of nuclear domains named PML nuclear bodies (PML-NBs). Defects in the formation of PML-NBs result in diseases such as acute promyelocytic leukemia (APL). Here, we discovered a new SUMO variant, SUMO5, with an ability to increase the volume and quantity of PML-NBs. However, characteristics and functions of SUMO5 in the biogenesis of PML-NBs remain unclear. In this study, we characterize SUMO5 and address the functions of SUMO5 in structuring PML-NBs under normal, pathological, and therapeutic conditions. We show that SUMO5 is a conserved member with the conjugation ability in human SUMO family. The formation of PML-NBs requires SUMO5 to conjugate and stabilize PML, and a replacement from SUMO5 conjugation to SUMO2/3 results in RNF4-mediated disruption of PML-NBs. The APL oncoprotein PML-RARα competes SUMO5 conjugation from PML, causing cytoplasmic translocation of PML and disruption of PML-NBs. During arsenite-mediated degradation of PML-RARα, SUMO5-conjugated PML translocates to detergent-insoluble fraction and regenerates functional PML-NBs. Our findings elucidate properties of the newly discovered SUMO5, and clarify the mechanisms of SUMO5 in the regulation of PML-NBs with complicated cooperations between SUMO variants under normal and oncogenic conditions.

2. The Functional Studies of Poly(ADP-ribose) Polymerase-2 in Transcriptional Regulation

Poly(ADP-ribose) polymerase-2 (PARP-2) catalyzes poly(ADP-ribosyl)ation (PARylation) and regulates numerous nuclear processes, including transcription regulation. Depletion of PARP-2 decreases transcriptional activity of transcription factors and globle gene expression. However, the molecular action of how PARP-2 controls transcription of target promoters remains unclear. Here we report that PARP-2 posseses transcriptional repression activity independently of its enzymatic activity. PARP-2 interacts and recruits histone deacetylases HDAC5 and HDAC7 and histone methyltransferase G9a to the promoters of cell cycle-related genes, generating repressive chromatin signatures. Our findings propose a novel mechanism of PARP-2 in transcriptional regulation involving protein-protein interactions, and highlight the importance of PARP-2 in the regulation of the cell cycle progression.
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