Naturally- and experimentally-designed restorations of the Parkin gene deficit in autosomal recessive juvenile parkinsonism
Naturally- and experimentally-designed restorations of the Parkin gene deficit in autosomal recessive juvenile parkinsonism
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DOI：
10.1016/j.bbrc.2009.11.141
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发表时间：
2010-01-01
影响因子：
3.1
通讯作者：
Ueno, Satoshi
中科院分区：
文献类型：
--
作者：
Asai, Hirohide;Hirano, Makito;Ueno, Satoshi
在常染色体隐性遗传性青少年帕金森综合征（ARJP）中，由于Parkin基因突变引起的核内事件仍然难以捉摸。我们在来自一对ARJP兄弟姐妹的成纤维细胞培养物中鉴定了突变的PARKIN蛋白，所述ARJP兄弟姐妹为外显子4缺失的Parkin基因纯合型。检测到的突变体，由缺乏外显子3以及外显子4的转录本编码，是一个框内缺失，删除121个氨基酸，导致344个氨基酸的蛋白质（PaDel 3A）。细胞培养和转染研究显示PaDel 3，4的表达水平与细胞周期蛋白，包括细胞周期蛋白E，CDK 2，ppRb和E2 F-1的表达水平之间呈负相关，并证明GFP-PaDel 3，4进入细胞核并作为患者细胞中SCFhSel-10连接酶复合物的一部分泛素化细胞周期蛋白E。此外，在转染的患者细胞中表达的核定位信号标记的PaDel 3，4最有效地泛素化细胞周期蛋白E和减少DNA损伤，保护细胞免受氧化应激。反义寡核苷酸处理促进外显子3的跳跃，从而产生PaDel 3，4，增加细胞存活。总的来说，我们提出，自然和实验诱导的外显子跳跃至少部分恢复了突变型帕金基因缺陷，为治疗性外显子跳跃的开发提供了分子基础。
Intranuclear events due to mutations in the Parkin gene remain elusive in autosomal recessive juvenile parkinsonism (ARJP) We identified a mutant PARKIN protein in fibroblast cultures from a pair of siblings with ARJP who were homozygous for the exon 4-deleted Parkin gene Disease was mild in one patient and debilitating in the other. The detected mutant, encoded by a transcript lacking exon 3 as well as exon 4, is an in-frame deletion that removes 121 aa, resulting in a 344-aa protein (PaDel3A). Cell culture and transfection studies revealed negative correlations between expression levels of PaDel3,4 and those of cell cycle proteins, including cyclin E, CDK2, ppRb, and E2F-1, and demonstrated that GFP-PaDel3,4 entered nucleus and ubiquitinated cyclin E as a part of SCFhSel-10 ligase complex in the patient cells. In addition, nuclear localization signal-tagged PaDel3,4 expressed in the transfected patient cells most effectively ubiquitinated cyclin E and reduced DNA damage, protecting cells from oxidative stress. Antisense-oligonucleotide treatment promoted skipping of exon 3 and thus generated PaDel3,4, increasing cell survival Collectively, we propose that naturally- and experimentally-induced exon skipping at least partly restores the mutant Parkin gene deficit, providing a molecular basis for the development of therapeutic exon skipping (C) 2009 Elsevier Inc All rights reserved,