Knockout of Mpv17-Like Protein (M-LPH) Gene in Human Hepatoma Cells Results in Impairment of mtDNA Integrity through Reduction of TFAM, OGG1, and LIG3 at the Protein Levels.
Knockout of Mpv17-Like Protein (M-LPH) Gene in Human Hepatoma Cells Results in Impairment of mtDNA Integrity through Reduction of TFAM, OGG1, and LIG3 at the Protein Levels.
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DOI：
10.1155/2018/6956414
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发表时间：
2018
影响因子：
--
通讯作者：
Yasuda T
中科院分区：
文献类型：
--
作者：
Iida R;Ueki M;Yasuda T
人Mpv 17样蛋白（M-LPH）被认为参与预防由线粒体DNA（mtDNA）损伤引起的线粒体功能障碍。为了阐明M-LPH功能的分子机制，我们使用CRISPR-Cas9技术敲除人肝癌HepG 2中的M-LPH。通过PCR定量和8-羟基脱氧鸟苷（8-OHdG）检测证实M-LPH-KO HepG 2细胞中线粒体DNA损伤增加。此外，共聚焦免疫荧光分析和线粒体提取物的蛋白质印迹分析表明，M-LPH-KO引起线粒体转录因子A（TFAM）的蛋白质水平降低，TFAM是转录和维持mtDNA的必需因子，以及两种DNA修复酶，8-氧代鸟嘌呤DNA糖基化酶（OGG 1）和DNA连接酶3（LIG 3），两者都参与线粒体碱基切除修复（BER）。因此，有人认为，mtDNA损伤的增加是由于TFAM缺乏导致的mtDNA不稳定性的累积效应和BER相关酶缺乏导致的BER能力降低。这些发现表明，M-LPH可能参与线粒体DNA的维持，因此线粒体功能，通过保护蛋白质至关重要的线粒体DNA的稳定性和维护，在一个综合的方式。
Human Mpv17-like protein (M-LPH) has been suggested to participate in prevention of mitochondrial dysfunction caused by mitochondrial DNA (mtDNA) damage. To clarify the molecular mechanism of M-LPH function, we knocked out M-LPH in human hepatoma HepG2 using CRISPR-Cas9 technology. An increase in mtDNA damage in M-LPH-KO HepG2 cells was demonstrated by PCR-based quantitation and 8-hydroxy-2′-deoxyguanosine (8-OHdG) measurement. Furthermore, confocal immunofluorescence analysis and Western blot analysis of mitochondrial extracts demonstrated that M-LPH-KO caused reductions in the protein levels of mitochondrial transcription factor A (TFAM), an essential factor for transcription and maintenance of mtDNA, and two DNA repair enzymes, 8-oxoguanine DNA glycosylase (OGG1) and DNA ligase 3 (LIG3), both involved in mitochondrial base excision repair (BER). Accordingly, it was suggested that the increase in mtDNA damage was due to a cumulative effect of mtDNA instability resulting from deficiencies of TFAM and diminished ability for BER arising from deficiencies in BER-related enzymes. These findings suggest that M-LPH could be involved in the maintenance of mtDNA, and therefore mitochondrial function, by protecting proteins essential for mtDNA stability and maintenance, in an integrated manner.
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影响因子：
4.2
作者：
Keeney, Paula M.;Quigley, Caitlin K.;Bennett, James P., Jr.
通讯作者：
Bennett, James P., Jr.
影响因子：
5.3
作者：
Iida, Reiko;Ueki, Misuzu;Yasuda, Toshihiro
通讯作者：
Yasuda, Toshihiro
DOI：
10.1016/j.bbrc.2006.04.008
发表时间：
2006-06-09
影响因子：
3.1
作者：
Iida, Reiko;Yasuda, Toshihiro;Kishi, Koichiro
通讯作者：
Kishi, Koichiro
影响因子：
4.6
作者：
Kunova, Nina;Ondrovicova, Gabriela;Pevala, Vladimir
通讯作者：
Pevala, Vladimir
影响因子：
4.8
作者：
Iida, R;Yasuda, T;Kishi, K
通讯作者：
Kishi, K