Abstract
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.
Highlights
Mitochondria are vital organelles in eukaryotic cells involved in essential functions such as energy production through oxidative phosphorylation (OXPHOS), maintenance of calcium homeostasis, regulation of apoptosis and necrosis, lipid metabolism, and immune responses [1]
As one possible explanation for this contradiction, we considered that mitochondrial dysfunction caused by Mitochondria possess their own double-stranded circular DNA (mtDNA) damage might be prevented by an increase in mtDNA copy number, as a number of studies have demonstrated the effectiveness of an increased mtDNA copy number for preservation of mitochondrial function [29, 30]
These findings strongly suggested that mitochondrial dysfunction due to mtDNA damage was prevented by an increase in mtDNA copy number, probably through a compensatory mechanism in HepG2 cells
Summary
Mitochondria are vital organelles in eukaryotic cells involved in essential functions such as energy production through oxidative phosphorylation (OXPHOS), maintenance of calcium homeostasis, regulation of apoptosis and necrosis, lipid metabolism, and immune responses [1]. Mitochondria possess their own double-stranded circular DNA (mtDNA), which encodes 22 tRNAs, 2 rRNAs, and 13 subunits of the OXPHOS complexes. There is evidence that BER proteins in mitochondria are localized to the inner membrane and to the nucleoid [12]. Details of the mechanism for regulation of these proteins involved in the maintenance of mtDNA integrity are not fully understood
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