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

Oxid Med Cell Longev. 2018 Sep 17;2018:6956414. doi: 10.1155/2018/6956414. eCollection 2018.

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.

MeSH terms

  • DNA Damage / physiology*
  • DNA Glycosylases / biosynthesis*
  • DNA Ligase ATP / biosynthesis*
  • DNA, Mitochondrial*
  • DNA-Binding Proteins / biosynthesis*
  • Gene Knockout Techniques
  • Hep G2 Cells
  • Humans
  • Membrane Proteins / metabolism*
  • Mitochondrial Proteins / biosynthesis*
  • Poly-ADP-Ribose Binding Proteins / biosynthesis*
  • Transcription Factors / biosynthesis*

Substances

  • DNA, Mitochondrial
  • DNA-Binding Proteins
  • MPV17L protein, human
  • Membrane Proteins
  • Mitochondrial Proteins
  • Poly-ADP-Ribose Binding Proteins
  • TFAM protein, human
  • Transcription Factors
  • DNA Glycosylases
  • oxoguanine glycosylase 1, human
  • DNA Ligase ATP
  • LIG3 protein, human