Down-regulation of the mitochondrial matrix peptidase ClpP in muscle cells causes mitochondrial dysfunction and decreases cell proliferation

Sathyaseelan S. Deepa, Shylesh Bhaskaran, Rojina Ranjit, Rizwan Qaisar, Binoj C. Nair, Yuhong Liu, Michael E. Walsh, Wilson C. Fok, Holly Van Remmen

Research output: Contribution to journalArticle

29 Scopus citations

Abstract

The caseinolytic peptidase P (ClpP) is the endopeptidase component of the mitochondrial matrix ATP-dependent ClpXP protease. ClpP degrades unfolded proteins to maintain mitochondrial protein homeostasis and is involved in the initiation of the mitochondrial unfolded protein response (UPRmt). Outside of an integral role in the UPRmt, the cellular function of ClpP is not well characterized in mammalian cells. To investigate the role of ClpP in mitochondrial function, we generated C2C12 muscle cells that are deficient in ClpP using siRNA or stable knockdown using lentiviral transduction. Reduction of ClpP levels by ~70% in C2C12 muscle cells resulted in a number of mitochondrial alterations including reduced mitochondrial respiration and reduced oxygen consumption rate in response to electron transport chain (ETC) complex I and II substrates. The reduction in ClpP altered mitochondrial morphology, changed the expression level of mitochondrial fission protein Drp1 and blunted UPRmt induction. In addition, ClpP deficient cells showed increased generation of reactive oxygen species (ROS) and decreased membrane potential. At the cellular level, reduction of ClpP impaired myoblast differentiation, cell proliferation and elevated phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) suggesting an inhibition of translation. Our study is the first to define the effects of ClpP deficiency on mitochondrial function in muscle cells in vitro. In addition, we have uncovered novel effects of ClpP on mitochondrial morphology, cell proliferation and protein translation pathways in muscle cells.

Original languageEnglish (US)
Pages (from-to)281-292
Number of pages12
JournalFree Radical Biology and Medicine
Volume91
DOIs
StatePublished - Feb 1 2016
Externally publishedYes

Keywords

  • Abbreviations ClpP caseinolytic peptidase P
  • DMEM Dulbecco's modified Eagle's medium
  • DOX doxycycline
  • ECAR extracellular acidification rate
  • eIF2α eukaryotic initiation factor 2 alpha
  • ETC electron transport chain
  • FCCP carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone
  • GCN2 general control nonderepressible 2
  • HO hydrogen peroxide
  • Hsp60 heat shock protein 60
  • Mfn mitofusin
  • MHC myosin heavy chain
  • mtDNA mitochondrial DNA
  • OCR oxygen consumption rate
  • PINK1 phosphatase and tensin homolog-induced kinase 1
  • PKR protein kinase R
  • QC quality control
  • ROS reactive oxygen species
  • shRNA short hairpin RNA
  • TFAM mitochondrial transcription factor A
  • UPR mitochondrial unfolded protein response

ASJC Scopus subject areas

  • Biochemistry
  • Physiology (medical)

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