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

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.

Loss of mitochondrial protease ClpP protects mice from diet‐induced obesity and insulin resistance

“…Previously, we found that knockdown of ClpP (70% reduction compared control cells) in C2C12 muscle cells increased the expression of mitochondrial fission protein dynamin‐related protein1, Drp1 . In gWAT of ClpP −/− mice, we did not see any change in the expression of fusion protein mitofusin 2 (Mfn2) or fission proteins Drp1 or Fis1 (data not shown).…”

“…In our previous study in C2C12 muscle cells, we performed an acute knockdown of ClpP and found that decline in ClpP (70% down-regulation) expression can cause mitochondrial dysfunction [13]. However, in ClpP À/À mice, such an effect was not observed in skeletal muscle.…”

Autophagy compensates impaired energy metabolism in CLPXP‐deficient Podospora anserina strains and extends healthspan

“…They certainly hold the key to understand the basis of the different responses. Apart from this basic relevance, our work provides novel data about the role of CLPXP, which emerges to be of important relevance for aging and specific adverse effects such as Perrault syndrome or different forms of human cancer (Fischer et al ., ; Gispert et al ., ; Jenkinson et al ., ; Cole et al ., ; Deepa et al ., ; Seo et al ., ). Studies using the P. anserina model system will continue to contribute to the elucidation of the cellular functions of this component of the mitochondrial quality control network and of its interactions with other cellular surveillance pathways.…”

Mitochondrial Quality Control in Age-Related Pulmonary Fibrosis

“…Several reports have demonstrated reduced activity of CLPP-1 in aged cells. As a result of deficient CLPP-1, cells were shown to have numerous alterations, such as a higher amount of ROS, decreased ATP production and downregulation of fusion markers, establishing a link between protease abnormalities, energy metabolism and mitochondrial dynamics [96,97]. In addition, CLPP-1-deficient aged cells were found to have an upregulation of mTORC1 signaling markers, suggesting that the increased activation of the mTORC1/PGC-1 axis in aged-AECII reported in our study could be a response to an upstream alteration in the mitochondrial matrix proteases [98,99].…”