Mitochondrial LON protease-dependent degradation of cytochrome c oxidase subunits under hypoxia and myocardial ischemia

Biochim Biophys Acta Bioenerg. 2017 Jul;1858(7):519-528. doi: 10.1016/j.bbabio.2017.04.003. Epub 2017 Apr 23.

Abstract

The mitochondrial ATP dependent matrix protease, Lon, is involved in the maintenance of mitochondrial DNA nucleoids and degradation of abnormal or misfolded proteins. The Lon protease regulates mitochondrial Tfam (mitochondrial transcription factor A) level and thus modulates mitochondrial DNA (mtDNA) content. We have previously shown that hypoxic stress induces the PKA-dependent phosphorylation of cytochrome c oxidase (CcO) subunits I, IVi1, and Vb and a time-dependent reduction of these subunits in RAW 264.7 murine macrophages subjected to hypoxia and rabbit hearts subjected to ischemia/reperfusion. Here, we show that Lon is involved in the preferential turnover of phosphorylated CcO subunits under hypoxic/ischemic stress. Induction of Lon protease occurs at 6 to 12 h of hypoxia and this increase coincides with lower CcO subunit contents. Over-expression of flag-tagged wild type and phosphorylation site mutant Vb and IVi1 subunits (S40A and T52A, respectively) caused marked degradation of wild type protein under hypoxia while the mutant proteins were relatively resistant. Furthermore, the recombinant purified Lon protease degraded the phosphorylated IVi1 and Vb subunits, while the phosphorylation-site mutant proteins were resistant to degradation. 3D structural modeling shows that the phosphorylation sites are exposed to the matrix compartment, accessible to matrix PKA and Lon protease. Hypoxic stress did not alter CcO subunit levels in Lon depleted cells, confirming its role in CcO turnover. Our results therefore suggest that Lon preferentially degrades the phosphorylated subunits of CcO and plays a role in the regulation of CcO activity in hypoxia and ischemia/reperfusion injury.

Keywords: 3D modeling; CcO subunits; Heart ischemia; Hypoxia; Mitochondrial LON; PKA dependent phosphorylation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP-Dependent Proteases / chemistry
  • ATP-Dependent Proteases / genetics
  • ATP-Dependent Proteases / metabolism*
  • Animals
  • Cell Hypoxia / physiology*
  • Cyclic AMP-Dependent Protein Kinases / metabolism
  • Electron Transport Complex IV / metabolism*
  • Humans
  • Male
  • Mice
  • Mitochondria, Heart / enzymology*
  • Mitochondrial Proteins / chemistry
  • Mitochondrial Proteins / genetics
  • Mitochondrial Proteins / metabolism*
  • Models, Molecular
  • Myocardial Ischemia / enzymology*
  • Phosphorylation
  • Protein Conformation
  • Protein Processing, Post-Translational
  • Protein Subunits
  • RAW 264.7 Cells
  • RNA Interference
  • RNA, Small Interfering / genetics
  • Rabbits
  • Recombinant Proteins / metabolism

Substances

  • Mitochondrial Proteins
  • Protein Subunits
  • RNA, Small Interfering
  • Recombinant Proteins
  • Electron Transport Complex IV
  • Cyclic AMP-Dependent Protein Kinases
  • ATP-Dependent Proteases
  • LONP1 protein, human
  • LONP1 protein, mouse