An impaired respiratory electron chain triggers down-regulation of the energy metabolism and de-ubiquitination of solute carrier amino acid transporters

Abstract

Hundreds of genes have been associated with respiratory chain disease (RCD), the most common inborn error of metabolism so far. Elimination of the respiratory electron chain by depleting the entire mitochondrial DNA (mtDNA, rho0 cells) has therefore one of the most severe impacts on the energy metabolism in eukaryotic cells. In this study, proteomic data sets including the post transcriptional modifications (PTMs) phosphorylation and ubiquitination were integrated with metabolomic data sets and selected enzyme activities in the osteosarcoma cell line 143B.TK. A shotgun based SILAC LC-MS proteomics and a targeted metabolomics approach was applied to elucidate the consequences of the rho0 state. Pathway and protein protein interaction (PPI) network analyses revealed a non-uniform down-regulation of the respiratory electron chain, the tricarboxylic acid (TCA) cycle and the pyruvate metabolism in rho0 cells. Metabolites of the TCA cycle were dysregulated, such as a reduction of citric acid and cis-aconitic acid (6- and 2.5-fold), and an increase of lactic acid, oxalacetic acid (both 2-fold), and succinic acid (5-fold) in rho0 cells. Signaling pathways such as GPCR, EGFR, G12/13 alpha and Rho GTPases were up-regulated in rho0 cells, which could be indicative for the mitochondrial retrograde response, a pathway of communication from mitochondria to the nucleus. This was supported by our phosphoproteome data, which revealed two main processes, GTPase-related signal transduction and cytoskeleton organization. Furthermore, a general de-ubiquitination in rho0 cells was observed, for example, 80S ribosomal proteins were in average 3-fold and SLC amino acid transporters 5-fold de-ubiquitinated. The latter might cause the observed significant increase of amino acids levels in rho0 cells. We conclude that an elimination of the respiratory electron chain, e.g. mtDNA depletion, not only leads to an uneven down-regulation of mitochondrial energy pathways, but also triggers the retrograde response.