We performed a comprehensive approach to determine the proteome of Saccharomyces cerevisiae mitochondria. The proteins of highly pure yeast mitochondria were separated by several independent methods and analyzed by tandem MS. From >20 million MS spectra, 750 different proteins were identified, indicating an involvement of mitochondria in numerous cellular processes. All known components of the oxidative phosphorylation machinery, the tricarboxylic acid cycle, and the stable mitochondria-encoded proteins were found. Based on the mitochondrial proteins described in the literature so far, we calculate that the identified proteins represent Ϸ90% of all mitochondrial proteins. The function of a quarter of the identified proteins is unknown. The mitochondrial proteome will provide an important database for the analysis of new mitochondrial and mitochondria-associated functions and the characterization of mitochondrial diseases.
We identified eukaryotic translation elongation factor 1A (eEF1A) Raf-mediated phosphorylation sites and defined their role in the regulation of eEF1A half-life and of apoptosis of human cancer cells. Mass spectrometry identified in vitro S21 and T88 as phosphorylation sites mediated by B-Raf but not C-Raf on eEF1A1 whereas S21 was phosphorylated on eEF1A2 by both B- and C-Raf. Interestingly, S21 belongs to the first eEF1A GTP/GDP-binding consensus sequence. Phosphorylation of S21 was strongly enhanced when both eEF1A isoforms were preincubated prior the assay with C-Raf, suggesting that the eEF1A isoforms can heterodimerize thus increasing the accessibility of S21 to the phosphate. Overexpression of eEF1A1 in COS 7 cells confirmed the phosphorylation of T88 also in vivo. Compared with wt, in COS 7 cells overexpressed phosphodeficient (A) and phospho-mimicking (D) mutants of eEF1A1 (S21A/D and T88A/D) and of eEF1A2 (S21A/D), resulted less stable and more rapidly proteasome degraded. Transfection of S21 A/D eEF1A mutants in H1355 cells increased apoptosis in comparison with the wt isoforms. It indicates that the blockage of S21 interferes with or even supports C-Raf induced apoptosis rather than cell survival. Raf-mediated regulation of this site could be a crucial mechanism involved in the functional switching of eEF1A between its role in protein biosynthesis and its participation in other cellular processes.
Mouse models are frequently used to study chronic liver diseases (CLDs). To assess their translational relevance, we quantified the similarity of commonly used mouse models to human CLDs based on transcriptome data. Geneexpression data from 372 patients were compared with data from acute and chronic mouse models consisting of 227 mice, and additionally to nine published gene sets of chronic mouse models. Genes consistently altered in humans and mice were mapped to liver cell types based on single-cell RNA-sequencing data and validated by immunostaining. Considering the top differentially expressed genes, the similarity between humans and mice varied among the mouse models and depended on the period of damage induction. The highest recall (0.4) and precision (0.33) were observed for the model with 12-months damage induction by CCl 4 and by a Western diet, respectively. Genes consistently upregulated between the chronic CCl 4 model and human CLDs were enriched in inflammatory and developmental processes, and mostly mapped to cholangiocytes, macrophages, and endothelial and mesenchymal cells. Down-regulated genes were enriched in metabolic processes and mapped to hepatocytes. Immunostaining confirmed the regulation of selected genes and their cell type specificity. Genes that were up-regulated in both acute and chronic models showed higher recall and precision with respect to human CLDs than exclusively acute or chronic genes. Conclusion: Similarly regulated genes in human and mouse CLDs were identified. Despite major interspecies differences, mouse models detected 40% of the genes significantly altered in human CLD. The translational relevance of individual genes can be assessed at https://saezl ab.shiny apps.io/liver disea seatl as/. (Hepatology Communications 2021;0:1-17).
Highlights d Agxt is hypermethylated and downregulated in mouse and human steatotic hepatocytes d Detoxification of mitochondrial glyoxylate is insufficient in the fatty liver d Challenge with dietary hydroxyproline leads to higher oxalate in the fatty liver d Rescuing Agxt expression in the fatty liver reduces hepatic oxalate production
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