Control of mitochondrial integrity in ageing and disease

Item does not contain fulltextVarious molecular and cellular pathways are active in eukaryotes to control the quality and integrity of mitochondria. These pathways are involved in keeping a 'healthy' population of this essential organelle during the lifetime of the organism. Quality control (QC) systems counteract processes that lead to organellar dysfunction manifesting as degenerative diseases and ageing. We discuss disease- and ageing-related pathways involved in mitochondrial QC: mtDNA repair and reorganization, regeneration of oxidized amino acids, refolding and degradation of severely damaged proteins, degradation of whole mitochondria by mitophagy and finally programmed cell death. The control of the integrity of mtDNA and regulation of its expression is essential to remodel single proteins as well as mitochondrial complexes that determine mitochondrial functions. The redundancy of components, such as proteases, and the hierarchies of the QC raise questions about crosstalk between systems and their precise regulation. The understanding of the underlying mechanisms on the genomic, proteomic, organellar and cellular levels holds the key for the development of interventions for mitochondrial dysfunctions, degenerative processes, ageing and age-related diseases resulting from impairments of mitochondria

Age-related changes in the mitochondrial proteome of the fungus Podospora anserina analyzed by 2D-DIGE and LC-MS/MS

Item does not contain fulltextMany questions concerning the molecular processes during biological aging remain unanswered. Since mitochondria are central players in aging, we applied quantitative two-dimensional difference gel electrophoresis (2D-DIGE) coupled to protein identification by mass spectrometry to study the age-dependent changes in the mitochondrial proteome of the fungus Podospora anserina - a well-established aging model. 67 gel spots exhibited significant, but remarkably moderate intensity changes. While typically the observed changes in protein abundance occurred progressively with age, for several proteins a pronounced change was observed at late age, sometimes inverting the trend observed at younger age. The identified proteins were assigned to a wide range of metabolic pathways including several implicated previously in biological aging. An overall decrease for subunits of complexes I and V of oxidative phosphorylation was confirmed by Western blot analysis and blue-native electrophoresis. Changes in several groups of proteins suggested a general increase in protein biosynthesis possibly reflecting a compensatory mechanism for increased quality control-related protein degradation at later age. Age-related augmentation in abundance of proteins involved in biosynthesis, folding, and protein degradation pathways sustain these observations. Furthermore, a significant decrease of two enzymes involved in the degradation of gamma-aminobutyrate (GABA) supported its previously suggested involvement in biological aging. BIOLOGICAL SIGNIFICANCE: We have followed the time course of changes in protein abundance during aging of the fungus P. anserina. The observed moderate but significant changes provide insight into the molecular adaptations to biological aging and highlight the metabolic pathways involved, thereby offering new leads for future research

Role of p38^MAPK and oxidative stress in copper-induced senescence

In the present work, we indicate that copper is involved in the senescence of human diploid fibroblasts and we describe mechanisms to explain it. Using different techniques, we show for the first time an accumulation of copper in cells during replicative senescence. This accumulation seems to be co-localized with lipofuscin. Second, we observed that an incubation of cells with copper sulfate induced oxidative stress, antioxidant response and premature senescence. Antioxidant molecules reduced the appearance of premature senescence. Third, we found that Nrf2 transcription factor was activated and regulated the expression of genes involved in antioxidant response while p38(MAPK) regulated the appearance of premature senescence. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11357-013-9521-3) contains supplementary material, which is available to authorized users

Structure and Biophysical Characterization of the S Adenosylmethionine dependent O Methyltransferase PaMTH1, a Putative Enzyme Accumulating during Senescence of Podospora anserina

Low levels of reactive oxygen species (ROS) act as important signaling molecules, but in excess they can damage biomolecules. ROS regulation is therefore of key importance. Several polyphenols in general and flavonoids in particular have the potential to generate hydroxyl radicals, the most hazardous among all ROS. However, the generation of a hydroxyl radical and subsequent ROS formation can be prevented by methylation of the hydroxyl group of the flavonoids. O-Methylation is performed by O-methyltransferases, members of the S-adenosyl-l-methionine (SAM)-dependent O-methyltransferase superfamily involved in the secondary metabolism of many species across all kingdoms. In the filamentous fungus Podospora anserina, a well established aging model, the O-methyltransferase (PaMTH1) was reported to accumulate in total and mitochondrial protein extracts during aging. In vitro functional studies revealed flavonoids and in particular myricetin as its potential substrate. The molecular architecture of PaMTH1 and the mechanism of the methyl transfer reaction remain unknown. Here, we report the crystal structures of PaMTH1 apoenzyme, PaMTH1-SAM (co-factor), and PaMTH1-S-adenosyl homocysteine (by-product) co-complexes refined to 2.0, 1.9, and 1.9 Å, respectively. PaMTH1 forms a tight dimer through swapping of the N termini. Each monomer adopts the Rossmann fold typical for many SAM-binding methyltransferases. Structural comparisons between different O-methyltransferases reveal a strikingly similar co-factor binding pocket but differences in the substrate binding pocket, indicating specific molecular determinants required for substrate selection. Furthermore, using NMR, mass spectrometry, and site-directed active site mutagenesis, we show that PaMTH1 catalyzes the transfer of the methyl group from SAM to one hydroxyl group of the myricetin in a cation-dependent manner

Association of mitochondrial antioxidant enzymes with mitochondrial DNA as integral nucleoid constituents

Mitochondrial DNA (mtDNA) is organized in protein-DNA macrocomplexes called nucleoids. Average nucleoids contain 2–8 mtDNA molecules, which are organized by the histone-like mitochondrial transcription factor A. Besides well-characterized constituents, such as single-stranded binding protein or polymerase γ (Polγ), various other proteins with ill-defined functions have been identified. We report for the first time that mammalian nucleoids contain essential enzymes of an integral antioxidant system. Intact nucleoids were isolated with sucrose density gradients from rat and bovine heart as well as human Jurkat cells. Manganese superoxide dismutase (SOD2) was detected by Western blot in the nucleoid fractions. DNA, mitochondrial glutathione peroxidase (GPx1), and Polγ were coimmunoprecipitated with SOD2 from nucleoid fractions, which suggests that an antioxidant system composed of SOD2 and GPx1 are integral constituents of nucleoids. Interestingly, in cultured bovine endothelial cells the association of SOD2 with mtDNA was absent. Using a sandwich filter-binding assay, direct association of SOD2 by salt-sensitive ionic forces with a chemically synthesized mtDNA fragment was demonstrated. Increasing salt concentrations during nucleoid isolation on sucrose density gradients disrupted the association of SOD2 with mitochondrial nucleoids. Our biochemical data reveal that nucleoids contain an integral antioxidant system that may protect mtDNA from superoxide-induced oxidative damage.—Kienhöfer, J., Häussler, D. J. F., Ruckelshausen, F., Muessig, E., Weber, K., Pimentel, D., Ullrich, V., Bürkle, A., Bachschmid, M. M. Association of mitochondrial antioxidant enzymes with mitochondrial DNA as integral nucleoid constituents