Persistent epigenetic modifications, such as histone acetylation, can change gene expression and the ageing process. Histone glycation was found to change with age in mice and may compete with acetylation since both modifications target the same amino acid. As young cells mainly get their energy from mitochondrial ATP generation, while senescent cells obtain ATP from glycolysis leading to increased formation of glycating agents such as dicarbonyls, we want to test the hypothesis that dicarbonyl-induced glycation has an impact on epigenetic regulation.
After identification of glycated nuclear proteins by mass spectrometry, the in vivo-relevance of these findings will be tested in our ageing mouse cohort and in patient samples. Depending on the proteins identified, their role in cell signaling, gene transcription / translation and regulation will be analyzed. Molecular consequence of the glycation on protein function and structure will be analyzed by structural biology methods including NMR spectroscopy.
SP9: Biological relevance of the H2S-producing selenium-binding protein 1 (SELENBP1)
Thilo Phillip (PhD):Protein glycation and glutathionylation as modulators of the activity of pro-ageing factors in C. elegans
Cenorhabditis elegans (C. elegans) harbors orthologs of human selenium-binding protein-1 with Y37A1B.5 (short: Y37) among them. We have found that Y37 is a pro-ageing factor attenuating stress resistance and life span, while at the same time providing worms with an advantage in selenium-containing environments. Whereas this provides an explanation for the use an organism may have for a life-shortening protein, it is still unclear how Y37 impairs stress resistance and lifespan. According to transcriptome analyses, Y37 is involved in sulfur and redox homeostasis, and its expression changes with age and is modulated by nutrient exposure. Our aims are (1) to identify the biological activity of selenium-binding protein orthologs such as Y37 protein and their contribution to C. elegans ageing; (2) to analyse the regulatory effect of glycation and glutathionylation, as markers of nutrient exposure and redox regulation, respectively, on expression and activity of these proteins as well as on C. elegans life span and stress resistance.
The mineralocorticoid receptor (MR) is an aldosterone-dependent transcription factor that physiologically regulates blood pressure but can also lead to inflammation, hypertrophy, fibrosis and endothelial dysfunctions in the cardiovascular system. Several studies indicate that a parainflammatory micro-milieu with oxidative and nitrosative stress represents a possible trigger to induce pathophysiological MR effects. During ageing or cardiovascular diseases, vascular cells are increasingly exposed to such a parainflammatory micro-milieu. We hypothesise that this leads to changes in the aldosterone-induced posttranslational modification (PTM) pattern of MR or associated proteins, resulting in an inadequately high MR activation and consequently leading to pathophysiological MR effects. The focus of our investigations is to explore the influence of nitrosative stress, especially nitric oxide (NO) and peroxynitrite (ONOO-), on the PTM pattern of the MR and on MR signalling and function.
SP12: Visualization of protein modifications and their impact on the electrical signalling
Principal Investigator: Stefan H. Heinemann (Dept. of Biophysics, CMB, FSU Jena and Jena University Hospital)
Oxidation of methionine residues in proteins gives rise to structural and functional modifications that can accumulate in aged tissue. While massive methionine oxidation results in loss of protein function and degradation, in select cases the protein modification Met(O) persists. We therefore develop and validate molecular fluorescence probes suited to monitor methionine oxidation and the incorporation of selenomethionine in living cells. We are particularly interested in such cases where Met oxidation affects voltage-gated ion channels to exert a gain-of-function effect, i.e., situations in which even a small fraction of modified proteins may cause significant changes in organ function. In NaV channels even minute impairment of inactivation leads to electrical hyperexcitability and an array of pathophysiological phenomena such as cardiac arrhythmia, myotonia, epilepsy, and altered pain sensation. We furthermore investigate how oxidative and nitrosative stress affects the signaling in insulin-secreting INS1 cells.
Marwa Ahmed (PhD):Visualization of protein modifications and their impact on the electrical signalling
Oxidation of methionine residues in proteins gives rise to structural and functional modifications that can accumulate in aged tissue. While massive methionine oxidation results in loss of protein function and degradation, in select cases the protein modification Met(O) persists. We therefore develop and validate molecular fluorescence probes suited to monitor methionine oxidation in living cells and organisms such as yeast and C. elegans. We are particularly interested in such cases where Met oxidation affects voltage-gated ion channels to exert a gain-of-function effect, i.e. situations in which even a small fraction of modified proteins may cause significant changes in organ function. In NaV channels, for example, even minute impairment of inactivation leads to electrical hyperexcitability and an array of pathophysiological phenomena such as cardiac arrhythmia, myotonia, epilepsy, and altered pain sensation. We furthermore investigate potential secondary modification steps, e.g. the formation of protein networks stabilised by sulfilimine bonds.
SP13: Functional characterization of CML modifications in endothelial cells
Principal Investigator: Regine Heller (Institute of Molecular Cell Biology, University Hospital Jena)
Rohama Zahid (PhD):Functional characterization of carboxymethylated proteins in endothelial cells
Ageing is known to be associated with the accumulation of advanced glycation end products such as carboxymethylated proteins in cells and tissues. However, little is known about how cells respond to glycative stress and how glycation of specific proteins influences cell signaling and function. Previous studies of our group revealed that treatment of endothelial cells with the reactive dicarbonyl glyoxal induced robust formation of carboxymethyllysine (CML) as well as alterations in protein abundance. More than 120 CML-modified proteins mainly related to metabolism and cytoskeleton regulation and the respective sites were identified by mass spectrometry analysis. One of the identified carboxymethylated proteins is tubulin whose modification seems to lead to altered microtubule dynamics. The current project is aimed at characterizing microtubule dynamics in living endothelial cells using fluorescence-labeled tubulin and relate it to possible microtubule dysfunction. Furthermore, CML modification of tubulin will be characterized in different models of endothelial ageing and
Previous studies of our group revealed that treatment of endothelial cells with glyoxal induced robust formation of carboxymethyllysine (CML), an advanced glycation end product (AGE) as well as alterations in the abundance of cellular proteins. Among others we found an increased expression and activity of AMP-activated protein kinase (AMPK), an important sensor and regulator of cellular energy metabolism, and an upregulation of heme oxygenase 1 (HO-1). The current project will reveal mechanisms and functional consequences of AMPK upregulation in response to enhanced AGE formation with a special focus on autophagy regulation by AMPK. In addition, we will test, which role the Nrf2-HO-1 pathway plays in counteracting glycative stress. In this context, we will link CML modification of selected proteins to their respective functions and test their role in replicative senescent or chronologically aged endothelial cells.
Julian Herpell (MD): Age-dependent alterations in the transcriptome and proteome of murine endothelial cells
Cardiovascular diseases are one of the leading causes of death in western countries. They are often associated with aging processes in the vascular system, in which the accumulation of senescent endothelial cells seems to play a role. Previous studies from our group have characterized replicative senescent endothelial cells in culture. Compared to young cells, they exhibit higher glycolytic activity, an increased oxidative glucose metabolism and accumulation of lipid droplets, which is linked to alterations in the expression pattern of metabolic enzymes. So far, however, chronologically aged endothelial cells have not been investigated. Thus, this project focuses on the characterization of transcriptomic and proteomic alterations in microvascular endothelial cells isolated from mice of increasing age (3 to 30 months) with a special focus on metabolic processes. Based on these studies, specific pathways will be selected to validate the obtained data by analyzing protein expression and function as well as metabolite profiles.
SP15: Comprehensive Landscape of Age-induced alterations in protein Ubiquitination and Deubiquitination
Amit Kumar Sahu (PhD):Comprehensive Landscape of Age-induced alterations in protein Ubiquitination and Deubiquitination
The ability to maintain a functional proteome declines during the aging process, which results in dysregulated protein homeostasis and the accumulation of damaged, misfolded, and partially ubiquitinated proteins and constitutes a major hallmark of the aging process. Protein degradation via the ubiquitin-proteasome system requires post-translational modification of the substrate with ubiquitin and subsequent formation of polyubiquitin chains via linkage type specific lysine residues within ubiquitin. We have previously shown that a subset of enzymes involved in the ubiquitin cycle are perturbed in their abundance with aging, this includes deubiquitinases (DUBs) like UCHL1 being more abundant in the brain of old killifish and mice. In this proposal, we aim (I) to understand the function of linkage type specific polyubiquitin chains in the context of aging, and (II) to systematically assess the activity and specificity of DUBs in the aging brain.
The homeostasis of the proteome of cells is required to maintain the function of organs and it was shown to decline during ageing. Advanced glycation end products (AGEs) are a family of non-enzymatic posttranslational modifications that have been shown to accumulate in ageing tissues. Although a handful of specific AGE-modified proteins have been identified, a detailed characterisation of the targets of AGEs is still missing. Previously, we have devised a mass spectrometry based approach to identify specific sites of carboxymethyllysine (CML, one of the most abundant AGEs) modifications in proteins. Using this approach, we identified over 1000 CML sites in cells treated with glycating agents and over 200 sites in primary tissues. In addition, we have established targeted proteomic assays based on isotopically labeled peptide standards for a subset of identified CML sites. In this project, we would like to investigate the functional role of these modifications and their relationships to the ageing process.
SP19: Bioinformatics analysis of transcriptional and epigenomic responses to posttranslational modifications
Tushar Patel (PhD):Development of novel epigenetic clocks based on posttranslational modifications of histones
Posttranslational modifications of histone proteins belong to key remodelers of chromatin. The chromatin state can be altered by environmental stimuli, which subsequently affects the expression of genes associated with aging and longevity. As shown in recent studies, various histone modifications undergo dynamic changes during aging. Some active (e.g., H3K9ac and H4K16ac) or repressive marks (e.g., H3K9me3 and H3K27me3) may be globally increased or decreased, respectively, during the aging process. In this project we will develop integrative bioinformatic approaches to model the dynamical changes of different histone modifications during aging. Using machine learning techniques, we aim to develop a novel epigenetic clock based on the posttranslational modifications of histones. The integration with further epigenomic layers such as open chromatin, DNA methylation together with the transcriptome will help us to better understand the epigenetic mechanisms of aging and longevity.
Elina Wiechens (PhD):Bioinformatics analysis of transcriptional and epigenomic responses to posttranslational modifications
Posttranslational modification (PTMs) of transcription factors (TFs) and histones are critical mechanisms for regulating the genomic activity. For some TFs, chemical alterations of lysines are known to influence the protein localisation and affect essential protein-protein interactions as well as promoter and transcription factor binding site (TFBS) affinities. We develop bioinformatic approaches to better investigate the influence of such modifications on genome regulation. In this project, we will develop computational methods to better detect differential binding of TFs as well as differential start site usage. We will also measure the impact of various PTMs on TF-binding and isoform-specific expression levels. The computational integration of the ChIP and RNA data within ProMoAge will allow us to better understand the impact of certain PTMs on genome activity and to identify relevant functional networks. Besides, we provide solutions to integrate other layers of information such as DNA methylation systematically.
SP22: Cross-talk of protein oxidation and protein phosphorylation in insulin secretion
Principal Investigator: Andrea Henze (Institute of Agricultural and Nutritional Science, MLU Halle-Wittenberg)
Insulin secretion is associated with changes in the cellular redox status and in this context, it has been demonstrated that hydrogen peroxide (H2O2) is essential for glucose stimulated insulin secretion. Since kinases and phosphatases are frequent targets of redox-modulation and additionally essentially involved in insulin secretion we hypothesize that oxidative posttranslational modifications (oxPTMs) of kinases and phosphatases represent a rational link mediating the effects of reactive oxygen species (ROS) in this context. Additionally, ROS-overflow is an important factor in β-cell loss and exhaustion during aging and in age-related diseases. Therefore, it is the specific aim of this project is to characterize the cross-talk of protein phosphorylation and oxidation during insulin secretion in vitro and in vivo considering physiological as well as pathophysiological aspects in ROS concentration and β-cell function.