Die Projekte mit den Projektnummern SP1, SP8, SP9, SP11, SP12, SP13, SP15, SP19 beschäftigen sich mit nicht-enzymatischen Proteinmodifikationen.
Die Projekte mit den Projektnummern SP2, SP3, SP7, SP10, SP14, SP16, SP17, SP18, SP20, SP21 beschäftigen sich mit enzymatischen Proteinmodifikationen.
Supervisor:: Marcus A. Glomb
PhD student: Christian Jokiel
Retinol and its derivatives regulate processes including vision, growth, cellular differentiation and proliferation. In addition, retinoids are known to regulate epigenetic phenomena such as DNA methylation and post-translational modification of histone proteins. They have therefore been suggested to influence the molecular biology of cellular ageing. Plasma retinol-binding protein (RBP4) is the principal carrier of vitamin A in the blood of vertebrates. In plasma, RBP4 is bound to a transthyretin (TTR) homotetramer, which is known to be the cause of senile systemic amyloidosis in elderly people. SP1 investigates the influence of glycation on major protein-based transport mechanisms such as the retinol-RBP4-TTR complex. Status of glycation in vitro and in vivo will be assessed by means of LC-MS/MS and binding characterised by equilibrium binding assays.
SP2: Acetylation and glycosylation in protein folding and misfolding: Impact on ageing-related diseases
Supervisor: Rüdiger Horstkorte
PhD student: Linda Kulka, Carolin Neu
Acetylation and glycosylation are widespread posttranslational modifications (PTMs) involved in diverse cellular processes and can affect the structure of proteins. We have demonstrated that proteins can misfold and form amyloid structures upon inhibition of histone deacetylases (HDACs) in clinically relevant concentrations. Acetylation plays a role in the aetiology of ageing-related proteinopathies such as in Alzheimer’s disease, Parkinson’s disease or Huntington’s disease. Furthermore, aggregate formation is fostered by mis-glycosylation of proteins; for instance, mutations in one key enzyme of terminal glycosylation (sialylation) are responsible for the age-dependent GNE (bifunctional UDP-N-acetylglucosamine 2-epimerase/N-acetyl-mannosamine kinase) myopathy. We aim to investigate how acetylation and glycosylation influence protein folding and misfolding, in particular proteins involved in ageing-related diseases. We also analyse, how protein shuttling factors can ameliorate protein misfolding and how a different PTM patterns can influence the shuttling process.
SP3: The function of the vertebrate GID-complex in the regulation of cellular energy homeostasis and lifespan extension
Supervisor: Thorsten Pfirrmann
PhD student: Lisa Fechtner (until 01/2022)
Dysregulation of AMPK activity is involved in a variety of age-related metabolic diseases and syndromes like type-2-diabetes (1-4). We discovered and recently published an ATP/AMP-independent regulation mechanism that depends on the ubiquitin-ligase function of the vertebrate GID-complex. Our data show that the GID-complex regulates AMPK activity by ubiquitination. Lack of AMPK ubiquitination leads to constitutive enzyme activation, to a subsequent shift from anabolic to catabolic pathways (branched chain amino acid (BCAA) degradation, beta-oxidation, autophagy), to an increase in organismal lifespan (5) and to a 40% reduction of cellular acetyl-CoA. We hypothesise that the GID-complex is a high potential drug target to affect AMPK activity. To pharmacologically interfere with GID-function in the future we need to obtain a better understanding of the molecular mechanism behind GID-dependent AMPK regulation. We plan to (I) further biochemically characterise AMPK ubiquitination-sites (Ub-sites) by immunoprecipitation and subsequent MS-analytics and (II) study the impact of acetyl-CoA levels on AMPK/GID-activity and organismal lifespan.
Supervisor: Anne Navarrete Santos
PhD student: Alicia Toto Nienguesso
Our interest focuses on changes in histone modifications due to maternal age and metabolic changes in the cell environment. A previous study showed that cell metabolites and age affect histone modifications in stem cells (Jung et al. 2019). We are investigating the potential molecular mechanisms based on the crosstalk of two important posttranslational modifications - histone methylation and methylase activation by O-GlcNAcylation (addition of N-acetylglucosamine (GlcNac) to serine/threonine residues). The O-GlcNAcylation of EZH2 at several serine and threonine moieties such as Ser75 is required for EZH2 protein stability and therefore facilitates the histone H3 trimethylation at K27 to form H3K27me3 (Chu et al. 2013, Lo et al. 2018). Both O-linked GlcNAc transferase (OGT)-mediated O-GlcNAcylation and EZH2-mediated H3K27me3 formation play a pivotal role in adipogenic development and stem cell maintenance. The hypothesis is that specific EZH2 O‑GlcNAcylation regulates stem cell properties and provides a new approach for cellular ageing in embryonic and adult stem cells.
Supervisor: Andreas Simm
PhD student: Shubhangi Karande, Yazan Dalilah
Persistent epigenetic modifications, such as histone modifications, can change gene expression and the ageing process. Glycation-induced histone modification in mice was found to change with age and as acetylation and glycation targets the same amino acid residues a competition for the respective amino acids is possible.
As young cells mainly get their energy from mitochondrial ATP generation, while senescent cells obtain ATP from glycolysis, we want to test the hypothesis that glycation based on dicarbonyls from the metabolism have an impact on epigenetic regulation.
After identification of glycated nuclear proteins by MS technology, the in-vivo relevance 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: Protein glycation and glutathionylation as modulators of the activity of pro-ageing factors in C. elegans
Supervisor: Lars-Oliver Klotz
PhD student: Thilo Philipp, Anne Sophie Scheller
Caenorhabditis elegans harbors orthologs of human selenium-binding protein-1, with Y37A1B.5 (short: Y37) among them. We have found that Y37 is a pro-aging 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 analyze 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.
Supervisor: Christian A. Hübner
PhD student: Andrea Bock (students representative), Mariam Kareli
Age-related sarcopenia is associated with a decrease in muscle mass, muscle contractile function, increased muscle cell apoptosis, and a diminished ability of the aged muscle to respond to hypertrophic stimuli. Similar alterations have been observed in myopathies that result from glycosylation defects of the dystrophin/dystroglycan complex. Therefore, we will address whether the glycosylation status of selected skeletal muscle proteins including members of dystrophin/dystroglycan complex changes during ageing. Because we could previously show that the glycosylation defects observed in a rare congenital disorder of glycosylation caused by defects in GDP-mannose-pyrophosphorylase-A (GMPPA) can be improved by dietary means, we will assess whether dietary habits can affect glycosylation in the ageing muscle.
Supervisor: Claudia Grossmann
PhD student: Yekaterina Gadasheva, Arooj Fatima
The mineralocorticoid receptor (MR) is an aldosterone-dependent transcription factor that physiologically regulates blood pressure but under certain circumstances 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 lies in exploring the influence of nitrosative stress, especially nitric oxide (NO) and peroxynitrite (ONOO-), on the PTM pattern of the MR and to explore the influence of nitrosative stress on MR signalling and function.
Supervisor: Stefan Heinemann
PhD student: Marwa Ahmed, Anogh Muthukumar
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: Principal investigator: Regine Heller / PhD student: Nasrin Haghnazari Sadaghiani - AGE modifications in endothelial cells – functional characterisation and relevance for senescence and ageing
Supervisor: Regine Heller
PhD student: Nasrin Haghnazari Sadaghiani, Shivani Prabhu
Previous studies of our group revealed that treatment of endothelial cells with glyoxal induced robust formation of advanced glycation end products (AGEs) and alterations in protein abundance. 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 (mice origin or human endothelial cells prepared from adipose tissue of patients (<30 years or >65 years).
Supervisor: Otmar Huber
PhD student: Dhanalakshmi Jothi, Pauline Förster
Ageing-associated changes in tissue homeostasis are a consequence of multiple molecular and cellular changes induced by alterations of signalling and metabolic pathways and accumulation of damaged molecules due to impaired repair mechanisms. We recently identified Nit1 as a new binding partner of b-catenin and repressor of the canonical Wnt-pathway disrupting the b-catenin/TCF-complex. Stress conditions were reported to redirect b-catenin from TCF to FOXO3a thereby activating FOXO3a transcriptional activity. Our results indicate that Nit1 is differentially regulated in stress responses and thereby could contribute to this effect. Interestingly, Nit1 has a second role as a metabolite repair enzyme acting as a dGSH (deamino-glutathione)-hydrolase. dGSH is generated as a side product of transaminase reactions. We here want to assess this dual role of Nit1 in stress response and investigate which posttranslational modifications modulate this activity during ageing and senescence.
Supervisor: Alessandro Ori
PhD student: Antonio Marino, Amit Kumar Sahu
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 post-translational 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 carboxymethyl lysine (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.
SP16: Glycosylation of Alzheimer-associated proteins in the context of microglia-mediated inflammation and disturbed blood-brain barrier functions
Supervisor: Thomas Hollemann, Dr. Jung
PhD student: Chaudry Luqman Abid, Maira Tariq
Age-dependent disturbed immune plasticity of microglia and disturbed blood-brain barrier BBB functionality may provide early phenotypes of late-onset Alzheimer's disease (LOAD). LOAD is age-related and the number of patients grows steadily not only in Germany. We published mutations (TREM2, ABCA7) and described an association (CD33) of microglia- and BBB-related genes that confer a risk for LOAD. These proteins undergo LOAD-associated post-translational modifications (PTMs) including glycosylation and particularly sialylation. We therefore ask how glycosylation of CD33, TREM2, and ABCA7 contribute to LOAD and how are alterations in these PTMs relate to cellular ageing mechanisms? With support of the RTG2155, we will analyse the impact of mutations on PTMs. We established LOAD-specific induced pluripotent stem (iPS) cell models for microglia and BBB cultures. We will apply these in vitro models for analysing the impact of PTMs on the functionality of TREM2, ABCA7, and CD33 and their contribution to the onset of LOAD.
Supervisor: Christoph Kaether
PhD student: Zahra Fanaei Kahrani, Tornike Nasrashvili
The anti-aging protein Klotho is strongly expressed in kidney and choroid plexus (CP). In kidney, Klotho full length functions as a co-receptor for FGF23. In addition, Klotho is shed from the surface and, together with a secreted splice form of Klotho, supplies the periphery with soluble Klotho (Klothos). In the CP the function of Klotho is not known, but Klothos is a constituent of cerebrospinal fluid (CSF) produced by the CP. In mice and humans, Klotho is involved in cognitive performance and is down-regulated in ageing. Klotho is heavily glycosylated and that could affect its stability or activity. In our project we want to test whether Klotho activity and/or stability is modulated by enzymatic and non-enzymatic PTM. We also analyse the role of Klotho in brain ageing using specific mouse knock-out models. Finally, we want to analyse the role of mammalian Klotho in IGF1/FOXO signalling and its modulation by PTMs.
Principal investigator: Julia von Maltzahn
During ageing and in models of premature ageing the number and functionality of muscle stem cells decreases dramatically. Different signalling pathways have been reported to be altered in aged muscle stem cells. Therefore, we are investigating which intrinsic changes in muscle stem cells occur during ageing that impair their functionality. Furthermore, we are examining the interaction of the muscle stem cell with its niche and how changes in the posttranslational modifications of membrane receptors such as glycosylation are changing in muscle stem cells during ageing. Additionally, we are analysing how these changes are affecting downstream signalling pathways.
SP19: Bioinformatics analysis of transcriptional and epigenomic responses to posttranslational modifications
Principal investigator: Steve Hoffmann
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 localization 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 contribute our wet-lab expertise to 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.
SP20: Identification of novel O-GlcNAcylated substrates in endothelial cells and investigation of their role in senescence and ageing
Principal investigator: Florian Meier, Darya Zibrova
The modification of proteins with O-linked N-acetylglucosamine (O-GlcNAc) is an essential post-translational modification that regulates protein function. Increased O-GlcNAcylation underlies the aetiology of age-related vascular pathologies via unknown mechanisms. However, O-GlcNAcylated substrates in vascular endothelium are barely studied and the identification of O-GlcNAc modification sites remains a technological challenge. We aim to establish a novel mass spectrometry-based approach for the identification of O-GlcNAcylated proteins in vascular endothelial cells using ion mobility spectrometry. With this workflow, we will study the role of O-GlcNAcylation in endothelial cell function and senescence. In particular, we will focus on the AMPK-GFAT1 signalling axis in this system.
Principal investigator: Patricia Franzka, Sonnhild Mittag, Christian Hübner
Glycosylation is the most common post-translational modification of proteins and lipids. It plays a prominent role in protein stability and conformation, cell-to-cell-communication, cell-matrix-interaction, adhesion, protein targeting and folding. We recently identified mutations in GDP-mannose-pyrophosphorylase-A (GMPPA) in patients suffering from muscle weakness, gait abnormalities, achalasia, alacrima and mental retardation (AAMR syndrome). We could subsequently show that GMPPA acts as an allosteric feedback inhibitor of GMPPB, which catalyzes the production of GDP-mannose, a key substrate for glycosylation. As a consequence, GMPPA defects result in the hyperglycosylation of several proteins. Here, we will use our GMPPA KO mouse model and different in vitro approaches to assess the consequences of GMPPA defects for brain development and ageing.
Principal investigator: Andrea Henze
PhD student: Theresa Hofmeister
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 and induce insulin secretion even in the absence of glucose (7, 8) though the exact mechanisms have not been evolved yet.
Since kinases and phosphatases are frequent targets of redox-modulation (9,10) 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.
Therefore, it is the specific aim of this project is to characterize the cross-talk of protein phosphorylation and oxidation during insulin secretion stimulated by different secretogogue in vitro and in vivo.
For this purpose, we will characterize changes in both PTMs during insulin secretion in permanent insulin secreting cell lines (i.e. INS-1E) as well as primary murine islets isolated from young as well as old animals. Additionally, we will analyze protein oxidation and phosphorylation in pancreatic tissue of rats considering effects of diet, age and gender.