Enzymatic PTMs

Principal Investigator: Rüdiger Horstkorte & Astrid Gesper & Kaya Bork (Institute for Physiological Chemistry, MLU Halle-Wittenberg) & Heidi Olzscha (Medical School Hamburg)

Carolin Neu (PhD): The impact of glycosylation and glycation on the generation and uptake of vehicles in aging cells

Tom Schneider (MD): Investigation of the influence of posttranslational modifications such as sialylation and glycation on protein half-life, cell adhesion and migration in wt and GNE-deficient HEK-293 cells

 (Poly)sialylation is integral to numerous cellular processes, including the regulation of cell-cell interactions and protein stability. The key enzyme of the sialic acid biosynthesis pathway is the bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), which catalyzes the epimerization and subsequent phosphorylation of UDP‑GlcNAc to ManNAc-6-phosphate. In this study, we will use HEK-293 cells and a variant lacking the GNE enzyme to examine how variations in sialylation levels affect the stability of downstream enzymes of the sialic acid biosynthesis pathway—NANS, NANP, and CMAS—as well as proteins that are typically polysialylated. Since a previous ProMoAge project demonstrated the influence of glycation on GNE activity, this study also aims to investigate the interplay between sialylation and glycation and their combined influence on the half-life of the aforementioned enzymes. Additionally, we will explore changes in cell behavior, particularly adhesion and migration, after glycation in these cells. The aim of this study is to elucidate the complex relationships between sialylation, glycation, and protein stability, together with their implications on cellular dynamics.

Principal Investigator: Anne Navarrete Santos (Institute of Anatomy and Cell Biology, MLU Halle-Wittenberg)

Silvia Leticia Monge Rodríguez (PhD): Role of the PPARy acetylation and the AMPK/ SIRT/ PPARy cascade in mesenchymal stem cell ageing

Adipose derived stem cells (ASC) reduce their adipogenic capacity when they become old, which leads to aging and increase the risk of obesity related diseases. How does the metabolic environment affect the capacity of differentiation of ASC? Our hypothesis is that gerometabolites and APOE affects the production of NAD+/NADH and AMPK activity, in consequence Sirtuin 1 —a deacetylase enzyme related with transcription factors regulation— and the peroxisome proliferator activated receptor gamma —essential for the plasticity and differentiation of mesenchymal stem cell— diminish their actions. The methodological approach of this project bases on two biological models - the New Zealand white APOE-/- rabbit and human mesenchymal stem cell lines from patients. ASCs from the different donors (grouped by age and metabolic status) will be analysed for AMPK-Sirt1-PPARy pathway activation, PPARy modification and adipogenic differentiation capacity.

Alicia Toto Nienguesso (PhD): Metabolic memory of cells as posttranslational modifications (PTMs) - PTMs as key regulators of metabolic ageing in adult stem cells

Our interest focuses on changes in histone modifications of stem cells, which were shown to depend on age and metabolic changes in the cell environment in a previous study. We are investigating the potential molecular mechanisms based on the crosstalk of two important posttranslational modifications - histone methylation and O-GlcNAcylation (addition of N-acetylglucosamine (GlcNac) to serine/threonine residues) of the methylase EZH2. The O-GlcNAcylation of EZH2 at several serine and threonine moieties such as serine 75 seems to be required for EZH2 protein stability in tumor cells and therefore may facilitate the histone H3 trimethylation at K27 to form H3K27me3. Both O-GlcNAcylation and EZH2-mediated H3K27me3 formation play a pivotal role in adipogenic development and stem cell maintenance. Our hypothesis is that specific O‑GlcNAcylation of EZH2 regulates stem cell properties and provides a new mechanism of cellular ageing in embryonic and adult stem cells.

Principal Investigator: Christian Hübner (Institute of Human Genetics, University Hospital Jena)

Abhijnan Chakraborty (PhD): Glycosylation in the ageing brain

Ageing is associated with a functional decline of the nervous system. This comes along with an overall decrease of brain mass and an altered synaptic function. Many proteins involved in synaptic function are glycosylated. Glycosylation plays a prominent role in protein stability and conformation, cell-to-cell communication, cell matrix interaction, adhesion, protein targeting and folding. Abnormal glycosylation of proteins can induce deleterious effects as observed in congenital disorders of glycosylation, which often result in serious, sometimes fatal malfunctions of different organ systems such as brain and muscle. While changes in protein glycosylation in the diseased brain are well characterized, it is yet unclear whether alterations in protein glycosylation may contribute to age-dependent changes in synaptic function and thus brain function. Therefore, we will assess the glycoproteome of the aging brain. For selected candidates we will then address how age-associated changes in glycosylation might contribute to the age-dependent decline of brain function.

Principal Investigator: Otmar Huber (Institute for Biochemistry II, Jena University Hospital)

Pauline Förster (PhD): The role of Nit1 in aging-associated stress response

In this project we want to identify effectors and pathways that are regulated by Nit1 and how these effectors affect aging-associated stress responses. Using a mass spectrometry-based approach to compare cells with a stable shRNA-mediated knock-down of Nit1 with scrambled shRNA-treated control cells, we want to screen for differentially regulated proteins. After bioinformatic analysis and verification of candidates we will study the role of Nit1 in the regulation of candidates specifically in the context of our previously observed crosstalk with FOXO3a and b-catenin signaling and how Nit-dependent regulation of these factors may affect senescence and aging.

Principal Investigator: Matthias Jung & Thomas Hollemann (Institute of Physiological Chemistry, MLU Halle Wittenberg)

Maira Tariq (PhD): Proteostasis at the old blood-brain barrier: Implications for late-onset Alzheimer’s disease

Ageing of the blood-brain barrier (BBB) results from an accumulation of deficiencies with contributions of senescence, increased inflammation, and oxidative stress. Though age related changes in BBB function may also represent an adaptation for healthy ageing, but results in consequence in a dysfunctional BBB, which often correlate with the progressive cause of brain diseases like the development of neural diseases, including late-onset Alzheimer's disease (LOAD). Ubiquitination and autophagy target proteins via post-translational modifications for regulating the protein homeostasis of amyloid precursor protein (APP) and its harmful byproduct amyloid beta (Aβ) in neurons, but also in other cells of the neurovascular unit including the BBB. Further, APP expression is considered as a neuroprotective response to stress factors with impact on healthy aging. Therefore, we ask how ubiquitination and autophagy in the BBB do change during ageing and how do these changes contribute to the development of LOAD. We study ubiquitination and autophagy in induced pluripotent stem cell-derived brain-capillary endothelial cells, pericytes, astrocytes, microglia, and neurons.

Judith Olex (MD): Endothelial Aging at the BBB: Impact of disturbed proteostasis on SARS-Cov-2 infection

The central nervous system is separated from the circulating blood by specialized endothelial cells that form a selective barrier to maintain proper conditions for neuronal function and cellular homeostasis, the so-called blood-brain barrier (BBB). However, it is not yet clear how aging disrupts the barrier function of the BBB and how these changes are related to various age-related diseases such as Alzheimer's disease or increased susceptibility to viral infections. There are currently various hypotheses that describe different mechanisms that also regulate ageing as the cause of Alzheimer's disease. One hypothesis states that disorders of the ubiquitin-proteasome system (UPS) promote the degradation of the BBB. The UPS is a selective protein degradation pathway in which the small protein ubiquitin is used as a marker to rapidly degrade proteins in the cell. When the UPS is disrupted, certain proteins can no longer be degraded correctly and amyloid beta (Aß) accumulates and damages the cells. Age- and/or disease-related dysfunction of the BBB has a major impact on whether SARS-CoV-2 infects the CNS. It is important to point out that elderly people suffered severely from the complications of COVID-19 infection, which manifested in long-lasting symptoms and permanent impairment. Recent studies suggest that infection of BCECs and transmigration of infected immune cells are crucial for CNS infiltration. It is therefore likely that changes to the BBB are of particular importance for the development and progression of COVID-19. Using induced pluripotent stem cells (iPSCs), we aim to better understand which changes in UPS-mediated Aß degradation are present in BCECs during aging and COVID-19. Therefore, iPSC-derived BCECs from young and old donors as well as from Alzheimer's patients will be analyzed. We will transfect BCECs with SARS-Cov-2 isolates and mimic SARS-CoV-2 infection by treatment with the corresponding spike proteins.

Principal Investigator: Christoph Kaether (FLI Jena)

Tornike Nasrashvili (PhD): Role of PTMs in senescent-induced transformation of the secretory pathway

One of the hallmarks of senescent cells is the senescence-associated secretory phenotype (SASP). The SASP-secretome is very well characterized, but the basis for the SASP is a rearrangement of the whole secretory pathway, indicated by morphological changes like dispersal of the Golgi, increase in lysosomal volume, increase in lysosomal lipofuscin aggregation and increased expression of the lysosomal enzyme b-galactosidase. Not surprisingly endoplasmatic reticulum (ER)-stress pathways including the UPR are up-regulated in senescent cells. Little is known about the molecular mechanisms mediating the rearrangement of the secretory pathway that ultimately results in SASP.

Specific aims of this project:

(1) To establish a comprehensive atlas of senescent-induced PTMs in the secretory pathway. (2) To mechanistically understand the impact on individual PTMs on proteins of the secretory pathway and their role in senescence and SASP-development. (3) To identify targets and prepare for screening assays for the development of future therapeutic interventions.

Zahra Fanaei Kahrani (PhD): Role of Klotho in brain ageing

The anti-aging protein Klotho is strongly expressed in kidney and choroid plexus (CP). In kidney, full length Klotho 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, which 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 (FLI Jena)

Christian Hayn (PhD): Molecular differences underlying functional impairment of aged muscle stem cells

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 and 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.

Principal Investigator: Florian Meier (Functional Proteomics, Jena University Hospital) & Daria Zibrova (Institute of Molecular Cell Biology, Jena University Hospital)

Felix Schneidmadel (PhD): Survey of the Post-Translational Modification Landscape in Ageing via
Trapped Ion Mobility Mass Spectrometry

Our current knowledge about biological processes involving protein post-translational modifications (PTMs) has been fueled by the widespread use of mass spectrometry-based proteomics. However, interactions between different PTMs remain largely elusive because state-of-the-art strategies typically analyze only one PTM at a time and involve tailored workflows to enrich a specific PTM of interest based on chemical or biochemical affinity. Conversely, all other peptides are discarded and, more critically, efficient enrichment strategies are lacking for most of the several hundreds of known in vivo modifications. Here, we propose to use an ‘open search’ bioinformatics approach combined with deep peptide fractionation to survey the PTM landscape of model systems in aging. We build on the recent development of rapid and sensitive trapped ion mobility mass spectrometry (TIMS-MS), which adds an additional dimension of separation as compared to conventional liquid chromatography – mass spectrometry.

Andreas Will (PhD): Identificatio of novel O-GlNAcylated substrates in endothelial cells and investigation of their role in senescence and ageing

The modification of proteins with O-linked N-acetylglucosamine (O-GlcNAc) is an essential posttranslational modification that regulates protein function. Increased O-GlcNAcylation underlies the aetiology of age-related vascular pathologies via unknown mechanisms. So far, O-GlcNAcylated substrates in vascular endothelium are barely studied and the identification of O-GlcNAc modification sites remains a technical 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.