Donnerstag, 25.09.2014

11:00 - 12:30 Uhr

Löwengebäude HS XIII

Vorsitz: Heller, Regine (Jena); Simm, Andreas (Halle (Saale))

Protein modifications and major degenerative diseases

Long-lasting protein modifications interfere with the function of regulatory networks in cells and tissues, thus contributing to age-related impairment of organ functions. For example, epigenetic mechanisms are based on long-lasting structural modifications of chromatin that modulate the availability of genes. They include DNA and histone methylation as well as histone acetylation / deacetylation, which control the transcription of genes crucial for cell viability and ageing. Epigenetic regulation of chromatin can be disrupted by age-associated non-enzymatic modifications, e.g. oxidation and/or glycation of histones. Protein oxidation is mediated by reactive oxygen species (ROS). Heavily oxidised proteins appear to first aggregate and then to form covalent cross-links that make them highly resistant to proteolysis which contribute to the accumulation of protein aggregates during diseases and the ageing process. Closely related to oxidative stress is the non-enzymatic glycation reaction. Reactive carbohydrates, such as glucose or fructose, can react non-enzymatically with free amino groups of proteins which lead to the formation of advanced glycation end products (AGEs). AGEs accumulate with age and foster for example tissue stiffening, a mechanism important for the dysfunction of the heart and /or vessel wall. Since enzymatic and non-enzymatic modifications target the same amino acid side chains, both processes can interfere. For example, a non-enzymatic AGE modification of lysine residues on histones may directly impact the epigenetic regulation by acetylation because both processes compete for the same site.

The symposium will demonstrate the impact of long-lasting protein modification on the development and outcome of major age-associated diseases like bronchial carcinomas, atherosclerosis, dementia and premature aging diseases.

11:00 Uhr

B. Bartling; H.-S. Hofmann1; A. Sohst; R.-E. Silber; V. Somoza2; A. Simm3
Universitätsklinik und Poliklinik für Innere Medizin, Herz- und Thoraxchirurgie, Universitätsklinikum, Martin-Luther-Universität Halle-Wittenberg, Halle (Saale); 1 Klinik für Thoraxchirurgie, Krankenhaus Barmherzige Brüder Regensburg, Regensburg; 2 Research Platform for Molecular Food Science, University of Vienna, Vienna/A; 3 Zentrum für Medizinische Grundlagenforschung (ZMG), Medizinische Fakultät, Martin-Luther-Universität Halle-Wittenberg, Halle (Saale);


Advanced glycation end-products impair the non-small cell lung carcinoma progression

Non-small cell lung carcinoma (NSCLC) is an age-related disease and, therefore, it occurs simultaneously with other age-related changes including the accumulation of advanced glycation end-products (AGEs) in the human body. Although many age-related changes might contribute to the NSCLC development, our clinical and experimental studies indicate an anti-tumorigenic effect of circulating and extracellular matrix-bound AGEs. This has been demonstrated in a clinical study showing that NSCLC patients with high AGE-related plasma fluorescence were characterized by a later reoccurrence of the tumor after curative surgery and a higher long-term survival rate compared to patients with low AGE fluorescence (25% vs. 47% 60-month-survival, P = 0.011). Moreover, another clinical study showed a better mid-term (20-month) survival of NSCLC patients with diabetes mellitus, which is associated with AGE increase, compared to patients without diabetes (76% vs. 59%, P = 0.048). To confirm the impact of circulating and matrix-bound AGEs on the NSCLC progression, we studied the in vivo NSCLC growth in mice of whom elevated circulating AGE level were induced by AGE-enriched nutrition and the in vitro NSCLC cell migration through collagen matrix increasingly modified with AGEs, respectively. The in vivo tumorigenicity assay demonstrated that mice with higher levels of circulating AGEs developed smaller tumors than mice with normal AGE levels, and the in vitro assay showed a reduced invasive cell migration through AGE-modified collagen matrix than non-modified collagen. Moreover, we found an inverse correlation between the in vitro NSCLC spheroid growth in plasma/serum of patients/mice and the plasma/serum AGE levels. In summary, our clinical and experimental studies indicate a protective effect of AGEs on the NSCLC progression.

11:20 Uhr

D. Zibrova; F. Vandermoere1; O. Göransson2; M.  Peggie3; S. Lindenmüller; K. Spengler; B. Viollet4; N. A. Morrice1; K. Sakamoto1; R. Heller
Institut für Molekulare Zellbiologie, Friedrich-Schiller-Universität Jena, Jena; $1 MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dundee/GB; $2 Department of Experimental Medical Sciences, Lund University, Lund/S; $3 Division of Signal Transduction Therapy, University of Dundee, Dundee/GB; $4 Institut Cochin, Université Paris Descartes, CNRS, Paris/F;


Regulation of angiogenesis by high-glucose-induced O-GlcNAcylation

O-GlcNAcylation is a posttranslational protein modification which is known to be increased in age-related degenerative diseases including diabetes. Hyperglycaemia enhances the activity of glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1), an enzyme controlling the abundance of this modification. We have identified GFAT1 as a novel substrate of adenosine monophosphate-activated protein kinase (AMPK). In the vasculature, AMPK regulates endothelial cell functions including angiogenesis. The current study was aimed at understanding the role of AMPK-GFAT1 axis in high glucose-induced vascular dysfunction.
Our data show that O-GlcNAc levels were elevated in endothelial cells with downregulated AMPK catalytic subunits and, oppositely, decreased in cells treated with chemical AMPK activator indicating that AMPK inhibits GFAT and downstream O-GlcNAcylation. We also demonstrate that AMPK-mediated inhibition of GFAT is critical for angiogenesis. Vascular endothelial growth factor (VEGF) increased GFAT1 phosphorylation in endothelial cells in an AMPK-dependent manner. Accordingly, GFAT inhibition by 6-diazo-5-oxonorleucine (DON) increased VEGF-induced angiogenesis. Furthermore, high (25 mM) glucose reduced VEGF-induced angiogenesis, which was rescued by DON. At the biochemical level, high glucose increased O-GlcNAcylation globally and on a range of proteins interacting with endothelial nitric oxide synthase (eNOS), which was associated with decreased eNOS phosphorylation; these effects of high glucose were counteracted by DON.
Taken together, we have shown for the first time that GFAT1 is a component of VEGF-AMPK signalling in endothelial cells. It is responsible for inhibitory effects of high glucose on angiogenesis via modulating O-GlcNAcylation of angiogenic proteins. AMPK may inhibit GFAT thereby counteracting adverse effects of high glucose and promoting angiogenesis.

11:40 Uhr

S. Stricker; A. Navarrete Santos1; H. Schrewe; D. Schlote2; A. Simm3; L. D. Schulz4; K. Hoffmann2
Max-Planck-Institut für Molekulare Genetik, Berlin; 1 Universitätsklinik und Poliklinik für Herz-und Thoraxchirurgie, 2 Institut für Humangenetik, 3 Zentrum für Medizinische Grundlagenforschung (ZMG), Medizinische Fakultät, Martin-Luther-Universität Halle-Wittenberg, Halle (Saale); 4 The Jackson Laboratory, Maine/USA;


Developmental defects and premature ageing in lamin B receptor deficient mice

The lamin B receptor (LBR) is a multifunctional inner nuclear membrane protein with structural impact on nuclear shape and chromatin organisation. Further, LBR belongs to the C14 sterol reductase family and has enzymatic activity in sterol metabolism. LBR mutations have been shown previously to cause dose-dependent hyposegmentation of granulocyte nuclei in heterozygous or homozygous Pelger-Huët-Anomaly.Heterozygous LBR mutations change blood morphology without causing any associated pathology. In contrast, homozygous mutations in LBR cause a spectrum of systemic malformations ranging from heart defects, brachydactyly and mental retardation, as occurs in Pelger-Anomaly, to severe cutaneous derangements, as seen in the recessive ichthyotic mice (ic), and finally to prenatal lethality, which is found in Greenberg dysplasia.
To elucidate the mechanisms responsible for this variable phenotype in individuals with lamin B receptor deficiency, we studied prenatal and postnatal development in ic/ic mice. We found first but modest differences in some of the homozygous embryos around mouse embryonic day E10. However, the highest mortality was perinatally and around weaning. The survival varied between a few hours and several weeks or months. Generally, all homozygous mice display growth retardation and severe skin defects. In addition to the previously described phenotypes ichthyosis, alopecia, nuclear hyposegmentation we observed other manifestations as increased frequency of hydrocephalus, abnormal histology of heart and muscle cells and an abnormal fat distribution. The latter manifestations of Lbr deficiency overlap with those of Lamin A diseases like Progeria, and with processes in physiological ageing. We therefore studied protein glycation as a biomarker of ageing. Compared to liver and heart of controls, ic/ic mice showed a significantly increased accumulation of intracellular glycated proteins Arg-pyrimidine, carboxyethyllysine and pentosidine.
Summarizing, manifestation in ic/ic mice start prenatally and are life threatening perinatally and around weaning. We conclude that the lamin B receptor is essential both for development and healthy ageing.

12:00 Uhr

A. Höhn
LS Ernährungstoxikologie, FSU Jena, Jena;


Protein Aggregates in Cellular Aging

Reactive oxygen species (ROS) are generated constantly within cells at low concentrations even under physiological conditions. During aging the levels of ROS can increase due to a limited capacity of antioxidant systems and repair mechanisms. Because of the abundance of proteins in cells, it is not surprising that they are major targets for oxidative modifications. Protein damage has an important influence on cellular viability since most protein damage is non-repairable, and has deleterious consequences on protein structure and function. In addition, damaged and modified proteins can form cross-links and provide a basis for many senescence-associated alterations and may contribute to a range of human pathologies One of the life limiting factors in postmitotic aging cells is the intracellular accumulation of lipofuscin, a highly oxidized aggregate of proteins and covalently cross-linked lipids, influencing the metabolism of a senescent cell.
The contribution of proteasomal inhibition as a cause of increasing protein oxidation and consequently elevated lipofuscin formation is well established; further the lysosomal system is considered to be involved. We could show that both, macroautophagy and the lysosomal system, are not mandatory for the formation of lipofuscin, since that material accumulates in the cytosol if autophagy or lysosomal activity is inhibited.
Furthermore we were able to present a biochemical lipofuscin-model explaining the mechanisms of proteasomal inhibition, derived from the altered characteristics of lipofuscin caused by partial proteolytic degradation. Besides this, it was proposed that lipofuscin is cytotoxic due to its ability to incorporate transition metals, resulting in a redox active surface. We could demonstrate a lipofuscin-mediated formation of oxidants and the role of iron in this process in a model of senescent fibroblasts, as well as with artificial lipofuscin in vitro.