Moderation: A. Simm, Halle (Saale); L.-O. Klotz, Jena
Ageing leads to impairment of tissue homeostasis and functional decline of organs and represents a major risk factor for prevalent diseases such as cardiovascular disease and neurodegeneration in developed countries. To devise therapies aimed at improving the health state of the elderly, a detailed knowledge of molecular mechanisms leading to the impairment of organ function with increasing age is essential. There is accumulating evidence that posttranslational modifications (PTMs) of proteins contribute to this decline.
PTMs occur either enzymatically catalyzed or non-enzymatically, with both types often targeting the same amino acid. Under physiological conditions enzymatic PTMs regulate protein activities, thereby controlling shape and function of cells. However, due to alterations of modifying enzymes or because of an altered cellular environment in aged organisms, PTM patterns may change, and, moreover, non-enzymatic modifications, such as oxidation or glycation, may compete with enzymatically regulated processes, such as acetylation and glycosylation. As a consequence, dysregulation of cellular processes occurs. The session will focus on such PTMs and their role in aging.
Glycosylation is the most abundant enzymatic posttranslational modification of proteins. Sialic acid represents the terminal monosaccharide of most glycoconjugates and is involved in a variety of cellular functions. Sialic acid is synthesized in the cytosol from UDP-N-acetylglucosamine (UDP-GlcNAc) by the UDP-GlcNAc-2-epimerase/ManNAc-kinase. UDP-GlcNAc-2-epimerase/ManNAc-kinase mutations cause hereditary inclusion body myopathy, a very serious age-related and late-onset disease of distal and proximal skeletal muscles. Recent studies have demonstrated that many intracellular proteins, which are normally non-glycosylated, are modified by a single O-linked GlcNAc on serine/threonine residues by the O-GlcNAc-transferase. O-GlcNAcylation influences the localization, activity or interaction of proteins. This is of special importance, since in age-dependent degenerating diseases false-regulated O-GlcNAcylation occurs.
FOXO transcription factors are involved in cellular adaptation to oxidative stimuli. FOXO activity is affected by the modulation of thiol levels; this is observed both in cultured mammalian cells and in the model organism C. elegans. Here, the consequences of genetically induced as well as of xenobiotic-induced thiol depletion for FOXO activity as well as for FOXO’s role in modulating aging processes will be discussed.
Formation of Advanced Glycated End Products (AGEs) is a result of glycation, which is non-enzymatic reaction between reducing sugars, or other α-dicarbonyl compounds, and amino groups of proteins, lipids and nucleic acids. AGEs accumulate during aging and have been implicated in the pathophysiology of numerous age-related diseases. Glycation of nuclear proteins at lysins or arginins may alter their function and may have an impact on epigenetic regulation of gene transcription.
Glycated nuclear proteins from young and replicative senescent cells, as well as from normal and tumor cells, are going to be compared, and identified modified proteins will be further analyzed.
Nuclei from human embryonic kidney cells 293A (HEK293A) and human kidney carcinoma cell line Caki-2 were isolated and AGE-modified nuclear proteins were compared via western blot using antibody against carboxymethyl-lysine (CML), hydroimidazolone MG-H1 and argpyrimidine. Different pattern of modified proteins in HEK293A and Caki-2 cell lines was observed. LC-MS/MS analysis revealed that in HEK293A cells 7 out of 3424 identified proteins are modified, while in Caki-2 cells among 2701 proteins 11 are modified. Two of them, Histone H4 and Myb-binding protein 1A, were identified in both cell lines. Amount of modified peptides (12 out of 32127 identified peptides in HEK293A cells and 14 out of 23528 in Caki-2 cells) is too low for reproducible quantification of differences in these modifications. Immunoprecipitation was used to enrich samples with modified proteins prior to analysis, enrichment steps are elucidated and optimized.
Identified AGE-modification of lysins/arginins within nuclear proteins indicate on a possibility to modify the epigenetic regulation of cells.
Impaired capacity to repair aging-associated damages by regeneration or stem cell activation is a typical sign of aging tissues and organs. Moreover, accumulation of advanced glycation endproducts (AGEs) is frequently observed in aging organisms. We here addressed if glycation may affect Wnt/ß-catenin signaling. In our studies we observed that glyoxal (GO)- or methylglyoxal (MGO)-induced glycation of Wnt3a-conditioned medium reduces Topflash reporter gene activity. In control experiments glycation of Wnt3a and not of other medium components were responsible for this effect. Moreover, it appears that AGEs per se can stimulate canonical Wnt signaling, whereas glycation of Wnt has an inhibitory effect. From our results we hypothesize that the final glycation-induced cellular changes depend on an interplay of RAGE- and Wnt-mediated effects.
Glycation is the non-enzymatic reaction of carbonyls with amine residues of proteins or amino acids, which lead to the production of a heterogeneous group of posttranslational modifications, known as advanced glycation end products (AGEs). This process takes place in the human body with sugar molecules like fructose or glucose or with side products of metabolic pathways like methylglyoxal and glyoxal. Elevated levels of AGEs can be found during normal aging but also play an important role in many age-related diseases, including diabetes mellitus, atherosclerosis, renal insufficiency, heart failure and Alzheimer’s disease. Besides, aging and age-related diseases are also associated with reduced immune function leading to impaired wound healing, tumor progression and a higher susceptibility to infections.
Because evidences were found that AGEs act negatively on the immune system, we investigated the role of glycation on the function of macrophages and natural killer cells. We could show that glycation indeed alters the function and phenotype of these innate immune cells and our findings might help to explain impairment in immune function in different diseases.