Background and previous work
AMPK represents an important regulator of cellular energy status and homeostasis and
integrates multiple longevity pathways such as FOXO, SIRT1 and inhibition of mTORC1. Reduced activity as well as hyperactivation of this enzyme has been observed during ageing. AMPK activation requires phosphorylation at threonine 172. In addition, it underlies acetylation at different lysine residues as well as oxidation and carbonylation of cysteine residues, which have been reported to decrease enzyme activation. In contrast, O-GlcNAcylation seems to positively regulate AMPK activity. Our previous work has characterised AMPK activation pathways and functions of AMPK in endothelial cells.
Recent data obtained in our group point to novel AMPK-dependent protective pathways counter-regulating adverse effects of high glucose and oxidative stress.
We hypothesise that age-related PTMs of AMPK and AMPK-regulating proteins contribute to endothelial dysfunction and cardiovascular ageing. Specifically, we want to address the following questions: (1) Does ageing affect the activity and expression of AMPK and its regulator proteins? Are the observed alterations linked to protein modifications? (2) Are protein modifications of AMPK/AMPK regulator proteins reversible? (3) Do protein modifications within the AMPK pathway affect endothelial functions? (4) Are age-dependent PTMs of AMPK suited as biomarkers?
Using models of replicative and stress-induced premature senescence (oxidative stress, hyperglycaemia) as well as endothelial cells prepared from young and aged mice, we will identify PTMs (glycosylation, acetylation, oxidation, AGE modification) of AMPK and its regulator proteins. We will study the effects of detected PTMs on AMPK phosphorylation and activation as well as on AMPK functions, such as regulation of autophagy, permeability, angiogenesis and glucose metabolism. Furthermore, we will investigate whether the observed PTMs are related to changes of the cellular redox state (e.g. increased generation of reactive oxygen species and/or decreased antioxidative capacity), to an altered histone acetyltransferase/histone deacetylase balance or to changes in glucose metabolisation through the hexosamine pathway. The reversibility or prevention of the identified PTMs will be tested by modifying the antioxidant defence (e.g. overexpression of thioredoxin), by modulating histone deacetylases such as SIRT1 and/or by affecting the key enzyme of the hexosamine pathway. We will also address the question of whether leukocytes can be used as surrogate cells for endothelial tissues and detection of AMPK modifications. We expect an improved understanding of age-related endothelial dysfunction and the characterisation of AMPK as a target against vascular ageing. Collaborations are planned with SP2, SP5 and SP8 with respect to identifications of PTMs, with SP9 and SP12 concerning ROS signalling and with SP9 and SP14 with regards to AMPK interconnection to FOXO and β-catenin pathways.