Background and previous work
Transcription factors of the FOXO family are major targets of insulin/IGF1 receptor (InsR) signalling as well as of the upstream regulator and anti-ageing hormone Klotho. The InsRFOXO pathway contributes to the regulation of longevity and oxidative stress resistance in C. elegans and mammals. It was hypothesised that Klotho modulates this cascade, stimulating FOXO activity by interfering with InsR signalling and thereby enhancing stress resistance and lifespan. We have tested the modulation of FOXO signalling in C. elegans and mammalian cells by exposure to alkylating and redox cycling agents, by thiol depletors and other stressful stimuli known to affect cellular redox regulation. Interestingly, several of these stressful stimuli imitate insulin at the level of FOXO activity, suggesting that stressful stimuli would mimic insulin signalling, thereby antagonising Klotho-induced extension of lifespan. In fact, the expression of FOXO-regulated metabolic and antioxidant proteins was shown to be attenuated upon exposure of mammalian cells to sources of oxidative stress.
(1) To test whether FOXO signalling – and FOXO-dependent stress resistance and lifespan – is modulated by non-enzymatic PTMs, such as glycation and alkylation of proteins involved in the FOXO pathway. (2) To analyse the effect of Klotho exposure or Klotho overexpression on insulin signalling and stress resistance in mammalian cells. (3) To test for a modulation of Klotho activity by non-enzymatic PTM.
We will expose human cells and C. elegans to glycating conditions/AGEs, to defined sources of reactive oxygen species, to reactive aldehydes generated during lipid peroxidation, or to xenobiotics metabolised to alkylating agents. We will also expose these cells to secreted Klotho that is either derived from senescent versus proliferating cells or that has undergone in vitro glycation. In C. elegans, the recently characterised Klotho ortholog will be both depleted and overexpressed. These model systems will then (1) be tested for the modulation of FOXO activity, shifts in FOXO/coregulator interactions, and FOXO target gene expression profiles in response to the aforementioned conditions, followed by (2) identification of PTM (glycation/alkylation) targets in the FOXO signalling cascade and (3) analysis of alterations in cellular stress resistance and lifespan.