Epigenetic upregulation of FKBP5 by aging and stress contributes to NF-kappa B-driven inflammation and cardiovascular risk

Aging and psychosocial stress are associated with increased inflammation and disease risk, but the underlying molecular mechanisms are unclear. Because both aging and stress are also associated with lasting epigenetic changes, a plausible hypothesis is that stress along the lifespan could confer disease risk through epigenetic effects on molecules involved in inflammatory processes. Here, by combining large-scale analyses in human cohorts with experiments in cells, we report that FKBP5, a protein implicated in stress physiology, contributes to these relations. Across independent human cohorts (total n > 3,000), aging synergized with stress-related phenotypes, measured with childhood trauma and major depression questionnaires, to epigenetically up-regulate FKBP5 expression. These age/stress-related epigenetic effects were recapitulated in a cellular model of replicative senescence, whereby we exposed replicating human fibroblasts to stress (glucocorticoid) hormones. Unbiased genome-wide analyses in human blood linked higher FKBP5 mRNA with a proinflammatory profile and altered NF-kappa B-related gene networks. Accordingly, experiments in immune cells showed that higher FKBP5 promotes inflammation by strengthening the interactions of NF-kappa B regulatory kinases, whereas opposing FKBP5 either by genetic deletion (CRISPR/Cas9-mediated) or selective pharmacological inhibition prevented the effects on NF-kappa B. Further, the age/stress-related epigenetic signature enhanced FKBP5 response to NF-kappa B through a positive feedback loop and was present in individuals with a history of acute myocardial infarction, a disease state linked to peripheral inflammation. These findings suggest that aging/stress-driven FKBP5-NF-kappa B signaling mediates inflammation, potentially contributing to cardiovascular risk, and may thus point to novel biomarker and treatment possibilities.Peer reviewe

Elucidation of the molecular and cellular mechanisms of FKBP51-selective inhibitors

The FK506 Binding Protein 51 (FKBP51) is an identified risk factor for a variety of psychiatric disorders such as major depressive disorder as well as obesity and chronic pain. Enzymatically, FKBP51 exhibits prolyl-peptidyl-isomerase activity located within a pocket in its FK1 domain. The natural ligand and immunosuppressive drug FK506 binds to this pocket. Nowadays, several new classes of FKBP51 ligands have been developed. These compounds that lack the immunosuppressive feature, show higher affinity for FKBP51 and partial selectivity towards other FKBP family members. The Selective Antagonist of FKBP51 by Induced Fit 1 (SAFit1) is highly selective for FKBP51 compared to FKBP52, a close homolog and partial functional counterplayer of FKBP51. Ligands of the SAFit class have been shown to reverse FKBP51 induced inhibition of neurite development of neuronal cells and are active in various mouse models. Mice treated with SAFit2 have an improved stress coping behavior, remain leaner during a high-fat diet and show decreased symptoms in a chronic pain state. The best described molecular and cellular activity of FKBP51 is linked to the Glucocorticoid Receptor (GR), a key player in the stress signaling cascade. Mutations of the fkbp5 gene were linked to increased FKBP51 levels and glucocorticoid resistance. Beside the GR, FKBP51 has been described as the interactor of many other proteins e.g. within the NF-κB and Akt signaling pathway. Although FKBP51 is an attractive drug target of continuous rising interest, the underlying molecular and cellular functions of FKBP51 ligands and the protein itself remain to be elucidated. This thesis contributes in closing the gap between molecular biology and clinical findings regarding FKBP51 and its ligands. The first approach was the investigation of a new class of FKBP51 ligands developed by Tianqi Mao in the Hausch lab. These FKBP51 PROTACs (proteolysis targeting chimeras) aim to chemically knock down their target. These PROTACs were screened and a promising structure was identified, which resulted in decreased endogenous FKBP51 levels in cells detected through Western blotting. This finding was confirmed by a decreased FKBP51-Luciferase fusion protein signal and the PROTACs action in a GR reporter gene assay. The later assay supports the model of FKBP51 inhibiting and FKBP52 enhancing GR signaling. However, conventional ligands did not impact the FKBP51 effect. The second approach elucidated the interaction of FKBPs and the E3 ligase regulator Glomulin (Glmn) in detail. FKBP12.6 has been identified as novel interactor of this protein utilizing the HTRF (Homogeneous Time Resolved Fluorescence) methology. This is the first in-vitro assay, which addresses the binding of FKBP51 and a protein interactor on the molecular biological level. The amino acids F67 and D68 have been identified to be essential to establish the binding of Glmn to FKBP51s FK1 domain. At last, the FKBP51-Glmn complex is the first discovered interaction that is sensitive to multiple classes of FKBP51 ligands. In conclusion, my findings indicate two distinct modes of action of FKBP51. One is ligand-dependent and PPIase-domain-dependent while the other is independent. My thesis points out the importance to increase the focus on the investigation on defined FKBP51 interactions and to raise the efforts to develop FKBP51 ligands that address both FKBP51 action modi