要旨
Homeostasis is a dynamic equilibrium fundamental for a healthy system. A major challenge to homeostasis is environmental stress to which the organism reacts with the stress response. The hypothalamic-pituitary-adrenal (HPA) axis is the main regulator of the stress response that, upon activation, leads to the release of glucocorticoids (GCs). GCs are steroid hormones that exert their function via glucocorticoid receptors (GR). They trigger on one hand the appropriate stress response in the periphery, and, on the other, inhibit the HPA axis itself via negative feedback to restore homeostasis. FK506-binding protein 51 (FKBP51) is a co-chaperone able to modulate the GR, and therefore the HPA axis. Furthermore the expression of FKBP5, the gene coding for FKBP51, is induced by GR activation. In the last decade, increasing evidence has unveiled additional roles of FKBP51 in the regulation of several cellular pathways and functions that are independent from its inhibitory role on GR. Among these, FKBP51 has been shown to link stress signaling to macroautophagy, a lytic type of autophagy pathway. Autophagy represents one of the main mechanisms regulating cellular homeostasis and response to stress. For this reason, in the first part of this doctoral thesis, the role of GR-mediated stress was investigated on two further autophagic pathways: 1) the chaperone-mediated autophagy (CMA), a selective type of lytic autophagy, and 2) the secretory autophagy, an unconventional secretory mechanism regulated by autophagy-related proteins and found to be involved in extracellular signaling of immune response. For this aim, an in vitro approach was adopted using human and murine cell lines that were treated with dexamethasone (Dex), a synthetic GR agonist.
For the first pathway, biochemical assays indicated that Dex-induced GR activation enhances CMA-mediated degradation of known CMA target proteins and that this process is dependent on FKBP51. Furthermore, the underlying molecular mechanism could be revealed by co-immunoprecipitation that displayed the co-localization of FKBP51, AKT and PHLPP on lysosomes. With a SILAC-based proteomics analysis, the proteome-wide effect of Dex-induced CMA could be observed and novel CMA targets were identified.
For the second pathway, interactome and co-immunoprecipitation analyses revealed the involvement of FKBP51 in the SNARE complex assembly essential for secretory autophagy. Furthermore, treatment with Dex lead to a strengthened interaction between the SNARE proteins and FKBP51, and to an increased secretion of IL1B, a well characterized cargo of secretory autophagy, as observed with in vitro ELISA experiments and in vivo hippocampal microdialyses. A global effect of Dex-induced secretory autophagy was finally observed with a secretome analysis.
The second part of my doctoral thesis focused on FKBP5/51 transcription variants and protein isoforms. In fact, despite its involvement in many cellular functions and disorders, very little is known about its four transcription variants and two isoforms. Thus, expression and degradation dynamics of FKBP51 isoforms and their differential functions in known molecular pathways were analyzed.
Overall this study highlighted FKBP51 as crucial mediator of the stress response on two autophagic pathways, which might contribute to the regulation of cell and protein homeostasis. Furthermore, this regulatory mechanism might underlie the link of stress to immune and psychiatric disorders.
