Medizinische Universität Graz - Research portal
Selected Publication:
Spreitzer, E.
Regulation of Cellular Senescence.
[ Diplomarbeit/Master Thesis (UNI) ] Technische Universität Graz; 2018. pp.92.
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- Authors Med Uni Graz:
- Advisor:
- Madl Tobias
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- Abstract:
- Aging is the process of getting older and is characterized by a decline of physiological mechanisms. It is also major risk factor for age-related diseases such as cancer, cardiovascular and neurodegenerative disorders. One reason that we age, is the accumulation of senescent cells over time. Upon DNA-damage they undergo senescence and are trapped in a stable cell cycle arrest. Pivotal part in deciding the cell’s fate is the interaction of the “guardian of the genome” cellular tumor antigen p53 (p53) and the “longevity gene” Forkhead box protein O4 (FOXO4). The de Keizer group designed a FOXO4-derived peptide, called FOXO4-DRI in order to dissociate the FOXO4 – p53 complex. Senescent IMR90 fibroblasts were efficiently and selectively targeted and eliminated by the peptide. Furthermore, treatment of fast-aging and naturally aged mice with FOXO4-DRI restored their fitness, fur density and renal function. Targeting the FOXO4 – p53 interaction is a promising way to cure age-related diseases and eventually “cure” aging. Molecular details of the FOXO4 – p53 interaction are essential to understand regulation of senescence and apoptosis and are key for optimizing or finding drugs to eliminate senescent cells. In this perspective, this thesis aims to identify the yet unknown binding site of FOXO4 on p53, which is necessary to determine the atomic structure of the complex and indispensable for understanding the regulatory mechanisms deciding the cell’s fate. An in-vitro approach using Nuclear Magnetic Resonance (NMR) spectroscopy, complemented with other molecular biological and biophysical methods was applied to gain insight into the p53 – FOXO4 interaction. Human protein constructs of p53 and FOXO4 were heterologously expressed in Escherichia coli cells and purified using immobilized metal ion affinity chromatography and Size Exclusion Chromatography. We identified the disordered N-terminal region of p53 as binding site for the FOXO4 Forkhead domain. Both of the subdomains, Transactivation domain 1 (TAD1) and Transactivation domain 2 (TAD2) are involved in binding. Based on chemical shift perturbation analysis the Transactivation domain 2 is the main binding site. Small-angle X-ray experiments revealed that two molecules of FOXO4 Forkhead bind to one molecule of the p53 N-terminal region. Within the main binding site is the phosphorylation site for Homeodomain-interacting protein kinase 2 (HIPK2), which was shown to promote apoptosis. Mutation of Serine 46 to Glutamine, which mimics the phosphorylation, did not alter binding of p53 transactivation domain to FOXO4 Forkhead domain. Furthermore, we demonstrate a proof-of-principle experiment using in-cell phosphorylation to identify two phosphorylation sites of p53 transactivation domain in HEK293T cell lysate. These findings allow to put the FOXO4 - p53 interaction in context with already known regulatory networks and interaction partners of both proteins. Additionally, the results obtained in this study can be used as starting point to determine the atomic resolution structure of the complex.