Studies of intramolecular interaction between Forkhead domain and Transactivation domain of Forkhead box protein O4

Abstract

Cellular senescence is the response induced by permanent cell cycle arrest regardless of the existence of growth factors. Abnormal accumulation of senescent cells which do not induce apoptosis leads to senescence-associated diseases such as type Ⅱ diabetes, osteoarthritis, and atherosclerosis and moreover cancers. Forkhead box protein O4 (FOXO4), a member of Forkhead box O subfamily, is a transcription factor which results in cellular senescence. p53 is a transcription factor which induces apoptosis of senescent cells. It is known that the FOXO4 binding to p53 contributes inhibiting apoptosis and maintaining senescent cells. Therefore, interruption of FOXO4-p53 interaction could induce a senescence cell-specific apoptosis. In order to target FOXO4-p53 interaction, the binding mechanism of FOXO4 and p53 should be elucidated. FOXO3, the most well studied homologous protein of FOXO4, has the forkhead domain (FHD) and the transactivation domain (TAD). Both domains participate in p53 binding and interact with each other. Sequence similarity of FOXO4 and FOXO3 suggests that FOXO4 also have the equivalent intramolecular interaction between FHD and TAD as well as binding of both domains with p53. In order to understand the details of FOXO4-p53 interaction, the intramolecular interaction of FOXO4 was investigated in this study. FOXO4 FHD and TAD domains were subcloned, overexpressed, and purified. TAD peptides were prepared by chemical synthesis. We identified intramolecular interaction between FOXO4 FHD and TAD by using isothermal titration calorimetry (ITC) analysis and nuclear magnetic resonance (NMR) experiments. ITC results show that TAD binds to FHD with a dissociation constant in the submicromolar range. 1H-15N HSQC titration data indicate that a hydrophobic cleft region of FHD was mostly perturbed by TAD peptides. The suggested intramolecular binding surfaces of FOXO4 in this study are partially overlapped with the TAD binding sites of FOXO3.