MODULATION OF P53 AGGREGATION BY THE INTRINSICALLY DISORDERED C-TERMINAL REGION

p53 is a protein that acts as a transcription factor playing a key role in cell cycle regulation and DNA damage repair, and consists of four main domains: the transactivation domain (TAD), the DNA-binding domain (DBD), the tetramerization domain (TET), and the C-terminal domain (CTD). Its intrinsically disordered regions (IDRs), the TAD and CTD, can influence p53 aggregation, a process that can impair its tumor-suppressor function and may even confer oncogenic-like function, contributing to tumor progression. Previous data from our group reveal that different p53 constructs, lacking one or both IDRs, exhibit distinct aggregation profiles, and suggest that, in wild-type p53, the TAD negatively regulates aggregation while the CTD promotes it. Thus, we hypothesize that the CTD acts as a positive modulator of WT p53 aggregation and this study aims to elucidate whether this action occurs through intermolecular or intramolecular interactions. Initially, pull-down assays were performed between the GST-p53CTD and p53-TDC (TADDBD) constructs using GSTrap resin to investigate possible TADDBD-CTD interactions. The fractions were collected for subsequent analysis by dot-blot using anti-p53 and anti-GST antibodies, and under the conditions evaluated, no coelution of p53-TDC with GST-p53CTD was observed, providing no evidence of interaction in this assay. Considering the possibility that the presence of GST, which is significantly larger than the CTD, might interfere with the interaction, the CTD was subsequently obtained by cleavage of the GST-p53CTD construct. In this process, different conditions were tested and evaluated by 22.5% SDS-PAGE to determine optimal cleavages. The results showed that efficient cleavage conditions had been established, enabling the isolation of the CTD using size-exclusion chromatography (SEC-HPLC), while monitoring absorbance at 280 nm and 214 nm. Once the CTD is isolated, we intend to conduct incubation assays with p53-TDC, using techniques such as circular dichroism, to evaluate the impact on protein aggregation. It is expected that the results will contribute to understanding the molecular mechanisms and the role of CTD in the p53 aggregation process.

This work was supported by the Universidade Federal do Rio de Janeiro (UFRJ) through the Programa Institucional de Bolsas de Iniciação Científica (PIBIC-UFRJ).