Journal
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
Volume 194, Issue -, Pages 24-31Publisher
ELSEVIER
DOI: 10.1016/j.ijbiomac.2021.11.160
Keywords
p53; Twist1; Nanobody; VHH; Intrabodies; Panning strategies
Funding
- TALENTS3 program (European Social Fund 2014/2020 Regional Operative Programme, Axis 3 -Education and Training) [782]
- Italian Association for Cancer Research (AIRC), (AIRC-IG grant)
- Ministero della Salute-Ricerca Finalizzata
- AIRC under IG 2020 [24589]
- CINECA [HP10CG7JPW, HP10BTJPER]
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The study showed that interfering with the p53:Twist1 complex could alleviate Twist1's inhibitory effect on p53, providing potential therapeutic tools for p53 wild type tumors. Using VHH nanobodies targeting the p53 C-terminal region, the research demonstrated the possibility of this intervention and validated its activity in human cell models. These findings provide insights into p53 biology and the development of targeted therapeutic approaches.
Twist1 promote the bypass of p53 response by interacting with p53 and facilitating its MDM2-mediated degradation. We reasoned that reagents able to interfere with the p53:Twist1 complex might alleviate Twist1 inhibitory effect over p53, thus representing potential therapeutic tools in p53 wild type tumors. From a preimmune library of llama nanobodies (VHH), we isolated binders targeting the p53 C-terminal region (p53CTD) involved in the interaction with Twist1 by using recombinant Twist1 as an epitope-specific competitor during elution. Positive hits were validated by proving their capacity to immunoprecipitate p53 and to inhibit Twist1:p53 binding in vitro. Molecular modeling confirmed a preferential docking of positive hits with p53-CTD. D11 VHH activity was validated in human cell models, succeeded in immunoprecipitating endogenous p53 and, similarly to Twist1 knock-down, interfered with p53 turnover, p53 phosphorylation at Serine 392 and affected cell viability. Despite the limited functional effect determined by D11 expression in target cells, our results provide the proof of principle that nanobodies ectopically expressed within a cell, have the capacity to target the assembly of the pro-tumorigenic Twist1:p53 complex. These results disclose novel tools for dissecting p53 biology and lay down the grounds for the development of innovative targeted therapeutic approaches.
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