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Intracellular displacement of p53 using transactivation domain (p53 TAD) specific nanobodies
Anneleen Steels1, Adriaan Verhelle1, Olivier Zwaenepoel1
1a Department of Biochemistry, Faculty of Medicine and Health Sciences , Ghent University , Ghent , Belgium.
Abstract:
The tumor suppressor p53 is of crucial importance in the prevention of cellular transformation. In the presence of cellular stress signals, the negative feedback loop between p53 and Mdm2, its main negative regulator, is disrupted, which results in the activation and stabilization of p53. Via a complex interplay between both transcription-dependent and - independent functions of p53, the cell will go through transient cell cycle arrest, cellular senescence or apoptosis. However, it remains difficult to completely fathom the mechanisms behind p53 regulation and its responses, considering the presence of multiple layers involved in fine-tuning them. In order to take the next step forward, novel research tools are urgently needed. We have developed single-domain antibodies, also known as nanobodies, that specifically bind with the N-terminal transactivation domain of wild type p53, but that leave the function of p53 as a transcriptional transactivator intact. When the nanobodies are equipped with a mitochondrial-outer-membrane (MOM)-tag, we can capture p53 at the mitochondria. This nanobody-induced mitochondrial delocalization of p53 is, in specific cases, associated with a decrease in cell viability and with morphological changes in the mitochondria. These findings underpin the potential of nanobodies as bona fide research tools to explore protein function and to unravel their biochemical pathways.
Insights
Researchers developed novel nanobodies targeting the p53 tumor suppressor protein. These nanobodies can alter p53 localization, impacting cell viability and mitochondrial morphology, offering new tools to study p53 pathways.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- The tumor suppressor p53 is vital for preventing cellular transformation.
- p53 activation and stabilization occur upon disruption of the p53-Mdm2 feedback loop during cellular stress.
- p53 regulates cell cycle arrest, senescence, and apoptosis through transcription-dependent and -independent functions.
Purpose of the Study:
- To develop novel research tools for investigating the complex mechanisms of p53 regulation and cellular responses.
- To create single-domain antibodies (nanobodies) that specifically target the N-terminal transactivation domain of wild-type p53 without impairing its transcriptional activity.
Main Methods:
- Development of single-domain antibodies (nanobodies) that bind to the N-terminal transactivation domain of wild-type p53.
- Functionalization of nanobodies with a mitochondrial-outer-membrane (MOM)-tag to induce p53 delocalization.
- Observation of cellular responses, including cell viability and mitochondrial morphology, following nanobody-induced p53 mitochondrial capture.
Main Results:
- Nanobodies specifically bind to the N-terminal transactivation domain of wild-type p53, preserving its transcriptional function.
- Mitochondrial-outer-membrane (MOM)-tagged nanobodies successfully captured p53 at the mitochondria.
- Nanobody-induced mitochondrial delocalization of p53 was associated with decreased cell viability and altered mitochondrial morphology in certain contexts.
Conclusions:
- Developed nanobodies serve as effective research tools for exploring protein function and biochemical pathways.
- Nanobody-mediated manipulation of p53 localization offers a novel approach to study p53's role in cellular processes.
- Further research using these nanobodies can help unravel the intricate mechanisms of p53 regulation and its downstream effects.
