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In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity
Published on: January 29, 2018
Pax8 protein stability is controlled by sumoylation
Tiziana de Cristofaro1, Anna Mascia, Andrea Pappalardo
1Istituto di Endocrinologia e Oncologia Sperimentale G. Salvatore-CNR and Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli Federico II, Via S. Pansini 5, 80131 Napoli, Italy.
Journal of Molecular Endocrinology
|November 1, 2008
Summary
This study reveals that the transcription factor Pax8 undergoes sumoylation, a post-translational modification that regulates its protein levels. This finding is crucial for understanding thyroid development and function.
Area of Science:
- Molecular Biology
- Cell Biology
- Endocrinology
Background:
- Pax8 is a key transcription factor in thyroid gland development and differentiation.
- Little is known about Pax8's post-translational modifications and their regulatory roles.
Purpose of the Study:
- To investigate the role of sumoylation as a post-translational modification of Pax8.
- To determine how sumoylation affects Pax8 stability, localization, and function.
Main Methods:
- Bioinformatic analysis of Pax8 sequence for sumoylation motifs.
- In vitro sumoylation assays using Pax8 and SUMO.
- Site-directed mutagenesis to create a non-sumoylatable Pax8 mutant (Pax8(K309R)).
- Co-immunoprecipitation assays to assess interactions with PIASy.
- Immunofluorescence to visualize Pax8 localization in SUMO nuclear bodies.
Main Results:
- Pax8 contains a conserved sumoylation motif and is sumoylated at lysine residue 309.
- The Pax8(K309R) mutant is resistant to sumoylation.
- PIASy enhances Pax8 sumoylation, acting as a SUMO ligase.
- Sumoylated Pax8 localizes to SUMO nuclear bodies.
- Sumoylation controls the steady-state protein levels of Pax8.
Conclusions:
- Sumoylation is a critical post-translational modification regulating Pax8 activity and stability.
- This modification impacts thyroid development and differentiated phenotype maintenance.
- Pax8 sumoylation influences its subcellular localization and protein turnover.
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