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In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity
Published on: January 29, 2018
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The conserved sumoylation consensus site in TRIM5α modulates its immune activation functions
Marie-Édith Nepveu-Traversy1, Lionel Berthoux1
1Laboratory of Retrovirology, Department of Medical Biology and BioMed Research Group, Université du Québec à Trois-Rivières, 3351 Boulevard des Forges, CP500, Trois-Rivières, QC G9A 5H7, Canada.
Virus Research
|March 4, 2014
Summary
The TRIM5α protein
Area of Science:
- Immunology
- Virology
- Molecular Biology
Background:
- TRIM5α is a primate protein that restricts retroviral infections.
- It functions by binding to viral cores and destabilizing them.
- TRIM5α also activates innate immune responses via NF-κB and AP-1.
Purpose of the Study:
- To investigate the role of a specific sumoylation site (K10) in TRIM5α function.
- To determine how K10 affects TRIM5α's anti-retroviral activity and immune activation.
Main Methods:
- Mutagenesis of the K10 site in Rhesus macaque TRIM5α (TRIM5αRh).
- Assays to measure K63-linked ubiquitin chain generation.
- Analysis of NF-κB and AP-1 activation.
- Western blotting for ubiquitylated TRIM5α.
- Nuclear export inhibition studies with leptomycin B.
Main Results:
- Mutating K10 to arginine (K10R) had minimal impact on direct retroviral restriction.
- K10R significantly reduced K63-linked ubiquitin chain formation and subsequent NF-κB/AP-1 activation.
- K10R increased levels of ubiquitylated TRIM5α.
- K10R enhanced nuclear localization of TRIM5αRh and reduced its association with nuclear SUMO bodies.
Conclusions:
- The TRIM5α sumoylation site (K10) modulates the E3 ubiquitin ligase activity of the RING domain.
- K10 promotes K63-linked ubiquitination for innate immune activation, while inhibiting auto-ubiquitylation.
- Lysine 10 is crucial for TRIM5α-mediated innate immune responses and regulates its nuclear transport.
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