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Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli
Published on: January 6, 2015
Membrane-associated RING-CH (MARCH) proteins down-regulate cell surface expression of the interleukin-6 receptor
Jeffrey J Babon1,2, Dina Stockwell1, Ladina DiRago1
1The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Vic 3052, Australia.
Insights
Membrane-Associated RING-CH E3 ubiquitin ligase MARCH2 inhibits Interleukin 6 (IL6) activity by down-regulating the IL6 receptor alpha chain. This highlights a novel immunosuppressive role for MARCH proteins in regulating inflammatory responses.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Interleukin 6 (IL6) is a critical cytokine involved in immune and inflammatory processes.
- Dysregulation of IL6 signaling is implicated in various diseases.
- Identifying regulators of IL6 activity is crucial for therapeutic development.
Purpose of the Study:
- To identify proteins that inhibit IL6 activity using a functional screen.
- To elucidate the mechanism by which identified proteins regulate IL6 signaling.
- To investigate the in vivo relevance of these regulators in immune cells.
Main Methods:
- A cDNA expression library screen was performed using IL6-mediated inhibition of M1 myeloid leukemia cell proliferation.
- Overexpression of candidate proteins in M1 cells was analyzed for IL6 responsiveness.
- Western blotting and genetic deletion studies in mice were used to assess receptor levels and protein function.
Main Results:
- The Membrane-Associated RING-CH E3 ubiquitin ligase MARCH2 was identified as an inhibitor of IL6 activity.
- MARCH2 selectively down-regulates the IL6 receptor alpha chain, not gp130.
- MARCH2, MARCH4, MARCH9, and an isoform of MARCH3 share this inhibitory function, requiring specific protein domains.
- Combined genetic deletion of MARCH2, 3, and 4 in mice leads to elevated IL6 receptor alpha levels in specific immune cells.
Conclusions:
- MARCH proteins, particularly MARCH2, play a significant role in regulating IL6 signaling through selective degradation of the IL6 receptor alpha chain.
- These findings reveal a novel mechanism for controlling inflammatory responses and suggest potential therapeutic applications for MARCH proteins in immune modulation.
Abstract:
Interleukin 6 (IL6) is a cytokine that regulates a number of important immune and inflammatory pathways. We used the ability of IL6 to inhibit the clonal proliferation of the mouse M1 myeloid leukemia cell line in agar to positively screen a cDNA expression library for proteins that inhibited IL6 activity. We found three clones completely resistant to IL6 that contained the cDNA for the Membrane-Associated RING-CH E3 ubiquitin ligase MARCH2. MARCH2 is a member of a family of membrane-bound E3 ubiquitin ligases that target cell surface receptors for degradation. MARCH2 overexpressing M1 clones retained responsiveness to the related cytokines leukemia inhibitory factor and oncostatin M and we showed that its inhibitory effect was a result of selective down-regulation of the IL6 receptor alpha chain and not the shared receptor subunit, gp130 or other signalling molecules. This activity of MARCH2 was also shared with related proteins MARCH4, MARCH9 and an isoform of MARCH3. The transmembrane domains and C-terminal domains, as well as a functional RING domain, of MARCH proteins were all required for substrate recognition and down-regulation. Genetic deletion of individual MARCH proteins in mice had no or little effect on IL6Rα levels but combined deletions of MARCH2,3 and 4 displayed elevated steady-state levels of IL6Rα in selected haemopoietic cell subsets including CD8+ and CD4+ T cells. These studies extend the potential immunosuppressive roles of MARCH proteins to include down-regulation of IL6 inflammatory responses.
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