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A BW Reporter System for Studying Receptor-Ligand Interactions
Published on: January 7, 2019
Selection of cDNA candidates that induce oligomerization of NLRP3 using a chimeric receptor approach
Shingo Honda1, Teruyuki Nagamune1, Masahiro Kawahara1
1Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Abstract:
Since diverse cellular events are regulated by protein oligomerization, identification of molecules that affect oligomerization of a target protein is important for understanding cellular physiology and developing therapeutics. In this study, we aimed to screen cDNA candidates that induce oligomerization of NLRP3, which is one of the important inflammatory sensor proteins, in mammalian cytoplasm. In our screening method, the chimera composed of NLRP3 and the kinase domain of c-kit, one of the receptor tyrosine kinases (RTKs) activated by oligomerization, is expressed in cytoplasm of an IL-3-dependent mammalian cell line. The cells are then transduced with a cDNA library, and cultured in the absence of IL-3. If the transduced cDNA is a NLRP3 activator, the kinase domain of the NLRP3-c-kit chimera is activated by oligomerization, which induces cell growth even in the absence of IL-3. Using this system, constitutive oligomers of two NLRP3 variants were clearly detected by cell growth. Furthermore, cDNA screening resulted in identification of three distinct cDNAs that are potential candidates of NLRP3 activators. These results demonstrate the utility of our chimeric receptor-based system for screening candidates that induce oligomerization of a target protein.
Insights
Researchers developed a novel screening system to identify molecules that trigger protein oligomerization. This method successfully detected NLRP3 activators, paving the way for new therapeutic strategies targeting inflammatory diseases.
Area of Science:
- Biochemistry
- Molecular Biology
- Immunology
Background:
- Protein oligomerization is crucial for diverse cellular functions.
- Identifying modulators of protein oligomerization is key for understanding cell physiology and developing therapeutics.
- The NLRP3 inflammasome is a critical sensor protein involved in inflammation.
Purpose of the Study:
- To screen for cDNA candidates that induce oligomerization of NLRP3 in mammalian cytoplasm.
- To develop and validate a novel screening system for identifying protein oligomerization inducers.
Main Methods:
- A chimeric receptor system was engineered, fusing NLRP3 with the kinase domain of c-kit (a receptor tyrosine kinase).
- This chimera was expressed in IL-3-dependent mammalian cells, which were then transduced with a cDNA library.
- Cell growth in the absence of IL-3 served as a readout for NLRP3 oligomerization and activation.
Main Results:
- The system successfully detected constitutive oligomers of two NLRP3 variants via cell growth.
- Screening of the cDNA library identified three distinct candidate genes that potentially activate NLRP3.
- The chimeric receptor-based system proved effective for screening protein oligomerization inducers.
Conclusions:
- A novel and effective chimeric receptor-based screening system for identifying protein oligomerization inducers was established.
- Three potential NLRP3 activators were identified, offering new avenues for therapeutic development.
- This system holds significant potential for advancing the study of protein oligomerization in cellular processes and disease.
