Protocol to profile spatially resolved NLRP3 inflammasome complexes using APEX2-based proximity labeling.

Area of Science:

• Immunology
• Molecular Biology
• Biochemistry

Background:

• The NLRP3 inflammasome is crucial for regulating inflammation and producing interleukin-1β (IL-1β).
• Understanding the spatial organization of inflammasome complexes is vital for deciphering inflammatory signaling pathways.

Purpose of the Study:

• To develop and present a protocol for spatially resolving NLRP3 inflammasome complexes.
• To enable detailed profiling of inflammasome components and their interactions.

Main Methods:

• Utilizing ascorbic peroxidase 2 (APEX2)-based proximity labeling to tag nearby proteins.
• Employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) for proteomic analysis.
• Developing a stable FLAG-NLRP3-APEX2 expression cell line and performing biotinylated proteome enrichment.

Main Results:

• The protocol allows for the characterization of the NLRP3 inflammasome complex with spatial resolution.
• Successful generation and validation of a stable cell line for proximity labeling.
• Enrichment and analysis of biotinylated proteins provide insights into the inflammasome interactome.

Conclusions:

• This APEX2-based proximity labeling method offers a powerful tool for studying inflammasome complex assembly and function.
• The protocol facilitates a deeper understanding of the spatial proteome of the NLRP3 inflammasome.
• This approach can be adapted for studying other multi-protein complexes involved in cellular signaling.