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Updated: Dec 10, 2025
Proteomic Profiling of Macrophages by 2D Electrophoresis
Published on: November 4, 2014
Resolving the Interactome of the Human Macrophage Immunometabolism Regulator (MACIR) with Enhanced Membrane Protein
Gavin McGauran1, Emma Dorris2, Razvan Borza3
1School of Biomolecular & Biomedical Science, University College Dublin, Belfield, Dublin, D04 V1W8, Ireland.
Abstract:
Expression of the macrophage immunometabolism regulator gene (MACIR) is associated with severity of autoimmune disease pathology and with the regulation of macrophage biology through unknown mechanisms. The encoded 206 amino acid protein lacks homology to any characterized protein sequence and is a disordered protein according to structure prediction algorithms. To identify interactions of MACIR with proteins from all subcellular compartments, a membrane solubilization buffer is employed, that together with a high affinity EF hand based pull down method, increases the resolution of quantitative mass spectrometry analysis with significant enrichment of interactions from membrane bound nuclear and mitochondrial compartments compared to samples prepared with radioimmunoprecipitation assay buffer. A total of 63 significant interacting proteins are identified and interaction with the nuclear transport receptor TNPO1 and the trafficking proteins UNC119 homolog A and B are validated by immunoprecipitation. Mutational analysis in two candidate nuclear localization signal motifs in the MACIR amino acid sequence shows the interaction with TNPO1 is likely via a non-classical proline/tyrosine-nuclear localization signal motif (aa98-117). It is shown that employing a highly specific and high affinity pull down method that performs efficiently in this glycerol and detergent rich buffer is a powerful approach for the analysis of uncharacterized protein interactomes.
Insights
Researchers identified proteins interacting with MACIR, a protein linked to autoimmune diseases. This discovery sheds light on MACIR
Area of Science:
- Immunology and Molecular Biology
- Protein-protein interactions
- Autoimmune disease research
Background:
- Macrophage immunometabolism regulator gene (MACIR) expression correlates with autoimmune disease severity.
- The MACIR protein's function and interactions are largely unknown.
- MACIR is a disordered protein lacking homology to known sequences.
Purpose of the Study:
- To identify proteins interacting with MACIR across all subcellular compartments.
- To elucidate the mechanisms by which MACIR regulates macrophage biology.
- To validate specific MACIR-protein interactions.
Main Methods:
- Utilized a novel membrane solubilization buffer and EF hand-based pull-down method.
- Employed quantitative mass spectrometry for high-resolution interactome analysis.
- Validated interactions using immunoprecipitation and mutational analysis.
Main Results:
- Identified 63 significant interacting proteins with MACIR.
- Validated interactions with TNPO1 (nuclear transport) and UNC119 homolog A/B (trafficking).
- Determined MACIR interacts with TNPO1 via a non-classical nuclear localization signal (aa98-117).
Conclusions:
- The developed pull-down method is effective for analyzing uncharacterized protein interactomes.
- Identified novel MACIR interactors provide insights into its role in macrophage biology.
- Findings contribute to understanding autoimmune disease pathogenesis.
