Spatiotemporal Profiling of Modification-specific Proteome Secretion Uncovers an Itaconation-activated Tyrosine Kinase

Title: Spatiotemporal profiling of modification-specific proteome secretion uncovers an itaconation-activated tyrosine kinase

Journal: Nature communications

Published: 2025

Background

Macrophages not only produce diverse signaling proteins for intercellular communication but also undergo extensive post-translational modifications (PTMs) upon activation. One such metabolite-driven PTM is itaconation (protein modification by itaconate), which has been shown to regulate intracellular pathways including the KEAP1-NRF2 antioxidant response, glycolysis, and pyroptosis by covalently modifying key cysteine residues on proteins. Despite these insights, the role of itaconation in the secreted proteome—proteins exported outside cells that mediate immune and signaling functions—was largely unexplored prior to this study. To address this gap, this study soughts to systematically characterize secreted proteins modified by itaconate and to understand how such modifications regulate extracellular signaling events in immune contexts.

Materials & Methods

Labeling of secreted proteins by distinct PTM probes

Raw264.7 cells were grown to 80% confluence and treated with either DMSO or distinct probes at specified concentrations and durations: ITalk (0.5–5 mM, 1–4 hours), Fumarate-Alkyne (2 mM, 6 hours), or HNE-Alkyne/1,6-Pr2GalNAz (100 μM, 6 hours). Cells were washed thrice with PBS, incubated in serum-free medium for 12 hours, and conditioned medium was collected. Proteins were concentrated using 10 kDa filters (Millipore, UFC801024), resuspended in 100 μL PBS with 0.4% SDS, and quantified via BCA assay. To assess labeling efficiency, 50 μL secreted proteins (2 mg/mL) underwent click chemistry with 0.5 mM CuSO₄, 1 mM BTTAA, 100 μM Rhodamine-Azide (for in-gel fluorescence) or Biotin-Azide (for streptavidin blotting; Biotin-Alkyne for 1,6-Pr2GalNAz samples), and 2.5 mM sodium ascorbate (2 hours, 25 °C). Excess reagents were removed by methanol: chloroform (4:1) precipitation.

Enrichment of labeled proteins by ITalk

.To validate novel itaconated proteins, Raw264.7 cells were grown to 80% confluence and treated with DMSO or 1 mM ITalk for 2 hours. For FYN mutation studies, HEK293T cells were grown to 70% confluence in 10 cm dishes and transiently transfected with FYN mutants for 16 hours, followed by treatment with 1 mM ITalk or DMSO for 6 hours. Cells were washed thrice with PBS and centrifuged at 1000 rpm (approx. 200 g) for 3 min. Cell pellets were lysed in 1 mL RIPA buffer (50 mM Tris pH 8, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100) containing 1× protease inhibitor cocktail (Roche) using sonication. Lysates were clarified by centrifugation (20,000 g, 30 min, 4 °C), and protein concentration was determined by BCA assay. For click chemistry, 1 mL lysate (2 mg/mL) was reacted with 0.5 mM CuSO₄, 1 mM BTTAA, 100 μM Biotin-Azide, and 2.5 mM sodium ascorbate (2 hours, 25 °C). Excess reagents were removed by methanol:chloroform (4:1) precipitation; samples were centrifuged (8000 g, 5 min, 4 °C) and washed twice with cold methanol.

Precipitated proteins were redissolved in 1 mL RIPA buffer. For each sample, 200 μL streptavidin magnetic beads were equilibrated with RIPA buffer (2 × 1 mL washes), then incubated with 2 mg protein in 1 mL RIPA buffer overnight at 4 °C. Post-enrichment, beads were collected magnetically and washed sequentially with: RIPA buffer (2×1 mL), 1 M KCl (1 × 1 mL), 0.1 M Na₂CO₃ (1 × 1 mL), 2 M urea/10 mM Tris-HCl pH 8.0 (1 × 1 mL), and RIPA buffer (2 × 1 mL).

Results

Itaconation Figure 1. a Comparison of itaconated proteins identified by PBSP with those previously identified through intracellular ITalk labeling and S-glycosylation-based competitive cysteine profiling. b Proportion of known secretory proteins among the itaconated secreted proteins identified by PBSP. c KEGG pathway analysis of the itaconated secreted proteins identified by PBSP. d KEGG pathway analysis of the newly identified itaconated proteins. e Enrichment ratios of representative itaconated secreted proteins involved in COVID-19, including the RNA sensor RIG-I, MAPK1, and MAPK3. f Enrichment ratios of representative itaconated secreted enzymes in the lysosome. g Enrichment ratios of representative itaconated secreted proteins involved in ubiquitination pathway.

Itaconation Figure 2. a Schematic of PBSP profiling under normal and exosome inhibition conditions. The exosome inhibitor GW4869 was used to block exosome secretion, reducing the enrichment of exosome-dependent itaconated secreted proteins. b Volcano plot showing the comparative enrichment of proteins between normal and exosome inhibition conditions. Exosome-dependent itaconated secreted proteins are highlighted in red. c Proportion of known exosome proteins within the exosome-dependent itaconated secreted proteins. d Enrichment ratios of exosome-dependent itaconated proteins (GSDMD) and exosome-independent itaconated proteins (LDHA) under normal and exosome inhibition conditions. e Gene Ontology (GO) biological process analysis of the exosome-dependent itaconated secreted proteins identified by PBSP. f Enrichment ratios of four components of the COP9 signalosome complex (CSN). g Enrichment ratios of representative proteins involved in apoptosis, including CDK1, CDK6, and CDK7, under normal and exosome inhibition conditions. h KEGG analysis of 204 novel itaconated proteins in the exosome-dependent itaconated secreted proteins.

Conclusions

This work demonstrates that itaconation is not restricted to intracellular proteins but also affects the secreted proteome, profoundly influencing extracellular signaling. The newly developed PBSP platform allows high-resolution profiling of PTM-regulated secreted proteins, revealing that itaconation modulates not only intracellular metabolic and immune pathways but also the secretory landscape, with functional consequences such as enhanced activity of key secreted kinases like FYN. This integrative chemoproteomic strategy thereby expands our understanding of how metabolite-driven modifications like itaconation shape immune communication and intercellular signaling networks.

Reference

Lu, W., Zhang, Y., Ni, X., Wang, P., Zhuang, S., & Qin, W. (2025). Spatiotemporal profiling of modification-specific proteome secretion uncovers an itaconation-activated tyrosine kinase. Nature communications, 16(1), 10924. https://doi.org/10.1038/s41467-025-66508-y

* For Research Use Only. Not for use in diagnostic procedures.

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