Temporal Quantitative Phosphoproteomics Profiling of Interleukin-33 Signaling Network Reveals Unique Modulators of Monocyte Activation

Abstract

Interleukin-33 (IL-33), a member of the IL-1 superfamily cytokines, is an endogenous danger signal and a nuclear-associated cytokine. It is one of the essential mediators of both innate and adaptive immune responses. Aberrant IL-33 signaling has been demonstrated to play a defensive role against various infectious and inflammatory diseases. Although the signaling responses mediated by IL-33 have been previously reported, the temporal signaling dynamics are yet to be explored. To this end, we applied quantitative temporal phosphoproteomics analysis to elucidate pathways and proteins induced by IL-33 in THP-1 monocytes. Employing a TMT labeling-based quantitation and titanium dioxide (TiO2)-based phosphopeptide enrichment strategy followed by mass spectrometry analysis, we identified and quantified 9448 unique phosphopeptides corresponding to 3392 proteins that showed differential regulation. Of these, 171 protein kinases, 60 phosphatases and 178 transcription factors were regulated at different phases of IL-33 signaling. In addition to the confirmed activation of canonical signaling modules including MAPK, NFκB, PI3K/AKT modules, pathway analysis of the time-dependent phosphorylation dynamics revealed enrichment of several cellular processes, including leukocyte adhesion, response to reactive oxygen species, cell cycle checkpoints, DNA damage and repair pathways. The detailed quantitative phosphoproteomic map of IL-33 signaling will serve as a potentially useful resource to study its function in the context of inflammatory and pathological conditions.

Molecular anatomy of eosinophil activation by IL5 and IL33

“…Proteomic and phosphoproteomic datasets were mapped onto templates for IL5 and IL33 signaling pathway ( Figure 5) using the KEGG Database, WikiPathways, and published networks 35–39 . We first identified eosinophil-expressed proteins identified in at least one of the three datasets ( Supplemental Spreadsheet 1 ) and then mapped phosphorylated proteins and significantly changing sites in the IL33-versus-IL5 phosphoproteomic dataset to pathways of interest ( Figure 5 ).…”

Comprehensive Overview of Bottom-Up Proteomics Using Mass Spectrometry

“…Special consideration is required during protein extraction where the cell lysis buffer should include phosphatase inhibitors such as sodium orthovanadate, sodium pyrophosphate and beta-glycerophosphate. 322 Enrichment can be done at the protein level before proteolysis. Phosphoprotein enrichment typically involves the use of immobilized metal-affinity chromatography (IMAC) to selectively capture phosphorylated proteins based on their high-affinity binding to metal ions such as Ga(III), Fe(III), Zn(II) and Al(III).…”

“…As with any proteomics experiment, phosphoproteomics studies require protein extraction, proteolytic enzyme digestion, phosphopeptide enrichment, peptide fractionation, LC-MS/MS, bioinformatics data analysis, and biological function inference. Special consideration is required during protein extraction where the cell lysis buffer should include phosphatase inhibitors such as sodium orthovanadate, sodium pyrophosphate and beta-glycerophosphate …”

“…Phosphopeptide enrichment often uses titanium dioxide (TiO 2 ) and/or IMAC such as Fe 3+ coupled to solid-phase materials. ,, The most common cost-effective beads for phosphopeptide extraction with Ti are ReSyn and GL Sciences, and CubeBio for Fe-based beads. Often organic acids such as glutamic acid, lactic acid, glycolic acid are added to compete with acidic non-phosphopeptides for binding to the metal-ions.…”

Dual Immune Regulatory Roles of Interleukin-33 in Pathological Conditions

“…The results of functional analysis suggested IL-33-induced Rho-dependent signaling. Further, this research group applied quantitative temporal phosphoproteomics analysis and identified several kinases and phosphatases regulated across timepoints; they found that IL-33 regulated phosphorylation sites on transcription factors, which revealed several cellular processes of IL-33 activation, including leukocyte adhesion, response to reactive oxygen species, cell cycle checkpoints, and DNA damage and repair pathways [ 139 ]. As technology advances, a more detailed and complete IL-33 signaling network will be achieved.…”