The Phagosomal Proteome in Interferon-γ-Activated Macrophages

Abstract

The ability of macrophages to clear pathogens and elicit a sustained immune response is regulated by various cytokines, including interferon-gamma (IFN-gamma). To investigate the molecular mechanisms by which IFN-gamma modulates phagosome functions, we profiled the changes in composition, abundance, and phosphorylation of phagosome proteins in resting and activated macrophages by using quantitative proteomics and bioinformatics approaches. We identified 2415 phagosome proteins together with 2975 unique phosphorylation sites with a high level of sensitivity. Using network analyses, we determined that IFN-gamma delays phagosomal acquisition of lysosomal hydrolases and peptidases for the gain of antigen presentation. Furthermore, this gain in antigen presentation is dependent on phagosomal networks of the actin cytoskeleton and vesicle-trafficking proteins, as well as Src kinases and calpain proteases. Major histocompatibility complex class I antigen-presentation assays validated the molecular participation of these networks in the enhanced capacity of IFN-gamma-activated macrophages to crosspresent exogenous antigens to CD8(+) T cells.

The Protein Tyrosine Phosphatase SHP-1 Regulates Phagolysosome Biogenesis

“…Indeed, characterization of the phagosome phosphoproteome and molecular analysis of the evolution of phagosomes highlighted the importance of phosphorylation in the regulation of phagolysosome biogenesis (4,12,22). This process is accomplished through transient, highly regulated interactions between the phagosome and the cytoskeleton, as well as the early, late, and recycling endocytic compartments (46)(47)(48).…”

“…In metazoans, the multiple possible combinations of these modules illustrate the complexity of phosphotyrosine signaling, which can regulate cellular functions either positively or negatively. A recent large-scale quantitative proteomic and phosphoproteomic analysis revealed that Tyr phosphorylation sites are overrepresented in phagosomes (12), as compared with global phosphoproteome (13). This finding suggested that Tyr phosphorylation plays an important role in phagosome signaling.…”

Regulation of Protein Kinase C function by phosphorylation on conserved and non-conserved sites

“…PKC : A recent phosphoproteomics study of Interferon-gamma treated mouse macrophages has revealed a two-fold decrease in abundance of PKC over a 24 hour period [99]. Furthermore, this decrease in protein abundance was paralleled by dephosphorylation at Ser-317 (11-fold downregulation) and Tyr-676 (3-fold downregulation).…”

High‐resolution quantitative proteome analysis reveals substantial differences between phagosomes of RAW 264.7 and bone marrow derived macrophages

“…In recent years, the cell line has also been used extensively in proteomics experiments including a large‐scale proteome 10, phagosome proteomics 4, 8, 11, responses to cytokines 12, 13, and identification of DNA receptors 14.…”

“…Phagocytosis was induced for 30 min using carboxylated 1 μm polystyrene beads (Estapor/EMD Millipore) and phagosomes were isolated using sucrose ultracentrifugation in which the phagosomes float above cell debris and other organelles 11, 20. Protein extracts from three biological replicates were resuspended in 1% sodium 3‐[(2‐methyl‐2‐undecyl‐1,3‐dioxolan‐4‐yl)methoxy]‐1‐propanesulfonate (commercially available as RapiGest, Waters) in 50 mM Tris‐HCl pH 8.0 with 5 mM tris(2‐carboxyethyl)phosphine (TCEP, Pierce), heated at 60°C for 5 min, then alkylated using 10 mM iodoacetamide, diluted to 0.1% RapiGest and finally trysinized overnight at 30°C.…”