Identification of proteins in activated human neutrophils susceptible to tyrosyl radical attack. A proteomic study using a tyrosylating fluorophore

Abstract

Tyrosyl radicals cross-linked to protein tyrosine residues (tyrosylated proteins) represent hallmarks of neutrophil-mediated injury at the inflammatory locus. Yet the proteins targeted by tyrosyl radicals in an intact cellular system remain to be elucidated. Here, we show that tyrosyl radicals generated by human neutrophils after activation by phorbol 12-myristate 13-acetate (PMA), interferon-g (IFN-g) or TNF-a could act in an autocrine manner by cross-linking to endogenous proteins. We have identified the tyrosylated proteins by using a membraneimpermeable tyrosine analogue, tyramine coupled to fluorescein (TyrFluo), in combination with proteomics techniques. Confocal microscopy images indicated that initially the tyrosylated proteins were localized in patches at the cell surface to become internalized subsequently. In the neutrophil membrane-associated proteome, lactoferrin was the prime target of tyrosylation. Out of three isoforms identified, an 80 kDa neutral isoform was tyrosylated more extensively than the 85 kD basic isoform, particularly after PMA activation. Although all three stimuli induced tyrosylation of the filamentous component vimentin, additional tyrosylated vimentin fragments were detected after IFN-g-and TNF-a-stimulation. Moreover, upon activation the bulk of vimentin behaved as a dimer (M r 120 kDa) being slightly tyrosylated, yet phosphorylated at Thr-425 possibly as a requirement for its externalization. Unexpectedly, bovine catalase added to end tyrosyl radicals formation was detected as a highly tyrosylated neutrophil-associated protein. A moderate stimulus-dependent tyrosylation of ATP synthaseb, a-enolase, glyceraldehyde 3-phosphate dehydrogenase, cytokeratin-10, filamin-A, and annexin-I was also observed. When the membrane-permeable probe (acetylTyrFluo) was used, protein tyrosylation was not observed indicating that the intracellular proteins were well protected against oxidative attack. This study shows that human neutrophils can modulate their proteome via a tyrosine oxidation pathway induced by pro-inflammatory mediators.

New Insights into the DT40 B Cell Receptor Cluster Using a Proteomic Proximity Labeling Assay

“…An early proteomic application of this concept exploited the presence of endogenous peroxidases combined with fluorescently labeled tyramine (49). Recently, several groups have generalized the method to deliver the peroxidase onto a predetermined target.…”

Cell surface expression of intermediate filament proteins vimentin and lamin B1 in human neutrophil spontaneous apoptosis

“…Neutrophils can generate tyrosyl radicals after activation with phorbol 12-myristate 13-acetate (PMA), IFN-␥, or tumor necrosis factor ␣ [43]. Those radicals act in an autocrine manner by cross-linking to endogenous proteins exposed to the medium.…”

Recent advances in blood‐related proteomics

“…The central pathway in the regulation of neutrophil function is the p38 mitogen-activated protein kinase signal transduction as shown by Singh et al [139]. Activation of neutrophil phorbol 12-myristate, tumor necrosis factor a or interferon g has been shown to induce tyrosylation of a number of endogenous proteins such as lactoferrin, catalase, vimentin, filamin A, myeloperoxidase, ATP synthetase b, annexin 1, cytokeratin 10, or glyceraldehyde 3-phosphate dehydrogenase after 2-DE and MS [140].…”