Peroxynitrite modulates acidic fibroblast growth factor (FGF-1) activity

Abstract

To establish peroxynitrite (ONOO −) as a mediator of acidic fibroblast growth factor (FGF-1) function, preparations of recombinant human FGF-1 were treated with the pro-oxidant in vitro and identified amino acid modifications were correlated with biologic activity. The sequence of FGF-1 amino acid modifications induced by increasing concentrations of ONOO − was from cysteine oxidation to dityrosine formation, and to tyrosine/tryptophan nitration. Low steady-state ONOO − concentrations (10–50 μM) induced formation of dityrosine, which involved less than 0.1% of the total tyrosines. Treatment of FGF-1 with ONOO − induced a dose-dependent (10–50 μM) loss of sulfhydryl groups that correlated with formation of reducible (dithiothreitol, arsenite) FGF-1 aggregates containing 50% latent biologic activity. Treatment with 0.1–0.5 mM ONOO − induced increasing formation of non-reducible, inactivated FGF-1 structures. Combination of real-time spectral analysis and electrospray mass spectroscopy revealed that six residues (Y29, Y69, Y108, Y111, Y139, and W121) were nitrated by ONOO −. ONOO − treatment (0.1 mM) of an active FGF-1 mutant (cysteines converted to serines) induced dose-dependent, non-reversible inhibition of biologic activity that correlated with nitration of Y108 and Y111, both of which reside within a conserved domain encompassing the putative FGF-1 receptor binding site. Collectively, these observations predict a role for low levels of ONOO − during secretion of FGF-1 as an extracellular complex containing latent biologic activity. High steady-state levels of ONOO − may induce extensive cysteine oxidation, critical tyrosine nitration, and non-reversible inactivation of FGF-1, a potential inhibitory feedback mechanism restoring cellular homeostatis during the resolution of inflammation and repair.