Peroxynitrite modulates acidic fibroblast growth factor (FGF-1) activity.
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 microM) 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 microM) loss of sulfhydryl groups that correlated with formation of reducible (dithiothreitol, arsenite) FGF-1 aggregates containing 50% latent biologic activity. Treatment with 0.1-0.5mM 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 (0.1mM) 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.
Bagnasco
Patricia
P
Department of Surgery, The University of Alabama at Birmingham, Birmingham, AL 35294, USA.
MacMillan-Crow
Lee Ann
LA
Greendorfer
Jessica S
JS
Young
Carlton J
CJ
Andrews
Lori
L
Thompson
John A
JA
D016475:3T3 Cells; D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D002452:Cell Count; D002455:Cell Division / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D016220:Fibroblast Growth Factor 1 / Q000737:chemistry* / Q000145:classification / Q000494:pharmacology*; D006801:Humans; D051379:Mice; D009566:Nitrates / Q000737:chemistry; D010084:Oxidation-Reduction; D030421:Peroxynitrous Acid / Q000737:chemistry* / Q000494:pharmacology*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000138:chemical synthesis
D000596:Amino Acids; D009566:Nitrates; D016220:Fibroblast Growth Factor 1; D030421:Peroxynitrous Acid; D014443:Tyrosine; C007543:dityrosine