Inhibition of tubulin polymerization by hypochlorous acid and chloramines.
Protein thiol oxidation and modification by nitric oxide and glutathione are emerging as common mechanisms to regulate protein function and to modify protein structure. Also, thiol oxidation is a probable outcome of cellular oxidative stress and is linked to degenerative disease progression. We assessed the effect of the oxidants hypochlorous acid and chloramines on the cytoskeletal protein tubulin. Total cysteine oxidation by the oxidants was monitored by labeling tubulin with the thiol-selective reagent 5-iodoacetamidofluorescein; by reaction with Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid); and by detecting interchain tubulin disulfides by Western blot under nonreducing conditions. Whereas HOCl induced both cysteine and methionine oxidation of tubulin, chloramines were predominantly cysteine oxidants. Cysteine oxidation of tubulin, rather than methionine oxidation, was associated with loss of microtubule polymerization activity, and treatment of oxidized tubulin with disulfide reducing agents restored a considerable portion of the polymerization activity that was lost after oxidation. By comparing the reactivity of hypochlorous acid and chloramines with the previously characterized oxidants, peroxynitrite and the nitroxyl donor Angeli's salt, we have identified tubulin thiol oxidation, not methionine oxidation or tyrosine nitration, as a common outcome responsible for decreased polymerization activity.
Department of Chemistry, The College of William and Mary, Williamsburg, VA 23187–8795, USA. lmland@wm.edu
Lisa M
LM
Landino
Tara D
TD
Hagedorn
Shannon B
SB
Kim
Katherine M
KM
Hogan
D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000187:drug effects / Q000378:metabolism; D002700:Chloramines / Q000494:pharmacology*; D006997:Hypochlorous Acid / Q000494:pharmacology*; D008715:Methionine / Q000378:metabolism; D010084:Oxidation-Reduction; D013552:Swine; D014404:Tubulin / Q000187:drug effects* / Q000378:metabolism
D002700:Chloramines; D014404:Tubulin; D006997:Hypochlorous Acid; D008715:Methionine; C030816:chloramine
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