Inhibition of tubulin polymerization by hypochlorous acid and chloramines

Abstract

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.

Decreased SIRT2 activity leads to altered microtubule dynamics in oxidatively-stressed neuronal cells: Implications for Parkinson's disease

“…However, possible mechanisms include conformational changes, altered sensitivity to MT lattice severing by katanin, or impaired incorporation into growing MTs (C. W. Chu et al, 2011; Janke and Bulinski, 2011; Sudo and Baas, 2010). Indeed, either acetylation or oxidative modification (Landino et al, 2011; Luduena and Roach, 1991; Mellon and Rebhun, 1976) of the tubulin heterodimer impairs MT polymerization, consistent with the observation that pre-lethal doses of 6OHDA increase levels of monomeric tubulin (V. P. Patel, et al, 2012). Acetylation sites that are predicted to face the outside surface of the MT lattice could also serve to regulate MT dynamics by affecting the binding of MAPs, such as EBs (Choudhary, et al, 2009; Janke and Bulinski, 2011).…”

Hypothiocyanous acid oxidation of tubulin cysteines inhibits microtubule polymerization

“…This is typical of the oxidants we have studied and no subunit specificity has been observed. [3,6,19]…”

“…[1,2,3] Tubulin, a heterodimer composed of similar 50 kDa α- and β-subunits, contains 20 reduced cysteines (12 in α-tubulin and 8 in β-tubulin). [4,5] Because some tubulin cysteine oxidation (~1–2 mol cys) by oxidants is tolerated before microtubule polymerization is compromised, microtubule protein thiols may protect other cellular targets from oxidation.…”

“…Our published work over the past decade has included oxidants that cause additional types of damage – for example, methionine oxidation, S-nitrosation and tyrosine nitration. [3,6,19] While Angeli’s salt is largely a cysteine oxidant, presumably via release of HNO, it produces nitrite as a byproduct which could yield nitrosation of cysteines. 2) MPO is aberrantly expressed in Alzheimer’s disease brain.…”

“…Total cysteine oxidation and effects on microtubule polymerization by HOSCN and previously characterized oxidants, HOCl, chloramines and Angeli’s salt, an HNO donor, are presented. [3]…”