Interactions of β tubulin isotypes with glutathione in differentiated neuroblastoma cells subject to oxidative stress

Abstract

Microtubules are a major component of the neuronal cytoskeleton. Tubulin, the subunit protein of microtubules, is an α/β heterodimer. Both α and β exist as families of isotypes, whose members are encoded by different genes and have different amino acid sequences. The βII and βIII isotypes are very prominent in the nervous system. Our previous work has suggested that βII may play a role in neuronal differentiation, but the role of βIII in neurons is not well understood. In the work reported here, we examined the roles of the different β-tubulin isotypes in response to glutamate/glycine treatment, and found that both βII and βIII bind to glutathione in the presence of ROS, especially βIII. In contrast, βI did not bind to glutathione. Our results suggest that βII and βIII, but especially βIII, may play an important role in the response of neuronal cells to stress. In view of the high levels of βII and βIII expressed in the nervous system it is conceivable that these tubulin isotypes may use their sulfhydryl groups to scavenge ROS and protect neuronal cells against oxidative stress.

Keywords: glutathionylation; microtubules; oxidative stress; tubulin.

FIGURE 1

Effect of silencing β tubulin isotypes on ROS formation in differentiated SK-N-SH cells treated with glutamate and glycine. Expression of βII (A, B) and βIII (C, D) transfected with isotype-specific siRNA (siRNAs were incubated with cells for 24h) was measured after glutamate/glycine treatment for 24h in differentiated SK-N-SH cells. Negative siRNA was used as a control. GAPDH was used as a loading control. Note that each β isotype decreases significantly. (E) Cells with knockdown β tubulin isotypes were treated with 500 μM glutamate, 100 μM glycine, and 2 mM CaCl₂ for the indicated times. Intracellular ROS production was determined by DCF fluorescence as described in Methods. Results are expressed as the log2 of fluorescence intensity normalized to the fluorescence intensity of undifferentiated SK-N-SH cells without any treatment, and are given as the mean ± SD. **ρ< 0.01; ***ρ<0.001 as compared with the control.

FIGURE 2

Enzymatic scavenging of reactive oxygen species (ROS). Hydrogen peroxide is converted into water; the cysteine-sulfhydryl moiety of glutathione (GSH) and protein sulfhydryls (protein-S⁻) are required for this reaction, in which GSH and protein-S⁻ are converted into their oxidized form, a GSS-protein.

FIGURE 3

Co-Immunoprecipitation of β tubulin isotypes with glutathione in differentiated SK-N-SH cells treated with glutamate/glycine. Differentiated SK-N-SH cells were treated with 500 μM glutamate, 100 μM glycine, and 2 mM CaCl₂ for the indicated times. Cell extracts were immunoprecipitated with monoclonal antibody to βI, βII, and βIII, respectively. The presence of glutathione was probed using mouse monoclonal glutathione antibody (upper row in each panel). The co-migration of glutathione with β tubulin isotypes was confirmed using the antibody to the isotypes (right image in each panel). The same nitrocellulose membrane was re-probed using β tubulin antibody to confirm the presence of β tubulin (lower row in each panel). The result showed that (A) no glutathione was detected at the position of βI, suggesting that βI did not interact with glutathione; (B) glutathione was detected at the position of βII, suggesting that βII interacted with glutathione; (C) glutathione was detected at the position of βIII, suggesting that βIII interacted with glutathione. These experiments were repeated twice. (D) Quantification of glutathione interacting with β tubulin isotypes. The figures shown in panels A, B and C were quantified using the ImageJ software available at http://rsb.info.nih.gov/ij/. Glutathione levels were normalized to β tubulin isotypes.