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.

Increasing glutathione levels by a novel posttranslational mechanism inhibits neuronal hyperexcitability

“…β-tubulin was chosen as the loading control based on a previously published study [ 114 ] which showed that the treatment of neuroblastoma cells with high concentrations of BSO (5 mM, or 12.5 mM) for 24 h affected the actin cytoskeletal network but not β-tubulin. Although β-tubulin has been shown by some studies to interact with GSH and form GSH-tubulin mixed disulfides, this interaction has been shown to occur at high (i) temperatures (30 °C) [ 115 ]; (ii) concentrations of GSH (∼5 mM) [ 116 ]; and (iii) levels of steady-state ROS [ 116 ]. The concentration and duration of treatment for PAO and DTT were selected from previous studies in literature where they were shown to be non-toxic [ 57 , 117 , 118 ].…”

Possible Roles of Specific Amino Acids in β-Tubulin Isotypes in the Growth and Maintenance of Neurons: Novel Insights From Cephalopod Mollusks

“…Since virtually no cell expresses only a single isotype of tubulin, one can probably assume that cellular microtubules rich in βIII are highly dynamic. Fourth, βIII responds to oxidative stress by binding to glutathione, much more so than do either βI or βII ( Guo et al, 2018 ). Finally, different approaches have shown that the presence of βIII protects the cell from oxidative or other stresses ( Gan et al, 2007 ; Guo et al, 2010 ).…”

“…The first set, called “HMBG βIII,” is based on the sequences of βIII from humans ( H omo) , mice ( M us ), cows ( B os ), and chickens ( G allus ). βIII-Tubulin from each of these is identical at the positions of interest and are, in a sense, being privileged because they may be the only organisms in which the functional properties of their βIII has been examined ( Lopata and Cleveland, 1987 ; Banerjee et al, 1992 ; Panda et al, 1994 ; Joe et al, 2008 ; Guo et al, 2010 , 2011 , 2018 ; Vemu et al, 2016 ). The second set, called “Vert βIII” refers to the 8 residues that appear to be conserved in all vertebrate βIII-tubulins, as described above.…”

Effect of Fetal Bovine Serum or Basic Fibroblast Growth Factor on Cell Survival and the Proliferation of Neural Stem Cells: The Influence of Homocysteine Treatment

“…Hcy in high concentrations acts as an oxidant due to its interaction with the heme proteins of the cell [ 39 , 40 ]. S-homocysteinylation could occur when Hcy directly reacts with β-3-tubulin cysteine residues, mimicking glutathione [ 41 ]. Furthermore, Chen et al have demonstrated that proteins in hela cell lysates undergo unspecific N-homocysteinylation of lysine residues, and that TUBB3 is among them [ 42 ].…”