Reversible Glutathionylation Regulates Actin Polymerization in A431 Cells

Abstract

In response to growth factor stimulation, many mammalian cells transiently generate reactive oxygen species (ROS) that lead to the elevation of tyrosine-phosphorylated and glutathionylated proteins. While investigating EGF-induced glutathionylation in A431 cells, paradoxically we found deglutathionylation of a major 42-kDa protein identified as actin. Mass spectrometric analysis revealed that the glutathionylation site is Cys-374. Deglutathionylation of the G-actin leads to about a 6-fold increase in the rate of polymerization. In vivo studies revealed a 12% increase in F-actin content 15 min after EGF treatment, and F-actin was found in the cell periphery suggesting that in response to growth factor, actin polymerization in vivo is regulated by a reversible glutathionylation mechanism. Deglutathionylation is most likely catalyzed by glutaredoxin (thioltranferase), because Cd(II), an inhibitor of glutaredoxin, inhibits intracellular actin deglutathionylation at 2 M, comparable with its IC 50 in vitro. Moreover, mass spectral analysis showed efficient transfer of GSH from immobilized S-glutathionylated actin to glutaredoxin. Overall, this study revealed a novel physiological relevance of actin polymerization regulated by reversible glutathionylation of the penultimate cysteine mediated by growth factor stimulation.

Targeted disruption of the glutaredoxin 1 gene does not sensitize adult mice to tissue injury induced by ischemia/reperfusion and hyperoxia

“…8A), indicating that Grx1 is an important cellular mechanism of de-glutathionylation, as shown in several previous studies [21,22,66,[68][69][70][71]. For example, de-glutathionylation of actin in NIH3T3 fibroblast cells, and its polymerization and migration to the cell periphery, in response to EGF-stimulation was shown to be abolished in cells in which Grx1 was knocked down by interference RNA [71].…”

“…8B). The current and other earlier studies suggest that Grx1 may preferentially de-glutathionylate certain cellular proteins including actin, a known cellular target for oxidant-induced S-glutathionylation [70,71]. Further studies to identify the protein targets of Grx1-mediated de-glutathionylation should advance our understanding in redox regulation of cellular function and oxidant-induced cell damage.…”

Oxidative imbalance in the aging inner ear

“…The accumulation of glutathionylated proteins is an indicator of protein oxidation arising from the formation of hydrogen peroxide [30,20,2]. The rise of 4-HNE signals lipid peroxidation, mainly due to the production of hydroxyl radical, and 3-NT suggests peroxynitrite reactions [26].…”

“…The extent of glutathione-conjugation to proteins is considered a measure of oxidative stress, especially due to H 2 O 2 [30]. Glutathionylated proteins, including a very prominent conjugate with actin at 42 kDa, increased in cochlear extracts with age.…”

Radical-free biology of oxidative stress

“…GS-ylation reactions are catalyzed by thiol transferases known as glutaredoxins (Grx). These reactions are reversible so that a highly reduced GSH/GSSG redox potential drives reduction of protein disulfides while an oxidizing potential drives protein GS-ylation (171,187). GSH is also used to protect against two-electron oxidants, such as peroxide reduction catalyzed by GSH peroxidases, and aldehydes and quinones catalyzed by GSH transferases.…”