Reversible glutathionylation regulates actin polymerization in A431 cells.

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 microM 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.

Overalkylation of a protein digest with iodoacetamide.

Cystine linkages in proteins are often opened with reducing agents, sometimes to improve their digestion, often to eliminate disulfide linkages from complicating analysis of the digest. After reduction, the sulfhydryls are usually reacted with iodoacetamide (IAM), iodoacetic acid (IAA), or another electrophile to prevent reformation of disulfide linkages in a random manner. When the amount of protein may be reliably estimated, side reactions from excess IAM or IAA can be avoided. When this is not so, removal of excess iodoalkane can be accomplished by HPLC, by dialysis, or simply by allowing a reducing thiol to consume any excess. In mass spectrometric analysis of proteins isolated by 1D or 2D gels, removal of the excess iodoalkane is often accomplished simply by washing the gel prior to proteolytic digestion. During a recent study of the glutathionylation site mapping of actin, IAM was used to block any residual sulfhydryl groups remaining on the protein so that they would not displace glutathione from its initial site. In addition, to avoid losses due to actin polymerization during dialysis, the IAM was allowed to remain during the digestion. This further ensured that any sulfhydryl groups liberated during the digestion would be similarly blocked by the IAM. Under these conditions, we observed the peptides to undergo N- as well as S-carbamidomethylation. In examining a series of other peptides alkylated with IAM in this way, we have found N-alkylation to be the rule rather than the exception and even O-alkylation was detected. The main sites to which the carbamidomethyl group attaches to the peptides have been located with LC-MS2 using an ion trap mass spectrometer and found to be the N-terminal amino group. A simple expedient to prevent such reactions when an excess of reducing agent must be avoided is to run the alkylation in the presence of a thioether such as 2,2'-thiodiethanol rather than a thiol.