A comparison of reversible versus irreversible protein glutathionylation

Abstract

Glutathionylation is generally a reversible posttranslational modification that occurs to cysteine residues that have been exposed to reactive oxygen species (P-SSG). This cyclical process can regulate various clusters of proteins, including those involved in critical cellular signaling functions. However, certain conditions can favor the formation of dehydroamino acids, such as 2,3-didehydroalanine (2,3-dehydroalanine, DHA) and 2,3-didehydrobutyrine (2,3-dehydrobutyrine), which can act as Michael acceptors. In turn, these can form Michael adducts with glutathione (GSH), resulting in the formation of a stable thioether conjugate, an irreversible process referred to as nonreducible glutathionylation. This is predicted to be prevalent in nature, particularly in more slowly turning over proteins. Such nonreducible glutathionylation can be distinguished from the more facile cycling signaling processes and is predicted to be of gerontological, toxicological, pharmacological, and oncological relevance. Here, we compare reversible and irreversible glutathionylation.

Keywords: Dehydroalanine; Glutaredoxin; Glutathione; Glutathione S-transferases; Glutathione disulfide; Irreversible protein glutathionylation; Lanthionine; Reversible glutathionylation; γ-Glutamyl-L-cysteine.

Figure 5.1

Chemical structure of glutathione in reduced (A) and oxidized (disulfide) forms (B).

Figure 5.2

Involvement of glutathione in the elimination of reactive oxygen and nitrogen species. Hydroxyl radical and nitric oxide (after oxidation to the NO⁺ form (nitrosyl cation)) or peroxynitrite (ONOO⁻) may interact directly with GSH leading to GSSG formation. Hydrogen peroxide may be removed by catalase or by glutathione peroxidase (GPx). The latter requires glutathione to reduce peroxide. GR, glutathione reductase; G6PDH, glucose-6-phosphate dehydrogenase; G6P, glucose-6-phosphate; 6PGL, 6-phosphogluconolactone.

Figure 5.3

S-Glutathionylation cycle. Proteins in which cysteine residues possess unusually low pKa values (redox sensors) are targets for oxidative or nitrosative stress. Cysteine residues within redox sensors can be oxidized to form protein sulfenic (P-SOH) and sulfinic (P-SOOH) acids. Some protein glutathionylation (P-SSG) reactions are mediated by GGT, Grx, or GSTP. P-SSG proteins have a wide variety of functions in cellular physiology/pathology, summaries of which can be found in Townsend (2007); the categories are depicted in the pie chart.

Figure 5.4

Formation of γ-glutamyldehydroalanylglycine (EdAG) from glutathione and busulfan. Glutathione reacts with busulfan [CH₃S(O)₂OCH₂CH₂CH₂CH₂OS(O)₂CH₃] in a reaction accelerated by GSTs. Two equivalents of CH₃S(O)₂H are released with the formation of the corresponding glutathione conjugate (GS⁺THT). This conjugate contains a cyclic sulfonium moiety, which is an excellent leaving group. The elimination of thiophene results in the formation of a glutathione analog (EdAG) in which the cysteine residue is converted to a dehydroalanine residue. EdAG can then participate in a Michael addition reaction with glutathione to generate the Michael adduct GSG that contains a stable thioether bond. From Younis et al. (2008) with permission.

Figure 5.5

Formation of a dehydroalanyl (DHA) residue in proteins and peptides followed by irreversible glutathionylation. A protein/peptide containing a leaving group (X) in the β position of an amino acid residue may undergo spontaneous β-elimination to generate a protein/peptide containing a DHA residue (center). Michael addition of the sulfhydryl group of glutathione will generate an adduct containing a nonreducible thioether bond, resulting in irreversible glutathionylation. Residues such as cysteine (X=–SH) and serine (X =–OH) are expected to be relatively stable. However, for proteins with very long turnover times, such as those found in the lens, β-elimination reactions may occur as a consequence of aging. Moreover, other residues such as selenocysteine, serine O-phosphate, and serine O-sulfate are expected to be more labile and amenable to β-elimination. Modified from Cooper, Pinto, et al. (2011).