S-sulfhydration: a cysteine posttranslational modification in plant systems

Abstract

Hydrogen sulfide is a highly reactive molecule that is currently accepted as a signaling compound. This molecule is as important as carbon monoxide in mammals and hydrogen peroxide in plants, as well as nitric oxide in both eukaryotic systems. Although many studies have been conducted on the physiological effects of hydrogen sulfide, the underlying mechanisms are poorly understood. One of the proposed mechanisms involves the posttranslational modification of protein cysteine residues, a process called S-sulfhydration. In this work, a modified biotin switch method was used for the detection of Arabidopsis (Arabidopsis thaliana) proteins modified by S-sulfhydration under physiological conditions. The presence of an S-sulfhydration-modified cysteine residue on cytosolic ascorbate peroxidase was demonstrated using liquid chromatography-tandem mass spectrometry analysis, and a total of 106 S-sulfhydrated proteins were identified. Immunoblot and enzyme activity analyses of some of these proteins showed that the sulfide added through S-sulfhydration reversibly regulates the functions of plant proteins in a manner similar to that described in mammalian systems.

Figure 1.

A, Schematic representation of the BSM for the detection of posttranslational modification of proteins by S-nitrosylation, where free thiols are blocked by MMTS, the S-NO bonds are reduced by ascorbate to form free thiols, and finally, these new thiols are ligated with the thiol-specific biotinylating agent biotin-HPDP to form biotin-labeled proteins. B, Schematic representation of the modified BSM for the detection of posttranslational modification of proteins by S-sulfhydration, where free thiol residues are first blocked with MMTS; the persulfide residues remain unreacted and available for subsequent reaction with biotin-HPDP to form biotin-labeled proteins. A sketch of a protein with different Cys residues is shown. Additional details are described in the text.

Figure 2.

Immunoblot analysis of the total S-sulfhydrated proteins. Protein cell extracts from 1 g of leaf tissue were exogenously untreated (L2) or treated (L3) using 200 µm hydrogen sulfide (Na₂S) for 30 min at 4°C and were subjected to the modified BSM. The labeled proteins were detected using protein-blot analysis with antibodies against biotin. Biotin-labeled cytochrome C protein (L1) and a protein cell extract that was not subjected to the modified BSM (L4) were used for the positive and negative control, respectively. Sypro Ruby fluorescent staining is shown as the protein loading control.

Figure 3.

Immunoblot analysis of specific S-sulfhydrated candidate proteins. Biotinylated proteins obtained from the leaf extracts subjected to the modified biotin switch assay were purified using streptavidin-agarose beads and analyzed using four different immunoblots with the following antibodies: antichloroplastic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies that recognized the chloroplastic isoforms A and B, anticytosolic GAPDH antibodies that recognized the cytosolic isoform C, anti-GS antibodies that recognized both the chloroplastic and cytosolic isoforms, and anticytosolic APX antibodies. Sypro Ruby fluorescent staining is shown as the protein loading control.

Figure 4.

Analysis of APX1 using mass spectrometry. A, The protein was identified with a sequence coverage of 74%; the identified peptides are shown in bold red, and the peptide containing S-sulfhydrated Cys-32 is shown underlined. B, LC-MS/MS analysis of the tryptic peptide containing Cys-32 of APX1. The table inside the spectrum contains the predicted ion types for the modified peptide, and the ions detected in the spectrum (Biemann, 1988) are highlighted in red color. Nomenclature of the fragment ions and types corresponds to that proposed by Roepstorff and Fohlman (1984) and modified by Biemann (1988).

Figure 5.

Enzyme activity regulation of Gln synthetase, APX, and GAPDH by S-sulfhydration in Arabidopsis. The protein leaf extracts were treated in the absence or presence of NaHS at the indicated concentrations for 30 min at 4°C (black bars), and an additional treatment with 50 mm DTT was performed for 10 min in some cases (gray bars). Then, Gln synthetase (A), APX (B), or GAPDH (C) enzyme activity was measured as described in “Materials and Methods.” All results are shown as the mean ± sd. a, Significant differences between treatments with and without NaHS (P < 0.05); b, significant differences between samples with or without DTT (P < 0.05).

Figure 6.

Enzyme activity regulation of recombinant cytosolic APX1 and cytosolic glyceraldehyde 3-phosphate dehydrogenase, isoform C1 (GAPC1) by S-sulfhydration. Purified proteins were treated in the absence or presence of NaHS at the indicated concentrations for 30 min at 4°C (black bars), and in some cases an additional treatment with 1 mm DTT was performed (gray bars). Then, APX1 (A) or GAPC1 (B) enzyme activity was measured as described in “Materials and Methods.” All results are shown as the mean ± sd. a, Significant differences between treatments with and without NaHS (P < 0.05); b, significant differences between samples with or without DTT (P < 0.05).