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. 2009 Nov 10;2(96):ra72.
doi: 10.1126/scisignal.2000464.

H2S signals through protein S-sulfhydration

Asif K Mustafa  1 , Moataz M Gadalla, Nilkantha Sen, Seyun Kim, Weitong Mu, Sadia K Gazi, Roxanne K Barrow, Guangdong Yang, Rui Wang, Solomon H Snyder
Affiliations

H2S signals through protein S-sulfhydration

Asif K Mustafa et al. Sci Signal. .

Abstract

Hydrogen sulfide (H2S), a messenger molecule generated by cystathionine gamma-lyase, acts as a physiologic vasorelaxant. Mechanisms whereby H2S signals have been elusive. We now show that H2S physiologically modifies cysteines in a large number of proteins by S-sulfhydration. About 10 to 25% of many liver proteins, including actin, tubulin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), are sulfhydrated under physiological conditions. Sulfhydration augments GAPDH activity and enhances actin polymerization. Sulfhydration thus appears to be a physiologic posttranslational modification for proteins.

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Figures

Fig. 1
Fig. 1
H2S covalently modifies proteins through S-sulfhydration of cysteine residues. (A) Liver lysates treated with 100 μM NaHS for 30 min at 37°C and subjected to the modified biotin switch assay with antibody against biotin (Anti-biotin Ab) to detect S-sulfhydration show numerous sulfhydrated proteins. (B) LC-MS/MS of a subset of the sulfhydrated proteins in (A) identifies 39 sulfhydrated proteins, including GAPDH, β-tubulin, and actin. (C) DTT treatment (1 mM) for 10 min reverses GAPDH, β-tubulin, and actin sulfhydration, detected with antibodies specific to each protein, implying a covalent sulfhydryl modification. (D) In HEK293 cells transfected with plasmids encoding CSE, exposure to 5 mM L-cysteine for 1 hour leads to GAPDH, β-tubulin, and actin sulfhydration, as assessed by the modified biotin switch assay with antibodies specific to each of the three proteins. Catalytically inactive CSE fails to sulfhydrate proteins.
Fig. 2
Fig. 2
Sulfhydration is a physiologic posttranslational modification absent in CSE−/− animals. (A) Measurements of H2S production with an H2S-selective electrode indicate that CSE−/− liver cannot generate H2S. All results are presented as the mean ± SEM. ***P < 0.001. (B) Modified biotin switch assay in liver shows the presence of endogenous physiologic sulfhydration of GAPDH, β-tubulin, and actin in wild-type but not CSE−/− animals. (C) Densitometric analysis quantitating basal protein sulfhydration in liver. Data are the mean ± SEM for five to seven independent experiments with representative data shown in (B) and (D). (D) Modified biotin switch assay in liver lysates incubated with increasing exogenous doses of the CSE substrate L-cysteine for 30 min at 37°C shows a dose-dependent increase in sulfhydration in wild-type but not CSE−/− animals. The untreated lanes represent endogenous liver sulfhydration, showing a substantially larger signal for GAPDH than for β-tubulin or actin, consistent with the mean values depicted in (C). (E) Densitometric analysis of (D) quantitating changes in GAPDH sulfhydration with increasing exogenous doses of L-cysteine. Data are the mean ± SEM for five to seven independent experiments.
Fig. 3
Fig. 3
Sulfhydration of GAPDH occurs at Cys150. (A) HPLC followed by LC-MS/MS on endogenous full-length GAPDH protein immunoprecipitated from mouse liver shows sulfhydration of Cys150 with an additional mass of ~32.058 daltons. m/z is the mass-to-charge ratio; the amino acid sequence surrounding Cys150 is shown at the bottom. (B) LC-MS/MS on purified full-length human GAPDH protein treated with 100 μM NaHS for 30 min at 37°C shows a mass shift consistent with Cys150 (152 in human) sulfhydration. Sulfhydration or sulfination could not be detected after treatment with 100 μM NaHS and 1 mM DTT or 500 μM H2O2, respectively. (C) Modified biotin switch assay in HEK293 cells treated with 100 μM NaHS for 30 min at 37°C. C150S mutant is not sulfhydrated. (D) Sulfhydration of GAPDH with radiolabeled [35S]cysteine and CSE at 37°C. GAPDH radiolabeling is reversed by the addition of 1 mM DTT or boiling of the CSE protein for 5 min. Data were quantified by Cherenkov scintillation counting. All results are the mean ± SEM. **P < 0.01. (E) Radiolabeling wild-type and C150S GAPDH with [35S]cysteine and CSE. C150S mutant is not labeled. All results are the mean ± SEM. ** P < 0.01.
Fig. 4
Fig. 4
Sulfhydration physiologically increases the catalytic activity of GAPDH. (A) GAPDH activity assayed in vitro at 37°C with increasing concentrations of NaHS. NaHS dose-dependently activates GAPDH. (B) DTT (1 mM) reverses in vitro GAPDH activation by 10 μM NaHS. All results are the mean ± SEM. **P < 0.01. (C) Wild-type versus C150S mutant GAPDH activity assayed in vitro with 15 μM NaHS. Wild-type (wt) but not C150S GAPDH is activated by NaHS. All results are the mean ± SEM. **P < 0.01. (D) GAPDH activity with increasing substrate G3P concentration with or without 10 μM NaHS. NaHS increases overall Vmax without affecting Km (~0.8 mM). (E) GAPDH activity in HEK293 cells transfected with nothing or plasmids encoding wild-type CSE or catalytically inactive CSE and incubated with increasing concentrations of L-cysteine in the media for 1 hour at 37°C. GAPDH is activated in a dose-dependent manner in the presence of wild-type CSE. (F) In vivo GAPDH activity from wild-type versus CSE−/− liver. Livers from CSE−/− mice show decreased GAPDH activity (n = 6 animals). All results are the mean ± SEM. *P < 0.05.
Fig. 5
Fig. 5
Sulfhydration enhances actin polymerization in vitro and in HEK293 cells. (A) Actin is sulfhydrated in vitro with 200 μM NaHS for 30 min at 37°C, an effect reversed by DTT (200 μM for 10 min). (B) NaHS (100 μM for 30 min at 37°C) sulfhydrates actin in HEK293 cells. (C) Immunocytochemical analysis of HEK293 cells treated with 100 μM NaHS for 1 hour at 37°C reveals rearrangement of the actin cytoskeleton (arrows show the extension of thin processes containing actin filaments in individual NaHS treated cells). Actin is stained in red. Scale bars, 20 μm. (D) NaHS (200 μM) enhances actin polymerization in vitro, an effect reversed by DTT (200 μM). (E) NaHS (200 μM) has no effect on actin depolymerization.
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