Supersulfide catalysis for nitric oxide and aldehyde metabolism

Figures

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  Fig. 1.. Illustration of the GSNOR and FDH activities of recombinant human WT ADH5 protein and Cys mutants of ADH5. (A) Schematic showing reactions catalyzed by ADH5 with dual enzymatic activities—GSNOR and FDH. NH2OH, hydroxylamine; formyl-SG, S-formylglutathione; HCOOH, formic acid; HCHO, formaldehyde. (B) Domain structure of human ADH5 (hADH5) protein. (C) Measurement of enzymatic activities of recombinant WT hADH5 and mutants of hADH5. GSNOR (left) and FDH (middle) activities of ADH5 (WT, C45S, and C174S) proteins. The ratio of GSNOR activities to FDH activities was calculated for each ADH5 protein (right). The results are presented as means ± SD of three independent enzyme assays. *P < 0.001 versus WT; one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test.

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  Fig. 2.. Supersulfidation of recombinant hADH5 protein and its C174S mutant. (A) Schematic showing the reaction mechanism in the original PMSA. In the initial step of the reaction, both thiol (─SH) and polysulfide [─(S)n-H] groups of the Cys residues in a protein are alkylated by the electrophiles (E). In the second reaction step, the PEGylation probe BPM selectively attacks alkylated polysulfides at proximal sulfur atoms. Polysulfide-rich proteins acquire PEGylation and demonstrate low gel mobility. (B) Detection of protein supersulfidation in WT ADH5 (top) and C174S mutant (bottom) proteins by using the original PMSA. Numbers on the gels and blots indicate expected numbers of PEG moieties in the protein, which were deduced from the mobility shift distance. Proteins were detected by means of CBB staining. 8NcG, 8-nitroguanosine 3′,5′-cyclic monophosphate; ASBT, 2-aminosulfonyl benzothiazole; MSBT, 2-methylsulfonyl benzothiazole; IAM, iodoacetamide; MBB, monobromobimane; MMTS, methyl methanethiosulfonate; NEM, N-ethylmaleimide; DTP, 4,4′-dithiopyridine; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); PCMB, p-chloromercuribenzoic acid. (C) Schematic drawing for the modified PMSA. In the initial…

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  Fig. 3.. Quantification of protein supersulfidation of recombinant ADH5 and detection of protein supersulfidation of ADH5 in mammalian cells. (A) Quantification of supersulfidated Cys residues in recombinant ADH5 (WT and C174S) by LC-ESI-MS/MS. Results are presented as means ± SD (n = 4). *P < 0.01 (unpaired Student’s t test). (B) Detection of protein supersulfidation in WT ADH5 and C174S mutant ADH5 proteins by using the modified PMSA with proteins expressed in HEK293T cells. Proteins were detected by means of immunoblot analysis with anti-FLAG antibody. Numbers on the gels and blots indicate expected numbers of PEG moieties in the protein, which were deduced from the mobility shift distance. WB, Western blotting.

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  Fig. 4.. Regulation of GSNOR by GSNOR/FDH reaction-dependent protein supersulfidation. (A) Schematic showing the reaction of the original PMSA. This PMSA used PEG48-MAL that selectively attacks polysulfides alkylated by IAM at proximal sulfur atoms. Polysulfide-rich proteins thus acquire PEGylation and demonstrate low gel mobility. (B) Detection of supersulfidation of recombinant WT hADH5 before and after GSNOR and FDH reactions by using the original PMSA.

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  Fig. 5.. Correlation of active site supersulfidation with GSNOR activity of ADH5. Recombinant WT ADH5 was immobilized on Sepharose beads and then underwent sequential GSNOR and FDH reactions in the presence of the indicated substrates. (A) Effects of ADH5 enzyme reactions on the GSNOR activity of recombinant ADH5. The ADH5 protein was first treated with the GSNOR reaction mixtures (GSNO plus NADH) for 60 min at 37°C and then subjected to sequential FDH reactions, followed by measurement of GSNOR activity. The FDH reaction mixture included 1 mM NAD+ and HM-SG (100 μM GSH + 500 μM formaldehyde), HM-(S)n-SG (100 μM GSH + 500 μM formaldehyde + 50 μM Na2S2), or a reducing agent (1 mM TCEP). (B) Effects of ADH5 enzyme reactions on the GSNOR activity of recombinant ADH5. The ADH5 protein was first treated with the FDH reaction mixtures containing 100 μM NAD+ and a substrate cocktail of HM-(S)n-SG (10 μM GSH + 50 μM formaldehyde + 5 μM Na2S4) and then subjected to sequential GSNOR reactions, followed by measurement of GSNOR activity. (C) High-precision sulfur proteome analysis of supersulfidated Cys residues of ADH5. The recombinant ADH5 that were treated with sequential FDH and GSNOR reactions,…

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  Fig. 6.. Glutathionylation of recombinant ADH5 protein and its C174S mutant. (A) Immunoblot analysis to detect glutathionylation of WT ADH5 and C174S mutant ADH5 after the GSNOR reaction. (B) Proteins were detected by means of CBB staining. (C) Immunoblot analysis to detect glutathionylation of WT ADH5 after the GSNOR and FDH reactions. The ADH5 alone (lane a) served as a control for other reactions (lanes b to f).

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  Fig. 7.. Characterization of ADH5 C174S in mammalian cells. (A) Immunoblot analysis to detect FLAG-tagged WT ADH5 and C174S mutant ADH5 that were transiently expressed in Adh5−/− MEFs. (B) Measurement of GSNOR (left) and FDH (middle) activities in lysates of Adh5−/− MEFs expressing WT ADH5 or C174S mutant ADH5. Right: The ratio of GSNOR activities to FDH activities for each ADH5 protein expressed in Adh5−/− MEFs. Data are means ± SD (n = 3). *P < 0.001, one-way ANOVA with Tukey’s multiple comparisons test. N.S., not significant. (C) Immunoblot analysis of WT and mutant ADH5 proteins expressed in Adh5+/+ and Adh5C174S/C174S MEFs. (D) Measurement of GSNOR (left) and FDH (middle) activities in lysates of Adh5+/+ and Adh5C174S/C174S MEFs. Right: The ratio of GSNOR activities to FDH activities for each sample. Data are means ± SD, n = 3. *P < 0.001, unpaired Student’s t test. (E) Viability of MEFs after NOC7 or HM-SG treatment. Data are means ± SD, n = 4. *P < 0.001 versus Mock Adh5−/−, #P < 0.001 versus Adh5−/−-expressing C174S mutant, ¶P < 0.01 versus Mock Adh5−/−, two-way ANOVA with Tukey’s multiple comparisons test.

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  Fig. 8.. Cardiac functions in ADH5 mutant mice and proposed supersulfide-mediated enzymatic reactions of ADH5. Improved cardiac functions of Adh5C174S/C174S mice. Basal LV pressures in Adh5+/+ (WT) (n = 8), Adh5C174S/C174S (n = 6), and Adh5−/− (n = 6) mice. (A) The maximal rate of LV pressure rise (LV dP/dtmax). (B) LVEDP. (C) The time constant of isovolumic relaxation (Tau). *P < 0.05, **P < 0.01, ***P < 0.001; one-way ANOVA with Tukey’s multiple comparisons test.

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  Fig. 9.. Proposed mechanisms of regulation of the GSNOR and FDH activities of ADH5 via protein supersulfidation. (A) ADH5 protein contains a zinc ion to which four residues (Cys45, His67, Glu68, and Cys174) coordinate. ADH5 contributes to NO signal regulation and formaldehyde detoxification by GSNOR and FDH activities using NADH and NAD+ respectively. Cys174 supersulfides enhance GSNOR activity. During the GSNOR reaction Cys174 loses supersulfides (Sx) and becomes glutathionylated. The coupled FDH reaction can restore GSNOR catalytic activity by removing the glutathionylation of Cys174. The additional presence of supersulfides in FDH reaction [HM-(S)x-G] can further enhance GSNOR catalytic activity by restoring Cys174 supersulfidation. [Sx], supersulfides; GR, glutathione reductase; GSNHOH, glutathione N-hydroxysulfenamide; HM-(S)x-G, S-hydroxymethylglutathionesupersulfide; formyl-SG, S-formylglutathione. (B) Inactivation of the C174 catalytic function by GSNOR and regeneration by FDH reaction. Proposed mechanism of inactivation of the C174 function during GSNOR reaction. Cys174 (C174) supersulfides are reduced during the GSNOR reaction and resulting persulfide (C174-SSH) is…