Towards Understanding the Pathway for Hydrogen Sulfide Metabolism

Hydrogen sulfide (H2S) is the most recently identified member of a small family of labile biological signaling molecules, termed gasotransmitters, which includes nitric oxide and carbon monoxide. H2S is the only gasotransmitter that is enzymatically metabolized a process that occurs in the mitochondria. H2S needs to be tightly regulated because it is toxic at high concentrations and leads to physiological defects at low concentrations. For example, a genetic defect that affects the metabolic pathway of H2S is ethylmalonic encephalopathy, a fatal disorder that is characterized by extremely high levels of H2S. On the other hand, animal model studies provide compelling evidence for a functional association between abnormally low levels of H2S and cardiovascular disease. In light of H2S’s critical role, the goal of this thesis was to identify and characterize two human enzymes that are proposed to comprise part of the metabolic pathway of H2S in mammals: Sulfide:quinone oxidoreductase (SQOR) and thiosulfate:glutathione sulfurtransferase (TST). The present study postulates that human sulfide:quinone oxidoreductase (SQOR), a membrane-bound enzyme, catalyzes the first step in the mitochondrial metabolism of H2S. The reaction involves a two-electron oxidation of H2S to S0 (sulfane sulfur) and uses coenzyme Q as an electron acceptor. The fact that SQOR is a membrane-associated protein has made its expression and isolation challenging. We successfully purified and characterized human SQOR. Cyanide, sulfite, or sulfide can act as the sulfane sulfur acceptor in reactions that produce thiocyanate, thiosulfate, or a putative sulfur analog of hydrogen peroxide (H2S2), respectively. Thiosulfate is a known intermediate in the oxidation of H2S within animals and the major product formed in glutathione-depleted cells or mitochondria. Importantly, oxidation of H2S by SQOR with sulfite as the sulfane sulfur acceptor is rapid and highly efficient at physiological pH (kcat/Km,H2S = 2.9 × 107 M-1 s-1). We propose that this highly efficient oxidation of H2S by SQOR is the predominant source of the thiosulfate in mammalian tissues and that sulfite is the physiological acceptor of the sulfane sulfur. Our proposal opposes an alternative hypothesis that glutathione is an acceptor of the sulfane sulfur, which we have compelling evidence against. The discovery that sulfite was the physiological acceptor of the sulfane sulfur and SQOR produced thiosulfate, led us to postulate a role in H2S metabolism for a TST that transfers the sulfane sulfur of thiosulfate to glutathione producing GSS- and sulfite. We postulate that the TST links together the SQOR and sulfur dioxygenase (SDO) steps in the pathway because it consumes the thiosulfate from the SQOR reaction and produces glutathione persulfide (GSS-), a substrate required for SDO. Although an active TST enzyme had been found in yeast, attempts by other laboratories to isolate and characterize the mammalian enzyme have been unsuccessful. We also discovered genes that encode for human and yeast TST (TSTD1 and RDL1, respectively). We demonstrated that GSS- was released into solution and consumed by SDO. Additionally, GSS- is a potent inhibitor of TSTD1 and RDL1, as judged by initial rate accelerations and ≥25-fold lower Km values for glutathione observed in the presence of SDO. Our studies support the conclusion that TST is the missing link between the SQOR and SDO reactions. The discovery of bacterial proteins that are fusions of SDO and TSTD1 provides phylogenetic evidence of the association of these enzymes. We successfully purified and characterized the fusion protein from Nitrosococcus oceani encoded by the gene Noc_2007. We showed that operationally, the fusion is a glutathione-dependent thiosulfate dioxygenase, which is the TST reaction followed by the SDO reaction. The thiosulfate dioxygenase reaction requires one mole of thiosulfate in the presence of oxygen to produce two moles of sulfite with a catalytic amount of glutathione. Lastly, the TST reaction is the apparent rate-limiting step in the thiosulfate dioxygenase reaction. From this study, we propose a new pathway for H2S metabolism, which opens the door to future research.Ph.D., Biochemistry -- Drexel University, 201

Biosynthesis of a Central Intermediate in Hydrogen Sulfide Metabolism by a Novel Human Sulfurtransferase and Its Yeast Ortholog

Human sulfide:quinone oxidoreductase (SQOR) catalyzes the conversion of H₂S to thiosulfate, the first step in mammalian H₂S metabolism. SQOR’s inability to produce the glutathione persulfide (GSS^–) substrate for sulfur dioxygenase (SDO) suggested that a thiosulfate:glutathione sulfurtransferase (TST) was required to provide the missing link between the SQOR and SDO reactions. Although TST could be purified from yeast, attempts to isolate the mammalian enzyme were not successful. We used bioinformatic approaches to identify genes likely to encode human TST (<i>TSTD1</i>) and its yeast ortholog (<i>RDL1</i>). Recombinant TSTD1 and RDL1 catalyze a predicted thiosulfate-dependent conversion of glutathione to GSS^–. Both enzymes contain a rhodanese homology domain and a single catalytically essential cysteine, which is converted to cysteine persulfide upon reaction with thiosulfate. GSS^– is a potent inhibitor of TSTD1 and RDL1, as judged by initial rate accelerations and ≥25-fold lower <i>K</i>_m values for glutathione observed in the presence of SDO. The combined action of GSS^– and SDO is likely to regulate the biosynthesis of the reactive metabolite. SDO drives to completion <i>p</i>-toluenethiosulfonate:glutathione sulfurtransferase reactions catalyzed by TSTD1 and RDL1. The thermodynamic coupling of the irreversible SDO and reversible TST reactions provides a model for the physiologically relevant reaction with thiosulfate as the sulfane donor. The discovery of bacterial Rosetta Stone proteins that comprise fusions of SDO and TSTD1 provides phylogenetic evidence of the association of these enzymes. The presence of adjacent bacterial genes encoding SDO–TSTD1 fusion proteins and human-like SQORs suggests these prokaryotes and mammals exhibit strikingly similar pathways for H₂S metabolism

Human Sulfide:Quinone Oxidoreductase Catalyzes the First Step in Hydrogen Sulfide Metabolism and Produces a Sulfane Sulfur Metabolite

Sulfide:quinone oxidoreductase (SQOR) is a membrane-bound enzyme that catalyzes the first step in the mitochondrial metabolism of H₂S. Human SQOR is successfully expressed at low temperature in <i>Escherichia coli</i> by using an optimized synthetic gene and cold-adapted chaperonins. Recombinant SQOR contains noncovalently bound FAD and catalyzes the two-electron oxidation of H₂S to S⁰ (sulfane sulfur) using CoQ₁ as an electron acceptor. The prosthetic group is reduced upon anaerobic addition of H₂S in a reaction that proceeds via a long-wavelength-absorbing intermediate (λ_max = 673 nm). Cyanide, sulfite, or sulfide can act as the sulfane sulfur acceptor in reactions that (i) exhibit pH optima at 8.5, 7.5, or 7.0, respectively, and (ii) produce thiocyanate, thiosulfate, or a putative sulfur analogue of hydrogen peroxide (H₂S₂), respectively. Importantly, thiosulfate is a known intermediate in the oxidation of H₂S by intact animals and the major product formed in glutathione-depleted cells or mitochondria. Oxidation of H₂S by SQOR with sulfite as the sulfane sulfur acceptor is rapid and highly efficient at physiological pH (<i>k</i>_cat/<i>K</i>_m,H₂S = 2.9 × 10⁷ M^–1 s^–1). A similar efficiency is observed with cyanide, a clearly artificial acceptor, at pH 8.5, whereas a 100-fold lower value is seen with sulfide as the acceptor at pH 7.0. The latter reaction is unlikely to occur in healthy individuals but may become significant under certain pathological conditions. We propose that sulfite is the physiological acceptor of the sulfane sulfur and that the SQOR reaction is the predominant source of the thiosulfate produced during H₂S oxidation by mammalian tissues

Eicosanoyl-5-hydroxytryptamide (EHT) prevents Alzheimer's disease-related cognitive and electrophysiological impairments in mice exposed to elevated concentrations of oligomeric beta-amyloid.

Soluble forms of oligomeric beta-amyloid (Aβ) are thought to play a central role in Alzheimer's disease (AD). Transgenic manipulation of methylation of the serine/threonine protein phosphatase, PP2A, was recently shown to alter the sensitivity of mice to AD-related impairments resulting from acute exposure to elevated levels of Aβ. In addition, eicosanoyl-5-hydroxytryptamide (EHT), a naturally occurring component from coffee beans that modulates PP2A methylation, was shown to confer therapeutic benefits in rodent models of AD and Parkinson's disease. Here, we tested the hypothesis that EHT protects animals from the pathological effects of exposure to elevated levels of soluble oligomeric Aβ. We treated mice with EHT-containing food at two different doses and assessed the sensitivity of these animals to Aβ-induced behavioral and electrophysiological impairments. We found that EHT administration protected animals from Aβ-induced cognitive impairments in both a radial-arm water maze and contextual fear conditioning task. We also found that both chronic and acute EHT administration prevented Aβ-induced impairments in long-term potentiation. These data add to the accumulating evidence suggesting that interventions with pharmacological agents, such as EHT, that target PP2A activity may be therapeutically beneficial for AD and other neurological conditions

Chronic EHT treatment prevents Aβ-induced impairment of long-term potentiation.

<p><b>(</b>A-B) Time course of averaged Schaffer collateral fEPSP responses (± SEM) in hippocampal slices prepared from animals fed control or EHT-containing diets and treated with either vehicle or 100 nM Aβ (horizontal bar) 20 min prior to delivery of theta-burst stimulation (arrow). (A) Aβ treatment significantly reduces potentiated responses following TBS in slices prepared from animals on control diets (2-way RM-ANOVA for treatment with time and treatment as factors: F(1,29) = 8.913, P = 0.0057). (B) Mice fed diets containing 0.01% EHT are resistant to Aβ-induced LTP impairment (2-way RM-ANOVA for treatment with time and treatment as factors: F(1,26) = 0.0943, P = 0.7612). C) Input/output (2-way RM-ANOVA for treatment with stimulus and treatment as factors: F(1,57) = 0.5466, P = 0.4628) (N = 31 control, 28 0.01% EHT).</p

EHT treatment reduces tau phosphorylation.

<p><b>(</b>A) Representative western blots for the indicated proteins and methylated PP2A/C performed on hippocampal homogenates prepared from animals fed control diet, or diets containing 0.01 or 0.1% EHT. (B) Histogram showing average ± SEM for tubulin-normalized band intensities expressed as average percent of control band intensity from replicate western blots in A show no significant differences in expression levels for any of the indicated proteins or for methylated PP2A/C (ANOVA for: PP2A/C: F(2,22) = 0.2533, P = 0.7784; PP2A/A: F(2,22) = 0.2588, P = 0.7743; B55α: F(2,22) = 0.06221, P = 0.9399; PME-1: F(2,22) = 0.4942, P = 0.6167; LCMT-1: F(2,22) = 0.2498, P = 0.7812; methyl-PP2A/C: F(2,22) = 0.1666, P = 0.8476). (C) Representative western blots for demethylated PP2A, and total PP2A/C performed on the homogenates described in A either treated (+) or mock treated (-) with 0.5 M sodium hydroxide. (D) Histogram of average demethylated PP2A/C (± SEM) in hippocampal homogenates prepared from animals fed control, or 0.01 or 0.1% EHT containing-diets show no significant differences in demethylated PP2A/C levels (ANOVA: F(2,22) = 0.1436, P = 0.8670). Values were calculated as ratios of demethyl-PP2A/C to total PP2A/C band intensities for -NaOH treated samples from replicate western blots shown in C and expressed as percent of the average of control. (E) Representative western blots performed on hippocampal homogenates prepared from animals fed control diet, or diets containing 0.01 or 0.1% EHT for phospho-Ser396/404 (PHF1), phospho-Ser202 (CP13) together with their corresponding total tau loading controls, as well as and total tau together with its corresponding β-actin loading control. (F) Histogram showing average band intensities ± SEM for phospho-Ser396/404-tau (PHF1), phospho-Ser202-tau (CP13) normalized to corresponding total tau loading control, and total tau normalized to corresponding β-actin loading control for replicate western blots shown in H show a trend for reduced phosphorylation at these sites in EHT-treated animals (ANOVA for: PHF1: F(2,22) = 5.147, P = 0.147, Bonferroni post-hoc for PHF1 0.1% EHT vs. Control: t = 3.154, P = 0.0092; CP13: F(2,22) = 1.433, P = 0.2599; total tau: F(2,22) = 0.1268, P = 0.8815). (G and H) Representative western blots for phospho-Ser9 and total GSK3B and phospho-Ser133 and total Creb performed on hippocampal homogenates prepared from animals fed control diet, or diets containing 0.01 or 0.1% EHT. (I) Histogram showing average ± SEM of phospho-GSK3β and phospho-Creb band intensities normalized to corresponding total GSK3β and Creb respectively for replicate western blots shown in G&H show no significant effect of EHT treatment on phosphorylation at these sites (ANOVA for: P-GSK3β F(2,22) = 1.761, P = 0.1952; P-Creb: F(2,22) = 0.1776, P = 0.8385). (N = 8 control, 8 0.01% EHT and 9 0.1% EHT treated animals for each measure).</p

EHT prevents Aβ-induced impairment of spatial learning and memory in a 2-day radial arm water maze task.

<p><b>(</b>A) Average number of errors committed (± SEM) during each 3-trial training block of a 2-day radial arm water maze task for the indicated treatment groups. 2-way RM-ANOVA for day 2 (blocks 6–10) with block and group as factors: F (5,69) = 4.424, P = 0.0015 for group; F (4,276) = 25.95, P<0.0001 for block,; F (20,276) = 0.5657, P = 0.9338 for interaction. Bonferroni post-hoc comparisons of the control + vehicle group to all other treatment groups show that only the control + Aβ group is significantly different than control + vehicle group. (N = 12 control + vehicle, 13 control + Aβ, 12 0.01% EHT + vehicle, 12 0.01% + Aβ, 13 0.1% + vehicle, 13 0.1% EHT + Aβ.) (B) Plot of the average escape latency (± SEM) for the indicated treatment groups during training on a visible platform Morris water maze task reveals no significant differences between groups (2-way RM-ANOVA with trial block and treatment group as factors: F(5,69) = 0.9766, P = 0.4384 for group, F(3,207) = 72.48, P<0.0001 for block, and F(15,207) = 0.8627, P = 0.6068 for interaction). (N = 12 control + vehicle, 13 control + Aβ, 12 0.01% EHT + vehicle, 12 0.01% + Aβ, 13 0.1% + vehicle, 13 0.1% EHT + Aβ). (C) Plot of the average swim speed (± SEM) for the indicated treatment groups during training on the visible platform Morris water maze task described in B reveals no significant differences between groups (2-way RM-ANOVA with trial block and treatment group as factors: F(5,69) = 1.232, P = 0.3035 for group, F(3,207) = 28.3, P<0.0001 for block, and F(15,207) = 0.9227, P = 0.5398 for interaction).</p

List of antibodies.

<p>List of antibodies.</p

Acute EHT treatment prevents Aβ-induced impairment of long-term potentiation.

<p><b>(</b>A-F) Time course of averaged Schaffer collateral fEPSP responses (± SEM) in hippocampal slices prepared from slices treated with vehicle or 0, 0.0001, 0.001, 0.01, 0.1, or 1 μM EHT +/- vehicle or 100 nM Aβ (horizontal bar) 20 min prior to delivery of theta-burst stimulation (arrow). (A) Aβ treatment significantly reduces potentiated responses following TBS in slices treated with 0 μM EHT (2-way RM-ANOVA for treatment with time and treatment as factors: F(1,19) = 8.827, P = 0.0078). (B) Aβ treatment significantly reduces potentiated responses following TBS in slices treated with 0.0001 μM EHT (2-way RM-ANOVA for treatment with time and treatment as factors: F(1,11) = 6.84, P = 0.0240). (B) Aβ treatment yields a non-significant trend for reduced potentiated responses following TBS in slices treated with 0.001 μM EHT (2-way RM-ANOVA for treatment with time and treatment as factors: F(1,16) = 2.123, P = 0.1645). (D-F) Aβ treatment does not significantly reduce potentiated responses following TBS in slices treated with 0.01, 0.1, or 1 μM EHT (2-way RM-ANOVA for treatment with time and treatment as factors: For 0.01 μM EHT: F(1,18) = 0.1646, P = 0.6898; For 0.1 μM EHT: F(1.21) = 0.0034, P = 0.9543; For 1 μM EHT: F(1,15) = 0.0014, P = 0.9702). Comparison of potentiated responses in the absence of Aβ revealed no effect of EHT treatment alone on TBS-induced LTP (2-way RM-ANOVA comparisons to 0 EHT + vehicle for treatment with time and treatment as factors: For 0.0001 μM EHT: F(1,15) = 0.0132, P = 0.9099; For 0.001 μM EHT: F(1,18) = 0.0539, P = 0.8191; For 0.01 μM EHT: F(1,18) = 0.16, P = 0.6939; For 0.1 μM EHT: F(1,21) = 0.2294, P = 0.6369; For 1 μM EHT: F(1,18) = 0.7098, P = 0.4106). (G) Plot of the average potentiated responses over the last 10 min of the recordings shown in B-F for slices treated with Aβ in the presence of the indicated concentrations of EHT. The upper and lower dashed lines indicate the mean potentiated response obtained in the absence of EHT for vehicle or Aβ treated slices respectively. (N = 11 0 μM EHT + vehicle, 10 0 μM EHT + Aβ, 6 0.0001 μM EHT + vehicle, 7 0.0001 μM EHT + Aβ, 9 0.0001 μM EHT + vehicle, 9 0.0001 μM EHT + Aβ, 9 0.01 μM EHT + vehicle, 11 0.01 μM EHT + Aβ, 12 0.1 μM EHT + vehicle, 11 0.1 μM EHT + Aβ, 9 1 μM EHT + vehicle, 8 1 μM EHT + Aβ slices).</p

Chronic EHT treatment prevents Aβ-induced impairment of long-term potentiation.

<p><b>(</b>A-B) Time course of averaged Schaffer collateral fEPSP responses (± SEM) in hippocampal slices prepared from animals fed control or EHT-containing diets and treated with either vehicle or 100 nM Aβ (horizontal bar) 20 min prior to delivery of theta-burst stimulation (arrow). (A) Aβ treatment significantly reduces potentiated responses following TBS in slices prepared from animals on control diets (2-way RM-ANOVA for treatment with time and treatment as factors: F(1,29) = 8.913, P = 0.0057). (B) Mice fed diets containing 0.01% EHT are resistant to Aβ-induced LTP impairment (2-way RM-ANOVA for treatment with time and treatment as factors: F(1,26) = 0.0943, P = 0.7612). C) Input/output (2-way RM-ANOVA for treatment with stimulus and treatment as factors: F(1,57) = 0.5466, P = 0.4628) (N = 31 control, 28 0.01% EHT).</p