Enzymology of H2S Biogenesis, Decay and Signaling

Significance: Hydrogen sulfide (H2S), produced by the desulfuration of cysteine or homocysteine, functions as a signaling molecule in an array of physiological processes including regulation of vascular tone, the cellular stress response, apoptosis, and inflammation. Recent Advances: The low steady-state levels of H2S in mammalian cells have been recently shown to reflect a balance between its synthesis and its clearance. The subversion of enzymes in the cytoplasmic trans-sulfuration pathway for producing H2S from cysteine and/or homocysteine versus producing cysteine from homocysteine, presents an interesting regulatory problem. Critical Issues: It is not known under what conditions the enzymes operate in the canonical trans-sulfuration pathway and how their specificity is switched to catalyze the alternative H2S-producing reactions. Similarly, it is not known if and whether the mitochondrial enzymes, which oxidize sulfide and persulfide (or sulfane sulfur), are regulated to increase or decrease H2S or sulfane-sulfur pools. Future Directions: In this review, we focus on the enzymology of H2S homeostasis and discuss H2S-based signaling via persulfidation and thionitrous acid. Antioxid. Redox Signal. 20, 770?782.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140254/1/ars.2013.5339.pd

High Turnover Rates for Hydrogen Sulfide Allow for Rapid Regulation of Its Tissue Concentrations

Abstract Aims: Hydrogen sulfide (H2S) is a signaling molecule, which influences many physiological processes. While H2S is produced and degraded in many cell types, the kinetics of its turnover in different tissues has not been reported. In this study, we have assessed the rates of H2S production in murine liver, kidney, and brain homogenates at pH 7.4, 37°C, and at physiologically relevant cysteine concentrations. We have also studied the kinetics of H2S clearance by liver, kidney, and brain homogenates under aerobic and anaerobic conditions. Results: We find that the rate of H2S production by these tissue homogenates is considerably higher than background rates observed in the absence of exogenous substrates. An exponential decay of H2S with time is observed and, as expected, is significantly faster under aerobic conditions. The half-life for H2S under aerobic conditions is 2.0, 2.8, and 10.0?min with liver, kidney, and brain homogenate, respectively. Western-blot analysis of the sulfur dioxygenase, ETHE1, involved in H2S catabolism, demonstrates higher steady-state protein levels in liver and kidney versus brain. Innovation: By combining experimental and simulation approaches, we demonstrate high rates of tissue H2S turnover and provide estimates of steady-state H2S levels. Conclusion: Our study reveals that tissues maintain a high metabolic flux of sulfur through H2S, providing a rationale for how H2S levels can be rapidly regulated. Antioxid. Redox Signal. 17, 22?31.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/98481/1/ars%2E2011%2E4310.pd

The Quantitative Significance of the Transsulfuration Enzymes for H2S Production in Murine Tissues

The enzymes of the transsulfuration pathway, cystathionine --synthase (CBS) and cystathionine --lyase (CSE), are important for the endogenous production of hydrogen sulfide (H2S), a gaseous signaling molecule. The relative contributions of CBS and CSE to H2S generation in different tissues are not known. In this study, we report quantification of CBS and CSE in murine liver and kidney and their contribution to H2S generation in these tissues and in brain at saturating substrate concentrations. We show that CBS protein levels are significantly lower than those of CSE; 60-fold and 20-fold in liver and kidney, respectively. Each enzyme is more abundant in liver compared with kidney, twofold and sixfold for CBS and CSE, respectively. At high substrate concentrations (20-mM each cysteine and homocysteine), the capacity for liver H2S production is approximately equal for CBS and CSE, whereas in kidney and brain, CBS constitutes the major source of H2S, accounting for -80% and -95%, respectively, of the total output. At physiologically relevant concentrations of substrate, and adjusting for the differences in CBS versus CSE levels, we estimate that CBS accounts for only 3% of H2S production by the transsulfuration pathway enzymes in liver. Antioxid. Redox Signal. 15, 363-372.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90506/1/ars-2E2010-2E3781.pd

Defective Thyroglobulin: Cell Biology of Disease

The primary functional units of the thyroid gland are follicles of various sizes comprised of a monolayer of epithelial cells (thyrocytes) surrounding an apical extracellular cavity known as the follicle lumen. In the normal thyroid gland, the follicle lumen is filled with secreted protein (referred to as colloid), comprised nearly exclusively of thyroglobulin with a half-life ranging from days to weeks. At the cellular boundary of the follicle lumen, secreted thyroglobulin becomes iodinated, resulting from the coordinated activities of enzymes localized to the thyrocyte apical plasma membrane. Thyroglobulin appearance in evolution is essentially synchronous with the appearance of the follicular architecture of the vertebrate thyroid gland. Thyroglobulin is the most highly expressed thyroid gene and represents the most abundantly expressed thyroid protein. Wildtype thyroglobulin protein is a large and complex glycoprotein that folds in the endoplasmic reticulum, leading to homodimerization and export via the classical secretory pathway to the follicle lumen. However, of the hundreds of human thyroglobulin genetic variants, most exhibit increased susceptibility to misfolding with defective export from the endoplasmic reticulum, triggering hypothyroidism as well as thyroidal endoplasmic reticulum stress. The human disease of hypothyroidism with defective thyroglobulin (either homozygous, or compound heterozygous) can be experimentally modeled in thyrocyte cell culture, or in whole animals, such as mice that are readily amenable to genetic manipulation. From a combination of approaches, it can be demonstrated that in the setting of thyroglobulin misfolding, thyrocytes under chronic continuous ER stress exhibit increased susceptibility to cell death, with interesting cell biological and pathophysiological consequences

GWAS of thyroid stimulating hormone highlights pleiotropic effects and inverse association with thyroid cancer

Correction: Volume12, Issue1 Article Number7354 DOI10.1038/s41467-021-27675-w PublishedDEC 16 2021Thyroid stimulating hormone (TSH) is critical for normal development and metabolism. To better understand the genetic contribution to TSH levels, we conduct a GWAS meta-analysis at 22.4 million genetic markers in up to 119,715 individuals and identify 74 genome-wide significant loci for TSH, of which 28 are previously unreported. Functional experiments show that the thyroglobulin protein-altering variants P118L and G67S impact thyroglobulin secretion. Phenome-wide association analysis in the UK Biobank demonstrates the pleiotropic effects of TSH-associated variants and a polygenic score for higher TSH levels is associated with a reduced risk of thyroid cancer in the UK Biobank and three other independent studies. Two-sample Mendelian randomization using TSH index variants as instrumental variables suggests a protective effect of higher TSH levels (indicating lower thyroid function) on risk of thyroid cancer and goiter. Our findings highlight the pleiotropic effects of TSH-associated variants on thyroid function and growth of malignant and benign thyroid tumors. Thyroid stimulating hormone (TSH) is critical for normal development and metabolism. Here, the authors conduct a GWAS and suggest protective effect of higher TSH on risk of thyroid cancer and goitre.Peer reviewe

Author Correction:GWAS of thyroid stimulating hormone highlights the pleiotropic effects and inverse association with thyroid cancer

The original version of this article contained an error in the results, in the second paragraph of the subsection entitled “Fine-mapping for potentially causal variants among TSH loci”, in which effect sizes for two variants were incorrectly reported

GWAS of thyroid stimulating hormone highlights pleiotropic effects and inverse association with thyroid cancer

Thyroid stimulating hormone (TSH) is critical for normal development and metabolism. To better understand the genetic contribution to TSH levels, we conduct a GWAS meta-analysis at 22.4 million genetic markers in up to 119,715 individuals and identify 74 genome-wide significant loci for TSH, of which 28 are previously unreported. Functional experiments show that the thyroglobulin protein-altering variants P118L and G67S impact thyroglobulin secretion. Phenome-wide association analysis in the UK Biobank demonstrates the pleiotropic effects of TSH-associated variants and a polygenic score for higher TSH levels is associated with a reduced risk of thyroid cancer in the UK Biobank and three other independent studies. Two-sample Mendelian randomization using TSH index variants as instrumental variables suggests a protective effect of higher TSH levels (indicating lower thyroid function) on risk of thyroid cancer and goiter. Our findings highlight the pleiotropic effects of TSH-associated variants on thyroid function and growth of malignant and benign thyroid tumors

Structural and spectroscopic characterization of human cystathionine β-synthase

Human cystathionine β-synthase (CBS), a pyridoxal 5′ -phosphate (PLP)-dependent hemeprotein, catalyzes the condensation of homocysteine and seine to give cystathionine. The CBS catalyzed reaction is the first committed step in the catabolic removal of the toxic metabolite, homocysteine, via the transsulfuration pathway. The latter also represents a pathway for the conversion of homocysteine to cysteine and ultimately to glutathione (GSH). The reaction catalyzed by CBS is of clinical importance. Mutations in CBS result in high levels of homocysteine and is the most common cause of hereditary homocystinuria. High homocysteine levels are an independent risk factor for cardiovascular diseases, neural tube defects and Alzheimer\u27s disease. Human CBS is a homotetramer of 63 kDa subunits and contains 1 mol of heme and 1 mol of PLP per subunit. While the dependence of CBS activity on PLP can be explained by the chemical similarity of the reaction it catalyses to β-replacement reactions catalyzed by other PLP enzymes, the role of the heme and its location relative to the active site was not known when this study was initiated. In this study, we have used a combination of spectroscopic methods to provide the first structural evidence for a regulatory role for the heme. The two cofactors, heme and PLP, were estimated to be grater than 10 Å apart indicating that a catalytic role for the heme is unlikely. However, changes in the heme oxidation state were found to be transmitted to PLP in the active site consistent with a regulatory role for this cofactor. We also purified and characterized a pathogenic CBS mutant, V168M, associated with a B6-responsive phenotype in patients and showed that the cofactor content of the enzyme is regulated by interactions between the C-terminal regulatory and the N-terminal catalytic domains. The defect introduced by the V168M substitution resulted in a 7-fold lower PLP and 2-fold lower heme content with a 13-fold decrease in enzyme activity and was completely alleviated by deletion of 143 amino acids from the C-terminal. Since some CBS mutations described in patients with high levels of homocysteine due to the CBS deficiency appear to result in normal enzyme activity, we hypothesized that CBS interacts with other proteins and that these interactions are disrupted in this subclass of pathogenic mutants. In order to address this question, we have used the yeast two-hybrid system and probed a human brain library with the CBS bait. These studies identified Ubc9 and PIAS1 which represents the E2 and E3 enzymes involved in the sumoylation pathway. CBS contains three consensus ΨKXE sequences that represent targets for sumoylation. All three sequences are surface exposed in the structure of the dimeric enzyme. In fact, K102 in one of the consensus sequences, LKCE, is mutated to a glutamine in a patient and raises the question as to whether sumoylation of the protein may be impaired. Although these studies are still in progress, our efforts to observe sumoylation of CBS is discussed

Increased transsulfuration mediates longevity and dietary restriction in \u3ci\u3eDrosophila\u3c/i\u3e

The mechanisms through which dietary restriction enhances health and longevity in diverse species are unclear. The transsulfuration pathway (TSP) is a highly conserved mechanism for metabolizing the sulfur-containing amino acids, methionine and cysteine. Here we show that Drosophila cystathionine β-synthase (dCBS), which catalyzes the rate-determining step in the TSP, is a positive regulator of lifespan in Drosophila and that the pathway is required for the effects of diet restriction on animal physiology and lifespan. dCBS activity was up-regulated in flies exposed to reduced nutrient conditions, and ubiquitous or neuron-specific transgenic overexpression of dCBS enhanced longevity in fully fed animals. Inhibition of the TSP abrogated the changes in lifespan, adiposity, and protein content that normally accompany diet restriction. RNAi-mediated knockdown of dCBS also limited lifespan extension by diet. Diet restriction reduced levels of protein translation in Drosophila, and we show that this is largely caused by increased metabolic commitment of methionine cycle intermediates to transsulfuration. However, dietary supplementation of methionine restored normal levels of protein synthesis to restricted animals without affecting lifespan, indicating that global reductions in translation alone are not required for diet-restriction longevity. Our results indicate a mechanism by which dietary restriction influences physiology and aging