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Increased Urinary 3-Mercaptolactate Excretion and Enhanced Passive Systemic Anaphylaxis in Mice Lacking Mercaptopyruvate Sulfurtransferase, a Model of Mercaptolactate-Cysteine Disulfiduria.

ABSTRACT: Mercaptopyruvate sulfurtransferase (Mpst) and its homolog thiosulfate sulfurtransferase (Tst = rhodanese) detoxify cyanide to thiocyanate. Mpst is attracting attention as one of the four endogenous hydrogen sulfide (H2S)/reactive sulfur species (RSS)-producing enzymes, along with cystathionine ?-synthase (Cbs), cystathionine ?-lyase (Cth), and cysteinyl-tRNA synthetase 2 (Cars2). MPST deficiency was found in 1960s among rare hereditary mercaptolactate-cysteine disulfiduria patients. Mpst-knockout (KO) mice with enhanced liver Tst expression were recently generated as its model; however, the physiological roles/significances of Mpst remain largely unknown. Here we generated three independent germ lines of Mpst-KO mice by CRISPR/Cas9 technology, all of which maintained normal hepatic Tst expression/activity. Mpst/Cth-double knockout (DKO) mice were generated via crossbreeding with our previously generated Cth-KO mice. Mpst-KO mice were born at the expected frequency and developed normally like Cth-KO mice, but displayed increased urinary 3-mercaptolactate excretion and enhanced passive systemic anaphylactic responses when compared to wild-type or Cth-KO mice. Mpst/Cth-DKO mice were also born at the expected frequency and developed normally, but excreted slightly more 3-mercaptolactate in urine compared to Mpst-KO or Cth-KO mice. Our Mpst-KO, Cth-KO, and Mpst/Cth-DKO mice, unlike semi-lethal Cbs-KO mice and lethal Cars2-KO mice, are useful tools for analyzing the unknown physiological roles of endogenous H2S/RSS production.

SUBMITTER: Akahoshi N

PROVIDER: S-EPMC7038117 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Increased Urinary 3-Mercaptolactate Excretion and Enhanced Passive Systemic Anaphylaxis in Mice Lacking Mercaptopyruvate Sulfurtransferase, a Model of Mercaptolactate-Cysteine Disulfiduria.

Akahoshi Noriyuki N Minakawa Tatsuro T Miyashita Masashi M Sugiyama Uran U Saito Chihiro C Takemoto Rintaro R Honda Akihiro A Kamichatani Waka W Kamata Shotaro S Anan Yasumi Y Ishii Isao I

International journal of molecular sciences 20200127 3

Mercaptopyruvate sulfurtransferase (Mpst) and its homolog thiosulfate sulfurtransferase (Tst = rhodanese) detoxify cyanide to thiocyanate. Mpst is attracting attention as one of the four endogenous hydrogen sulfide (H<sub>2</sub>S)/reactive sulfur species (RSS)-producing enzymes, along with cystathionine β-synthase (Cbs), cystathionine γ-lyase (Cth), and cysteinyl-tRNA synthetase 2 (Cars2). MPST deficiency was found in 1960s among rare hereditary mercaptolactate-cysteine disulfiduria patients. M ...[more]

PMID: 32012740

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Project description:Background and purposeDuring angiogenesis, quiescent endothelial cells (ECs) are activated by various stimuli to form new blood vessels from pre-existing ones in physiological and pathological conditions. Many research groups have shown that hydrogen sulfide (H2 S), the newest member of the gasotransmitter family, acts as a proangiogenic factor. To date, very little is known about the regulatory role of 3-mercaptopyruvate sulfurtransferase (3-MST), an important H2 S-producing enzyme in ECs. The aim of our study was to explore the potential role of 3-MST in human EC bioenergetics, metabolism, and angiogenesis.Experimental approachTo assess in vitro angiogenic responses, we used EA.hy926 human vascular ECs subjected to shRNA-mediated 3-MST attenuation and pharmacological inhibition of proliferation, migration, and tube-like network formation. To evaluate bioenergetic parameters, cell respiration, glycolysis, glucose uptake, and mitochondrial/glycolytic ATP production were measured. Finally, global metabolomic profiling was performed to determine the level of 669 metabolic compounds.Key results3-MST-attenuated ECs subjected to shRNA or pharmacological inhibition of 3-MST significantly reduced EC proliferation, migration, and tube-like network formation. 3-MST silencing also suppressed VEGF-induced EC migration. From bioenergetic and metabolic standpoints, 3-MST attenuation decreased mitochondrial respiration and mitochondrial ATP production, increased glucose uptake, and perturbed the entire EC metabolome.Conclusion and implications3-MST regulates bioenergetics and morphological angiogenic functions in human ECs. The data presented in the current report support the view that 3-MST pathway may be a potential candidate for therapeutic modulation of angiogenesis.Linked articlesThis article is part of a themed section on Hydrogen Sulfide in Biology & Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.4/issuetoc.

Project description:Endogenous hydrogen sulfide (H2S), which is primarily generated by 3-mercaptopyruvate sulfurtransferase (3-MST) in Escherichia coli (E. coli) under aerobic conditions, renders bacteria highly resistant to oxidative stress. However, the biosynthetic pathway and physiological role of this gas under anaerobic conditions remains largely unknown. In the present study, we demonstrate that cysteine desulfurase (IscS), not 3-MST, is the primary source of endogenous H2S in E. coli under anaerobic conditions. A significant decrease in H2S production under anaerobic conditions was observed in E. coli upon deletion of IscS, but not in 3-MST-deficient bacteria (?mstA). Furthermore, the H2S-producing activity of recombinant IscS using L-cysteine as a substrate exhibited an approximately 2.6-fold increase in the presence of dithiothreitol (DTT), indicating that H2S production catalyzed by IscS was greatly increased under reducing conditions. The activity of IscS was regulated under the different redox conditions and the midpoint redox potential was determined to be -329 ± 1.6 mV. Moreover, in E. coli cells H2S production from IscS is regulated under oxidative and reductive stress. A mutant E. coli (?iscS) strain lacking a chromosomal copy of the IscS-encoding gene iscS showed significant growth defects and low levels of ATP under both aerobic and anaerobic conditions. The growth defects could be fully restored after addition of 500 ?M Na2S (an H2S donor) under anaerobic conditions, but not by the addition of cysteine, sodium sulfite or sodium sulfate. We also showed that the addition of 500 ?M Na2S to culture medium stimulates ATP synthesis in the mutant E. coli (?iscS) strain in the logarithmic growth phase but suppresses ATP synthesis in wild-type E. coli. Our results reveal a new H2S-producing pathway in E. coli under anaerobic conditions and show that hydrogen sulfide from IscS contributes to sustaining cell growth and bioenergetics under oxygen-deficient conditions.

Dataset Information

Increased Urinary 3-Mercaptolactate Excretion and Enhanced Passive Systemic Anaphylaxis in Mice Lacking Mercaptopyruvate Sulfurtransferase, a Model of Mercaptolactate-Cysteine Disulfiduria.

Publications

Increased Urinary 3-Mercaptolactate Excretion and Enhanced Passive Systemic Anaphylaxis in Mice Lacking Mercaptopyruvate Sulfurtransferase, a Model of Mercaptolactate-Cysteine Disulfiduria.

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