11 research outputs found
Supplementary material for the article: Wedmann, R.; Onderka, C.; Wei, S.; Szijártó, I. A.; Miljkovic, J. L.; Mitrovic, A.; Lange, M.; Savitsky, S.; Yadav, P. K.; Torregrossa, R.; et al. Improved Tag-Switch Method Reveals That Thioredoxin Acts as Depersulfidase and Controls the Intracellular Levels of Protein Persulfidation. Chemical Science 2016, 7 (5), 3414–3426. https://doi.org/10.1039/c5sc04818d
Supplementary material for: [https://doi.org/10.1039/c5sc04818d]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/1925
Insights into the mechanism of the reaction between hydrogen sulfide and peroxynitrite
Hydrogen sulfide and peroxynitrite are endogenously generated molecules that participate in biologically relevant pathways. A revision of the kinetic features of the reaction between peroxynitrite and hydrogen sulfide revealed a complex process. The rate constant of peroxynitrite decay, (6.65 ± 0.08) × 103 M-1 s-1 in 0.05 M sodium phosphate buffer (pH 7.4, 37 °C), was affected by the concentration of buffer. Theoretical modeling suggested that, as in the case of thiols, the reaction is initiated by the nucleophilic attack of HS- on the peroxide group of ONOOH by a typical bimolecular nucleophilic substitution, yielding HSOH and NO2 -. In contrast to thiols, the reaction then proceeds to the formation of distinct products that absorb near 408 nm. Experiments in the presence of scavengers and carbon dioxide showed that free radicals are unlikely to be involved in the formation of these products. The results are consistent with product formation involving the reactive intermediate HSSH and its fast reaction with a second peroxynitrite molecule. Mass spectrometry and UV-Vis absorption spectra predictions suggest that at least one of the products is HSNO2 or its isomer HSONO.Fil: Cuevasanta, Ernesto. Universidad de la República; UruguayFil: Zeida Camacho, Ari Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Carballal, Sebastián. Universidad de la República; UruguayFil: Wedmann, Rudolf. Universitat Erlangen-Nuremberg; AlemaniaFil: Morzan, Uriel N.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Trujillo, Madia. Universidad de la República; UruguayFil: Radi, Rafael. Universidad de la República; UruguayFil: Estrin, Dario Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Filipovic, Milos R.. Universitat Erlangen-Nuremberg; AlemaniaFil: Alvarez, Beatriz. Universidad de la República; Urugua
Biosynthesis and Reactivity of Cysteine Persulfides in Signaling
Hydrogen
sulfide (H<sub>2</sub>S) elicits pleiotropic physiological
effects ranging from modulation of cardiovascular to CNS functions.
A dominant method for transmission of sulfide-based signals is via
posttranslational modification of reactive cysteine thiols to persulfides.
However, the source of the persulfide donor and whether its relationship
to H<sub>2</sub>S is as a product or precursor is controversial. The
transsulfuration pathway enzymes can synthesize cysteine persulfide
(Cys–SSH) from cystine and H<sub>2</sub>S from cysteine and/or
homocysteine. Recently, Cys–SSH was proposed as the primary
product of the transsulfuration pathway with H<sub>2</sub>S representing
a decomposition product of Cys–SSH. Our detailed kinetic analyses
demonstrate a robust capacity for Cys–SSH production by the
human transsulfuration pathway enzymes, cystathionine beta-synthase
and γ-cystathionase (CSE) and for homocysteine persulfide synthesis
from homocystine by CSE only. However, in the reducing cytoplasmic
milieu where the concentration of reduced thiols is significantly
higher than of disulfides, substrate level regulation favors the synthesis
of H<sub>2</sub>S over persulfides. Mathematical modeling at physiologically
relevant hepatic substrate concentrations predicts that H<sub>2</sub>S rather than Cys–SSH is the primary product of the transsulfuration
enzymes with CSE being the dominant producer. The half-life of the
metastable Cys–SSH product is short and decomposition leads
to a mixture of polysulfides (Cys–S–(S)<sub><i>n</i></sub>–S–Cys). These in vitro data, together
with the intrinsic reactivity of Cys–SSH for cysteinyl versus
sulfur transfer, are consistent with the absence of an observable
increase in protein persulfidation in cells in response to exogenous
cystine and evidence for the formation of polysulfides under these
conditions
Does Perthionitrite (SSNO<sup>–</sup>) Account for Sustained Bioactivity of NO? A (Bio)chemical Characterization
Hydrogen sulfide (H<sub>2</sub>S)
and nitric oxide (NO) are important signaling molecules that regulate
several physiological functions. Understanding the chemistry behind
their interplay is important for explaining these functions. The reaction
of H<sub>2</sub>S with <i>S</i>-nitrosothiols to form the
smallest <i>S</i>-nitrosothiol, thionitrous acid (HSNO),
is one example of physiologically relevant cross-talk between H<sub>2</sub>S and nitrogen species. Perthionitrite (SSNO<sup>–</sup>) has recently been considered as an important biological source
of NO that is far more stable and longer living than HSNO. In order
to experimentally address this issue here, we prepared SSNO<sup>–</sup> by two different approaches, which lead to two distinct species:
SSNO<sup>–</sup> and dithionitric acid [HON(S)S/HSN(O)S]. (H)S<sub>2</sub>NO species and their reactivity were studied by <sup>15</sup>N NMR, IR, electron paramagnetic resonance and high-resolution electrospray
ionization time-of-flight mass spectrometry, as well as by X-ray structure
analysis and cyclic voltammetry. The obtained results pointed toward
the inherent instability of SSNO<sup>–</sup> in water solutions.
SSNO<sup>–</sup> decomposed readily in the presence of light,
water, or acid, with concomitant formation of elemental sulfur and
HNO. Furthermore, SSNO<sup>−</sup> reacted with H<sub>2</sub>S to generate HSNO. Computational studies on (H)SSNO provided additional
explanations for its instability. Thus, on the basis of our data,
it seems to be less probable that SSNO<sup>–</sup> can serve
as a signaling molecule and biological source of NO. SSNO<sup>–</sup> salts could, however, be used as fast generators of HNO in water
solutions
Synthesis and Pharmacological Evaluation of Novel Adenine–Hydrogen Sulfide Slow Release Hybrids Designed as Multitarget Cardioprotective Agents
This
work deals with the design, synthesis, and evaluation of the
cardioprotective properties of a number of novel hybrid compounds
combining the adenine nucleus with a suitable H<sub>2</sub>S slow-releasing
moiety, coupled via a stable ether bond. The H<sub>2</sub>S release
rate of the hybrids and their ability to increase cGMP were estimated
in vitro. The most promising derivatives <b>4</b> and <b>11</b>, both containing 4-hydroxythiobenzamide
moiety as H<sub>2</sub>S donor, were selected for further in vivo
evaluation. Their ability to release H<sub>2</sub>S in vivo was recorded
using a new fully validated UPLC-DAD method. Both compounds reduced
significantly the infarct size when administered at the end of sustained
ischemia. Mechanistic studies showed that they conferred enhanced
cardioprotection compared to adenine or 4-hydroxythiobenzamide. They
activate the PKG/PLN pathway in the ischemic myocardium, suggesting
that the combination of both pharmacophores results in synergistic
cardioprotective activity through the combination of both molecular
pathways that trigger cardioprotection
Improved tag-switch method reveals that thioredoxin acts as depersulfidase and controls the intracellular levels of protein persulfidation
Hydrogen sulfide (H
S) has emerged as a signalling molecule capable of regulating several important physiological functions such as blood pressure, neurotransmission and inflammation. The mechanisms behind these effects are still largely elusive and oxidative posttranslational modification of cysteine residues (protein persulfidation or
-sulfhydration) has been proposed as the main pathway for H
S-induced biological and pharmacological effects. As a signalling mechanism, persulfidation has to be controlled. Using an improved tag-switch assay for persulfide detection we show here that protein persulfide levels are controlled by the thioredoxin system. Recombinant thioredoxin showed an almost 10-fold higher reactivity towards cysteine persulfide than towards cystine and readily cleaved protein persulfides as well. This reaction resulted in H
S release suggesting that thioredoxin could be an important regulator of H
S levels from persulfide pools. Inhibition of the thioredoxin system caused an increase in intracellular persulfides, highlighting thioredoxin as a major protein depersulfidase that controls H
S signalling. Finally, using plasma from HIV-1 patients that have higher circulatory levels of thioredoxin, we could prove depersulfidase role
Improved tag-switch method reveals that thioredoxin acts as depersulfidase and controls the intracellular levels of protein persulfidation
Hydrogen sulfide (H2S) has emerged as a signalling molecule capable of regulating several important physiological functions such as blood pressure, neurotransmission and inflammation. The mechanisms behind these effects are still largely elusive and oxidative posttranslational modification of cysteine residues (protein persulfidation or S-sulfhydration) has been proposed as the main pathway for H2S-induced biological and pharmacological effects. As a signalling mechanism, persulfidation has to be controlled. Using an improved tag-switch assay for persulfide detection we show here that protein persulfide levels are controlled by the thioredoxin system. Recombinant thioredoxin showed an almost 10-fold higher reactivity towards cysteine persulfide than towards cystine and readily cleaved protein persulfides as well. This reaction resulted in H2S release suggesting that thioredoxin could be an important regulator of H2S levels from persulfide pools. Inhibition of the thioredoxin system caused an increase in intracellular persulfides, highlighting thioredoxin as a major protein depersulfidase that controls H2S signalling. Finally, using plasma from HIV-1 patients that have higher circulatory levels of thioredoxin, we could prove depersulfidase role in vivo.Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3543