Acidity and nucleophilic reactivity of glutathione persulfide

Persulfides (RSSH/RSS2) participate in sulfur trafficking and metabolic processes, and are proposed to mediate the signaling effects of hydrogen sulfide (H2S). Despite their growing relevance, their chemical properties are poorly understood. Herein, we studied experimentally and computationally the formation, acidity, and nucleophilicity of glutathione persulfide (GSSH/ GSS2), the derivative of the abundant cellular thiol glutathione (GSH). We characterized the kinetics and equilibrium of GSSH formation from glutathione disulfide and H2S. A pKa of 5.45 for GSSH was determined, which is 3.49 units below that of GSH. The reactions of GSSH with the physiologically relevant electrophiles peroxynitrite and hydrogen peroxide, and with the probe monobromobimane, were studied and compared with those of thiols. These reactions occurred through SN2 mechanisms. At neutral pH, GSSH reacted faster than GSH because of increased availability of the anion and, depending on the electrophile, increased reactivity. In addition, GSS2 presented higher nucleophilicity with respect to a thiolate with similar basicity. This can be interpreted in terms of the so-called a effect, i.e. the increased reactivity of a nucleophile when the atom adjacent to the nucleophilic atom has high electron density. The magnitude of the a effect correlated with the Brønsted nucleophilic factor, bnuc, for the reactions with thiolates and with the ability of the leaving group. Our study constitutes the first determination of the pKa of a biological persulfide and the first examination of the a effect in sulfur nucleophiles, and sheds light on the chemical basis of the biological properties of persulfides.Fil: Benchoam, Dayana. Universidad de la República; UruguayFil: Semelak, Jonathan Alexis. 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: Cuevasanta, Ernesto. Universidad de la República; UruguayFil: Mastrogiovanni, Mauricio. Universidad de la Republica; UruguayFil: Grassano, Juan S.. Universidad de Buenos Aires; ArgentinaFil: Ferrer-Sueta, Gerardo. Universidad de la Republica; UruguayFil: Zeida Camacho, Ari Fernando. Universidad de la Republica; UruguayFil: Trujillo, Madia. Universidad de la Republica; UruguayFil: Möller, Matías N.. Universidad de la Republica; 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: Alvarez, Beatriz. Universidad de la Republica; Urugua

Colouration in amphibians as a reflection of nutritional status : the case of tree frogs in Costa Rica

Colouration has been considered a cue for mating success in many species; ornaments in males often are related to carotenoid mobilization towards feathers and/or skin and can signal general health and nutrition status. However, there are several factors that can also link with status, such as physiological blood parameters and body condition, but there is not substantial evidence which supports the existence of these relationships and interactions in anurans. This study evaluated how body score and blood values interact with colouration in free-range Agalychnis callidryas and Agalychnis annae males. We found significant associations between body condition and plasmatic proteins and haematocrit, as well as between body condition and colour values from the chromaticity diagram. We also demonstrated that there is a significant relation between the glucose and plasmatic protein values that were reflected in the ventral colours of the animals, and haematocrit inversely affected most of those colour values. Significant differences were found between species as well as between populations of A. callidryas, suggesting that despite colour variation, there are also biochemical differences within animals from the same species located in different regions. These data provide information on underlying factors for colouration of male tree frogs in nature, provide insights about the dynamics of several nutrients in the amphibian model and how this could affect the reproductive output of the animals

Kinetics studies reveal a key role of a redox-active glutaredoxin in the evolution of the thiol-redox metabolism of trypanosomatid parasites

Trypanosomes are flagellated protozoan parasites (kinetoplastids) that have a unique redox metabolism based on the small dithiol trypanothione (T(SH)2). Although GSH may still play a biological role in trypanosomatid parasites beyond being a building block of T(SH)2, most of its functions are replaced by T(SH)2 in these organisms. Consequently, trypanosomes have several enzymes adapted to using T(SH)2 instead of GSH, including the glutaredoxins (Grxs). However, the mechanistic basis of Grx specificity for T(SH)2 is unknown. Here, we combined fast-kinetic and biophysical approaches, including NMR, MS, and fluorescent tagging, to study the redox function of Grx1, the only cytosolic redox-active Grx in trypanosomes. We observed that Grx1 reduces glutathione-containing disulfides (including oxidized trypanothione) in very fast reactions (k > 5 7 105 M-1 s-1). We also noted that disulfides without a GSH are much slower oxidants, suggesting a strongly selective binding of the GSH molecule. Not surprisingly, oxidized Grx1 was also reduced very fast by T(SH)2 (4.8 7 106 M-1 s-1); however, GSH-mediated reduction was extremely slow (39 M-1 s-1). This kinetic selectivity in the reduction step of the catalytic cycle suggests that Grx1 uses preferentially a dithiol mechanism, forming a disulfide on the active site during the oxidative half of the catalytic cycle and then being rapidly reduced by T(SH)2 in the reductive half. Thus, the reduction of glutathionylated substrates avoids GSSG accumulation in an organism lacking glutathione reductase. These findings suggest that Grx1 has played an important adaptive role during the rewiring of the thiol-redox metabolism of kinetoplastids

Fluorescence Lifetime Phasor Analysis of the Decamer–Dimer Equilibrium of Human Peroxiredoxin 1

Protein self-assembly is a common feature in biology and is often required for a myriad of fundamental processes, such as enzyme activity, signal transduction, and transport of solutes across membranes, among others. There are several techniques to find and assess homo-oligomer formation in proteins. Naturally, all these methods have their limitations, meaning that at least two or more different approaches are needed to characterize a case study. Herein, we present a new method to study protein associations using intrinsic fluorescence lifetime with phasors. In this case, the method is applied to determine the equilibrium dissociation constant (KD) of human peroxiredoxin 1 (hPrx1), an efficient cysteine-dependent peroxidase, that has a quaternary structure comprised of five head-to-tail homodimers non-covalently arranged in a decamer. The hPrx1 oligomeric state not only affects its activity but also its association with other proteins. The excited state lifetime of hPrx1 has distinct values at high and low concentrations, suggesting the presence of two different species. Phasor analysis of hPrx1 emission lifetime allowed for the identification and quantification of hPrx1 decamers, dimers, and their mixture at diverse protein concentrations. Using phasor algebra, we calculated the fraction of hPrx1 decamers at different concentrations and obtained KD (1.1 × 10−24 M4) and C0.5 (1.36 μM) values for the decamer–dimer equilibrium. The results were validated and compared with size exclusion chromatography. In addition, spectral phasors provided similar results despite the small differences in emission spectra as a function of hPrx1 concentration. The phasor approach was shown to be a highly sensitive and quantitative method to assess protein oligomerization and an attractive addition to the biophysicist’s toolkit

Regulatory de novo mutations underlying intellectual disability

The genetic aetiology of a major fraction of patients with intellectual disability (ID) remains unknown. De novo mutations (DNMs) in protein-coding genes explain up to 40% of cases, but the potential role of regulatory DNMs is still poorly understood. We sequenced 63 whole genomes from 21 ID probands and their unaffected parents. In addition, we analysed 30 previously sequenced genomes from exome-negative ID probands. We found that regulatory DNMs were selectively enriched in fetal brain-specific enhancers as compared with adult brain enhancers. DNM-containing enhancers were associated with genes that show preferential expression in the prefrontal cortex. Furthermore, we identified recurrently mutated enhancer clusters that regulate genes involved in nervous system development (CSMD1, OLFM1, and POU3F3). Most of the DNMs from ID probands showed allele-specific enhancer activity when tested using luciferase assay. Using CRISPR-mediated mutation and editing of epigenomic marks, we show that DNMs at regulatory elements affect the expression of putative target genes. Our results, therefore, provide new evidence to indicate that DNMs in fetal brain-specific enhancers play an essential role in the aetiology of ID.This work was funded by grants from the Wellcome Trust Institute Strategic Support and National Institute for Health Research (NIHR) Imperial Biomedical Research Centre, Institute for Translational Medicine and Therapeutics (P70888) obtained by SS Atanur. J Ferrer and MG De Vas’s work was funded by grants from the Wellcome Trust (WT101033 to J Ferrer), Medical Research Council (MR/L02036X/1 to J Ferrer), and European Research Council Advanced Grant (789055 to J Ferrer). MM Pradeepa’s lab is funded by the UKRI/MRC (MR/T000783/1), and Barts charity (MGU0475) grants. TN Khan was partially supported by the Government of Pakistan under the PSDP project “Development of National University of Medical Sciences (NUMS), Rawalpindi.