Permeability of human red blood cell membranes to hydrogen peroxide

Resumen del Conference paper presentado a 64th Annual Meeting of the Biophysical Society, San Diego, CA. 2020Hydrogen peroxide (H2O2) and other reactive species are important physiological mediators in the vascular system. Enzymatic production of H2O2 is involved in regulating cell growth, proliferation and vasodilation. Whereas endothelial cells are important sources of H2O2, red blood cells (RBC) are considered the most important sinks of H2O2 in the vasculature. However, little is known about the permeability of their membrane to H2O2. The permeability coefficient of human RBC membranes to H2O2 was determined using the enzyme latency method, based on measuring the rate of H2O2 decomposition in lysed vs whole cells. If the passage through the membrane is the rate limiting step in H2O2 decomposition, then a difference is observed that can be used to calculate the permeability coefficient. Additional experiments were done to differentiate between simple diffusion through the lipid fraction and facilitated diffusion through protein channels. The lack of reported permeability coefficients for lipid membranes prompted us to do experiments with phospholipid-cholesterol liposome membranes that indicated that simple diffusion is a slow process. Determination of partition coefficients in different solvents mimicking different depths of the membrane indicate that the low permeability of lipid membranes to H2O2 is caused mainly by its very low solubility in the acyl region of the bilayer. The activation energy of permeation through RBC membranes suggested that protein channels were involved in facilitating H2O2 diffusion through the membrane. Inhibitors of hAQP3 and hAQP1 had no effect in H2O2 consumption rate, suggesting that other membrane proteins may be involved. Although the RBC membrane presents a significant barrier to H2O2 passage, especially in comparison with other solutes such as oxygen and nitric oxide, the permeability is still high enough to support the role of RBC as sinks of H2O2 in circulation.ANII: FMV_1_2019_15559

Detection and quantification of nitric oxide–derived oxidants in biological systems

The free radical nitric oxide (NO) exerts biological effects through the direct and reversible interaction with specific targets (e.g. soluble guanylate cyclase) or through the generation of secondary species, many of which can oxidize, nitrosate or nitrate biomolecules. The NO -derived reactive species are typically short-lived, and their preferential fates depend on kinetic and compartmentalization aspects. Their detection and quantification are technically challenging. In general, the strategies employed are based either on the detection of relatively stable end products or on the use of synthetic probes, and they are not always selective for a particular species. In this study, we describe the biologically relevant characteristics of the reactive species formed downstream from NO , and we discuss the approaches currently available for the analysis of NO , nitrogen dioxide (NO2 ), dinitrogen trioxide (N2O3), nitroxyl (HNO), and peroxynitrite (ONOO /ONOOH), as well as peroxynitrite-derived hydroxyl (HO) and carbonate anion (CO3) radicals. We also discuss the biological origins of and analytical tools for detecting nitrite (NO2), nitrate (NO3), nitrosyl–metal complexes, S-nitrosothiols, and 3-nitrotyrosine. Moreover, we highlight state– of–the–art methods, alert readers to caveats of widely used techniques, and encourage retirement of approaches that have been supplanted by more reliable and selective tools for detecting and measuring NO -derived oxidants. We emphasize that the use of appropriate analytical methods needs to be strongly grounded in a chemical and biochemical understanding of the species and mechanistic pathways involveAgencia Nacional de Investigación e Innovación FCE_1_2017_1_136043Comisión Sectorial de Investigación Científica (CSIC)Universidad de la República. Espacio Interdisciplinari

Permeability of lipid membranes to hydrogen peroxide

Resumen del Conference paper presentado a SfRBM 27th Annual Conferenc

Possible molecular basis of the biochemical effects of cysteine-derived persulfides

Persulfides (RSSH/RSS−) are species closely related to thiols (RSH/RS−) and hydrogen sulfide (H2S/HS−), and can be formed in biological systems in both low and high molecular weight cysteine-containing compounds. They are key intermediates in catabolic and biosynthetic processes, and have been proposed to participate in the transduction of hydrogen sulfide effects. Persulfides are acidic, more acidic than thiols, and the persulfide anions are expected to be the predominant species at neutral pH. The persulfide anion has high nucleophilicity, due in part to the alpha effect, i.e., the increased reactivity of a nucleophile when the neighboring atom has high electron density. In addition, persulfides have electrophilic character, a property that is absent in both thiols and hydrogen sulfide. In this article, the biochemistry of persulfides is described, and the possible ways in which the formation of a persulfide could impact on the properties of the biomolecule involved are discussed

Leukoreduction of red blood cell concentrates for transfusion

Resumen del Conference Paper presentado a SfRBM 27th Annual Conferenc

Permeability of phospholipid membranes and human red blood cell membranes to hydrogen peroxide

Resumen del Conference paper presentado a SfRBM 28th Annual ConferenceHydrogen peroxide (H2O2) is an oxygen-derived oxidant involved in multiple redox processes in the cell, ranging from physiological signaling pathways to oxidative damage reactions when it is found at higher concentrations. In the vascular system, H2O2 is metabolized mainly by red blood cells (RBC) due to their very efficient antioxidant systems and high membrane permeability. However, the information regarding H2O2 transport in the human RBC membrane is limited, as neither the exact value of the permeability coefficient (Pm) nor the permeation mechanisms are known. To explore whether H2O2 permeates through the lipid fraction or protein channels, we studied H2O2 solubility in organic solvents and its permeability in lipid membranes, in order to compare with the RBC membrane. Through measurements of partition constants, we found that H2O2 is 14 and 122000 times less soluble in octanol and hexadecane than in water, anticipating a large thermodynamic barrier to H2O2 permeation by lipid membranes. The Pm in phospholipid membranes of different compositions, determined using the catalase-latency method, varied from 4×10-4 to 5×10-3 cm s-1, at 37°C. On the other hand, in human RBC we determined a Pm of 1.6×10-3 cm s-1. After obtaining these results, we evaluated the potential role of aquaporins as H2O2 transporters by checking the effect of aquaporin inhibitors in H2O2 consumption by RBC, and also by studying H2O2 permeability in RBC devoid of either aquaporin 1 or aquaporin 3. Surprisingly, we could not detect any differences in H2O2 permeability in any case. Altogether, these results provide new information on lipid membrane permeability to H2O2 and a new value for the Pm in human RBC, which was previously unknown. Additionally, they indicate that H2O2 is not transported by aquaporins in human RBC membranes, suggesting simple diffusion or a still unidentified membrane protein as a more probable pathway.ANII: ANII: FMV_1_2019_15559

Human papillomavirus type 18 E5 oncoprotein cooperates with E6 and E7 in promoting cell viability and invasion and in modulating the cellular redox state

BACKGROUND High-risk human papillomaviruses (HR-HPVs) are the etiological agents of cervical cancer. Among them, types 16 and 18 are the most prevalent worldwide. The HPV genome encodes three oncoproteins (E5, E6, and E7) that possess a high transformation potential in culture cells when transduced simultaneously. In the present study, we analysed how these oncoproteins cooperate to boost key cancer cell features such as uncontrolled cell proliferation, invasion potential, and cellular redox state imbalance. Oxidative stress is known to contribute to the carcinogenic process, as reactive oxygen species (ROS) constitute a potentially harmful by-product of many cellular reactions, and an efficient clearance mechanism is therefore required. Cells infected with HR-HPVs can adapt to oxidative stress conditions by upregulating the formation of endogenous antioxidants such as catalase, glutathione (GSH), and peroxiredoxin (PRX). OBJECTIVES The primary aim of this work was to study how these oncoproteins cooperate to promote the development of certain cancer cell features such as uncontrolled cell proliferation, invasion potential, and oxidative stress that are known to aid in the carcinogenic process. METHODS To perform this study, we generated three different HaCaT cell lines using retroviral transduction that stably expressed combinations of HPV-18 oncogenes that included HaCaT E5-18, HaCaT E6/E7-18, and HaCaT E5/E6/E7-18. FINDINGS Our results revealed a statistically significant increment in cell viability as measured by MTT assay, cell proliferation, and invasion assays in the cell line containing the three viral oncogenes. Additionally, we observed that cells expressing HPV-18 E5/E6/E7 exhibited a decrease in catalase activity and a significant augmentation of GSH and PRX1 levels relative to those of E5, E6/E7, and HaCaT cells. MAIN CONCLUSIONS This study demonstrates for the first time that HPV-18 E5, E6, and E7 oncoproteins can cooperate to enhance malignant transformationANII: PD_NAC_2016_1_13332

The chemical basis of thiol addition to nitro-conjugated linoleic acid, a protective cell-signaling lipid

Nitroalkene fatty acids are formed in vivo and exert protective and anti-inflammatory effects via reversible Michael addition to thiol-containing proteins in key signaling pathways. Nitro-conjugated linoleic acid (NO2-CLA) is preferentially formed, constitutes the most abundant nitrated fatty acid in humans, and contains two carbons that could potentially react with thiols, modulating signaling actions and levels. In this work, we examined the reactions of NO2-CLA with low molecular weight thiols (glutathione, cysteine, homocysteine, cysteinylglycine, and β-mercaptoethanol) and human serum albumin. Reactions followed reversible biphasic kinetics, consistent with the presence of two electrophilic centers in NO2-CLA located on the β- and δ-carbons with respect to the nitro group. The differential reactivity was confirmed by computational modeling of the electronic structure. The rates (kon and koff) and equilibrium constants for both reactions were determined for different thiols. LC-UV-Visible and LC-MS analyses showed that the fast reaction corresponds to β-adduct formation (the kinetic product), while the slow reaction corresponds to the formation of the δ-adduct (the thermodynamic product). The pH dependence of the rate constants, the correlation between intrinsic reactivity and thiol pKa, and the absence of deuterium solvent kinetic isotope effects suggested stepwise mechanisms with thiolate attack on NO2-CLA as rate-controlling step. Computational modeling supported the mechanism and revealed additional features of the transition states, anionic intermediates, and final neutral products. Importantly, the detection of cysteine-δ-adducts in human urine provided evidence for the biological relevance of this reaction. Finally, human serum albumin was found to bind NO2-CLA both non-covalently and to form covalent adducts at Cys-34, suggesting potential modes for systemic distribution. These results provide new insights into the chemical basis of NO2-CLA signaling actions

Fluorescent detection of hydrogen sulfide (H2S) through the formation of pyrene excimers enhances H2S quantification in biochemical systems

Hydrogen sulfide (H2S) is produced endogenously by several enzymatic pathways and modulates physiological functions in mammals. Quantification of H2S in biochemical systems remains challenging because of the presence of interferents with similar reactivity, particularly thiols. Herein, we present a new quantification method based on the formation of pyrene excimers in solution. We synthesized the probe 2-(maleimido)ethyl 4-pyrenylbutanoate (MEPB) and determined that MEPB reacted with H2S in a two-step reaction to yield the thioether-linked dimer (MEPB)2S, which formed excimers upon excitation, with a broad peak of fluorescence emission centered at 480 nm. In contrast, we found that the products formed with thiols showed peaks at 378 and 398 nm. The difference in emission between the products prevented the interference. Furthermore, we showed that the excimer fluorescence signal yielded a linear response to H2S, with a limit of detection of 54 nM in a fluorometer. Our quantification method with MEPB was successfully applied to follow the reaction of H2S with glutathione disulfide and to quantify the production of H2S from cysteine by Escherichia coli. In conclusion, this method represents an addition to the toolkit of biochemists to quantify H2S specifically and sensitively in biochemical systems.MEC: I/FVF2017/069ANII: FCE_1_2017_1_136043CSIC: I+D 2017; I+D 202