Ana Denicola

Universidad de la República de Uruguay | UdelaR · Instituto de Química Biológica

Hydrogen peroxide (H2O2) is an oxidant produced endogenously by several enzymatic pathways. While it can cause molecular damage, H2O2 also plays a role in regulating cell proliferation and survival through redox signaling pathways. In the vascular system, red blood cells (RBCs) are notably efficient at metabolizing H2O2. In addition to a robust ant...

Peroxynitrite is a powerful oxidant formed in vivo in sites where superoxide and nitric oxide coincide. Peroxynitrite is cytotoxic through oxidative modification of target biomolecules that can occur by direct or indirect reactions. Indirect reactions usually involve the generation of peroxynitrite-derived radicals that include nitrogen dioxide, hy...

We have studied the reduction reactions of two cytosolic human peroxiredoxins (Prx) in their disulfide form by three thioredoxins (Trx; two human and one bacterial), with the aim of better understanding the rate and mechanism of those reactions, and their relevance in the context of the catalytic cycle of Prx. We have developed a new methodology ba...

Peroxiredoxins (Prx), thiol-dependent peroxidases, were first identified as H2O2 detoxifiers, and more recently as H2O2 sensors, intermediates in redox-signaling pathways, metabolism modulators, and chaperones. The multifaceted nature of Prx is not only dependent on their peroxidase activity but also strongly associated with specific protein–prote...

Peroxiredoxins (Prx) are ubiquitous, highly conserved peroxidases whose activity depends on catalytic cysteine residues. The Prx1-class of the peroxiredoxin family, also called typical 2-Cys Prx, organize as head-to-tail homodimers containing two active sites. The peroxidatic cysteine CP of one monomer reacts with the peroxide substrate to form sul...

Red blood cells (RBCs) are exposed to both external and internal sources of oxidants that challenge their integrity and compromise their physiological function and supply of oxygen to tissues. Autoxidation of oxyhemoglobin is the main source of endogenous RBC oxidant production, yielding superoxide radical and then hydrogen peroxide. In addition,...

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 o...

The formation S-nitrosothiols (RSNO) is observed in vivo and depends on the enzymatic synthesis of nitric oxide. However, generation of RSNO does not occur by direct reaction between thiols and nitric oxide, but requires oxidation of thiols or nitric oxide. Different pathways could lead to the biological formation of RSNO, including nitric oxide au...

We have centered our discussion on the role of blood thiols on the general homeostasis of oxygen- and nitrogen-derived oxidants. The central thiol participation in redox homeostasis is supported by the high relative concentration of thiol antioxidants both in plasma and blood cells together with their preferential reaction with oxidants of diverse...

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 limitati...

Hydrogen peroxide (H2O2) is an oxidant, but also an important signaling molecule in vascular biology, mediating several physiological functions. Red blood cells (RBC) have been proposed to be the primary sink of H2O2 in the vasculature because they are the main cellular component of blood with a robust antioxidant defense and a high membrane permea...

In July 2021, I had the honor of becoming an elected member of the Council of the International Union for Pure and Applied Biophysics (IUPAB) and joined the Editorial Board of Biophysical Reviews. This is a short biographical sketch to introduce myself to the readers of the journal.

Significance: The systematic investigation of oxidative modification of proteins by reactive oxygen species started in 1980. Later, it was shown that reactive nitrogen species could also modify proteins. Some protein oxidative modifications promote loss of protein function, cleavage or aggregation, and some result in proteotoxicity and cellular ho...

Cell signaling events mediated by the oxidation of specific thiol-containing proteins, is now, one of the trending topics in redox biology. Oxidants such as hydrogen peroxide (H2O2), have demonstrated to be important signaling molecules under physiological conditions. Nevertheless, the slow reactions between H2O2 and the thiols of redox-regulated p...

Sirtuin 6, SIRT6, is critical for both glucose and lipid homeostasis and is involved in maintaining genomic stability under conditions of oxidative DNA damage such as those observed in age-related diseases. There is an intense search for modulators of SIRT6 activity, however, not many specific activators have been reported. Long acyl-chain fatty ac...

Salinity is a key variable influencing the life history of aquatic organisms. Environmental variability in salinity has been associated with increases in the formation of reactive species, ending up in a state of oxidative stress. In copepods, information about effects of salinity variations mostly includes data from short-term experiments where th...

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Life on Earth evolved in the presence of hydrogen peroxide, and other peroxides also emerged before and with the rise of aerobic metabolism. They were considered only as toxic byproducts for many years. Nowadays, peroxides are also regarded as metabolic products that play essential physiological cellular roles. Organisms have developed efficient me...

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 preferen...

Peroxiredoxins (Prx) are enzymes that efficiently reduce hydroperoxides through active participation of cysteine residues (CP, CR). The first step in catalysis, the reduction of peroxide substrate, is fast, 107 - 108 M-1s-1 for human Prx2. In addition, the high intracellular concentration of Prx positions them not only as good antioxidants but also...

This chapter includes an overview of the structure of cell membranes and a review of the permeability of membranes to biologically relevant oxygen and nitrogen reactive species, namely oxygen, singlet oxygen, superoxide, hydrogen peroxide, hydroxyl radical, nitric oxide, nitrogen dioxide, peroxynitrite and also hydrogen sulfide. Physical interactio...

Lipoproteins and lipid membranes accelerate •NO autoxidation by increasing local concentration of •NO and O2. Although the idea that proteins could also accelerate this reaction was presented some time ago, it was largely criticized and dismissed. Herein the effect of proteins on •NO autoxidation rates was studied following •NO disappearance with a...

Carbonate radicals (CO3·-) are generated by the bicarbonate-dependent peroxidase activity of cytosolic superoxide dismutase (Cu,Zn-SOD, SOD-1). The present work explored the use of bleaching of pyrogallol red (PGR) dye to quantify the rate of CO3%- formation from bovine and human SOD-1 (bSOD-1 and hSOD-1, respectively). This approach was compared t...

Peroxiredoxins are thiol‐dependent peroxidases that function in peroxide detoxification and H2O2 induced signaling. Among the six isoforms expressed in humans, PRDX1 and PRDX2 share 97% sequence similarity, 77% sequence identity including the active site, subcellular localization (cytosolic) but they hold different biological functions albeit assoc...

Sirtuins are NAD⁺-dependent deacetylases reported as key factors in metabolism, regulation, inflammation and stress response. Human sirtuin 6 (hSIRT6) has been shown in vivo to efficiently deacetylase histone 3, however, in vitro displays low deacetylase activity and possess long-chain fatty acid deacylase activity. So far, only a few compounds hav...

PknG from Mycobacterium tuberculosis is a multidomain Serine/Threonine protein kinase that regulates bacterial metabolism as well as the pathogen's ability to survive inside the host by still uncertain mechanisms. To uncover PknG interactome we developed an affinity purification-mass spectrometry strategy to stepwise recover PknG substrates and int...

Red blood cells (RBC) are considered as a circulating sink of H2O2, but a significant debate remains over the role of the different intraerythocyte peroxidases. Herein we examined the kinetic of decomposition of exogenous H2O2 by human RBC at different cell densities, using fluorescent and oxymetric methods, contrasting the results against a mathem...

Red blood cells (RBC) are considered as a circulating sink of H2O2, but a significant debate remains over the role of the different erythrocyte peroxidases. Herein we examined the kinetic of decomposition of exogenous H2O2 by human RBC at different cell densities, using fluorescent and oxymetric methods, contrasting the results against a mathematic...

Oxygen and nitric oxide are small hydrophobic molecules that usually need to diffuse a considerable distance to accomplish their biological functions and necessarily need to traverse several lipid membranes. Different methods have been used to study the diffusion of these molecules in membranes and herein we focus in the quenching of fluorescence o...

Two-cysteine peroxiredoxins (Prx) have a three-step catalytic cycle consisting of (1) reduction of peroxide and formation of sulfenic acid on the enzyme, (2) condensation of the sulfenic acid with a thiol to form disulfide, also known as resolution, and (3) reduction of the disulfide by a reductant protein. By following changes in protein fluoresce...

Sirtuins are NAD⁺-dependent deacetylases reported as key factors in metabolism regulation, inflammation and stress response. Human sirtuin 6 (hSIRT6) is known as a histone 3 deacetylase despite of its low activity in vitro. Its deacetylase activity increases in the presence of fatty acids and possess long chain fatty acid deacylase activity. Studie...

Storage inflicts a series of changes on red blood cells (RBC) that compromise the cell survival and functionality; largely these alterations (storage lesions) are due to oxidative modifications. The possibility of improving the quality of packed RBC stored for transfusion including N-acetylcysteine (NAC) in the preservation solution was explored. R...

Oxidative damage occurs as a natural process in organisms, but recently several studies suggested that oxidative damage may be enhanced by environmental stress. Here, levels of oxidative damage (protein carbonylation) and vital rates performances of Acartia tonsa were compared in two estuaries with different degree of anthropogenic impact through f...

Peroxiredoxins (Prx) are a large family of thiol-dependent peroxidases that reduce hydroperoxides at expense of thioredoxin or other disulfide reductases. Human Prx2 is well expressed in the cytosol, a 2-Cys typical Prx. The first reaction in the catalytic cycle, the peroxidatic cysteine with H2O2, is extraordinary fast (10⁸ M⁻¹ s⁻¹) placing Prx as...

Sirtuins are a conserved family of NAD-dependent protein deacylases. Initially proposed as histone deacetylases, it is now known that they act on a variety of proteins including transcription factors and metabolic enzymes, having a key role in the regulation of cellular homeostasis. Seven isoforms are identified in mammals (SIRT1–7), all of them sh...

This practical class activity was designed to introduce students to recombinant protein expression and purification. The principal goal is to shed light on basic aspects concerning recombinant protein production, in particular protein expression, chromatography methods for protein purification, and enzyme activity as a tool to evaluate purity and c...

Además de lípidos, proteínas y azúcares, los alimentos poseen componentes no esenciales en la dieta que son beneficiosos para la salud, que son componentes distintivos de los llamados «functional-foods» fundamentales para la buena nutrición. Combinado entonces un estilo de vida saludable, el consumo de alimentos funcionales puede ser una muy buena...

In the present work we studied the reaction under gastric conditions of pyrogallol red (PGR), a polyphenolic dye, with nitrous acid (HONO). PGR has been used as a model polyphenol due to its strong UV-visible absorption and its high reactivity towards reactive species (radicals and non-radicals, RS). The reaction was followed by UV-visible spectros...

Peroxiredoxins (Prx) are efficient thiol-dependent peroxidases and key players in the mechanism of H2O2-induced redox signaling. Any structural change that could affect their redox state, oligomeric structure, and/or interaction with other proteins could have a significant impact on the cascade of signaling events. Several post-translational modifi...

Profuse epidemiological evidence supports a higher risk for coronary heart disease and stroke in smoker women than in men with the same habit. Although it is already known that cigarette smoking alters the redox state, is unknown if the imbalance in the normal equilibrium between oxidants and antioxidants is responsible for the elevated female susc...

Evidence has accumulated showing that hydrogen peroxide (H2O2) acts as a signaling molecule via oxidation of critical cysteine residues on target proteins. The reaction of H2O2 with thiols is thermodynamically favored, but its selectivity is imposed by differences in reaction kinetics. Previously proposed signal relaying mechanisms, such as the flo...

PknG from Mycobacterium tuberculosis is a Ser/Thr protein kinase that regulates key metabolic processes within the bacterial cell as well as signaling pathways from the infected host cell. This multi-domain protein has a conserved canonical kinase domain with N- and C-terminal flanking regions of unclear functional roles. The N-terminus harbors a r...

Oxidative stress is associated with several pathologies like cardiovascular, neurodegenerative, cancer and even aging. It has been suggested that a diet rich in antioxidants would be beneficial to human health and a lot of interest is focused on the determination of antioxidant capacity of natural products. Different chemical methods have been deve...

Volatile anesthetics have been widely used for more than 170 years. However, the mechanisms underlying the effects of anesthetics on membrane dynamics and structure are still under debate. Herein we study this problem for first time using fluorescence confocal microscopy experiments of giant Unilamellar Vesicles (GUVs). This approach allows obtaini...

Hydrogen sulfide (H(2)S) is mainly known for its toxicity but has recently been shown to be produced endogenously in mammalian tissues and to be associated with physiological regulatory functions. To better understand the role of biomembranes in modulating its biological distribution and effects; we measured the partition coefficient of H(2)S in mo...

Permeation in liposomes. Experimental details and equations. (DOC)

Diffusional spread dependence on membrane resistance. A) Concentration profiles of total sulfide (H2S+HS−) as a function of time and distance considering a spherical and continuous source (“cell”) with unhindered diffusion (r = 5 µm, D = 2.32×10−5 cm2 s−1); B) with a resistance of 20 membranes per cell (D = 2.02×10−5 cm2 s−1); and C) with a resista...

Typical experimental values used to calculate KPmem/w in liposomes. (DOC)

Derivation of Equation 1 . (DOC)

Permeation of hydrogen sulfide through liposome phospholipid membranes. A) Reaction scheme showing the transport of protons across the membrane by H2S, leading to intravesicular acidification. B) Decrease in intravesicular HPTS fluorescence caused by H2S, indicating entrance of H2S into the vesicle and intravesicular acidification. HPTS is shown in...

Pulmonary surfactant (PS) is a lipid-protein mixture lining the alveolar air-water interface. By lowering surface tension, PS stabilizes the respiratory surface, thereby impeding alveolar collapse. Lack or alterations of PS properties are associated with acute lung injury (ALI). Sevoflurane is widely used in medical practice; however, some adverse...

The antioxidant capacity of propolis from the southern region of Uruguay was evaluated using in vitro as well as cellular assays. Free radical scavenging capacity was assessed by ORAC, obtaining values significantly higher than those of other natural products (8000 μmol Trolox equiv/g propolis). ORAC values correlated well with total polyphenol con...

Production of recombinant proteins is a process intensively used in the research laboratory. In addition, the main biotechnology market products are recombinant proteins and monoclonal antibodies. The biological (and clinical) properties of the protein product strongly depend on the conformation of the polypeptide. Therefore, assessment of the corr...

The reaction of 5-aminosalicylic acid (5-ASA) with nitrous acid has been studied at low pH under conditions that simulate a gastric environment. The course of the reaction was followed by UV–visible and fluorescence spectroscopy and the products were analyzed by high performance liquid chromatography (HPLC) with UV–visible and mass spectroscopic de...

Pulmonary surfactant (PS) is a complex mixture of lipids and proteins responsible to protect the lung tissue from mechanical stress during ventilation, by decreasing alveolar surface tension. Indeed, an appropriate PS function is critical in order to avoid alveolar collapse. There are many causes that produce PS dysfunction, lung damage and alveola...

Protein thiol reactivity generally involves the nucleophilic attack of the thiolate on an electrophile. A low pK(a) means higher availability of the thiolate at neutral pH but often a lower nucleophilicity. Protein structural factors contribute to increasing the reactivity of the thiol in very specific reactions, but these factors do not provide an...

In most organisms, thioredoxin (Trx) and/or glutathione (GSH) systems are essential for redox homeostasis and deoxyribonucleotide synthesis. Platyhelminth parasites have a unique and simplified thiol-based redox system, in which the selenoprotein thioredoxin-glutathione reductase (TGR), a fusion of a glutaredoxin (Grx) domain to canonical thioredox...

The physicochemical properties of nitrogen monoxide (NO) and its derived reactive species determine the efficiency of the reactions that can take place in the lipid environment relative to the aqueous solution. The higher solubility of NO in membranes than in water leads to an acceleration of the rate of NO autoxidation in the membrane, while the l...

In most organisms, thioredoxin (Trx) and/or glutathione (GSH) systems are essential for redox homeostasis and deoxyribonucleotide synthesis. Platyhelminth parasites have a unique and simplified thiol-based redox system, in which the selenoprotein thioredoxin-glutathione reductase (TGR), a fusion of a glutaredoxin (Grx) domain to canonical thioredox...

The fast reaction of (•)NO and O(2)(•-) to give ONOO(-) has been extensively studied at irreversible conditions, but the reasons for the wide variations in observed forward rate constants (3.8 ≤ k(f) ≤ 20 × 10(9) M(-1) s(-1)) remain unexplained. We characterized the diffusion-dependent aqueous (pH > 12) chemical equilibrium of the form (•)NO + O(2)...

Hydrogen sulfide (H(2)S) is an endogenously generated gas that can also be administered exogenously. It modulates physiological functions and has reported cytoprotective effects. To evaluate a possible antioxidant role, we investigated the reactivity of hydrogen sulfide with several one- and two-electron oxidants. The rate constant of the direct re...

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