RESUMEN
Morpheeins are proteins that reversibly assemble into different oligomers, whose architectures are governed by conformational changes of the subunits. This property could be utilized in bionanotechnology where the building of nanometric and new high-ordered structures is required. By capitalizing on the adaptability of morpheeins to create patterned structures and exploiting their inborn affinity toward inorganic and living matter, "bottom-up" creation of nanostructures could be achieved using a single protein building block, which may be useful as such or as scaffolds for more complex materials. Peroxiredoxins represent the paradigm of a morpheein that can be applied to bionanotechnology. This review describes the structural and functional transitions that peroxiredoxins undergo to form high-order oligomers, e.g., rings, tubes, particles, and catenanes, and reports on the chemical and genetic engineering approaches to employ them in the generation of responsive nanostructures and nanodevices. The usefulness of the morpheeins' behavior is emphasized, supporting their use in future applications.
Asunto(s)
Nanoestructuras/química , Peroxirredoxinas/química , Proteínas/química , Biopolímeros/química , Peroxirredoxinas/metabolismo , Proteínas/metabolismo , Relación Estructura-ActividadRESUMEN
The brain tissue partial oxygen pressure (PbtO2) and near-infrared spectroscopy (NIRS) neuromonitoring are frequently compared in the management of acute moderate and severe traumatic brain injury patients; however, the relationship between their respective output parameters flows from the complex pathogenesis of tissue respiration after brain trauma. NIRS neuromonitoring overcomes certain limitations related to the heterogeneity of the pathology across the brain that cannot be adequately addressed by local-sample invasive neuromonitoring (e.g., PbtO2 neuromonitoring, microdialysis), and it allows clinicians to assess parameters that cannot otherwise be scanned. The anatomical co-registration of an NIRS signal with axial imaging (e.g., computerized tomography scan) enhances the optical signal, which can be changed by the anatomy of the lesions and the significance of the radiological assessment. These arguments led us to conclude that rather than aiming to substitute PbtO2 with tissue saturation, multiple types of NIRS should be included via multimodal systemic- and neuro-monitoring, whose values then are incorporated into biosignatures linked to patient status and prognosis. Discussion on the abnormalities in tissue respiration due to brain trauma and how they affect the PbtO2 and NIRS neuromonitoring is given.
Asunto(s)
Lesiones Traumáticas del Encéfalo/diagnóstico por imagen , Lesiones Traumáticas del Encéfalo/metabolismo , Encéfalo/diagnóstico por imagen , Oxígeno/metabolismo , Espectroscopía Infrarroja Corta/métodos , Análisis de los Gases de la Sangre , Encéfalo/irrigación sanguínea , Encéfalo/fisiopatología , Lesiones Traumáticas del Encéfalo/fisiopatología , Circulación Cerebrovascular , Glicocálix , Hematócrito , Hemoglobinas/metabolismo , Humanos , Imagen por Resonancia Magnética/métodos , Microcirculación , Neuroimagen , Tomografía Óptica/métodosRESUMEN
Peroxiredoxins (Prxs) and glutathione peroxidases (Gpxs) provide the majority of peroxides reducing activity in the cytoplasm. Both are peroxidases but differences in the chemical mechanism of reduction of oxidative agents, as well as in the reactivity of the catalytically active residues, confer peculiar features on them. Ultimately, Gpx should be regarded as an efficient peroxides scavenger having a high-reactive selenocysteine (Sec) residue. Prx, by having a low pKa cysteine, is less efficient than Gpx in reduction of peroxides under physiological conditions, but the chemistry of the sulfur together with the peculiar structural arrangement of the active site, in typical Prxs, make it suitable to sense a redox environment and to switch-in-function so as to exert holdase activity under redox-stress conditions. The complex macromolecular assembly would have evolved the chaperone holdase function and the moonlighting behaviour typical of many Prxs.
Asunto(s)
Evolución Biológica , Cisteína/metabolismo , Peroxirredoxinas/fisiología , Selenocisteína/metabolismo , Biocatálisis , Modelos Moleculares , Oxidación-Reducción , Peroxirredoxinas/química , Conformación ProteicaRESUMEN
Allostery is an important property of biological macromolecules which regulates diverse biological functions such as catalysis, signal transduction, transport, and molecular recognition. However, the concept was expressed using two different definitions by J. Monod and, over time, more have been added by different authors, making it fuzzy. Here, we reviewed the different meanings of allostery in the current literature and found that it has been used to indicate that the function of a protein is regulated by heterotropic ligands, and/or that the binding of ligands and substrates presents homotropic positive or negative cooperativity, whatever the hypothesized or demonstrated reaction mechanism might be. Thus, proteins defined to be allosteric include not only those that obey the two-state concerted model, but also those that obey different reaction mechanisms such as ligand-induced fit, possibly coupled to sequential structure changes, and ligand-linked dissociation-association. Since each reaction mechanism requires its own mathematical description and is defined by it, there are many possible 'allosteries'. This lack of clarity is made even fuzzier by the fact that the reaction mechanism is often assigned imprecisely and/or implicitly in the absence of the necessary experimental evidence. In this review, we examine a list of proteins that have been defined to be allosteric and attempt to assign a reaction mechanism to as many as possible.
Asunto(s)
Proteínas , Regulación Alostérica , Ligandos , Proteínas/metabolismo , Proteínas/química , Humanos , Unión Proteica , Modelos MolecularesRESUMEN
The two-state allosteric model of Monod, Wyman and Changeux (1965) offers a simple and elegant, yet very powerful and comprehensive, description of the functional behavior of hemoglobin. Although the extensive body of structural and functional information available is by-and-large consistent with this conceptual framework, some discrepancies between theory and experiment have been extensively discussed and considered to demand modifications of the original hypothesis. More recently the role of tertiary structural changes has been re-analyzed leading to extended kinetic models or indicating that powerful heterotropic effectors may be of paramount importance in controlling the function of human hemoglobin. The aim of this review is to analyze, and possibly reconcile, some discrepancies. We always felt that by looking at hemoglobins other than human HbA, the relative role of tertiary and quaternary allosteric effects may be better understood. The model systems illustrated below are the different hemoglobins from trout's blood, since they are characterized by the most striking variability of heterotropic effects, ranging from totally absent to very extreme with dominant contributions of tertiary effects. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.
Asunto(s)
Hemoglobinas/química , Oxígeno/química , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Animales , Sitios de Unión , Hemoglobinas/metabolismo , Humanos , Cinética , Modelos Químicos , Modelos Moleculares , Oxígeno/metabolismo , Unión ProteicaRESUMEN
Plasmodium falciparum is the vector of the most prevalent and deadly form of malaria, and, among the Plasmodium species, it is the one with the highest rate of drug resistance. At the basis of a rational drug design project there is the selection and characterization of suitable target(s). Thioredoxin reductase, the first protection against reactive oxygen species in the erythrocytic phase of the parasite, is essential for its survival. Hence it represents a good target for the design of new anti-malarial active compounds. In this paper we present the first crystal structure of recombinant P. falciparum thioredoxin reductase (PfTrxR) at 2.9Å and discuss its differences with respect to the human orthologue. The most important one resides in the dimer interface, which offers a good binding site for selective non competitive inhibitors. The striking conservation of this feature among the Plasmodium parasites, but not among other Apicomplexa parasites neither in mammals, boosts its exploitability.
Asunto(s)
Plasmodium falciparum/enzimología , Reductasa de Tiorredoxina-Disulfuro/química , Antimaláricos/química , Antimaláricos/farmacología , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
The oxygen demands of the human body require the constant circulation of blood carrying an enormous concentration of hemoglobin (Hb). Oxygen transport depends not only on the amount of Hb, but also on the control over the affinity of the protein for the gas, which can be optimized for the environmental conditions by changes in the concentration of effectors (hydrogen ions, chloride, CO2, and DPG) inside the red cell. Some pathological conditions affecting Hb may benefit from pharmacological interventions to increase or decrease its affinity for oxygen, or otherwise modify its properties, or alter its biosynthesis. Examples of such conditions include sickle cell anemia, thalassemias and inherited hemoglobinopathies. Effective and safe drugs such as voxelotor, bezafibrate and efaproxiral are available that significantly increase or decrease Hb oxygen affinity. Some medical conditions not directly affecting the blood or its oxygen carrying capacity may also be relieved by the manipulation of Hb. For example, the standard treatment of acute cyanide poisoning requires the oxidation of a fraction of the Hb in the bloodstream so that it efficiently scavenges cyanide. Tumors are often extremely hypoxic and therefore strongly resistant to radiotherapy; the sensitivity of cancerous tissue to X-rays may be increased by improved oxygenation through drugs binding Hb. This review attempts to provide a systematic exploration of the pharmacology of Hb, its molecular basis, and its intended and possible uses.
Asunto(s)
Anemia de Células Falciformes , Hemoglobinas , Anemia de Células Falciformes/tratamiento farmacológico , Anemia de Células Falciformes/metabolismo , Hemoglobinas/metabolismo , Humanos , Hipoxia/metabolismo , OxígenoRESUMEN
Allostery is a property of biological macromolecules featuring cooperative ligand binding and regulation of ligand affinity by effectors. The definition was introduced by Monod and Jacob in 1963, and formally developed as the "concerted model" by Monod, Wyman, and Changeux in 1965. Since its inception, this model of cooperativity was seen as distinct from and not reducible to the "sequential model" originally formulated by Pauling in 1935, which was developed further by Koshland, Nemethy, and Filmer in 1966. However, it is difficult to decide which model is more appropriate from equilibrium or kinetics measurements alone. In this paper, we examine several cooperative proteins whose functional behavior, whether sequential or concerted, is established, and offer a combined approach based on functional and structural analysis. We find that isologous, mostly helical interfaces are common in cooperative proteins regardless of their mechanism. On the other hand, the relative contribution of tertiary and quaternary structural changes, as well as the asymmetry in the liganded state, may help distinguish between the two mechanisms.
Asunto(s)
Proteínas , Ligandos , Regulación Alostérica , CinéticaRESUMEN
Schistosomiasis is the second most widespread human parasitic disease. It is principally treated with one drug, praziquantel, that is administered to 100 million people each year; less sensitive strains of schistosomes are emerging. One of the most appealing drug targets against schistosomiasis is thioredoxin glutathione reductase (TGR). This natural chimeric enzyme is a peculiar fusion of a glutaredoxin domain with a thioredoxin selenocysteine (U)-containing reductase domain. Selenocysteine is located on a flexible C-terminal arm that is usually disordered in the available structures of the protein and is essential for the full catalytic activity of TGR. In this study, we dissect the catalytic cycle of Schistosoma mansoni TGR by structural and functional analysis of the U597C mutant. The crystallographic data presented herein include the following: the oxidized form (at 1.9 Å resolution); the NADPH- and GSH-bound forms (2.3 and 1.9 Å, respectively); and a different crystal form of the (partially) reduced enzyme (3.1 Å), showing the physiological dimer and the entire C terminus of one subunit. Whenever possible, we determined the rate constants for the interconversion between the different oxidation states of TGR by kinetic methods. By combining the crystallographic analysis with computer modeling, we were able to throw further light on the mechanism of action of S. mansoni TGR. In particular, we hereby propose the putative functionally relevant conformational change of the C terminus after the transfer of reducing equivalents from NADPH to the redox sites of the enzyme.
Asunto(s)
Cristalografía por Rayos X/métodos , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , NADH NADPH Oxidorreductasas/química , NADH NADPH Oxidorreductasas/metabolismo , Estructura Terciaria de Proteína , Schistosoma mansoni/enzimología , Animales , Catálisis , Electrones , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , NADP/metabolismo , Oxidación-ReducciónRESUMEN
Nitric oxide (NO) inhibits mitochondrial respiration by decreasing the apparent affinity of cytochrome c oxidase (CcO) for oxygen. Using iNOS-transfected HEK 293 cells to achieve regulated intracellular NO production, we determined NO and O(2) concentrations and mitochondrial O(2) consumption by high-resolution respirometry over a range of O(2) concentrations down to nanomolar. Inhibition of respiration by NO was reversible, and complete NO removal recovered cell respiration above its routine reference values. Respiration was observed even at high NO concentrations, and the dependence of IC(50) on [O(2)] exhibits a characteristic but puzzling parabolic shape; both these features imply that CcO is protected from complete inactivation by NO and are likely to be physiologically relevant. We present a kinetic model of CcO inhibition by NO that efficiently predicts experimentally determined respiration at physiological O(2) and NO concentrations and under hypoxia, and accurately predicts the respiratory responses under hyperoxia. The model invokes competitive and uncompetitive inhibition by binding of NO to the reduced and oxidized forms of CcO, respectively, and suggests that dissociation of NO from reduced CcO may involve its O(2)-dependent oxidation. It also explains the non-linear dependence of IC(50) on O(2) concentration, and the hyperbolic increase of c(50) as a function of NO concentration.
Asunto(s)
Complejo IV de Transporte de Electrones/metabolismo , Depuradores de Radicales Libres/farmacología , Óxido Nítrico/fisiología , Oxígeno/metabolismo , Respiración de la Célula , Células Cultivadas , Humanos , Riñón/efectos de los fármacos , Riñón/embriología , Riñón/enzimología , Cinética , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Oxidación-Reducción , Consumo de OxígenoRESUMEN
Human erythrocytes (RBCs), stored at 4 degrees C under nominal absence of external energy sources and calcium ions, show a gradual decrease in membrane roughness (R(rms)) at the end of which the appearance of morphological phenomena (spicules, vesicles and spherocytes) is observed on the cell membrane, phenomena that can mainly be ascribed to the ATP-dependent disconnection of the cortical cytoskeleton from the lipid bilayer. After depletion of the intracellular energy sources obtained under the extreme conditions chosen, treatment with a minimal rejuvenation solution makes the following remarks possible: (i) RBCs are able to regenerate adenosine triphosphate (ATP) and 2,3-bisphosphoglycerate only up to 4 days of storage at 4 degrees C, whereas from the eighth day energy stocks cannot be replenished because of a disorder in the transmembrane mechanisms of transport; (ii) the RBCs' roughness may be restored to the initial value (i.e. that observed in fresh RBCs) only in samples stored up to 4-5 days, whereas after the eighth day of storage the rejuvenation procedure appears to be inefficient; (iii) membrane physical properties - as measured by R(rms) - are actually controlled by the metabolic production of ATP, necessary to perform the RBCs' basic functions; (iv) once energy stores cannot be replenished, a regulated sequence of the morphological events (represented by local buckles that lead to formation of spicules and vesicles of the lipid bilayer with generation of spherocytes) is reminiscent of the RBCs' apoptotic final stages; (v) the morphological phenomenology of the final apoptotic stages is passive (i.e. determined by simple mechanical forces) and encoded in the mechanical properties of the membrane-skeleton; and (vi) necrotic aspects (e.g. disruption of cell membrane integrity, so that intracellular protein content is easily released) ensue when RBCs are almost totally (> or =90%) depleted in an irreversible way of the energetic stores.
Asunto(s)
2,3-Difosfoglicerato/metabolismo , Adenosina Trifosfato/metabolismo , Calcio , Metabolismo Energético , Membrana Eritrocítica/metabolismo , Muerte Celular , Frío , Membrana Eritrocítica/patología , Humanos , Preservación Biológica , Factores de TiempoRESUMEN
The oxidation of the pseudohalide thiocyanate (SCN(-)) by Euphorbia peroxidase, in the presence or absence of added calcium, is investigated. After incubation of the native enzyme with hydrogen peroxide, the formation of Compound I occurs and serves to catalyze the thiocyanate oxidation pathways. The addition of a stoichiometric amount of SCN(-) to Compound I leads to the native enzyme spectrum; this process clearly occurs via two electron transfers from pseudohalide to Compound I. In the presence of 10 mM calcium ions, the addition of a stoichiometric amount of SCN(-) to Compound I leads to the formation of Compound II that returns to the native enzyme after addition of a successive stoichiometric amount of SCN(-), indicating that the oxidation occurs via two consecutive one-electron transfer steps. Moreover, different reaction products can be detected when the enzyme-hydrogen peroxide-thiocyanate reaction is performed in the absence or presence of 10 mM Ca(2+) ions. The formation of hypothiocyanous acid is easy demonstrated in the absence of added calcium, whereas in the presence of this ion, CN(-) is formed as a reaction product that leads to the formation of an inactive species identified as the peroxidase-CN(-) complex. Thus, although monomeric, Euphorbia peroxidase is an allosteric enzyme, finely tuned by Ca(2+) ions. These ions either can enhance the catalytic efficiency of the enzyme toward some substrates or can regulate the ability of the enzyme to exploit different metabolic pathways toward the same substrate.
Asunto(s)
Calcio/metabolismo , Euphorbia/enzimología , Peroxidasa/metabolismo , Tiocianatos/metabolismo , Benzotiazoles/metabolismo , Cianuros/metabolismo , Dianisidina/metabolismo , Peróxido de Hidrógeno/metabolismo , Concentración de Iones de Hidrógeno , Resonancia Magnética Nuclear Biomolecular , Oxidación-Reducción , Espectrofotometría , Ácidos Sulfónicos/metabolismoRESUMEN
Schistosomiasis is a parasitic disease affecting over 200 million people currently treated with one drug, praziquantel. A possible drug target is the seleno-protein thioredoxin-glutathione reductase (TGR), a key enzyme in the pathway of the parasite for detoxification of reactive oxygen species. The enzyme is a unique fusion of a glutaredoxin domain with a thioredoxin reductase domain, which contains a selenocysteine (Sec) as the penultimate amino acid. Auranofin (AF), a gold-containing compound already in clinical use as an anti-arthritic drug, has been shown to inhibit TGR and to substantially reduce worm burden in mice. Using x-ray crystallography we solved (at 2.5 A resolution) the structure of wild type TGR incubated with AF. The electron density maps show that the actual inhibitor is gold, released from AF. Gold is bound at three different sites not directly involving the C-terminal Sec residue; however, because the C terminus in the electron density maps is disordered, we cannot exclude the possibility that gold may also bind to Sec. To investigate the possible role of Sec in the inactivation kinetics, we tested the effect of AF on a model enzyme of the same superfamily, i.e. the naturally Sec-lacking glutathione reductase, and on truncated TGR. We demonstrate that the role of selenium in the onset of inhibition by AF is catalytic and can be mimicked by an external source of selenium (benzeneselenol). Therefore, we propose that Sec mediates the transfer of gold from its ligands in AF to the redox-active Cys couples of TGR.
Asunto(s)
Antirreumáticos/química , Auranofina/química , Regulación de la Expresión Génica , Proteínas del Helminto/química , Complejos Multienzimáticos/química , NADH NADPH Oxidorreductasas/química , Schistosoma mansoni/metabolismo , Animales , Antirreumáticos/farmacología , Auranofina/farmacología , Catálisis , Cristalografía por Rayos X/métodos , Cisteína/química , Relación Dosis-Respuesta a Droga , Cinética , Modelos Moleculares , Complejos Multienzimáticos/antagonistas & inhibidores , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , Oxidación-Reducción , Estructura Terciaria de Proteína , Selenio/químicaRESUMEN
Oxidative stress is a widespread challenge for living organisms, and especially so for parasitic ones, given the fact that their hosts can produce reactive oxygen species (ROS) as a mechanism of defense. Thus, long lived parasites, such as the flatworm Schistosomes, have evolved refined enzymatic systems capable of detoxifying ROS. Among these, glutathione peroxidases (Gpx) are a family of sulfur or selenium-dependent isozymes sharing the ability to reduce peroxides using the reducing equivalents provided by glutathione or possibly small proteins such as thioredoxin. As for other frontline antioxidant enzymatic systems, Gpxs are localized in the tegument of the Schistosomes, the outermost defense layer. In this article, we present the first crystal structure at 1.0 and 1.7 A resolution of two recombinant SmGpxs, carrying the active site mutations Sec43Cys and Sec43Ser, respectively. The structures confirm that this enzyme belongs to the monomeric class 4 (phospholipid hydroperoxide) Gpx. In the case of the Sec to Cys mutant, the catalytic Cys residue is oxidized to sulfonic acid. By combining static crystallography with molecular dynamics simulations, we obtained insight into the substrate binding sites and the conformational changes relevant to catalysis, proposing a role for the unusual reactivity of the catalytic residue.
Asunto(s)
Cristalografía por Rayos X , Glutatión Peroxidasa/química , Simulación de Dinámica Molecular , Schistosoma mansoni/enzimología , Esquistosomiasis mansoni/parasitología , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Humanos , Datos de Secuencia Molecular , Mutación Puntual , Unión Proteica , Conformación Proteica , Alineación de SecuenciaRESUMEN
We recently developed an atomic force microscopy-based protocol to use the roughness of the plasma membrane of erythrocytes (red blood cells, RBCs) as a morphological parameter, independently from the cell shape, to investigate the membrane-skeleton integrity in healthy and pathological cells. Here we apply the method to investigate a complex physiological phenomenon, the RBCs aging, that plays a major role in the regulation of the RBCs' turnover. The aging, monitored morphologically and biochemically, has been accelerated and modulated by preventing oxidative stresses as well as the effects of proteases and divalent cations, and by artificially consuming the intracellular adenosine triphosphate. The collected data evidence that the progression of aging causes a drastic decrease of the measured roughness that is diagnostic of a progressive, adenosine triphosphate-dependent alteration of the membrane-skeleton properties. Finally, the degree of reversibility of such effects has been investigated as a function of aging time, enabling the detection of irreversible transformation in the RBCs' structure and metabolism.
Asunto(s)
Senescencia Celular/fisiología , Membrana Eritrocítica/metabolismo , Microscopía de Fuerza Atómica/métodos , Células Cultivadas , Membrana Eritrocítica/ultraestructura , Humanos , Propiedades de SuperficieRESUMEN
Ligand-linked changes in the aggregation state of biological macromolecules occur and have importance in several physiological processes, e.g., the response of hormone receptors, cooperative ligand binding, and others. The mathematical formalisms that express the thermodynamics governing these processes are complex, as they are required to describe observations made under experimental conditions in which many parameters may be simultaneously varied. The description of the functional behaviour of proteins that present ligand-linked association-dissociation events must accommodate cases where both the binding stoichiometries and reaction mechanisms are variable. In this paper, we review some paradigmatic cases that cover different structural arrangements and binding modes, with special attention to the case of dissociating homodimeric transport proteins and receptors. Even though we cannot pretend to be comprehensive on the proteins presenting this behaviour, we believe that we can attempt to be comprehensive on the structural arrangements and thermodynamic properties of these systems, which fall into a limited set of possible types.
Asunto(s)
Monóxido de Carbono/química , Citocromos c/química , Factor de Crecimiento Epidérmico/química , Hemoglobinas/química , Factor de Crecimiento Derivado de Plaquetas/química , Animales , Sitios de Unión , Monóxido de Carbono/metabolismo , Chromatiaceae/metabolismo , Citocromos c/genética , Citocromos c/metabolismo , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/química , Receptores ErbB/genética , Receptores ErbB/metabolismo , Hemoglobinas/genética , Hemoglobinas/metabolismo , Humanos , Cinética , Ligandos , Factor de Crecimiento Derivado de Plaquetas/genética , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Unión Proteica , Multimerización de Proteína , Scapharca/metabolismo , Estereoisomerismo , TermodinámicaRESUMEN
Hemoglobin and myoglobin have been considered for a long time the paradigmatic model systems for protein function, to the point of being defined the "hydrogen atom[s] of biology". Given this privileged position and the huge amount of quantitative information available on these proteins, the red blood cell might appear as the model system and"hydrogen atom" of system biology. Indeed, since the red cell's main function is O2 transport by hemoglobin, the gap between the protein and the cell may appear quite small. Yet, a surprisingly large amount of detailed biochemical information is required for the modelization of the respiratory properties of the erythrocyte. This problem is compounded if modelization aims at uncovering or explaining evolutionarily selected functional properties of hemoglobin. The foremost difficulty lies in the fact that hemoglobins having different intrinsic properties and relatively ancient evolutionary divergence may behave similarly in the complex milieu of blood, whereas very similar hemoglobins sharing a substantial sequence similarity may present important functional differences because of the mutation of a few key residues. Thus, the functional properties of hemoglobin and blood may reflect more closely the recent environmental challenges than the remote evolutionary history of the animal. We summarize in this review the case of hemoglobins from mammals, in an attempt to provide a reasoned summary of their complexity that, we hope, may be of help to scientists interested in the quantitative exploration of the evolutionary physiology of respiration. Indeed the basis of a meaningful modelization of the red cell requires a large amount of information collected in painstaking and often forgotten studies of the biochemical properties of hemoglobin carried out over more than a century.
Asunto(s)
Dióxido de Carbono/metabolismo , Eritrocitos/fisiología , Evolución Molecular , Hemoglobinas/metabolismo , Oxígeno/metabolismo , Respiración , Regulación Alostérica , Animales , Transporte Biológico , Eritrocitos/citología , Hemoglobinas/genética , Humanos , Cinética , Mamíferos , Mutación , Especificidad de la Especie , Biología de Sistemas/métodosRESUMEN
Selective suicide inhibitors represent a seductively attractive approach for inactivation of therapeutically relevant enzymes since they are generally devoid of off-target toxicity in vivo. While most suicide inhibitors are converted to reactive species at enzyme active sites, theoretically bioactivation can also occur in ectopic (secondary) sites that have no known function. Here, we report an example of such an "ectopic suicide inhibition", an unprecedented bioactivation mechanism of a suicide inhibitor carried out by a non-catalytic site of thioredoxin glutathione reductase (TGR). TGR is a promising drug target to treat schistosomiasis, a devastating human parasitic disease. Utilizing hits selected from a high throughput screening campaign, time-resolved X-ray crystallography, molecular dynamics, mass spectrometry, molecular modeling, protein mutagenesis and functional studies, we find that 2-naphtholmethylamino derivatives bound to this novel ectopic site of Schistosoma mansoni (Sm)TGR are transformed to covalent modifiers and react with its mobile selenocysteine-containing C-terminal arm. In particular, one 2-naphtholmethylamino compound is able to specifically induce the pro-oxidant activity in the inhibited enzyme. Since some 2-naphtholmethylamino analogues show worm killing activity and the ectopic site is not conserved in human orthologues, a general approach to development of novel and selective anti-parasitic therapeutics against schistosoma is proposed.
Asunto(s)
Complejos Multienzimáticos , NADH NADPH Oxidorreductasas , Animales , Cristalografía por Rayos X , Glutatión Reductasa , Humanos , NADH NADPH Oxidorreductasas/genética , Schistosoma mansoni , Reductasa de Tiorredoxina-DisulfuroRESUMEN
Hemoglobin-based blood substitutes are one of the options available to derive a resuscitating fluid taking into account clinical and physiological demands. In this paper we investigated a novel protein, Hb(alphaalpha,betabeta) obtained as a combination of two homodimers alpha(2) and beta(2) both derived from a fusion gene containing two alfa chains or two beta chains, each respectively coupled via a specific linker. The construct here described is thus a novel heterodimeric hemoglobin carrying four heme groups. The protein cannot dissociate into dimers, as demonstrated by its absence of reactivity versus haptoglobin, and is expected to have a relatively long circulating half-life. The modification does not increase the autoxidation rate, but increases the oxygen affinity, due to a destabilization of the T quaternary state. Characterization of the biochemical properties of this protein in comparison with HbA is reported.
Asunto(s)
Sustitutos Sanguíneos/uso terapéutico , Hemoglobina A/metabolismo , Hemoglobinas/genética , Oxihemoglobinas/uso terapéutico , Automatización , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , Dimerización , Expresión Génica , Hemoglobina A/genética , Hemoglobina A/uso terapéutico , Hemoglobinas/química , Hemoglobinas/metabolismo , Hemoglobinas/uso terapéutico , Humanos , Cinética , Oxígeno/sangre , Plásmidos , Proteínas Recombinantes/química , Proteínas Recombinantes/uso terapéuticoRESUMEN
Cooperative ligand binding is a fundamental property of many biological macromolecules, notably transport proteins, hormone receptors, and enzymes. Positive homotropic cooperativity, the form of cooperativity that has greatest physiological relevance, causes the ligand affinity to increase as ligation proceeds, thus increasing the steepness of the ligand-binding isotherm. The measurement of the extent of cooperativity has proven difficult, and the most commonly employed marker of cooperativity, the Hill coefficient, originates from a structural hypothesis that has long been disproved. However, a wealth of relevant biochemical data has been interpreted using the Hill coefficient and is being used in studies on evolution and comparative physiology. Even a cursory analysis of the pertinent literature shows that several authors tried to derive more sound biochemical information from the Hill coefficient, often unaware of each other. As a result, a perplexing array of equations interpreting the Hill coefficient is available in the literature, each responding to specific simplifications or assumptions. In this work, we summarize and try to order these attempts, and demonstrate that the Hill coefficient (i) provides a minimum estimate of the free energy of interaction, the other parameter used to measure cooperativity, and (ii) bears a robust statistical correlation to the population of incompletely saturated ligation intermediates. Our aim is to critically evaluate the different analyses that have been advanced to provide a physical meaning to the Hill coefficient, and possibly to select the most reliable ones to be used in comparative studies that may make use of the extensive but elusive information available in the literature.