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Ovarian cancer often develops resistance to conventional therapies, hampering their effectiveness. Here, using ex vivo paired ovarian cancer ascites obtained before and after chemotherapy and in vitro therapy-induced secretomes, we show that molecules secreted by ovarian cancer cells upon therapy promote cisplatin resistance and enhance DNA damage repair in recipient cancer cells. Even a short-term incubation of chemonaive ovarian cancer cells with therapy-induced secretomes induces changes resembling those that are observed in chemoresistant patient-derived tumor cells after long-term therapy. Using integrative omics techniques, we find that both ex vivo and in vitro therapy-induced secretomes are enriched with spliceosomal components, which relocalize from the nucleus to the cytoplasm and subsequently into the extracellular vesicles upon treatment. We demonstrate that these molecules substantially contribute to the phenotypic effects of therapy-induced secretomes. Thus, SNU13 and SYNCRIP spliceosomal proteins promote therapy resistance, while the exogenous U12 and U6atac snRNAs stimulate tumor growth. These findings demonstrate the significance of spliceosomal network perturbation during therapy and further highlight that extracellular signaling might be a key factor contributing to the emergence of ovarian cancer therapy resistance.
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Cisplatino , Resistencia a Antineoplásicos , Neoplasias Ováricas , Empalmosomas , Femenino , Humanos , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Neoplasias Ováricas/genética , Neoplasias Ováricas/tratamiento farmacológico , Empalmosomas/metabolismo , Cisplatino/farmacología , Línea Celular Tumoral , Animales , Ratones , Vesículas Extracelulares/metabolismo , Supervivencia Celular/efectos de los fármacos , Antineoplásicos/farmacología , ARN Nuclear Pequeño/metabolismo , ARN Nuclear Pequeño/genética , Reparación del ADNRESUMEN
One of the hallmarks of Alzheimer's disease (AD) is the accumulation of beta-amyloid peptide (Aß) leading to formation of soluble neurotoxic Aß oligomers and insoluble amyloid plaques in various parts of the brain. Aß undergoes post-translational modifications that alter its pathogenic properties. Aß is produced not only in brain, but also in the peripheral tissues. Such Aß, including its post-translationally modified forms, can enter the brain from circulation by binding to RAGE and contribute to the pathology of AD. However, the transport of modified forms of Aß across the blood-brain barrier (BBB) has not been investigated. Here, we used a transwell BBB model as a controlled environment for permeability studies. We found that Aß42 containing isomerized Asp7 residue (iso-Aß42) and Aß42 containing phosphorylated Ser8 residue (pS8-Aß42) crossed the BBB better than unmodified Aß42, which correlated with different contribution of endocytosis mechanisms to the transport of these isoforms. Using microscale thermophoresis, we observed that RAGE binds to iso-Aß42 an order of magnitude weaker than to Aß42. Thus, post-translational modifications of Aß increase the rate of its transport across the BBB and modify the mechanisms of the transport, which may be important for AD pathology and treatment.
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Inactivation of enzymes responsible for biosynthesis of the cell wall component of ADP-glycero-manno-heptose causes the development of oxidative stress and sensitivity of bacteria to antibiotics of a hydrophobic nature. The metabolic precursor of ADP-heptose is sedoheptulose-7-phosphate (S7P), an intermediate of the non-oxidative branch of the pentose phosphate pathway (PPP), in which ribose-5-phosphate and NADPH are generated. Inactivation of the first stage of ADP-heptose synthesis (ΔgmhA) prevents the outflow of S7P from the PPP, and this mutant is characterized by a reduced biosynthesis of NADPH and of the Glu-Cys-Gly tripeptide, glutathione, molecules known to be involved in the resistance to oxidative stress. We found that the derepression of purine biosynthesis (∆purR) normalizes the metabolic equilibrium in PPP in ΔgmhA mutants, suppressing the negative effects of gmhA mutation likely via the over-expression of the glycine-serine pathway that is under the negative control of PurR and might be responsible for the enhanced synthesis of NADPH and glutathione. Consistently, the activity of the soxRS system, as well as the level of glutathionylation and oxidation of proteins, indicative of oxidative stress, were reduced in the double ΔgmhAΔpurR mutant compared to the ΔgmhA mutant.
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Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Antibacterianos/farmacología , Antibacterianos/metabolismo , NADP/metabolismo , Purinas/farmacología , Purinas/metabolismo , Heptosas/química , Heptosas/metabolismo , Glutatión/metabolismo , Vía de Pentosa FosfatoRESUMEN
Hemoglobin is the main protein of red blood cells that provides oxygen transport to all cells of the human body. The ability of hemoglobin to bind the main low-molecular-weight thiol of the cell glutathione, both covalently and noncovalently, is not only an important part of the antioxidant protection of red blood cells, but also affects its affinity for oxygen in both cases. In this study, the properties of oxyhemoglobin in complex with reduced glutathione (GSH) and properties of glutathionylated hemoglobin bound to glutathione via an SS bond were characterized. For this purpose, the methods of circular dichroism, Raman spectroscopy, infrared spectroscopy, tryptophan fluorescence, differential scanning fluorimetry, and molecular modeling were used. It was found that the glutathionylation of oxyhemoglobin caused changes in the secondary structure of the protein, reducing the alpha helicity, but did not affect the heme environment, tryptophan fluorescence, and the thermostability of the protein. In the noncovalent complex of oxyhemoglobin with reduced glutathione, the secondary structure of hemoglobin remained almost unchanged; however, changes in the heme environment and the microenvironment of tryptophans, as well as a decrease in the protein's thermal stability, were observed. Thus, the formation of a noncovalent complex of hemoglobin with glutathione makes a more significant effect on the tertiary and quaternary structure of hemoglobin than glutathionylation, which mainly affects the secondary structure of the protein. The obtained data are important for understanding the functioning of glutathionylated hemoglobin, which is a marker of oxidative stress, and hemoglobin in complex with GSH, which appears to deposit GSH and release it during deoxygenation to increase the antioxidant protection of cells.
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Antioxidantes , Oxihemoglobinas , Humanos , Triptófano , Hemoglobinas , Glutatión , Hemo , OxígenoRESUMEN
The deposition of beta-amyloid (Aß) aggregates in the brain, accompanied by impaired cognitive function, is a characteristic feature of Alzheimer's disease (AD). An important role in this process is played by vascular disorders, in particular, a disturbance of the blood-brain barrier (BBB). The BBB controls the entry of Aß from plasma to the brain via the receptor for advanced glycation end products (RAGE) and the removal of brain-derived Aß via the low-density lipoprotein receptor-related protein (LRP1). The balance between the input of Aß to the brain from the periphery and its output is disturbed during AD. Aß changes the redox-status of BBB cells, which in turn changes the functioning of mitochondria and disrupts the barrier function of endothelial cells by affecting tight junction proteins. Aß oligomers have the greatest toxic effect on BBB cells, and oligomers are most rapidly transferred by transcytosis from the brain side of the BBB to the blood side. Both the cytotoxic effect of Aß and the impairment of barrier function are partly due to the interaction of Aß monomers and oligomers with membrane-bound RAGE. AD therapies based on the disruption of this interaction or the creation of decoys for Aß are being developed. The question of the transfer of various Aß isoforms through the BBB is important, since it can influence the development of AD. It is shown that the rate of input of Aß40 and Aß42 from the blood into the brain is different. The actual question of the transfer of pathogenic Aß isoforms with post-translational modifications or mutations through the BBB still remains open.
RESUMEN
Ovarian cancer is known to be the most lethal malignancy among all gynecological cancers affecting a large number of women worldwide. The treatment of ovarian cancer is challenging due to the high recurrence rate of the disease and is further complicated by acquired chemoresistance. Most ovarian cancer deaths are the result of the metastatic spread of drug-resistant cells. The theory of cancer stem cells (CSC) suggests that both tumor initiation and progression are driven by a population of undifferentiated capable of self-renewal, tumor initiation and development of chemoresistance. The CD117 mast/stem cell growth factor receptor (KIT) is the most commonly used marker for ovarian CSCs. Here, we analyze the correlation between CD117 expression and histological tumor type in ovarian cancer cell lines (SK-OV-3 and MES-OV) and in small/medium extracellular vesicles (EVs) isolated from the urine of ovarian cancer patients. We have demonstrated that the abundance of CD117 on cells and EVs is correlated with tumor grade and therapy resistance status. Moreover, using small EVs isolated from ovarian cancer ascites, it was shown that recurrent disease is characterized by a much higher abundance of CD117 on EVs than primary tumor.
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Intracellular tripeptide glutathione is an important agent of cell survival under hypoxia. Glutathione covalently binds to SH groups of hemoglobin cysteine residues, protecting them from irreversible oxidation, and changes its affinity to oxygen. Reduced glutathione (GSH) can also form a noncovalent complex with hemoglobin. Previously, we showed that hemoglobin tetramer has four noncovalent binding sites of glutathione GSH molecules inside, two of which are released during hemoglobin transition to deoxy form. In this study, we characterized the conserved cysteine residues and residues of noncovalent glutathione binding sites in the sequences of a number of hypoxia-tolerant and hypoxia-sensitive mammals. The solvent accessibility of all HbA and HbB residues in oxy and deoxy forms was analyzed. The alpha subunit of all species considered was shown to have no conserved cysteines, whereas the beta subunit contains Cys93 residue, which is conserved across species and whose glutathionylation changes the affinity of hemoglobin for oxygen 5-6-fold. It was found that the key residues of noncovalent glutathione binding sites in both alpha and beta subunits are absolutely conserved in all species considered, suggesting a common mechanism of hemoglobin redox regulation for both hypoxia-sensitive and hypoxia-tolerant mammals.
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Cisteína , Hipoxia , Animales , Oxígeno , Sitios de Unión , Glutatión , Hemoglobinas , MamíferosRESUMEN
Fast changes in environmental oxygen availability translate into shifts in mitochondrial free radical production. An increase in intraerythrocytic reduced glutathione (GSH) during deoxygenation would support the detoxification of exogenous oxidants released into the circulation from hypoxic peripheral tissues. Although reported, the mechanism behind this acute oxygen-dependent regulation of GSH in red blood cells remains unknown. This study explores the role of hemoglobin (Hb) in the oxygen-dependent modulation of GSH levels in red blood cells. We have demonstrated that a decrease in Hb O2 saturation to 50% or less observed in healthy humans while at high altitude, or in red blood cell suspensions results in rising of the intraerythrocytic GSH level that is proportional to the reduction in Hb O2 saturation. This effect was not caused by the stimulation of GSH de novo synthesis or its release during deglutathionylation of Hb's cysteines. Using isothermal titration calorimetry and in silico modeling, we observed the non-covalent binding of four molecules of GSH to oxy-Hb and the release of two of them upon deoxygenation. Localization of the GSH binding sites within the Hb molecule was identified. Oxygen-dependent binding of GSH to oxy-Hb and its release upon deoxygenation occurred reciprocally to the binding and release of 2,3-bisphosphoglycerate. Furthermore, noncovalent binding of GSH to Hb moderately increased Hb oxygen affinity. Taken together, our findings have identified an adaptive mechanism by which red blood cells may provide an advanced antioxidant defense to respond to oxidative challenges immediately upon deoxygenation.
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Glutatión , Oxígeno , Humanos , Oxígeno/metabolismo , Glutatión/metabolismo , Hemoglobinas/metabolismo , Eritrocitos/metabolismo , Oxihemoglobinas/metabolismoRESUMEN
Beta-amyloid peptide (Aß) is a ligand associated with RAGE (Advanced glycosylation end product-specific receptor). Aß is translocated in complexes with RAGE from the blood to brain across the blood-brain barrier (BBB) by transcytosis. Aß and its isoforms are important factors in the Alzheimer's disease (AD) pathogenesis. However, interaction with RAGE was previously studied for Aß but not for its isoforms. The present study has been directed at identifying the key interaction interfaces between RAGE and Aß isoforms (Aß40, Aß42, phosphorylated and isomerized isoforms pS8-Aß42, isoD7-Aß42). Two interfaces have been identified by docking: they are represented by an extended area at the junction of RAGE domains V and C1 and a smaller area linking C1 and C2 domains. Molecular dynamics (MD) simulations have shown that all Aß isoforms form stable and tightly bound complexes. This indicates that all Aß isoforms potentially can be transported through the cell as part of a complex with RAGE. Modeling of RAGE interaction interfaces with Aß indicates which chemical compounds can potentially be capable of blocking this interaction, and impair the associated pathogenic cascades. The ability of three RAGE inhibitors (RAP, FPS-ZM1 and RP-1) to disrupt the RAGE:Aß interaction has been probed by docking and subsequently the complexes' stability verified by MD. The RP-1 and Aß interaction areas coincide and therefore this inhibitor is very promising for the RAGE:Aß interaction inhibition.
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Enfermedad de Alzheimer , Péptidos beta-Amiloides , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Barrera Hematoencefálica/metabolismo , Humanos , Ligandos , Simulación de Dinámica Molecular , Fragmentos de Péptidos/metabolismo , Isoformas de Proteínas/metabolismo , Receptor para Productos Finales de Glicación Avanzada/metabolismoRESUMEN
Impaired lipopolysaccharide biosynthesis in Gram-negative bacteria results in the "deep rough" phenotype, which is characterized by increased sensitivity of cells to various hydrophobic compounds, including antibiotics novobiocin, actinomycin D, erythromycin, etc. The present study showed that E. coli mutants carrying deletions of the ADP-heptose biosynthesis genes became hypersensitive to a wide range of antibacterial drugs: DNA gyrase inhibitors, protein biosynthesis inhibitors (aminoglycosides, tetracycline), RNA polymerase inhibitors (rifampicin), and ß-lactams (carbenicillin). In addition, it was found that inactivation of the gmhA, hldE, rfaD, and waaC genes led to dramatic changes in the redox status of cells: a decrease in the pool of reducing NADPH and ATP equivalents, the concentration of intracellular cysteine, a change in thiol homeostasis, and a deficiency in the formation of hydrogen sulfide. In "deep rough" mutants, intensive formation of reactive oxygen species was observed, which, along with a lack of reducing agents, such as reactive sulfur species or NADPH, leads to oxidative stress and an increase in the number of dead cells in the population. Within the framework of modern ideas about the role of oxidative stress as a universal mechanism of the bactericidal action of antibiotics, inhibition of the enzymes of ADP-heptose biosynthesis is a promising direction for increasing the effectiveness of existing antibiotics and solving the problem of multidrug resistance.
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Proteínas de Escherichia coli , Escherichia coli , Adenosina Difosfato/metabolismo , Antibacterianos/farmacología , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Heptosas/química , Heptosas/metabolismo , Lipopolisacáridos/química , Lipopolisacáridos/farmacología , NADP/metabolismo , Estrés OxidativoRESUMEN
Beta-amyloid (Aß) has a dual role, both as an important factor in the pathology of Alzheimer's disease and as a regulator in brain physiology. The inhibitory effect of Aß42 oligomers on Na,K-ATPase contributes to neuronal dysfunction in Alzheimer's disease. Still, the physiological role of the monomeric form of Aß42 interaction with Na,K-ATPase remains unclear. We report that Na,K-ATPase serves as a receptor for Aß42 monomer, triggering Src kinase activation. The co-localization of Aß42 with α1- and ß1-subunits of Na,K-ATPase, and Na,K-ATPase with Src kinase in SH-SY5Y neuroblastoma cells, was observed. Treatment of cells with 100 nM Aß42 causes Src kinase activation, but does not alter Na,K-ATPase transport activity. The interaction of Aß42 with α1ß1 Na,K-ATPase isozyme leads to activation of Src kinase associated with the enzyme. Notably, prevention of Na,K-ATPase:Src kinase interaction by a specific inhibitor pNaKtide disrupts the Aß-induced Src kinase activation. Stimulatory effect of Aß42 on Src kinase was lost under hypoxic conditions, which was similar to the effect of specific Na,K-ATPase ligands, the cardiotonic steroids. Our findings identify Na,K-ATPase as a Aß42 receptor, thus opening a prospect on exploring the physiological and pathological Src kinase activation caused by Aß42 in the nervous system.
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Péptidos beta-Amiloides , ATPasa Intercambiadora de Sodio-Potasio , Familia-src Quinasas , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Humanos , Neuroblastoma , Sodio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
Alzheimer's disease (AD) is a neurodegenerative disease accompanied by progressive cognitive and memory dysfunction due to disruption of normal electrotonic properties of neurons and neuronal loss. The Na,K-ATPase interaction with beta amyloid (Aß) plays an important role in AD pathogenesis. It has been shown that Na,K-ATPase activity in the AD brain was significantly lower than those in age-matched control brain. The interaction of Aß42 with Na,K-ATPase and subsequent oligomerization leads to inhibition of the enzyme activity. In this study interaction interfaces between three common Aß42 isoforms, and different conformations of human Na,K-ATPase (α1ß1) have been obtained using molecular modeling, including docking and molecular dynamics (MD). Interaction sites of Na,K-ATPase with Aß42 are localized between extracellular parts of α- and ß- subunits and are practically identical for Na,K-ATPase at different conformations. Thermodynamic parameters for the formation of Na,K-ATPase:Aß42 complex at different conformations acquired by isothermal titration calorimetry (ITC) are similar, which is in line with the data of molecular modeling. Similarity of Na,K-ATPase interaction interfaces with Aß in all conformations allowed us to cross-screen potential inhibitors for this interaction and find pharmaceutical compounds that could block it.
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The Alzheimer's disease (AD)-associated breakdown of the blood-brain barrier (BBB) promotes the accumulation of beta-amyloid peptide (Aß) in the brain as the BBB cells provide Aß transport from the brain parenchyma to the blood, and vice versa. The breakdown of the BBB during AD may be caused by the emergence of blood-borne Aß pathogenic forms, such as structurally and chemically modified Aß species; their effect on the BBB cells has not yet been studied. Here, we report that the effects of Aß42, Aß42, containing isomerized Asp7 residue (iso-Aß42) or phosphorylated Ser8 residue (p-Aß42) on the mitochondrial potential and respiration are closely related to the redox status changes in the mouse brain endothelial cells bEnd.3. Aß42 and iso-Aß42 cause a significant increase in nitric oxide, reactive oxygen species, glutathione, cytosolic calcium and the mitochondrial potential after 4 h of incubation. P-Aß42 either does not affect or its effect develops after 24 h of incubation. Aß42 and iso-Aß42 activate mitochondrial respiration compared to p-Aß42. The isomerized form promotes a greater cytotoxicity and mitochondrial dysfunction, causing maximum oxidative stress. Thus, Aß42, p-Aß42 and iso-Aß42 isoforms differently affect the BBBs' cell redox parameters, significantly modulating the functioning of the mitochondria. The changes in the level of modified Aß forms can contribute to the BBBs' breakdown during AD.
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Enfermedad de Alzheimer , Péptidos beta-Amiloides , Animales , Ratones , Péptidos beta-Amiloides/metabolismo , Barrera Hematoencefálica/metabolismo , Células Endoteliales/metabolismo , Enfermedad de Alzheimer/metabolismo , Oxidación-Reducción , Endotelio/metabolismo , Fragmentos de Péptidos/metabolismoRESUMEN
BACKGROUND: Analyzing the local sequence content in proteins, earlier we found that amino acid residue frequencies differ on various distances between amino acid positions in the sequence, assuming the existence of structural units. METHODS: We used informational entropy of protein sequences to find that the structural unit of proteins is a block of adjacent amino acid residues-"information unit". The ANIS (ANalysis of Informational Structure) method uses these information units for revealing hierarchically organized Elements of the Information Structure (ELIS) in amino acid sequences. RESULTS: The developed mathematical apparatus gives stable results on the structural unit description even with a significant variation in the parameters. The optimal length of the information unit is five, and the number of allowed substitutions is one. Examples of the application of the method for the design of protein molecules, intermolecular interactions analysis, and the study of the mechanisms of functioning of protein molecular machines are given. CONCLUSIONS: ANIS method makes it possible not only to analyze native proteins but also to design artificial polypeptide chains with a given spatial organization and, possibly, function.
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Cardiotonic steroids (CTSs) are specific inhibitors of Na,K-ATPase (NKA). They induce diverse physiological effects and were investigated as potential drugs in heart diseases, hypertension, neuroinflammation, antiviral and cancer therapy. Here, we compared the inhibition mode and binding of CTSs, such as ouabain, digoxin and marinobufagenin to NKA from pig and rat kidneys, containing CTSs-sensitive (α1S) and -resistant (α1R) α1-subunit, respectively. Marinobufagenin in contrast to ouabain and digoxin interacted with α1S-NKA reversibly, and its binding constant was reduced due to the decrease in the deepening in the CTSs-binding site and a lower number of contacts between the site and the inhibitor. The formation of a hydrogen bond between Arg111 and Asp122 in α1R-NKA induced the reduction in CTSs' steroid core deepening that led to the reversible inhibition of α1R-NKA by ouabain and digoxin and the absence of marinobufagenin's effect on α1R-NKA activity. Our results elucidate that the difference in signaling, and cytotoxic effects of CTSs may be due to the distinction in the deepening of CTSs into the binding side that, in turn, is a result of a bent-in inhibitor steroid core (marinobufagenin in α1S-NKA) or the change of the width of CTSs-binding cavity (all CTSs in α1R-NKA).
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Bufanólidos/farmacología , Digoxina/farmacología , Riñón/enzimología , Ouabaína/farmacología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Sitios de Unión , Glicósidos Cardíacos/farmacología , Enlace de Hidrógeno , Riñón/efectos de los fármacos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Ratas , ATPasa Intercambiadora de Sodio-Potasio/química , PorcinosRESUMEN
The hemoglobin concentration of 35 g/dl of human red blood cells is close to the solubility threshold. Using microwave dielectric spectroscopy, we have assessed the amount of water associated with hydration shells of methemoglobin as a function of its concentration in the presence or absence of ions. We estimated water-hemoglobin interactions to interpret the obtained data. Within the concentration range of 5-10 g/dl of methemoglobin, ions play an important role in defining the free-to-bound water ratio competing with hemoglobin to recruit water molecules for the hydration shell. At higher concentrations, hemoglobin is a major contributor to the recruitment of water to its hydration shell. Furthermore, the amount of bound water does not change as the hemoglobin concentration is increased from 15 to 30 g/dl, remaining at the level of â¼20% of the total intracellular water pool. The theoretical evaluation of the ratio of free and bound water for the hemoglobin concentration in the absence of ions corresponds with the experimental results and shows that the methemoglobin molecule binds about 1400 water molecules. These observations suggest that within the concentration range close to the physiological one, hemoglobin molecules are so close to each other that their hydration shells interact. In this case, the orientation of the hemoglobin molecules is most likely not stochastic, but rather supports partial neutralization of positive and negative charges at the protein surface. Furthermore, deformation of the red blood cell shape results in the rearrangement of these structures.
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Metahemoglobina/química , Espectroscopía Dieléctrica , Humanos , Microondas , Modelos Moleculares , Agua/químicaRESUMEN
The ability of red blood cells (RBCs) to transport gases, their lifespan as well as their rheological properties invariably depend on the deformability, hydration, and membrane stability of these cells, which can be measured by Laser optical rotational red cell analyser (Lorrca® Maxsis, RR Mechatronics). The osmoscan mode of Lorrca is currently used in diagnosis of rare anemias in clinical laboratories. However, a broad range of normal values for healthy subjects reduces the sensitivity of this method for diagnosis of mild disease phenotype. In this pilot study, we explored the impact of age and gender of 45 healthy donors, as well as RBC age on the Lorrca indices. Whereas gender did not affect the Lorrca indices in our study, the age donors had a profound effect on the O_hyper parameter. To study the impact of RBC age on the osmoscan parameters, we have isolated low (L)-, medium (M)-, or high (H)- density fractions enriched with young, mature, and senescent RBCs, respectively, and evaluated the influence of RBC age-related properties, such as density, morphology, and redox state, on the osmoscan indices. As before, O_hyper was the most sensitive parameter, dropping markedly with an increase in RBC density and age. Senescence was associated with a decrease in deformability (EI_max) and tolerability to low and high osmolatites (Area). L-fraction was enriched with reticulocytes and cells with high projected area and EMA staining, but also contained a small number of cells small in projected area and most likely, terminally senescent. L-fraction was on average slightly less deformable than mature cells. The cells from the L-fraction produced more oxidants and NO than all other fractions. However, RBCs from the L-fraction contained maximal levels of reduced thiols compared to other fractions. Our study suggests that reference values for O_hyper should be age-stratified, and, most probably, corrected for the average RBC age. Further multi-center study is required to validate these suggestions before implementing them into clinical practice.
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This study focuses on assessing the possible impact of changes in hemoglobin (Hb) oxygenation on the state of water in its hydration shell as it contributes to red blood cell deformability. Microwave Dielectric Spectroscopy (MDS) was used to monitor the changes in interactions between water molecules and Hb, the number of water molecules in the protein hydration shell, and the dynamics of pre-protein water in response to the transition of Hb from the tense (T) to the relaxed (R) state, and vice versa. Measurements were performed for Hb solutions of different concentrations (5 g/dl-30 g/dl) in phosphate-buffered saline buffer. Cole-Cole parameters of the main water relaxation peak in terms of interactions of water molecules (dipole-dipole/ionic dipole) during the oxygenation-deoxygenation cycle were used to analyze the obtained data. The water mobility-represented by α as a function of ln τ-differed dramatically between the R (oxygenated) state and the T (deoxygenated) state of Hb at physiologically relevant concentrations (30 g/dl-35 g/dl or 4.5 mM-5.5 mM). At these concentrations, oxygenated hemoglobin was characterized by substantially lower mobility of water in the hydration shell, measured as an increase in relaxation time, compared to deoxyhemoglobin. This change indicated an increase in red blood cell cytosolic viscosity when cells were oxygenated and a decrease in viscosity upon deoxygenation. Information provided by MDS on the intraerythrocytic water state of intact red blood cells reflects its interaction with all of the cytosolic components, making these measurements powerful predictors of the changes in the rheological properties of red blood cells, regardless of the cause.
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Oxihemoglobinas/química , Agua/química , Espectroscopía Dieléctrica , Ditionita/química , Humanos , Microondas , Oxidación-Reducción , Conformación Proteica , ViscosidadRESUMEN
BACKGROUND: S-glutathionylation is the formation of disulfide bonds between the tripeptide glutathione and cysteine residues of the protein, protecting them from irreversible oxidation and in some cases causing change in their functions. Regulatory glutathionylation of proteins is a controllable and reversible process associated with cell response to the changing redox status. Prediction of cysteine residues that undergo glutathionylation allows us to find new target proteins, which function can be altered in pathologies associated with impaired redox status. We set out to analyze this issue and create new tool for predicting S-glutathionylated cysteine residues. RESULTS: One hundred forty proteins with experimentally proven S-glutathionylated cysteine residues were found in the literature and the RedoxDB database. These proteins contain 1018 non-S-glutathionylated cysteines and 235 S-glutathionylated ones. Based on 235 S-glutathionylated cysteines, non-redundant positive dataset of 221 heptapeptide sequences of S-glutathionylated cysteines was made. Based on 221 heptapeptide sequences, a position-specific matrix was created by analyzing the protein sequence near the cysteine residue (three amino acid residues before and three after the cysteine). We propose the method for calculating the glutathionylation propensity score, which utilizes the position-specific matrix and a criterion for predicting glutathionylated peptides. CONCLUSION: Non-S-glutathionylated sites were enriched by cysteines in - 3 and + 3 positions. The proposed prediction method demonstrates 76.6% of correct predictions of S-glutathionylated cysteines. This method can be used for detecting new glutathionylation sites, especially in proteins with an unknown structure.