RESUMEN
The Na+/K+-ATPase is an integral plasma membrane glycoprotein of all animal cells that couples the exchange of intracellular Na+ for extracellular K+ to the hydrolysis of ATP. The asymmetric distribution of Na+ and K+ is essential for cellular life and constitutes the physical basis of a series of fundamental biological phenomena. The pumping mechanism is explained by the Albers-Post model. It involves the presence of gates alternatively exposing Na+/K+-ATPase transport sites to the intracellular and extracellular sides and includes occluded states in which both gates are simultaneously closed. Unlike for K+, information is lacking about Na+-occluded intermediates, as occluded Na+ was only detected in states incapable of performing a catalytic cycle, including two Na+-containing crystallographic structures. The current knowledge is that intracellular Na+ must bind to the transport sites and become occluded upon phosphorylation by ATP to be transported to the extracellular medium. Here, taking advantage of epigallocatechin-3-gallate to instantaneously stabilize native Na+-occluded intermediates, we isolated species with tightly bound Na+ in an enzyme able to perform a catalytic cycle, consistent with a genuine occluded state. We found that Na+ becomes spontaneously occluded in the E1 dephosphorylated form of the Na+/K+-ATPase, exhibiting positive interactions between binding sites. In fact, the addition of ATP does not produce an increase in Na+ occlusion as it would have been expected; on the contrary, occluded Na+ transiently decreases, whereas ATP lasts. These results reveal new properties of E1 intermediates of the Albers-Post model for explaining the Na+ transport pathway.
Asunto(s)
Biocatálisis , ATPasa Intercambiadora de Sodio-Potasio , Sodio , Animales , Adenosina Trifosfato/metabolismo , Membrana Celular/metabolismo , Cinética , Potasio/metabolismo , Sodio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Transporte Iónico , Fosforilación , Cationes Monovalentes/metabolismoRESUMEN
There is growing consensus that under physiological conditions, collecting duct H+ secretion is independent of epithelial Na+ channel (ENaC) activity. We have recently shown that the direct ENaC inhibitor benzamil acutely impairs H+ excretion by blocking renal H+-K+-ATPase. However, the question remains whether inhibition of ENaC per se causes alterations in renal H+ excretion. To revisit this question, we studied the effect of the antibiotic trimethoprim (TMP), which is well known to cause K+ retention by direct ENaC inhibition. The acute effect of TMP (5 µg/g body wt) was assessed in bladder-catheterized mice, allowing real-time measurement of urinary pH, electrolyte, and acid excretion. Dietary K+ depletion was used to increase renal H+-K+-ATPase activity. In addition, the effect of TMP was investigated in vitro using pig gastric H+-K+-ATPase-enriched membrane vesicles. TMP acutely increased natriuresis and decreased kaliuresis, confirming its ENaC-inhibiting property. Under control diet conditions, TMP had no effect on urinary pH or acid excretion. Interestingly, K+ depletion unmasked an acute urine alkalizing effect of TMP. This finding was corroborated by in vitro experiments showing that TMP inhibits H+-K+-ATPase activity, albeit at much higher concentrations than benzamil. In conclusion, under control diet conditions, TMP inhibited ENaC function without changing urinary H+ excretion. This finding further supports the hypothesis that the inhibition of ENaC per se does not impair H+ excretion in the collecting duct. Moreover, TMP-induced urinary alkalization in animals fed a low-K+ diet highlights the importance of renal H+-K+-ATPase-mediated H+ secretion in states of K+ depletion.NEW & NOTEWORTHY The antibiotic trimethoprim (TMP) often mediates K+ retention and metabolic acidosis. We suggest a revision of the underlying mechanism that causes metabolic acidosis. Our results indicate that TMP-induced metabolic acidosis is secondary to epithelial Na+ channel-dependent K+ retention. Under control dietary conditions, TMP does not per se inhibit collecting duct H+ secretion. These findings add further argument against a physiologically relevant voltage-dependent mechanism of collecting duct H+ excretion.
Asunto(s)
Acidosis , Túbulos Renales Colectores , Ratones , Animales , Porcinos , Trimetoprim/farmacología , Trimetoprim/metabolismo , Túbulos Renales Colectores/metabolismo , Canales Epiteliales de Sodio/metabolismo , Sodio/metabolismo , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Antibacterianos/farmacología , Acidosis/metabolismoRESUMEN
In the present work, we evaluated the antifungal activities of two novel ebselen analogs, N-allyl-benzisoselenazol-3(2H)-one (N-allyl-bs) and N-3-methylbutylbenzisoselenazol-3(2H)-one (N-3mb-bs). Colorimetric and turbidity assays were performed to determine the minimum inhibitory concentration (MIC) of these compounds in S1 (fluconazole-sensitive) and S2 (fluconazole-resistant) strains of C. albicans. N-3mb-bs was more active than the N-allyl-bs compound. It is noteworthy that the concentration of N-3mb-bs observed to inhibit fungal growth by 50% (18.2 µM) was similar to the concentration observed to inhibit the activity of the yeast plasma membrane H+-ATPase (Pma1p) by 50% (19.6 µM). We next implemented a mouse model of vulvovaginal candidiasis (VVC) using the S1 strain and examined the mouse and yeast proteins present in the vaginal lavage fluid using proteomics. The yeast proteins detected were predominately glycolytic enzymes or virulence factors associated with C. albicans while the mouse proteins present in the lavage fluid included eosinophil peroxidase, desmocollin-1, and gasdermin-A. We then utilized the N-3mb-bs compound (12.5 mg/kg) in the mouse VVC model and observed that it significantly reduced the vaginal fungal burden, histopathological changes in vagina tissue, and expression of myeloperoxidase (MPO). All in all, the present work has identified a potentially promising drug candidate for VVC treatment.
RESUMEN
The Na+,K+-ATPase generates electrochemical gradients of Na+ and K+ across the plasma membrane via a functional cycle that includes various phosphoenzyme intermediates. However, the structure and function of these intermediates and how metal fluorides mimick them require further investigation. Here, we describe a 4.0 Å resolution crystal structure and functional properties of the pig kidney Na+,K+-ATPase stabilized by the inhibitor beryllium fluoride (denoted E2-BeFx). E2-BeFx is expected to mimic properties of the E2P phosphoenzyme, yet with unknown characteristics of ion and ligand binding. The structure resembles the E2P form obtained by phosphorylation from inorganic phosphate (Pi) and stabilized by cardiotonic steroids, including a low-affinity Mg2+ site near ion binding site II. Our anomalous Fourier analysis of the crystals soaked in Rb+ (a K+ congener) followed by a low-resolution rigid-body refinement (6.9-7.5 Å) revealed preocclusion transitions leading to activation of the dephosphorylation reaction. We show that the Mg2+ location indicates a site of initial K+ recognition and acceptance upon binding to the outward-open E2P state after Na+ release. Furthermore, using binding and activity studies, we find that the BeFx-inhibited enzyme is also able to bind ADP/ATP and Na+. These results relate the E2-BeFx complex to a transient K+- and ADP-sensitive E∗P intermediate of the functional cycle of the Na+,K+-ATPase, prior to E2P.
Asunto(s)
Berilio , Glicósidos Cardíacos , Fluoruros , Riñón , ATPasa Intercambiadora de Sodio-Potasio , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Berilio/química , Glicósidos Cardíacos/química , Fluoruros/química , Riñón/enzimología , Cinética , Fosfatos/metabolismo , Fosforilación , Dominios Proteicos , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/química , PorcinosRESUMEN
In the present work, a series of N-terpenyl organoselenium compounds (CHB1-6) were evaluated for antimycotic activity by determining the minimum inhibitory concentration (MIC) for each compound in fluconazole (FLU)-sensitive (S1) and FLU-resistant (S2) strains of Candida albicans (C. albicans). The most active compounds in the MIC screen were CHB4 and CHB6, which were then evaluated for cytotoxicity in human cervical cancer cells (KB-3-1) and found to be selective for fungi. Next, CHB4 and CHB6 were investigated for skin irritation using a reconstructed 3D human epidermis and both compounds were considered safe to the epidermis. Using a mouse model of vulvovaginal candidiasis (VVC), CHB4 and CHB6 both exhibited antimycotic efficacy by reducing yeast colonization of the vaginal tract, alleviating injury to the vaginal mucosa, and decreasing the abundance of myeloperoxidase (MPO) expression in the tissue, indicating a reduced inflammatory response. In conclusion, CHB4 and CHB6 demonstrate antifungal activity in vitro and in the mouse model of VVC and represent two new promising antifungal agents.
Asunto(s)
Candidiasis Vulvovaginal , Femenino , Humanos , Candidiasis Vulvovaginal/tratamiento farmacológico , Candidiasis Vulvovaginal/metabolismo , Candidiasis Vulvovaginal/microbiología , Antifúngicos/metabolismo , Fluconazol/farmacología , Candida albicans , Vagina/microbiología , Pruebas de Sensibilidad MicrobianaRESUMEN
Spin labels based on cinobufagin, a specific inhibitor of the Na,K-ATPase, have proved valuable tools to characterize the binding site of cardiotonic steroids (CTSs), which also constitutes the extracellular cation pathway. Because existing literature suggests variations in the physiological responses caused by binding of different CTSs, we extended the original set of spin-labeled inhibitors to the more potent bufalin derivatives. Positioning of the spin labels within the Na,K-ATPase site was defined and visualized by molecular docking. Although the original cinobufagin labels exhibited lower affinity, continuous-wave electron paramagnetic resonance spectra of spin-labeled bufalins and cinobufagins revealed a high degree of pairwise similarity, implying that these two types of CTS bind in the same way. Further analysis of the spectral lineshapes of bound spin labels was performed with emphasis on their structure (PROXYL vs. TEMPO), as well as length and rigidity of the linkers. For comparable structures, the dynamic flexibility increased in parallel with linker length, with the longest linker placing the spin label at the entrance to the binding site. Temperature-related changes in spectral lineshapes indicate that six-membered nitroxide rings undergo boat-chair transitions, showing that the binding-site cross section can accommodate the accompanying changes in methyl-group orientation. D2O-electron spin echo envelope modulation in pulse-electron paramagnetic resonance measurements revealed high water accessibilities and similar polarity profiles for all bound spin labels, implying that the vestibule leading to steroid-binding site and cation-binding sites is relatively wide and water-filled.
Asunto(s)
ATPasa Intercambiadora de Sodio-Potasio , Agua , Sitios de Unión , Espectroscopía de Resonancia por Spin del Electrón , Simulación del Acoplamiento Molecular , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Marcadores de SpinRESUMEN
Epithelial Na+ channel (ENaC) blockers elicit acute and substantial increases of urinary pH. The underlying mechanism remains to be understood. Here, we evaluated if benzamil-induced urine alkalization is mediated by an acute reduction in H+ secretion via renal H+-K+-ATPases (HKAs). Experiments were performed in vivo on HKA double-knockout and wild-type mice. Alterations in dietary K+ intake were used to change renal HKA and ENaC activity. The acute effects of benzamil (0.2 µg/g body wt, sufficient to block ENaC) on urine flow rate and urinary electrolyte and acid excretion were monitored in anesthetized, bladder-catheterized animals. We observed that benzamil acutely increased urinary pH (ΔpH: 0.33 ± 0.07) and reduced NH4+ and titratable acid excretion and that these effects were distinctly enhanced in animals fed a low-K+ diet (ΔpH: 0.74 ± 0.12), a condition when ENaC activity is low. In contrast, benzamil did not affect urine acid excretion in animals kept on a high-K+ diet (i.e., during high ENaC activity). Thus, urine alkalization appeared completely uncoupled from ENaC function. The absence of benzamil-induced urinary alkalization in HKA double-knockout mice confirmed the direct involvement of these enzymes. The inhibitory effect of benzamil was also shown in vitro for the pig α1-isoform of HKA. These results suggest a revised explanation of the benzamil effect on renal acid-base excretion. Considering the conditions used here, we suggest that it is caused by a direct inhibition of HKAs in the collecting duct and not by inhibition of the ENaC function.NEW & NOTEWORTHY Bolus application of epithelial Na+ channel (EnaC) blockers causes marked and acute increases of urine pH. Here, we provide evidence that the underlying mechanism involves direct inhibition of the H+-K+ pump in the collecting duct. This could provide a fundamental revision of the previously assumed mechanism that suggested a key role of ENaC inhibition in this response.
Asunto(s)
Amilorida/análogos & derivados , Canales Epiteliales de Sodio/efectos de los fármacos , ATPasa Intercambiadora de Hidrógeno-Potásio/efectos de los fármacos , Sodio/metabolismo , Amilorida/farmacología , Animales , Canales Epiteliales de Sodio/metabolismo , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Túbulos Renales Colectores/metabolismo , Ratones , Natriuresis/efectos de los fármacos , Eliminación Renal/efectos de los fármacos , Eliminación Renal/fisiología , Sodio en la Dieta/metabolismoRESUMEN
Kinetic properties and crystal structures of the Na+,K+-ATPase in complex with cardiotonic steroids (CTS) revealed significant differences between CTS subfamilies (Laursen et al.). Thus, we found beneficial effects of K+ on bufadienolide binding, which strongly contrasted with the well-known antagonism between K+ and cardenolides. In order to understand this peculiarity of bufalin interactions, we used docking and molecular dynamics simulations of the complexes involving Na+,K+-ATPase, bufadienolides (bufalin, cinobufagin), and ions (K+, Na+, Mg2+). The results revealed that bufadienolide binding is affected by (i) electrostatic attraction of the lactone ring by a cation and (ii) the ability of a cation to stabilize and "shape" the site constituted by transmembrane helices of the α-subunit (αM1-6). The latter effect was due to varying coordination patterns involving amino acid residues from helix bundles αM1-4 and αM5-10. Substituents on the steroid core of a bufadienolide add to and modify the cation effects. The above rationale is fully consistent with the ion effects on the kinetics of Na+,K+-ATPase/bufadienolide interactions.
Asunto(s)
Bufanólidos , Ouabaína , Cationes , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
Cardiotonic steroids (CTSs) are specific and potent inhibitors of the Na(+),K(+)-ATPase, with highest affinity to the phosphoenzyme (E2P) forms. CTSs are comprised of a steroid core, which can be glycosylated, and a varying number of substituents, including a five- or six-membered lactone. These functionalities have specific influence on the binding properties. We report crystal structures of the Na(+),K(+)-ATPase in the E2P form in complex with bufalin (a nonglycosylated CTS with a six-membered lactone) and digoxin (a trisaccharide-conjugated CTS with a five-membered lactone) and compare their characteristics and binding kinetics with the previously described E2P-ouabain complex to derive specific details and the general mechanism of CTS binding and inhibition. CTSs block the extracellular cation exchange pathway, and cation-binding sites I and II are differently occupied: A single Mg(2+) is bound in site II of the digoxin and ouabain complexes, whereas both sites are occupied by K(+) in the E2P-bufalin complex. In all complexes, αM4 adopts a wound form, characteristic for the E2P state and favorable for high-affinity CTS binding. We conclude that the occupants of the cation-binding site and the type of the lactone substituent determine the arrangement of αM4 and hypothesize that winding/unwinding of αM4 represents a trigger for high-affinity CTS binding. We find that the level of glycosylation affects the depth of CTS binding and that the steroid core substituents fine tune the configuration of transmembrane helices αM1-2.
Asunto(s)
Bufanólidos/metabolismo , Digoxina/metabolismo , Modelos Moleculares , Ouabaína/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Bufanólidos/química , Cristalografía por Rayos X , Digoxina/química , Fluorescencia , Glicosilación , Cinética , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , Porcinos , Difracción de Rayos XRESUMEN
Urea interacts with the Na,K-ATPase, leading to reversible as well as irreversible inhibition of the hydrolytic activity. The enzyme purified from shark rectal glands is more sensitive to urea than Na,K-ATPase purified from pig kidney. An immediate and reversible inhibition under steady-state conditions of hydrolytic activity at 37°C is demonstrated for the three reactions studied: the overall Na,K-ATPase activity, the Na-ATPase activity observed in the absence of K+ as well as the K+-dependent phosphatase reaction (K-pNPPase) seen in the absence of Na+. Half-maximal inhibition is seen with about 1M urea for shark enzyme and about 2M urea for pig enzyme. In the presence of substrates there is also an irreversible inhibition in addition to the reversible process, and we show that ATP protects against the irreversible inhibition for both the Na,K-ATPase and Na-ATPase reaction, whereas the substrate paranitrophenylphosphate leads to a slight increase in the rate of irreversible inhibition of the K-pNPPase. The rate of the irreversible inactivation in the absence of substrates is much more rapid for shark enzyme than for pig enzyme. The larger number of potentially urea-sensitive hydrogen bonds in shark enzyme compared to pig enzyme suggests that interference with the extensive hydrogen bonding network might account for the higher urea sensitivity of shark enzyme. The reversible inactivation is interpreted in terms of domain interactions and domain accessibilities using as templates the available crystal structures of Na,K-ATPase. It is suggested that a few interdomain hydrogen bonds are those mainly affected by urea during reversible inactivation.
Asunto(s)
Adenosina Trifosfato/metabolismo , Inhibidores Enzimáticos/química , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Urea/química , Animales , Inhibidores Enzimáticos/farmacología , Enlace de Hidrógeno , Hidrólisis , Cinética , Modelos Biológicos , Modelos Moleculares , Nitrofenoles/metabolismo , Compuestos Organofosforados/metabolismo , Conformación Proteica , Desnaturalización Proteica , Estructura Terciaria de Proteína , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/química , Squalus acanthias , Relación Estructura-Actividad , Especificidad por Sustrato , Porcinos , Temperatura , Urea/farmacologíaRESUMEN
The Na(+),K(+)-ATPase maintains electrochemical gradients for Na(+) and K(+) that are critical for animal cells. Cardiotonic steroids (CTSs), widely used in the clinic and recently assigned a role as endogenous regulators of intracellular processes, are highly specific inhibitors of the Na(+),K(+)-ATPase. Here we describe a crystal structure of the phosphorylated pig kidney Na(+),K(+)-ATPase in complex with the CTS representative ouabain, extending to 3.4 Å resolution. The structure provides key details on CTS binding, revealing an extensive hydrogen bonding network formed by the ß-surface of the steroid core of ouabain and the side chains of αM1, αM2, and αM6. Furthermore, the structure reveals that cation transport site II is occupied by Mg(2+), and crystallographic studies indicate that Rb(+) and Mn(2+), but not Na(+), bind to this site. Comparison with the low-affinity [K2]E2-MgF(x)-ouabain structure [Ogawa et al. (2009) Proc Natl Acad Sci USA 106(33):13742-13747) shows that the CTS binding pocket of [Mg]E2P allows deep ouabain binding with possible long-range interactions between its polarized five-membered lactone ring and the Mg(2+). K(+) binding at the same site unwinds a turn of αM4, dragging residues Ile318-Val325 toward the cation site and thereby hindering deep ouabain binding. Thus, the structural data establish a basis for the interpretation of the biochemical evidence pointing at direct K(+)-Mg(2+) competition and explain the well-known antagonistic effect of K(+) on CTS binding.
Asunto(s)
Ouabaína/química , ATPasa Intercambiadora de Sodio-Potasio/química , Animales , Sitios de Unión , Cardiotónicos/química , Cardiotónicos/metabolismo , Cristalografía por Rayos X , Enlace de Hidrógeno , Magnesio/química , Magnesio/metabolismo , Modelos Moleculares , Ouabaína/metabolismo , Potasio/metabolismo , Conformación Proteica , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , PorcinosRESUMEN
This paper addresses the question of long-range interactions between the intramembranous cation binding sites and the cytoplasmic nucleotide binding site of the ubiquitous ion-transporting Na,K-ATPase using (13)C cross-polarization magic-angle spinning (CP-MAS) solid-state nuclear magnetic resonance. High-affinity ATP binding is induced by the presence of Na(+) as well as of Na-like substances such as Tris(+), and these ions are equally efficient promoters of nucleotide binding. CP-MAS analysis of bound ATP with Na,K-ATPase purified from pig kidney membranes reveals subtle differences in the nucleotide interactions within the nucleotide site depending on whether Na(+) or Tris(+) is used to induce binding. Differences in chemical shifts for ATP atoms C1' and C5' observed in the presence of Na(+) or Tris(+) suggest alterations in the residues surrounding the bound nucleotide, hydrogen bonding, and/or conformation of the ribose ring. This is taken as evidence of a long-distance communication between the Na(+)-filled ion sites in the membrane interior and the nucleotide binding site in the cytoplasmic domain and reflects the first conformational change ultimately leading to phosphorylation of the enzyme. Stopped-flow fluorescence measurements with the nucleotide analogue eosin show that the dissociation rate constant for eosin is larger in Tris(+) than in Na(+), giving kinetic evidence of the difference in structural effects of Na(+) and Tris(+). According to the recent crystal structure of the E1·AlF4(-)·ADP·3Na(+) form, the coupling between the ion binding sites and the nucleotide side is mediated by, among others, the M5 helix.
Asunto(s)
Adenosina Trifosfato/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Sodio/metabolismo , Adenosina Difosfato/metabolismo , Animales , Sitios de Unión , Cationes Monovalentes/metabolismo , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Potasio/metabolismo , Unión Proteica , ATPasa Intercambiadora de Sodio-Potasio/química , PorcinosRESUMEN
Tricyclic clerodane diterpenes (TCDs) are natural compounds that often show potent cytotoxicity for cancer cells, but their mode of action remains elusive. A computationally based similarity search (CDRUG), combined with principal component analysis (ChemGPS-NP) and docking calculations (GOLD 5.2), suggested TCDs to be inhibitors of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) pump, which is also the target of the sesquiterpene lactone thapsigargin. Biochemical studies were performed with 11 TCDs on purified rabbit skeletal muscle sarcoplasmic reticulum membranes, which are highly enriched with the SERCA1a isoform. Casearborin D (2) exhibited the highest affinity, with a KD value of 2 µM and giving rise to complete inhibition of SERCA1a activity. Structure-activity relationships revealed that functionalization of two acyl side chains (R1 and R4) and the hydrophobicity imparted by the aliphatic chain at C-9, as well as a C-3,C-4 double bond, play crucial roles for inhibitory activity. Docking studies also suggested that hydrophobic interactions in the binding site, especially with Phe256 and Phe834, may be important for a strong inhibitory activity of the TCDs. In conclusion, a novel class of SERCA inhibitory compounds is presented.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Diterpenos de Tipo Clerodano/aislamiento & purificación , Diterpenos de Tipo Clerodano/farmacología , Errores Innatos del Metabolismo de los Aminoácidos , Animales , Sitios de Unión , Diterpenos de Tipo Clerodano/química , Ensayos de Selección de Medicamentos Antitumorales , Retículo Endoplásmico/metabolismo , Humanos , Enfermedades Mitocondriales , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Conejos , Retículo Sarcoplasmático/metabolismo , Sarcosina-Deshidrogenasa/deficiencia , Relación Estructura-Actividad , Tapsigargina/farmacologíaRESUMEN
The RNase D-type 3'-5' exonuclease Rrp6p from Saccharomyces cerevisiae is a nuclear-specific cofactor of the RNA exosome and associates in vivo with Rrp47p (Lrp1p). Here, we show using biochemistry and small-angle X-ray scattering (SAXS) that Rrp6p and Rrp47p associate into a stable, heterodimeric complex with an elongated shape consistent with binding of Rrp47p to the nuclease domain and opposite of the HRDC domain of Rrp6p. Rrp47p reduces the exonucleolytic activity of Rrp6p on both single-stranded and structured RNA substrates without significantly altering the affinity towards RNA or the ability of Rrp6p to degrade RNA secondary structure.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/ultraestructura , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/ultraestructura , Proteínas Nucleares/metabolismo , Proteínas Nucleares/ultraestructura , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/ultraestructura , ARN/metabolismo , ARN/ultraestructura , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestructura , Secuencia de Aminoácidos , Sitios de Unión , Simulación por Computador , Proteínas de Unión al ADN/química , Complejo Multienzimático de Ribonucleasas del Exosoma/química , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Nucleares/química , Unión Proteica , Conformación Proteica , ARN/química , Proteínas de Unión al ARN/química , Proteínas de Saccharomyces cerevisiae/química , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
Digitalis-like compounds (DLCs) comprise a diverse group of molecules characterized by a cis-trans-cis ring-fused steroid core linked to a lactone. They have been used in the treatment of different medical problems including heart failure, where their inotropic effect on heart muscle is attributed to potent Na(+),K(+)-ATPase inhibition. Their application as drugs, however, has declined in recent past years due to their small safety margin. Since human Na(+),K(+)-ATPase is represented by four different isoforms expressed in a tissue-specific manner, one of the possibilities to improve the therapeutic index of DLCs is to exploit and amend their isoform selectivity. Here, we aimed to reveal the determinants of selectivity of the ubiquitously expressed α1 isoform and the more restricted α2 isoform toward several well-known DLCs and their hydrogenated forms. Using baculovirus to express various mutants of the α2 isoform, we were able to link residues Met(119) and Ser(124) to differences in affinity between the α1 and α2 isoforms to ouabain, dihydro-ouabain, digoxin, and dihydro-digoxin. We speculate that the interactions between these amino acids and DLCs affect the initial binding of these DLCs. Also, we observed isoform selectivity for DLCs containing no sugar groups.
Asunto(s)
Aminoácidos/metabolismo , Glicósidos Digitálicos/metabolismo , Isoenzimas/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Aminoácidos/química , Isoenzimas/química , Isoenzimas/genética , Mutación , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/genética , Especificidad por SustratoRESUMEN
Five new cardenolide lactates (15) and one new dioxane double linked cardenolide glycoside (17) along with 15 known compounds (616 and 1821) were isolated from the ornamental milkweed Asclepias curassavica. Their structures were elucidated by extensive spectroscopic methods (IR, UV, MS, 1D- and 2D-NMR). The molecular structures and absolute configurations of 13 and 17 were further confirmed by single-crystal X-ray diffraction analysis. Simultaneous isolation of dioxane double linked cardenolide glycosides (1721) and cardenolide lactates (15) provided unique chemotaxonomic markers for this genus. Compounds 121 were evaluated for the inhibitory activities against DU145 prostate cancer cells. The dioxane double linked cardenolide glycosides showed the most potent cytotoxic effect followed by normal cardenolides and cardenolide lactates, while the C21 steroids were non-cytotoxic. Enzymatic assay established a correlation between the cytotoxic effects in DU145 cancer cells and the Ki for the inhibition of Na(+),K(+)-ATPase. Molecular docking analysis revealed relatively strong H-bond interactions between the bottom of the binding cavity and compounds 18 or 20, and explained why the dioxane double linked cardenolide glycosides possessed higher inhibitory potency on Na(+),K(+)-ATPase than the cardenolide lactate.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Asclepias/química , Cardenólidos/farmacología , Inhibidores Enzimáticos/farmacología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/aislamiento & purificación , Cardenólidos/química , Cardenólidos/aislamiento & purificación , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Humanos , Modelos Moleculares , Conformación Molecular , Estructura Molecular , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Relación Estructura-ActividadRESUMEN
Digitalis-like compounds (DLCs), specific inhibitors of Na,K-ATPase, are implicated in cellular signaling. Exposure of cell cultures to ouabain, a well-known DLC, leads to up- or down regulation of various processes and involves activation of Src kinase. Since Na,K-ATPase is the only known target for DLC binding an in vitro experimental setup using highly purified Na,K-ATPase from pig kidney and commercially available recombinant Src was used to investigate the mechanism of coupling between the Na,K-ATPase and Src. Digoxin was used as a representative DLC for inhibition of Na,K-ATPase. The activation of Src kinase was measured as the degree of its autophosphorylation. It was observed that in addition to digoxin, Src activation was dependent on concentrations of other specific ligands of Na,K-ATPase: Na(+), K(+), vanadate, ATP and ADP. The magnitude of the steady-state ATPase activity therefore seemed to affect Src activation. Further experiments with an ATP regenerating system showed that the ATP/ADP ratio determined the extent of Src activation. Thus, our model system which represents the proposed very proximal part of the Na,K-ATPase-Src signaling cascade, shows that Src kinase activity is regulated by both ATP and ADP concentrations and provides no evidence for a direct interaction between Na,K-ATPase and Src.
Asunto(s)
Adenosina Difosfato/química , Adenosina Trifosfato/química , Ouabaína/química , ATPasa Intercambiadora de Sodio-Potasio/química , Familia-src Quinasas/química , Adenosina Difosfato/genética , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Animales , Glicósidos Digitálicos/química , Digoxina/química , Activación Enzimática/fisiología , Humanos , Riñón/química , Riñón/metabolismo , Fosforilación/fisiología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal/fisiología , ATPasa Intercambiadora de Sodio-Potasio/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Porcinos , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
Cardiotonic steroids such as ouabain bind with high affinity to the membrane-bound cation-transporting P-type Na,K-ATPase, leading to complete inhibition of the enzyme. Using synchrotron radiation circular dichroism spectroscopy we show that the enzyme-ouabain complex is less susceptible to thermal denaturation (unfolding) than the ouabain-free enzyme, and this protection is observed with Na,K-ATPase purified from pig kidney as well as from shark rectal glands. It is also shown that detergent-solubilised preparations of Na,K-ATPase are stabilised by ouabain, which could account for the successful crystallisation of Na,K-ATPase in the ouabain-bound form. The secondary structure is not significantly affected by the binding of ouabain. Ouabain appears however, to induce a reorganization of the tertiary structure towards a more compact protein structure which is less prone to unfolding; recent crystal structures of the two enzymes are consistent with this interpretation. These circular dichroism spectroscopic studies in solution therefore provide complementary information to that provided by crystallography.
Asunto(s)
Membrana Celular/química , Ouabaína/química , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/ultraestructura , Cardiotónicos , Activación Enzimática , Estabilidad de Enzimas , Conformación Proteica , Relación Estructura-Actividad , Especificidad por Sustrato , TemperaturaRESUMEN
Structures of membrane proteins determined by X-ray crystallography and, increasingly, by cryo-electron microscopy often fail to resolve the structural details of unstable or reactive small molecular ligands in their physiological sites. This work demonstrates that 13C chemical shifts measured by magic-angle spinning (MAS) solid-state NMR (SSNMR) provide unique information on the conformation of a labile ligand in the physiological site of a functional protein in its native membrane, by exploiting freeze-trapping to stabilise the complex. We examine the ribose conformation of ATP in a high affinity complex with Na,K-ATPase (NKA), an enzyme that rapidly hydrolyses ATP to ADP and inorganic phosphate under physiological conditions. The 13C SSNMR spectrum of the frozen complex exhibits peaks from all ATP ribose carbon sites and some adenine base carbons. Comparison of experimental chemical shifts with density functional theory (DFT) calculations of ATP in different conformations and protein environments reveals that the ATP ribose ring adopts an C3'-endo (N) conformation when bound with high affinity to NKA in the E1Na state, in contrast to the C2'-endo (S) ribose conformations of ATP bound to the E2P state and AMPPCP in the E1 complex. Additional dipolar coupling-mediated measurements of H-C-C-H torsional angles are used to eliminate possible relative orientations of the ribose and adenine rings. The utilization of chemical shifts to determine membrane protein ligand conformations has been underexploited to date and here we demonstrate this approach to be a powerful tool for resolving the fine details of ligand-protein interactions.
RESUMEN
We have characterized a putative Ca(2+)-ATPase from the pathogenic bacterium Listeria monocytogenes with the locus tag lmo0841. The purified and detergent-solubilized protein, which we have named Listeria monocytogenes Ca(2+)-ATPase 1 (LMCA1), performs a Ca(2+)-dependent ATP hydrolysis and actively transports Ca(2+) after reconstitution in dioleoylphosphatidyl-choline vesicles. Despite a high sequence similarity to the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) and plasma membrane Ca(2+)-ATPase (PMCA), LMCA1 exhibits important biochemical differences such as a low Ca(2+) affinity (K(0.5) â¼80 µm) and a high pH optimum (pH â¼9). Mutational studies indicate that the unusually high pH optimum can be partially ascribed to the presence of an arginine residue (Arg-795), corresponding in sequence alignments to the Glu-908 position at Ca(2+) binding site I of rabbit SERCA1a, but probably with an exposed position in LMCA1. The arginine is characteristic of a large group of putative bacterial Ca(2+)-ATPases. Moreover, we demonstrate that H(+) is countertransported with a transport stoichiometry of 1 Ca(2+) out and 1 H(+) in per ATP hydrolyzed. The ATPase may serve an important function by removing Ca(2+) from the microorganism in environmental conditions when e.g. stressed by high Ca(2+) and alkaline pH.