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Patients with high ALDH1A3-expressing glioblastoma (ALDH1A3hi GBM) show limited benefit from postoperative chemoradiotherapy. Understanding the mechanisms underlying such resistance in these patients is crucial for the development of new treatments. Here, we show that the interaction between ALDH1A3 and PKM2 enhances the latter's tetramerization and promotes lactate accumulation in glioblastoma stem cells (GSCs). By scanning the lactylated proteome in lactate-accumulating GSCs, we show that XRCC1 undergoes lactylation at lysine 247 (K247). Lactylated XRCC1 shows a stronger affinity for importin α, allowing for greater nuclear transposition of XRCC1 and enhanced DNA repair. Through high-throughput screening of a small-molecule library, we show that D34-919 potently disrupts the ALDH1A3-PKM2 interaction, preventing the ALDH1A3-mediated enhancement of PKM2 tetramerization. In vitro and in vivo treatment with D34-919 enhanced chemoradiotherapy-induced apoptosis of GBM cells. Together, our findings show that ALDH1A3-mediated PKM2 tetramerization is a potential therapeutic target to improve the response to chemoradiotherapy in ALDH1A3hi GBM.
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Glioblastoma , Proteínas de Unión a Hormona Tiroide , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X , Glioblastoma/metabolismo , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Humanos , Animales , Línea Celular Tumoral , Ratones , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/metabolismo , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Ratones Desnudos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Proteínas de la Membrana/metabolismo , Proteínas Portadoras/metabolismo , Hormonas Tiroideas/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Aldehído Oxidorreductasas , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NHRESUMEN
Cement production is a primary source of global carbon emissions. As a hazardous waste, municipal solid waste incineration fly ash (MSWI-FA) can be pretreated as a cementitious and effective carbon capture material. This study proposes an efficient carbonation dechlorination pretreatment and resource recovery strategy using flue gas micro-nano bubble (MNB) to wash MSWI-FA. The results showed that the flue gas MNB water washing reaction solution inhibited CaCO3 boundary layer blocking and adsorption on NaCl and KCl leaching. Under low water-to-solid ratio and CO2 concentration conditions, two-step washing reduced the MSWI-FA chlorine content to <1%, improving the dechlorination effect by 19.72% compared to conventional carbonation. The flue gas MNB water accelerated the precipitation of Ca2+ and Ca(ClO)2 in the form of calcite. The higher the CO2 concentration in the flue gas MNB, the better the fragmentation and purification of the MSWI-FA shell, leading to improved dechlorination and CO2 fixation. Under optimized conditions, the mean particle size of MSWI-FA decreased by 47.82%, and the CO2 fixation rate reached 73.80%, with a 58.35% increase in the washing carbonation rate. MSWI-FA pretreated by flue gas MNB washing was used as both the raw material and supplementary cementitious material for sulfoaluminate cementitious (SAC) material, exhibiting excellent compressive strength and heavy metal stabilization. The maximum compressive strength of the MSWI-FA-based SAC material cured for 28 d reached 130 MPa. Cr leaching was inhibited with increased hydration time, and the leaching concentration was far below the standard limit.
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Metales Pesados , Eliminación de Residuos , Residuos Sólidos/análisis , Ceniza del Carbón , Eliminación de Residuos/métodos , Dióxido de Carbono , Material Particulado , Incineración/métodos , Metales Pesados/análisis , Carbonatos , Carbono , AguaRESUMEN
To enhance the utilization of solid waste in cement kiln co-processing, this study analyzed the multifaceted synergy of pyrolysis and mineralization processes of iron-rich sludge (SS) and waste incineration fly ash (FA) at optimal blending ratios. Based on the physicochemical properties of SS and co-pyrolysis experiments, it was found that Fe acted as a positive catalyst in pyrolysis between 700 and 1000 °C, while the endogenous polymerization effect of Fe(III) mineral groups dominated above 800 °C. Additionally, the study investigated the solidification and migration of heavy metals and the transformation of harmful elements (S, Cl, and P). Results indicated that the best mixture ratios for SS and FA were 6:4 and 9:1, respectively, and synergistic pyrolysis and mineral co-curing effects were observed in the pyrolysis temperature range of 50-1000 °C. The synergy between SS and FA allowed for the decomposition and solidification of harmful organic components and heavy metals, reducing environmental risks. Furthermore, in actual production, by mixing 100 tons of SS and FA with Portland cement with a daily output of 2500 tons, the compressive strength during early hydration stages can reach 34.52 MPa on the third day, exceeding the highest performance of Portland cement (62.5R) strength index specified in the standard.
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BACKGROUND AND OBJECTIVES: Histopathological features and molecular biomarkers have been studied as potential prognostic factors. This study aimed to investigate the clinical features, molecular phenotypes, and survival prognosis of isocitrate dehydrogenase (IDH)-mutant (IDHmt) gliomas with histone H3 alterations (H3-alterations). METHODS: A total of 236 and 657 patients with whole-exome sequencing data were separately collected from the Chinese Glioma Genome Atlas and The Cancer Genome Atlas databases. Survival analysis of patients with glioma was performed using Kaplan-Meier survival curves stratified by histone H3 status. Univariate and multivariate analyses were used to identify the associations between histone H3 status and other clinicopathological factors with survival in patients with IDH-mutant gliomas. RESULTS: Diffuse gliomas with H3 alterations are more likely to be high grade in 2 cohorts ( P = .025 and P = .021, respectively). IDHmt glioma patients with H3-alteration had significantly less life expectancy than histone H3 wild-type ( P = .041 and P = .008, respectively). In the Chinese Glioma Genome Atlas cohort, Karnofsky performance scores ≤ 80 (HR 2.394, 95% CI 1.257-4.559, P = .008), extent of resection (HR 0.971, 95% CI 0.957-0.986, P < .001), high WHO grade (HR 6.938, 95% CI 2.787-17.269, P < .001), H3-alteration (HR 2.482, 95% CI 1.183-4.981, P = .016), and 1p/19q codeletion (HR 0.169, 95% CI 0.073-0.390, P < .001) were independently associated with IDHmt gliomas. In the The Cancer Genome Atlas cohort, age (HR 1.034, 95% CI 1.008-1.061, P = .010), high WHO grade (HR 2.365, 95% CI 1.263-4.427, P = .007), and H3-alteration (HR 2.501, 95% CI 1.312-4.766, P = .005) were independently associated with IDHmt gliomas. CONCLUSION: Identification and assessment of histone H3 status in clinical practice might help improve prognostic prediction and develop therapeutic strategies for these patient subgroups.
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Neoplasias Encefálicas , Glioma , Humanos , Histonas/genética , Isocitrato Deshidrogenasa/genética , Neoplasias Encefálicas/cirugía , Mutación/genética , Glioma/patología , PronósticoRESUMEN
BACKGROUND: Diagnosis and treatment of patients with glioblastoma (GBM) who are also diagnosed with primary non-central nervous system (CNS) tumors remain a challenge, yet little is known about the clinical characteristics and prognosis of these patients. The data presented here compared the clinical and pathological features between glioblastoma patients with or without primary non-CNS tumors, trying to further explore this complex situation. METHODS: Statistical analysis was based on the clinical and pathological data of 45 patients who were diagnosed with isocitrate dehydrogenase (IDH) wild-type glioblastoma accompanied by non-CNS tumors between January 2019 and February 2022 in Beijing Tiantan Hospital. Univariate COX proportional hazard regression model was used to determine risk factors for overall survival. RESULTS: It turned out to be no significant difference in the overall survival (OS) of the 45 patients with IDH-wild-type GBM plus non-CNS tumors, compared with the 112 patients who were only diagnosed with IDH-wild-type GBM. However, there was a significant difference in OS of GBM patients with benign tumors compared to those with malignant tumors. CONCLUSIONS: Implications for the non-central nervous system tumors on survival of glioblastomas were not found in this research. However, glioblastomas complicated with other malignant tumors still showed worse clinical outcomes.
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Nowadays the formation mechanism of anodic TiO2 nanotubes has attracted extensive attention. Field-assisted dissolution (TiO2 + 6F- + 4H+ â [TiF6]2- + 2H2O) has been considered as the causal link to the formation and growth of nanotubes. But it is hard for this theory to explain three stages of the current-time curve. Here, the anodization of titanium was studied by adding different concentrations of H3PO4 (0%, 4 wt%, 6 wt%, 8 wt%, and 10 wt%) in ethylene glycol containing the same concentration of NH4F (0.5 wt%). The results prove that under the action of the same concentration of NH4F, the growth rate of nanotubes decreases obviously with the increase of H3PO4 concentration, and the second stage of the current-time curve is also prolonged simultaneously. These experimental facts cannot be interpreted by field-assisted dissolution theory and the viscous flow model. Here, an anion layer formed by H3PO4 and the electronic current theory are ably used to explain these facts reasonably for the first time.
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A new co-processing mode of waste liquid from anaerobic fermentation of organic wastes and municipal solid waste incineration fly ash (MSWI-FA) dechlorination is reported in this paper. Taking acetic acid, the most common organic acid in anaerobic fermentation systems, as the representative of anaerobic fermentation organic acids, the improvement of the dechlorination effect and the mechanism of washing MSWI-FA with low concentrations of organic acid lotion were explored. The chlorine content of MSWI-FA was reduced to 0.82% after the optimal process washing pretreatment. Three anaerobic fermentation waste liquids (AFWLs) were used to verify that the chlorine content of MSWI-FA could be reduced to less than 1%, and the dechlorination effect of brewery wastewater, which reduced the chlorine content of MSWI-FA to 0.91%, was the best at this. The influence of the washing process on MSWI-FA pyrolysis was reflected in the whole process. The release of chloride decreased and the weight loss was mainly due to the release of CO2. The melting point of MSWI-FA, washed by the optimal process, was reduced by nearly 30 â, and only 0.06% chlorine remained after calcination at 1100 â, which was extremely beneficial in reducing the release of trace elements in MSWI-FA during heat treatment, and for the preparation of cement raw meal.
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Metales Pesados , Eliminación de Residuos , Anaerobiosis , Carbono , Cloro , Ceniza del Carbón , Fermentación , Incineración , Metales Pesados/análisis , Material Particulado , Residuos Sólidos/análisisRESUMEN
Microneedle permits transdermal biosensing and drug delivery with minor pain. However, accurate microneedle transdermal positioning with minimal skin deformation remains a significant technical challenge due to inhomogeneous skin topology and discontinuous force applied to the microneedle. Here, we introduce bioinspired rotation microneedles for in vivo accurate microneedle positioning as inspired by honeybees' stingers. We demonstrate the benefits of rotation microneedles in alleviating skin resistance through finite element analysis, full-thickness porcine validations, and mathematical derivations of microneedle-skin interaction stress fields. The max penetration force was mitigated by up to 45.7% and the force attenuation rate increased to 2.73 times in the holding stage after penetration. A decrease in max skin deflection and a faster deformation recovery introduced by rotation microneedles implied a more precise penetration depth. Furthermore, we applied the rotation microneedles in psoriasis mice, a monogenic disorder animal model, for minimally invasive biological sample extraction and proinflammatory cytokine monitoring. An ultrasensitive detection method is realized by using only one microneedle to achieve cytokine mRNA level determination compared to commonly required biopsies or blood collection. Thus, rotation microneedles permit a simple, rapid, and ultraminimal-invasive method for subcutaneous trace biological sample acquisition and subsequent point-of-care diagnostics with minimal damage to both microneedles and skins.
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The Chagan Depression is an important oil and gas exploration tectonic unit in the Yingen-Ejinaqi Basin, north central China. It has been revealed that the Chagan Depression has abundant oil and gas resources, but the study of hydrocarbon kitchens has not been carried out. The Early Cretaceous Bayingebi 2 Formation has the most important source rocks in the Chagan Depression. In this paper, the Bayingebi 2 Formation was selected to study the hydrocarbon kitchen evolution. The thermal maturity evolution of the source rocks and the locations and geological time of the development of hydrocarbon kitchens were revealed. The results show that the maturity of source rocks in the Bayingebi 2 Formation has reached the maximum during the middle depositional period of the Yingen Formation, and the hydrocarbon generation has ceased since the Late Cretaceous. The source rocks of the Bayingebi 2 Formation in the Chagan Depression have two hydrocarbon kitchens, namely, the western subdepression and the eastern subdepression hydrocarbon kitchens. The western subdepression hydrocarbon kitchen was formed in the Suhongtu 1 Formation depositional period and ended in the Yingen Formation depositional period. The location of the hydrocarbon kitchen was relatively stable and developed in the central and southern parts. However, the eastern subdepression hydrocarbon kitchen developed only during the Yingen Formation depositional period and was located in the north subsag of the Hantamiao sag zone. Finally, the evolution of the hydrocarbon kitchen reveals that oil and gas exploration still needs to be carried out around the western subdepression hydrocarbon kitchen and it may be considered to abandon the exploration in the eastern subdepression.
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In order to leverage the unique advantages of redundant manipulators, avoiding the singularity during motion planning and control should be considered as a fundamental issue to handle. In this article, a distributed scheme is proposed to improve the manipulability of redundant manipulators in a group. To this end, the manipulability index is incorporated into the cooperative control of multiple manipulators in a distributed network, which is used to guide manipulators to adjust to the optimal spatial position. Moreover, from the perspective of game theory, this article formulates the problem into a Nash equilibrium. Then, a neural network with anti-noise ability is constructed to seek and approximate the optimal strategy profile of the Nash equilibrium problem with time-varying parameters. Theoretical analyses show that the neural network model has the superior global convergence and noise immunity. Finally, simulation results demonstrate that the neural network is effective in real-time cooperative motion generation of multiple redundant manipulators under perturbations in distributed networks.
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PURPOSE: This retrospective study investigates the accuracy of freehand implant placement and whether the factors of presence of an adjacent tooth, implant quadrant, number of missing teeth, and location of the implant site influence direction and angulation deviations. MATERIALS AND METHODS: According to specific inclusion and exclusion criteria, a total of 112 implants from 75 partially edentulous patients were recruited for this retrospective study. The implants were inserted using a freehand approach by one experienced clinician (right-handed). The full thickness flap was elevated to expose the alveolar bone in the implant surgery, and the implant crown consisted of an all-ceramic restoration retained by cement. The planned implant position was preoperatively determined using implant planning software. The postoperative implant position was determined by analyzing the alignment after optically scanning the dentition using a specifically designed registration model in Geomagic Studio software. The deviations between the planned and postoperative implant positions were then calculated. The outcomes included direction and angulation deviations between the planned and postoperative implant positions. All data were analyzed by ANOVA, Bonferroni correction, regression analysis, and one-sample t-tests conducted using SPSS. RESULTS: The 3D deviations between planned and postoperative implant positions were 1.22 ± 0.63 mm at the entrance point, 1.91 ± 1.17 mm at the apical point, and 7.93 ± 5.56° in angulation. The presence of adjacent teeth influenced deviations in the mesiodistal (F = 4.338, p = 0.006) and buccolingual directions (F = 3.017, p = 0.033) at the entrance point and mesiodistal angulation (F = 7.979, p < 0.001). The quadrant influenced deviation in the buccolingual direction at the apical point (F = 6.093, p = 0.001) and buccolingual angulation (F = 6.457, p < 0.001). The number of missing teeth had no effect on deviations of direction and angulation of implants. The location of the implant site affected the deviation in the buccolingual direction at the entrance point (F = 3.096, p = 0.049) and the mesiodistal direction at the apical point (F = 3.724, p = 0.027). CONCLUSION: The 3D accuracy of freehand-placed implants could be acceptable in clinical situations. The results showed that the presence of an adjacent tooth and the quadrant and the location of the implant site influenced the direction and angulation deviations of the implant position; however, the factor of number of missing teeth did not.
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Implantes Dentales , Cirugía Asistida por Computador , Tomografía Computarizada de Haz Cónico , Implantación Dental Endoósea , Humanos , Estudios RetrospectivosRESUMEN
The motility of the Dictyostelium discoideum (DD) cell is studied by video microscopy when the cells are plated on top of an agar plate at different densities, n. It is found that the fluctuating kinetics of the cells can be divided into two normal directions: the cell's forward-moving direction and its normal direction. Along the forward-moving direction, the slope of the amplitude of fluctuation vs. velocity (R||(v)) increases with n, while along the normal direction the slope of R⥠is independent of n. Both R|| and R⥠are functions of the cell speed v. The observed linearity in Râ¥(v) indicated that the amplitude of orientational fluctuation (κ) of DD cells is a constant independent of v. The independence of the slope of Râ¥(v) on n indicated that κ is also not affected by cellular interactions. The independence of κ on both v and n suggests that orientational fluctuation originates from the intrinsic property of motion fluctuations in DD.
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Dictyostelium/fisiología , Algoritmos , Cinética , Microscopía por Video , MovimientoRESUMEN
A fragment of RyR1 (amino acids 4064-4210) is predicted to fold to at least one lobe of calmodulin and to bind Ca(2+). This fragment of RyR1 (R4064-4210) was subcloned, expressed, refolded, and purified. Consistent with the predicted folding pattern, R4064-4210 was found to bind two molecules of Ca(2+) and undergo a structural change upon binding Ca(2+) that exposes hydrophobic amino acids. R4064-4210 also binds to RyR1, the L-type Ca(2+) channel (Cav(1.1)), and several synthetic calmodulin binding peptides. Both R4064-4210 and a peptide representing the calmodulin-binding region of RyR1 (R3614-3643) alter the Ca(2+) dependence of ((3)H)ryanodine binding to RyR1, suggesting that they may both be interfering with an intramolecular interaction between amino acids 4064-4210 and amino acids 3614-3643 in the native RyR1 to alter or regulate the response of the channel to changes in Ca(2+) concentration. The finding that a domain within RyR1 binds Ca(2+) and interacts with calmodulin-binding motifs may provide insights into the mechanism for calcium- and calmodulin-dependent regulation of this channel and perhaps for its regulation by the L-type Ca(2+) channel.
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Calcio/metabolismo , Calmodulina/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Aminoácidos/química , Animales , Sitios de Unión , Calcio/química , Canales de Calcio Tipo L/química , Dicroismo Circular , ADN/química , Escherichia coli/metabolismo , Imagenología Tridimensional , Conformación Molecular , Datos de Secuencia Molecular , Paramecium , Péptidos/química , Unión Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Conejos , Rianodina/química , Homología de Secuencia de Aminoácido , Espectrometría de FluorescenciaRESUMEN
The purpose of this study was to determine whether there are alterations in the dihydropyridine and/or ryanodine receptors that might explain the excitation-contraction uncoupling associated with eccentric contraction-induced skeletal muscle injury. The left anterior crural muscles (i.e., tibialis anterior, extensor digitorum longus, and extensor hallucis longus) of mice were injured in vivo by 150 eccentric contractions. Peak isometric tetanic torque of the anterior crural muscles was reduced approximately 45% immediately and 3 days after the eccentric contractions. Partial restoration of peak isometric tetanic and subtetanic forces of injured extensor digitorum longus muscles by 10 mM caffeine indicated the presence of excitation-contraction uncoupling. Scatchard analysis of [3H]ryanodine binding indicated that the number of ryanodine receptor binding sites was not altered immediately postinjury but decreased 16% 3 days later. Dihydropyridine receptor binding sites increased approximately 20% immediately after and were elevated to the same extent 3 days after the injury protocol. Muscle injury did not alter the sensitivity of either receptor. These data suggest that a loss or altered sensitivity of the dihydropyridine and ryanodine receptors does not contribute to the excitation-contraction uncoupling immediately after contraction-induced muscle injury. We also concluded that the loss in ryanodine receptors 3 days after injury is not the primary cause of excitation-contraction uncoupling at that time.
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Canales de Calcio Tipo L/metabolismo , Contracción Muscular , Músculo Esquelético/lesiones , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Animales , Sitios de Unión , Cafeína/farmacología , Femenino , Contracción Isométrica , Isradipino/metabolismo , Ratones , Ratones Endogámicos ICR , Músculo Esquelético/efectos de los fármacos , Tarso Animal , Torque , Heridas y Lesiones/metabolismo , Heridas y Lesiones/fisiopatologíaRESUMEN
The cardiac L-type voltage-dependent calcium channel is responsible for initiating excitation-contraction coupling. Three sequences (amino acids 1609-1628, 1627-1652, and 1665-1685, designated A, C, and IQ, respectively) of its alpha(1) subunit contribute to calmodulin (CaM) binding and Ca(2+)-dependent inactivation. Peptides matching the A, C, and IQ sequences all bind Ca(2+)CaM. Longer peptides representing A plus C (A-C) or C plus IQ (C-IQ) bind only a single molecule of Ca(2+)CaM. Apocalmodulin (ApoCaM) binds with low affinity to the IQ peptide and with higher affinity to the C-IQ peptide. Binding to the IQ and C peptides increases the Ca(2+) affinity of the C-lobe of CaM, but only the IQ peptide alters the Ca(2+) affinity of the N-lobe. Conversion of the isoleucine and glutamine residues of the IQ motif to alanines in the channel destroys inactivation (Zühlke et al., 2000). The double mutation in the peptide reduces the interaction with apoCaM. A mutant CaM unable to bind Ca(2+) at sites 3 and 4 (which abolishes the ability of CaM to inactivate the channel) binds to the IQ, but not to the C or A peptide. Our data are consistent with a model in which apoCaM binding to the region around the IQ motif is necessary for the rapid binding of Ca(2+) to the C-lobe of CaM. Upon Ca(2+) binding, this lobe is likely to engage the A-C region.
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Canales de Calcio Tipo L/química , Calcio/química , Calmodulina/química , Alanina/química , Animales , Sitios de Unión , Calcio/metabolismo , Canales de Calcio Tipo L/metabolismo , Calmodulina/genética , Bovinos , Relación Dosis-Respuesta a Droga , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Glutamina/química , Isoleucina/química , Mutación , Péptidos/química , Unión Proteica , Estructura Terciaria de Proteína , Espectrometría de FluorescenciaRESUMEN
Both apocalmodulin (Ca(2+)-free calmodulin) and Ca(2+)-calmodulin bind to and regulate the activity of skeletal muscle Ca(2+) release channel (ryanodine receptor, RYR1). Both forms of calmodulin protect sites after amino acids 3630 and 3637 on RYR1 from trypsin cleavage. Only apocalmodulin protects sites after amino acids 1982 and 1999 from trypsin cleavage. Ca(2+)-calmodulin and apocalmodulin both bind to two different synthetic peptides representing amino acids 3614-3643 and 1975-1999 of RYR1, but Ca(2+)-calmodulin has a higher affinity than apocalmodulin for both peptides. Cysteine 3635, within the 3614-3643 sequence of RYR1, can form a disulfide bond with a cysteine on an adjacent subunit within the RYR1 tetramer. The second cysteine is now shown to be between amino acids 2000 and 2401. The close proximity of the cysteines forming the intersubunit disulfide to the two sites that bind calmodulin suggests that calmodulin is binding at a site of intersubunit contact, perhaps with one lobe bound between amino acids 3614 and 3643 on one subunit and the second lobe bound between amino acids 1975 and 1999 on an adjacent subunit. This model is consistent with the finding that Ca(2+)-calmodulin and apocalmodulin each bind to a single site per RYR1 subunit (Rodney, G. G., Williams, B. Y., Strasburg, G. M., Beckingham, K., and Hamilton, S. L. (2000) Biochemistry 39, 7807-7812).
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Calmodulina/metabolismo , Músculo Esquelético/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Hidrólisis , Datos de Secuencia Molecular , Conejos , Canal Liberador de Calcio Receptor de Rianodina/química , Tripsina/metabolismoRESUMEN
Calmodulin activates the skeletal muscle Ca(2+) release channel RYR1 at nm Ca(2+) concentrations and inhibits the channel at microm Ca(2+) concentrations. Using a deletion mutant of calmodulin, we demonstrate that amino acids 2-8 are required for high affinity binding of calmodulin to RYR1 at both nm and microm Ca(2+) concentrations and are required for maximum inhibition of the channel at microm Ca(2+) concentrations. In contrast, the addition of three amino acids to the N terminus of calmodulin increased the affinity for RYR1 at both nm and microm Ca(2+) concentrations, but destroyed its functional effects on RYR1 at nm Ca(2+). Using both full-length RYR1 and synthetic peptides, we demonstrate that the calmodulin-binding site on RYR1 is likely to be noncontiguous, with the C-terminal lobe of both apocalmodulin and Ca(2+)-calmodulin binding to amino acids between positions 3614 and 3643 and the N-terminal lobe binding at sites that are not proximal in the primary sequence. Ca(2+) binding to the C-terminal lobe of calmodulin converted it from an activator to an inhibitor, but an interaction with the N-terminal lobe was required for a maximum effect on RYR1. This interaction apparently depends on the native sequence or structure of the first few amino acids at the N terminus of calmodulin.