RESUMEN
A key player in energy metabolism is phosphofructokinase-1 (PFK1) whose activity and behavior strongly influence glycolysis and thus have implications in many areas. In this research, PFK1 assays were performed to convert F6P and ATP into F-1,6-P and ADP for varied pH and ATP concentrations. PFK1 activity was assessed by evaluating F-1,6-P generation velocity in two ways: (1) directly calculating the time slope from the first two or more datapoints of measured product concentration (the initial-velocity method), and (2) by fitting all the datapoints with a differential equation explicitly representing the effects of ATP and pH (the modeling method). Similar general trends of inhibition were shown by both methods, but the former gives only a qualitative picture while the modeling method yields the degree of inhibition because the model can separate the two simultaneous roles of ATP as both a substrate of reaction and an inhibitor of PFK1. Analysis based on the model suggests that the ATP affinity is much greater to the PFK1 catalytic site than to the inhibitory site, but the inhibited ATP-PFK1-ATP complex is much slower than the uninhibited PFK1-ATP complex in product generation, leading to reduced overall reaction velocity when ATP concentration increases. The initial-velocity method is simple and useful for general observation of enzyme activity while the modeling method has advantages in quantifying the inhibition effects and providing insights into the process.
Asunto(s)
Adenosina Trifosfato , Fosfofructoquinasa-1 , Adenosina Trifosfato/metabolismo , Fosfofructoquinasa-1/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Fructosafosfatos/metabolismo , Adenosina Difosfato/metabolismo , GlucólisisRESUMEN
Cells depend on a continuous supply of adenosine triphosphate (ATP), the universal energy currency. In mitochondria, ATP is produced by a series of redox reactions, whereby an electrochemical gradient is established across the inner mitochondrial membrane. The ATP synthase harnesses the energy of the gradient to generate ATP from adenosine diphosphate (ADP) and inorganic phosphate. We determined the structure of ATP synthase within mitochondria of the unicellular flagellate Polytomella by electron cryo-tomography and subtomogram averaging at up to 4.2-angstrom resolution, revealing six rotary positions of the central stalk, subclassified into 21 substates of the F1 head. The Polytomella ATP synthase forms helical arrays with multiple adjacent rows defining the cristae ridges. The structure of ATP synthase under native operating conditions in the presence of a membrane potential represents a pivotal step toward the analysis of membrane protein complexes in situ.
Asunto(s)
Chlorophyceae , Mitocondrias , ATPasas de Translocación de Protón Mitocondriales , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Microscopía por Crioelectrón , Tomografía con Microscopio Electrónico , Mitocondrias/enzimología , Mitocondrias/ultraestructura , Membranas Mitocondriales/enzimología , Membranas Mitocondriales/metabolismo , ATPasas de Translocación de Protón Mitocondriales/química , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Rotación , Chlorophyceae/enzimologíaRESUMEN
Phosphoryl transfer is a fundamental reaction in cellular signaling and metabolism that requires Mg2+ as an essential cofactor. While the primary function of Mg2+ is electrostatic activation of substrates, such as ATP, the full spectrum of catalytic mechanisms exerted by Mg2+ is not known. In this study, we integrate structural biology methods, molecular dynamic (MD) simulations, phylogeny, and enzymology assays to provide molecular insights into Mg2+-dependent structural reorganization in the active site of the metabolic enzyme adenylate kinase. Our results demonstrate that Mg2+ induces a conformational rearrangement of the substrates (ATP and ADP), resulting in a 30° adjustment of the angle essential for reversible phosphoryl transfer, thereby optimizing it for catalysis. MD simulations revealed transitions between conformational substates that link the fluctuation of the angle to large-scale enzyme dynamics. The findings contribute detailed insight into Mg2+ activation of enzymes and may be relevant for reversible and irreversible phosphoryl transfer reactions.
Asunto(s)
Adenilato Quinasa , Dominio Catalítico , Magnesio , Simulación de Dinámica Molecular , Magnesio/metabolismo , Magnesio/química , Adenilato Quinasa/metabolismo , Adenilato Quinasa/química , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/química , Conformación Proteica , Adenosina Difosfato/metabolismo , Adenosina Difosfato/químicaRESUMEN
Hsp70 belongs to a family of molecular chaperones ubiquitous through organisms that assist client protein folding and prevent aggregation. It works through a tightly ATP-regulated allosteric cycle mechanism, which organizes its two NBD and SBD into alternate open and closed arrangements that facilitate loading and unloading of client proteins. The two cytosolic human isoforms Hsc70 and HspA1 are relevant targets for neurodegenerative diseases and cancer. Illuminating the molecular details of Hsp70 functional dynamics is essential to rationalize differences among the well-characterized bacterial homologue DnaK and the less explored human forms and develop subtype- or species-selective allosteric drugs. We present here a molecular dynamics-based analysis of the conformational dynamics of HspA1. By using an "allosterically impaired" mutant for comparison, we can reconstruct the impact of the ADP-ATP swap on interdomain contacts and dynamic coordination in full-length HspA1, supporting previous predictions that were, however, limited to the NBD. We model the initial onset of the conformational cycle by proposing a sequence of structural steps, which reveal the role of a specific human sequence insertion at the linker, and a modulation of the angle formed by the two NBD lobes during the progression of docking. Our findings pinpoint functionally relevant conformations and set the basis for a selective structure-based drug discovery approach targeting allosteric sites in human Hsp70.
Asunto(s)
Adenosina Difosfato , Adenosina Trifosfato , Proteínas HSP70 de Choque Térmico , Simulación de Dinámica Molecular , Mutación , Humanos , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/química , Adenosina Difosfato/metabolismo , Adenosina Difosfato/química , Conformación ProteicaRESUMEN
The ClC-3 chloride/proton exchanger is both physiologically and pathologically critical, as it is potentiated by ATP to detect metabolic energy level and point mutations in ClC-3 lead to severe neurodegenerative diseases in human. However, why this exchanger is differentially modulated by ATP, ADP or AMP and how mutations caused gain-of-function remains largely unknow. Here we determine the high-resolution structures of dimeric wildtype ClC-3 in the apo state and in complex with ATP, ADP and AMP, and the disease-causing I607T mutant in the apo and ATP-bounded state by cryo-electron microscopy. In combination with patch-clamp recordings and molecular dynamic simulations, we reveal how the adenine nucleotides binds to ClC-3 and changes in ion occupancy between apo and ATP-bounded state. We further observe I607T mutation induced conformational changes and augments in current. Therefore, our study not only lays the structural basis of adenine nucleotides regulation in ClC-3, but also clearly indicates the target region for drug discovery against ClC-3 mediated neurodegenerative diseases.
Asunto(s)
Adenosina Trifosfato , Canales de Cloruro , Microscopía por Crioelectrón , Simulación de Dinámica Molecular , Enfermedades Neurodegenerativas , Canales de Cloruro/metabolismo , Canales de Cloruro/genética , Canales de Cloruro/química , Humanos , Adenosina Trifosfato/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/patología , Nucleótidos de Adenina/metabolismo , Técnicas de Placa-Clamp , Mutación , Adenosina Difosfato/metabolismo , Células HEK293 , Adenosina Monofosfato/metabolismo , Animales , Conformación ProteicaRESUMEN
Cell-to-cell mitochondrial transfer has recently been shown to play a role in maintaining physiological functions of cell. We previously illustrated that mitochondrial transfer within osteocyte dendritic network regulates bone tissue homeostasis. However, the mechanism of triggering this process has not been explored. Here, we showed that stressed osteocytes in mice release adenosine diphosphate (ADP), resulting in triggering mitochondrial transfer from healthy osteocytes to restore the oxygen consumption rate (OCR) and to alleviate reactive oxygen species accumulation. Furthermore, we identified that P2Y2 and P2Y6 transduced the ADP signal to regulate osteocyte mitochondrial transfer. We showed that mitochondrial metabolism is impaired in aged osteocytes, and there were more extracellular nucleotides release into the matrix in aged cortical bone due to compromised membrane integrity. Conditioned medium from aged osteocytes triggered mitochondrial transfer between osteocytes to enhance the energy metabolism. Together, using osteocyte as an example, this study showed new insights into how extracellular ADP triggers healthy cells to rescue energy metabolism crisis in stressed cells via mitochondrial transfer in tissue homeostasis.
Asunto(s)
Adenosina Difosfato , Homeostasis , Mitocondrias , Osteocitos , Animales , Osteocitos/metabolismo , Mitocondrias/metabolismo , Ratones , Adenosina Difosfato/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Consumo de Oxígeno , Metabolismo Energético , Ratones Endogámicos C57BL , Estrés FisiológicoRESUMEN
Understanding muscle contraction mechanisms is a standing challenge, and one of the approaches has been to create models of the sarcomere-the basic contractile unit of striated muscle. While these models have been successful in elucidating many aspects of muscle contraction, they fall short in explaining the energetics of functional phenomena, such as rigor, and in particular, their dependence on the concentrations of the biomolecules involved in the cross-bridge cycle. Our hypothesis posits that the stochastic time delay between ATP adsorption and ADP/Pi release in the cross-bridge cycle necessitates a modeling approach where the rates of these two reaction steps are controlled by two independent parts of the total free energy change of the hydrolysis reaction. To test this hypothesis, we built a two-filament, stochastic-mechanical half-sarcomere model that separates the energetic roles of ATP and ADP/Pi in the cross-bridge cycle's free energy landscape. Our results clearly demonstrate that there is a nontrivial dependence of the cross-bridge cycle's kinetics on the independent concentrations of ATP, ADP, and Pi. The simplicity of the proposed model allows for analytical solutions of the more basic systems, which provide novel insight into the dominant mechanisms driving some of the experimentally observed contractile phenomena.
Asunto(s)
Adenosina Difosfato , Adenosina Trifosfato , Modelos Biológicos , Sarcómeros , Adenosina Difosfato/metabolismo , Sarcómeros/fisiología , Sarcómeros/metabolismo , Adenosina Trifosfato/metabolismo , Cinética , Contracción Muscular/fisiología , Biología Computacional , AnimalesRESUMEN
The ATP-binding cassette (ABC) transporter, MsbA, plays a pivotal role in lipopolysaccharide (LPS) biogenesis by facilitating the transport of the LPS precursor lipooligosaccharide (LOS) from the cytoplasmic to the periplasmic leaflet of the inner membrane. Despite multiple studies shedding light on MsbA, the role of lipids in modulating MsbA-nucleotide interactions remains poorly understood. Here we use native mass spectrometry (MS) to investigate and resolve nucleotide and lipid binding to MsbA, demonstrating that the transporter has a higher affinity for adenosine 5'-diphosphate (ADP). Moreover, native MS shows the LPS-precursor 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo)2-lipid A (KDL) can tune the selectivity of MsbA for adenosine 5'-triphosphate (ATP) over ADP. Guided by these studies, four open, inward-facing structures of MsbA are determined that vary in their openness. We also report a 2.7 Å-resolution structure of MsbA in an open, outward-facing conformation that is not only bound to KDL at the exterior site, but with the nucleotide binding domains (NBDs) adopting a distinct nucleotide-free structure. The results obtained from this study offer valuable insight and snapshots of MsbA during the transport cycle.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Adenosina Difosfato , Adenosina Trifosfato , Espectrometría de Masas , Transportadoras de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Adenosina Trifosfato/metabolismo , Adenosina Difosfato/metabolismo , Espectrometría de Masas/métodos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Lipopolisacáridos/metabolismo , Lípido A/metabolismo , Lípido A/química , Unión Proteica , Modelos Moleculares , Cristalografía por Rayos X , Lípidos/química , Escherichia coli/metabolismo , Conformación ProteicaRESUMEN
BACKGROUND: Leukocyte-platelet aggregates comprise a pathogenic link between hemostasis and immunity, but the prerequisites and mechanisms of their formation remain not understood. AIMS: To quantify the formation, composition, and morphology of leukocyte-platelet aggregates in vitro under the influence of various cellular activators. METHODS: Phorbol-12-myristate-13-acetate (PMA), lipopolysaccharide (LPS), thrombin receptor-activating peptide (TRAP-6), and adenosine diphosphate (ADP) were used as cellular activators. Flow cytometry was utilized to identify and quantify aggregates in whole human blood and platelet-rich plasma. Cell types and cellular aggregates were identified using fluorescently labeled antibodies against the appropriate cellular markers, and cell activation was assessed by the expression of appropriate surface markers. For confocal fluorescent microscopy, cell membranes and nuclei were labeled. Neutrophil-platelet aggregates were studied using scanning electron microscopy. RESULTS: In the presence of PMA, ADP or TRAP-6, about 17-38 % of neutrophils and 61-77 % of monocytes formed aggregates with platelets in whole blood, whereas LPS did not induce platelet aggregation with either neutrophils or monocytes due the inability to activate platelets. Similar results were obtained when isolated neutrophils were added to platelet-rich plasma. All the cell types involved in the heterotypic aggregation expressed molecular markers of activation. Fluorescent and electron microscopy of the aggregates showed that the predominant platelet/leukocyte ratios were 1:1 and 2:1. CONCLUSIONS: Formation of leukocyte-platelet aggregates depends on the nature of the cellular activator and the spectrum of its cell-activating ability. An indispensable condition for formation of leukocyte-platelet aggregates is activation of all cell types including platelets, which is the restrictive step.
Asunto(s)
Plaquetas , Leucocitos , Lipopolisacáridos , Agregación Plaquetaria , Acetato de Tetradecanoilforbol , Humanos , Plaquetas/metabolismo , Plaquetas/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Agregación Plaquetaria/efectos de los fármacos , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Lipopolisacáridos/farmacología , Adenosina Difosfato/farmacología , Adenosina Difosfato/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Fragmentos de Péptidos/farmacología , Citometría de Flujo , Monocitos/efectos de los fármacos , Monocitos/metabolismoRESUMEN
Genome segregation is a fundamental process that preserves the genetic integrity of all organisms, but the mechanisms driving genome segregation in archaea remain enigmatic. This study delved into the unknown function of SegC (SSO0033), a novel protein thought to be involved in chromosome segregation in archaea. Using fluorescence polarization DNA binding assays, we discovered the ability of SegC to bind DNA without any sequence preference. Furthermore, we determined the crystal structure of SegC at 2.8 Å resolution, revealing the multimeric configuration and forming a large positively charged surface that can bind DNA. SegC has a tertiary structure folding similar to those of the ThDP-binding fold superfamily, but SegC shares only 5-15% sequence identity with those proteins. Unexpectedly, we found that SegC has nucleotide triphosphatase (NTPase) activity. We also determined the SegC-ADP complex structure, identifying the NTP binding pocket and relative SegC residues involved in the interaction. Interestingly, images from negative-stain electron microscopy revealed that SegC forms filamentous structures in the presence of DNA and NTPs. Further, more uniform and larger SegC-filaments are observed, when SegA-ATP was added. Notably, the introduction of SegB disrupts these oligomers, with ATP being essential for regulating filament formation. These findings provide insights into the functional and structural role of SegC in archaeal chromosome segregation.
Asunto(s)
Proteínas Arqueales , Segregación Cromosómica , Modelos Moleculares , Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Proteínas Arqueales/genética , Unión Proteica , Cristalografía por Rayos X , Adenosina Difosfato/metabolismo , Adenosina Difosfato/química , Sitios de Unión , ADN de Archaea/metabolismo , ADN de Archaea/química , ADN de Archaea/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/ultraestructuraRESUMEN
Precise modulation of host-guest interactions between programmable Ln-MOFs (lanthanide metal-organic frameworks) and phosphate analytes holds immense promise for enabling novel functionalities in biosensing. However, the intricate relationship between these functionalities and structures remains largely elusive. Understanding this correlation is crucial for advancing the rational design of fluorescent biosensor technology. Presently, there exists a large research gap concerning the utilization of Ln-MOFsto monitor the conversion of ATP to ADP, which poses a limitation for kinase detection. In this work, we delve into the potential of Ln-MOFs to amplify the fluorescence response during the kinase-mediated ATP-to-ADP conversion. Six Eu-MOFs were synthesized and Eu-TPTC ([1,1':4',1â³]-terphenyl-3,3'',5,5''-tetracarboxylic acid) was selected as a ratiometric fluorescent probe, which is most suitable for high-precision detection of creatine kinase activity through the differential response from ATP to ADP. The molecular -level mechanism was confirmed by density functional theory. Furthermore, a simple paper chip-based platform was constructed to realize the fast (20 min) and sensitive (limit of detection is 0.34 U/L) creatine kinase activity detection in biological samples. Ln-MOF-phosphate interactions offer promising avenues for kinase activity assays and hold the potential for precise customization of analytical chemistry.
Asunto(s)
Adenosina Difosfato , Adenosina Trifosfato , Estructuras Metalorgánicas , Adenosina Trifosfato/análisis , Adenosina Trifosfato/metabolismo , Estructuras Metalorgánicas/química , Adenosina Difosfato/análisis , Adenosina Difosfato/metabolismo , Adenosina Difosfato/química , Creatina Quinasa/metabolismo , Creatina Quinasa/análisis , Creatina Quinasa/química , Técnicas Biosensibles/métodos , Colorantes Fluorescentes/química , Elementos de la Serie de los Lantanoides/química , AnimalesRESUMEN
The HSPA5 protein (BiP/Grp78) serves as a pivotal chaperone in maintaining cellular protein quality control. As a member of the human HSP70 family, HSPA5 comprises two distinct domains: a nucleotide-binding domain (NBD) and a peptide-binding domain (PBD). In this study, we investigated the interdomain interactions of HSPA5, aiming to elucidate how these domains regulate its function as a chaperone. Our findings revealed that HSPA5-FL, HSPA5-T, and HSPA5-N exhibit varying affinities for ATP and ADP, with a noticeable dependency on Mg2+ for optimal interactions. Interestingly, in ADP assays, the presence of the metal ion seems to enhance NBD binding only for HSPA5-FL and HSPA5-T. Moreover, while the truncation of the C-terminus does not significantly impact the thermal stability of HSPA5, experiments involving MgATP underscore its essential role in mediating interactions and nucleotide hydrolysis. Thermal stability assays further suggested that the NBD-PBD interface enhances the stability of the NBD, more pronounced for HSPA5 than for the orthologous HSPA1A, and prevents self-aggregation through interdomain coupling. Enzymatic analyses indicated that the presence of PBD enhances NBD ATPase activity and augments its nucleotide affinity. Notably, the intrinsic chaperone activity of the PBD is dependent on the presence of the NBD, potentially due to the propensity of the PBD for self-oligomerization. Collectively, our data highlight the pivotal role of allosteric mechanisms in modulating thermal stability, nucleotide interaction, and ATPase activity of HSPA5, underscoring its significance in protein quality control within cellular environments.
Asunto(s)
Adenosina Trifosfato , Chaperón BiP del Retículo Endoplásmico , Proteínas de Choque Térmico , Estabilidad Proteica , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Humanos , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/química , Adenosina Difosfato/metabolismo , Adenosina Difosfato/química , Unión Proteica , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/química , Dominios Proteicos , Magnesio/metabolismo , Magnesio/químicaRESUMEN
The Tn7 family of transposons is notable for its highly regulated integration mechanisms, including programmable RNA-guided transposition. The targeting pathways rely on dedicated target selection proteins from the TniQ family and the AAA+ adaptor TnsC to recruit and activate the transposase at specific target sites. Here, we report the cryoelectron microscopy (cryo-EM) structures of TnsC bound to the TniQ domain of TnsD from prototypical Tn7 and unveil key regulatory steps stemming from unique behaviors of ATP- versus ADP-bound TnsC. We show that TnsD recruits ADP-bound dimers of TnsC and acts as an exchange factor to release one protomer with exchange to ATP. This loading process explains how TnsC assembles a heptameric ring unidirectionally from the target site. This unique loading process results in functionally distinct TnsC protomers within the ring, providing a checkpoint for target immunity and explaining how insertions at programmed sites precisely occur in a specific orientation across Tn7 elements.
Asunto(s)
Adenosina Difosfato , Adenosina Trifosfato , Microscopía por Crioelectrón , Elementos Transponibles de ADN , Transposasas , Elementos Transponibles de ADN/genética , Adenosina Trifosfato/metabolismo , Transposasas/metabolismo , Transposasas/genética , Transposasas/química , Adenosina Difosfato/metabolismo , Unión Proteica , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Modelos Moleculares , Multimerización de Proteína , Sitios de UniónRESUMEN
The Orthoflavivirus NS3 helicase (NS3h) is crucial in virus replication, representing a potential drug target for pathogenesis. NS3h utilizes nucleotide triphosphate (ATP) for hydrolysis energy to translocate on single-stranded nucleic acids, which is an important step in the unwinding of double-stranded nucleic acids. Intermediate states along the ATP hydrolysis cycle and conformational changes between these states, represent important yet difficult-to-identify targets for potential inhibitors. Extensive molecular dynamics simulations of West Nile virus NS3h+ssRNA in the apo, ATP, ADP+Pi and ADP bound states were used to model the conformational ensembles along this cycle. Energetic and structural clustering analyses depict a clear trend of differential enthalpic affinity of NS3h with ADP, demonstrating a probable mechanism of hydrolysis turnover regulated by the motif-VI loop (MVIL). Based on these results, MVIL mutants (D471L, D471N and D471E) were found to have a substantial reduction in ATPase activity and RNA replication compared to the wild-type. Simulations of the mutants in the apo state indicate a shift in MVIL populations favoring either a closed or open 'valve' conformation, affecting ATP entry or stabilization, respectively. Combining our molecular modeling with experimental evidence highlights a conformation-dependent role for MVIL as a 'valve' for the ATP-pocket, presenting a promising target for antiviral development.
Asunto(s)
Adenosina Trifosfato , Simulación de Dinámica Molecular , ARN Helicasas , Proteínas no Estructurales Virales , Virus del Nilo Occidental , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Virus del Nilo Occidental/enzimología , Virus del Nilo Occidental/genética , ARN Helicasas/metabolismo , ARN Helicasas/química , ARN Helicasas/genética , Adenosina Trifosfato/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Difosfato/química , Secuencias de Aminoácidos , Mutación , Nucleótidos/metabolismo , Nucleótidos/química , Hidrólisis , Replicación Viral/genética , Conformación Proteica , Proteasas Virales , Serina Endopeptidasas , Nucleósido-Trifosfatasa , ARN Helicasas DEAD-boxRESUMEN
BACKGROUND: Lactobacillus plantarum has been found to play a significant role in maintaining the balance of intestinal flora in the human gut. However, it is sensitive to commonly used antibiotics and is often incidentally killed during treatment. We attempted to identify a means to protect L. plantarum ATCC14917 from the metabolic changes caused by two commonly used antibiotics, ampicillin, and doxycycline. We examined the metabolic changes under ampicillin and doxycycline treatment and assessed the protective effects of adding key exogenous metabolites. RESULTS: Using metabolomics, we found that under the stress of ampicillin or doxycycline, L. plantarum ATCC14917 exhibited reduced metabolic activity, with purine metabolism a key metabolic pathway involved in this change. We then screened the key biomarkers in this metabolic pathway, guanine and adenosine diphosphate (ADP). The exogenous addition of each of these two metabolites significantly reduced the lethality of ampicillin and doxycycline on L. plantarum ATCC14917. Because purine metabolism is closely related to the production of reactive oxygen species (ROS), the results showed that the addition of guanine or ADP reduced intracellular ROS levels in L. plantarum ATCC14917. Moreover, the killing effects of ampicillin and doxycycline on L. plantarum ATCC14917 were restored by the addition of a ROS accelerator in the presence of guanine or ADP. CONCLUSIONS: The metabolic changes of L. plantarum ATCC14917 under antibiotic treatments were determined. Moreover, the metabolome information that was elucidated can be used to help L. plantarum cope with adverse stress, which will help probiotics become less vulnerable to antibiotics during clinical treatment.
Asunto(s)
Ampicilina , Antibacterianos , Doxiciclina , Lactobacillus plantarum , Metabolómica , Lactobacillus plantarum/metabolismo , Lactobacillus plantarum/efectos de los fármacos , Antibacterianos/farmacología , Ampicilina/farmacología , Doxiciclina/farmacología , Especies Reactivas de Oxígeno/metabolismo , Purinas/metabolismo , Estrés Fisiológico/efectos de los fármacos , Redes y Vías Metabólicas/efectos de los fármacos , Adenosina Difosfato/metabolismo , HumanosRESUMEN
In in vitro model of short-term therapeutic inhalation of Xe/O2 mixture, xenon in millimolar concentrations led to a pronounced decrease in induced platelet aggregation in the platelet-enriched blood plasma. The maximum and statistically significant decrease occurred in response to induction by collagen (by ≈30%, p≤0.01) and ADP (by ≈25%, p≤0.01). A slightly weaker but statistically significant reduction in aggregation appeared in response to ristocetin (by ≈12%, p≤0.01) and epinephrine (by ≈9%, p≤0.01). It should be noted that the spontaneous aggregation exceeded the reference values in the control group. Nevertheless, even at minimal absolute values, spontaneous platelet aggregation decreased by 2 times in response to xenon (p≤0.01). The reasons for the decrease of spontaneous and induced aggregation are xenon accumulation in the lipid bilayer of the membrane with subsequent nonspecific (mechanical) disassociation of membrane platelet structures and specific block of its distinct from neuronal NMDA receptor.
Asunto(s)
Agregación Plaquetaria , Xenón , Xenón/farmacología , Agregación Plaquetaria/efectos de los fármacos , Humanos , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Adenosina Difosfato/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Plasma Rico en Plaquetas/metabolismo , Epinefrina/farmacología , Epinefrina/sangre , Colágeno/metabolismoRESUMEN
Platelets play essential roles in the formation of blood clots by clumping with coagulation factors at the site of vascular injury to stop bleeding; therefore, a reduction in the platelet number or disorder in their function causes bleeding risk. In our research, we developed a method to assess platelet aggregation using an optical approach within a microfluidic chip's channel by evaluating the size of laser speckles. These speckles, associated with slowed blood flow in the microfluidic channel, had a baseline size of 28.54 ± 0.72 µm in whole blood. Removing platelets from the sample led to a notable decrease in speckle size to 27.04 ± 1.23 µm. Moreover, the addition of an ADP-containing agonist, which activates platelets, resulted in an increased speckle size of 32.89 ± 1.69 µm. This finding may provide a simple optical method via microfluidics that could be utilized to assess platelet functionality in diagnosing bleeding disorders and potentially in monitoring therapies that target platelets.
Asunto(s)
Plaquetas , Agregación Plaquetaria , Plaquetas/efectos de los fármacos , Humanos , Agregación Plaquetaria/efectos de los fármacos , Pruebas de Función Plaquetaria/métodos , Pruebas de Función Plaquetaria/instrumentación , Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/métodos , Adenosina Difosfato/farmacologíaRESUMEN
Regulatory cystathionine ß-synthase (CBS) domains are widespread in proteins; however, difficulty in structure determination prevents a comprehensive understanding of the underlying regulation mechanism. Tetrameric microbial inorganic pyrophosphatase containing such domains (CBS-PPase) is allosterically inhibited by AMP and ADP and activated by ATP and cell alarmones diadenosine polyphosphates. Each CBS-PPase subunit contains a pair of CBS domains but binds cooperatively to only one molecule of the mono-adenosine derivatives. We used site-directed mutagenesis of Desulfitobacterium hafniense CBS-PPase to identify the key elements determining the direction of the effect (activation or inhibition) and the "half-of-the-sites" ligand binding stoichiometry. Seven amino acid residues were selected in the CBS1 domain, based on the available X-ray structure of the regulatory domains, and substituted by alanine and other residues. The interaction of 11 CBS-PPase variants with the regulating ligands was characterized by activity measurements and isothermal titration calorimetry. Lys100 replacement reversed the effect of ADP from inhibition to activation, whereas Lys95 and Gly118 replacements made ADP an activator at low concentrations but an inhibitor at high concentrations. Replacement of these residues for alanine increased the stoichiometry of mono-adenosine phosphate binding by twofold. These findings identified several key protein residues and suggested a "two non-interacting pairs of interacting regulatory sites" concept in CBS-PPase regulation.
Asunto(s)
Cistationina betasintasa , Cistationina betasintasa/metabolismo , Cistationina betasintasa/química , Cistationina betasintasa/genética , Mutación , Unión Proteica , Mutagénesis Sitio-Dirigida , Nucleótidos de Adenina/metabolismo , Nucleótidos de Adenina/química , Dominios Proteicos , Pirofosfatasas/metabolismo , Pirofosfatasas/química , Pirofosfatasas/genética , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Pirofosfatasa Inorgánica/metabolismo , Pirofosfatasa Inorgánica/química , Pirofosfatasa Inorgánica/genética , Modelos Moleculares , Sitios de UniónRESUMEN
Peripheral mononuclear cells (PBMCs) exhibit robust changes in mitochondrial respiratory capacity in response to health and disease. While these changes do not always reflect what occurs in other tissues, such as skeletal muscle, these cells are an accessible and valuable source of viable mitochondria from human subjects. PBMCs are exposed to systemic signals that impact their bioenergetic state. Thus, expanding our tools to interrogate mitochondrial metabolism in this population will elucidate mechanisms related to disease progression. Functional assays of mitochondria are often limited to using respiratory outputs following maximal substrate, inhibitor, and uncoupler concentrations to determine the full range of respiratory capacity, which may not be achievable in vivo. The conversion of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) by ATP-synthase results in a decrease in mitochondrial membrane potential (mMP) and an increase in oxygen consumption. To provide a more integrated analysis of mitochondrial dynamics, this article describes the use of high-resolution fluorespirometry to measure the simultaneous response of oxygen consumption and mitochondrial membrane potential (mMP) to physiologically relevant concentrations of ADP. This technique uses tetramethylrhodamine methylester (TMRM) to measure mMP polarization in response to ADP titrations following maximal hyperpolarization with complex I and II substrates. This technique can be used to quantify how changes in health status, such as aging and metabolic disease, affect the sensitivity of mitochondrial response to energy demand in PBMCs, T-cells, and monocytes from human subjects.
Asunto(s)
Leucocitos Mononucleares , Potencial de la Membrana Mitocondrial , Humanos , Potencial de la Membrana Mitocondrial/fisiología , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/citología , Rodaminas/química , Adenosina Difosfato/metabolismo , Adenosina Difosfato/farmacología , Consumo de Oxígeno/fisiología , Mitocondrias/metabolismo , Colorantes Fluorescentes/químicaRESUMEN
BACKGROUND AND OBJECTIVES: The total thrombus-formation analysis system (T-TAS) can quantitatively analyse the contribution of platelets to haemostasis using reconstituted blood samples. However, it is unsuitable in cases with low platelet counts. We introduced a haemodilution (HD) chip with a shallow chamber depth, adapted to low platelet counts and high shear conditions (1500 s-1). MATERIALS AND METHODS: Blood samples were prepared by mixing red blood cell products, standard human plasma and platelet products; the final platelet count was 50 × 103/µL. Aggregation tests were performed by using the aggregation inducers collagen, adenosine diphosphate (ADP) and ristocetin. Samples with 2-, 4- and 9-day-old platelet products (N = 10) were evaluated. RESULTS: The HD chip enabled the stable analysis of the haemostatic function of all samples at a platelet count of 50 × 103/µL. Haemostatic function was correlated with ADP aggregation (time to 10 kPa [T10]: r = -0.53; area under the curve for 30 min: r = 0.40) and storage period (T10: r = 0.44). CONCLUSION: The HD chip-mounted T-TAS can stably analyse haemostatic function under low platelet counts and high shear conditions; this approach is expected to serve as a bridge to in vivo haemostatic tests with experimental animals.