RESUMO
Pannexin1 hemichannels (Panx1 HCs) are found in the membrane of most mammalian cells and communicate the intracellular and extracellular spaces, enabling the passive transfer of ions and small molecules. They are involved in physiological and pathophysiological conditions. During apoptosis, the C-terminal tail of Panx1 is proteolytically cleaved, but the permeability features of hemichannels and their role in cell death remain elusive. To address these topics, HeLa cells transfected with full-length human Panx1 (fl-hPanx1) or C-terminal truncated hPanx1 (Δ371hPanx1) were exposed to alkaline extracellular saline solution, increasing the activity of Panx1 HCs. The Δ371hPanx1 HC was permeable to DAPI and Etd+, but not to propidium iodide, whereas fl-hPanx1 HC was only permeable to DAPI. Furthermore, the cytoplasmic Ca2+ signal increased only in Δ371hPanx1 cells, which was supported by bioinformatics approaches. The influx of Ca2+ through Δ371hPanx1 HCs was necessary to promote cell death up to about 95% of cells, whereas the exposure to alkaline saline solution without Ca2+ failed to induce cell death, and the Ca2+ ionophore A23187 promoted more than 80% cell death even in fl-hPanx1 transfectants. Moreover, cell death was prevented with carbenoxolone or 10Panx1 in Δ371hPanx1 cells, whereas it was undetectable in HeLa Panx1-/- cells. Pretreatment with Ferrostatin-1 and necrostatin-1 did not prevent cell death, suggesting that ferroptosis or necroptosis was not involved. In comparison, zVAD-FMK, a pancaspase inhibitor, reduced death by ~60%, suggesting the involvement of apoptosis. Therefore, alkaline pH increases the activity of Δ371hPanx1HCs, leading to a critical intracellular free-Ca2+ overload that promotes cell death.
Assuntos
Cálcio , Conexinas , Proteínas do Tecido Nervoso , Humanos , Conexinas/metabolismo , Conexinas/genética , Células HeLa , Cálcio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Apoptose , Morte Celular , Sinalização do CálcioRESUMO
Thyroxine (T4) is a drug extensively utilized for the treatment of hypothyroidism. However, the oral absorption of T4 presents certain limitations. This research investigates the efficacy of CO2 nanobubbles in water as a potential oral carrier for T4 administration to C57BL/6 hypothyroid mice. Following 18 h of fasting, the formulation was administered to the mice, demonstrating that the combination of CO2 nanobubbles and T4 enhanced the drug's absorption in blood serum by approximately 40%. To comprehend this observation at a molecular level, we explored the interaction mechanism through which T4 engages with the CO2 nanobubbles, employing molecular simulations, semi-empirical quantum mechanics, and PMF calculations. Our simulations revealed a high affinity of T4 for the water-gas interface, driven by additive interactions between the hydrophobic region of T4 and the gas phase and electrostatic interactions of the polar groups of T4 with water at the water-gas interface. Concurrently, we observed that at the water-gas interface, the cluster of T4 formed in the water region disassembles, contributing to the drug's bioavailability. Furthermore, we examined how the gas within the nanobubbles aids in facilitating the drug's translocation through cell membranes. This research contributes to a deeper understanding of the role of CO2 nanobubbles in drug absorption and subsequent release into the bloodstream. The findings suggest that utilizing CO2 nanobubbles could enhance T4 bioavailability and cell permeability, leading to more efficient transport into cells. Additional research opens the possibility of employing lower concentrations of this class of drugs, thereby potentially reducing the associated side effects due to poor absorption.
Assuntos
Dióxido de Carbono , Modelos Animais de Doenças , Hipotireoidismo , Tiroxina , Água , Animais , Hipotireoidismo/tratamento farmacológico , Hipotireoidismo/metabolismo , Camundongos , Dióxido de Carbono/química , Água/química , Camundongos Endogâmicos C57BL , Administração Oral , Nanopartículas/química , Portadores de Fármacos/químicaRESUMO
In neurosecretion, allosteric communication between voltage sensors and Ca2+ binding in BK channels is crucially involved in damping excitatory stimuli. Nevertheless, the voltage-sensing mechanism of BK channels is still under debate. Here, based on gating current measurements, we demonstrate that two arginines in the transmembrane segment S4 (R210 and R213) function as the BK gating charges. Significantly, the energy landscape of the gating particles is electrostatically tuned by a network of salt bridges contained in the voltage sensor domain (VSD). Molecular dynamics simulations and proton transport experiments in the hyperpolarization-activated R210H mutant suggest that the electric field drops off within a narrow septum whose boundaries are defined by the gating charges. Unlike Kv channels, the charge movement in BK appears to be limited to a small displacement of the guanidinium moieties of R210 and R213, without significant movement of the S4.
Assuntos
Ativação do Canal Iônico , Canais de Potássio Ativados por Cálcio de Condutância Alta , Arginina/metabolismo , Ativação do Canal Iônico/genética , Simulação de Dinâmica Molecular , MutaçãoRESUMO
The occurrence of intercellular channels formed by pannexin1 has been challenged for more than a decade. Here, we provide an electrophysiological characterization of exogenous human pannexin1 (hPanx1) cellcell channels expressed in HeLa cells knocked out for connexin45. The observed hPanx1 cellcell channels show two phenotypes: O-state and S-state. The former displayed low transjunctional voltage (Vj) sensitivity and single-channel conductance of â¼175 pS, with a substate of â¼35 pS; the latter showed a peculiar dynamic asymmetry in Vj dependence and single-channel conductance identical to the substate conductance of the O-state. S-state hPanx1 cellcell channels were also identified between TC620 cells, a human oligodendroglioma cell line that endogenously expresses hPanx1. In these cells, dye and electrical coupling increased with temperature and were strongly reduced after hPanx1 expression was knocked down by small interfering RNA or inhibited with Panx1 mimetic inhibitory peptide. Moreover, cellcell coupling was augmented when hPanx1 levels were increased with a doxycycline-inducible expression system. Application of octanol, a connexin gap junction (GJ) channel inhibitor, was not sufficient to block electrical coupling between HeLa KO Cx45-hPanx1 or TC620 cell pairs. In silico studies suggest that several arginine residues inside the channel pore may be neutralized by hydrophobic interactions, allowing the passage of DAPI, consistent with dye coupling observed between TC620 cells. These findings demonstrate that endogenously expressed hPanx1 forms intercellular cellcell channels and their unique properties resemble those described in innexin-based GJ channels. Since Panx1 is ubiquitously expressed, finding conditions to recognize Panx1 cellcell channels in different cell types might require special attention.
Assuntos
Conexinas , Proteínas do Tecido Nervoso , Animais , Conexinas/metabolismo , Humanos , Canais Iônicos , Mamíferos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismoRESUMO
Endoxylanases belonging to family 10 of the glycoside hydrolases (GH10) are versatile in the use of different substrates. Thus, an understanding of the molecular mechanisms underlying substrate specificities could be very useful in the engineering of GH10 endoxylanases for biotechnological purposes. Herein, we analyzed XynA, an endoxylanase that contains a (ß/α)8-barrel domain and an intrinsically disordered region (IDR) of 29 amino acids at its amino end. Enzyme activity assays revealed that the elimination of the IDR resulted in a mutant enzyme (XynAΔ29) in which two new activities emerged: the ability to release xylose from xylan, and the ability to hydrolyze p-nitrophenyl-ß-d-xylopyranoside (pNPXyl), a substrate that wild-type enzyme cannot hydrolyze. Circular dichroism and tryptophan fluorescence quenching by acrylamide showed changes in secondary structure and increased flexibility of XynAΔ29. Molecular dynamics simulations revealed that the emergence of the pNPXyl-hydrolyzing activity correlated with a dynamic behavior not previously observed in GH10 endoxylanases: a hinge-bending motion of two symmetric regions within the (ß/α)8-barrel domain, whose hinge point is the active cleft. The hinge-bending motion is more intense in XynAΔ29 than in XynA and promotes the formation of a wider active site that allows the accommodation and hydrolysis of pNPXyl. Our results open new avenues for the study of the relationship between IDRs, dynamics and activity of endoxylanases, and other enzymes containing (ß/α)8-barrel domain.
Assuntos
Endo-1,4-beta-Xilanases/metabolismo , Glicosídeo Hidrolases/metabolismo , Sequência de Aminoácidos , Catálise , Domínio Catalítico/fisiologia , Hidrólise , Especificidade por Substrato/fisiologia , Xilanos/metabolismo , Xilose/metabolismoRESUMO
Pannexin1 (Panx1) channels are ubiquitously expressed in vertebrate cells and are widely accepted as adenosine triphosphate (ATP)-releasing membrane channels. Activation of Panx1 has been associated with phosphorylation in a specific tyrosine residue or cleavage of its C-terminal domains. In the present work, we identified a residue (S394) as a putative phosphorylation site by Ca2+/calmodulin-dependent kinase II (CaMKII). In HeLa cells transfected with rat Panx1 (rPanx1), membrane stretch (MS)-induced activation-measured by changes in DAPI uptake rate-was drastically reduced by either knockdown of Piezo1 or pharmacological inhibition of calmodulin or CaMKII. By site-directed mutagenesis we generated rPanx1S394A-EGFP (enhanced green fluorescent protein), which lost its sensitivity to MS, and rPanx1S394D-EGFP, mimicking phosphorylation, which shows high DAPI uptake rate without MS stimulation or cleavage of the C terminus. Using whole-cell patch-clamp and outside-out excised patch configurations, we found that rPanx1-EGFP and rPanx1S394D-EGFP channels showed current at all voltages between ±100 mV, similar single channel currents with outward rectification, and unitary conductance (â¼30 to 70 pS). However, using cell-attached configuration we found that rPanx1S394D-EGFP channels show increased spontaneous unitary events independent of MS stimulation. In silico studies revealed that phosphorylation of S394 caused conformational changes in the selectivity filter and increased the average volume of lateral tunnels, allowing ATP to be released via these conduits and DAPI uptake directly from the channel mouth to the cytoplasmic space. These results could explain one possible mechanism for activation of rPanx1 upon increase in cytoplasmic Ca2+ signal elicited by diverse physiological conditions in which the C-terminal domain is not cleaved.
Assuntos
Sinalização do Cálcio , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Conexinas/química , Conexinas/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Cálcio/metabolismo , Calmodulina/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Conexinas/genética , Citoplasma/metabolismo , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Indóis/farmacocinética , Canais Iônicos/genética , Canais Iônicos/metabolismo , Simulação de Dinâmica Molecular , Proteínas do Tecido Nervoso/genética , Técnicas de Patch-Clamp , Fosforilação , Serina/genética , Serina/metabolismoRESUMO
High order oligomers are crucial for normal cell physiology, and protein function perturbed by missense mutations underlies several autosomal dominant diseases. Dynamin-2 is one of such protein forming helical oligomers that catalyze membrane fission. Mutations in this protein, where R465W is the most frequent, cause dominant centronuclear myopathy, but the molecular mechanisms underpinning the functional modifications remain to be investigated. To unveil the structural impact of this mutation in dynamin-2, we used full-atom molecular dynamics simulations and coarse-grained models and built dimers and helices of wild-type (WT) monomers, mutant monomers, or both WT and mutant monomers combined. Our results show that the mutation R465W causes changes in the interactions with neighbor amino acids that propagate through the oligomer. These new interactions perturb the contact between monomers and favor an extended conformation of the bundle signaling element (BSE), a dynamin region that transmits the conformational changes from the GTPase domain to the rest of the protein. This extended configuration of the BSE that is only relevant in the helices illustrates how a small change in the microenvironment surrounding a single residue can propagate through the oligomer structures of dynamin explaining how dominance emerges in large protein complexes.
Assuntos
Dinamina II/genética , Miopatias Congênitas Estruturais/patologia , Domínios Proteicos/genética , Multimerização Proteica/genética , Arginina/genética , Cristalografia por Raios X , Dinamina II/metabolismo , Dinamina II/ultraestrutura , Humanos , Simulação de Dinâmica Molecular , Mutação de Sentido Incorreto , Miopatias Congênitas Estruturais/genética , Conformação Proteica em alfa-Hélice/genética , Triptofano/genéticaRESUMO
SARS-CoV-2 ORF3a is believed to form ion channels, which may be involved in the modulation of virus release, and has been implicated in various cellular processes like the up-regulation of fibrinogen expression in lung epithelial cells, downregulation of type 1 interferon receptor, caspase-dependent apoptosis, and increasing IFNAR1 ubiquitination. ORF3a assemblies as homotetramers, which are stabilized by residue C133. A recent cryoEM structure of a homodimeric complex of ORF3a has been released. A lower-resolution cryoEM map of the tetramer suggests two dimers form it, arranged side by side. The dimer's cryoEM structure revealed that each protomer contains three transmembrane helices arranged in a clockwise configuration forming a six helices transmembrane domain. This domain's potential permeation pathway has six constrictions narrowing to about 1 Å in radius, suggesting the structure solved is in a closed or inactivated state. At the cytosol end, the permeation pathway encounters a large and polar cavity formed by multiple beta strands from both protomers, which opens to the cytosolic milieu. We modeled the tetramer following the arrangement suggested by the low-resolution tetramer cryoEM map. Molecular dynamics simulations of the tetramer embedded in a membrane and solvated with 0.5 M of KCl were performed. Our simulations show the cytosolic cavity is quickly populated by both K+ and Cl-, yet with different dynamics. K+ ions moved relatively free inside the cavity without forming proper coordination sites. In contrast, Cl- ions enter the cavity, and three of them can become stably coordinated near the intracellular entrance of the potential permeation pathway by an inter-subunit network of positively charged amino acids. Consequently, the central cavity's electrostatic potential changed from being entirely positive at the beginning of the simulation to more electronegative at the end.
RESUMO
In mammals, temperature-sensitive TRP channels make membrane conductance of cells extremely temperature dependent, allowing the detection of temperature ranging from noxious cold to noxious heat. We progressively deleted the distal carboxyl terminus domain (CTD) of the cold-activated melastatin receptor channel, TRPM8. We found that the enthalpy change associated with channel gating is proportional to the length of the CTD. Deletion of the last 36 amino acids of the CTD transforms TRPM8 into a reduced temperature-sensitivity channel (Q10 â¼4). Exposing the intracellular domain to a denaturing agent increases the energy required to open the channel indicating that cold drives channel gating by stabilizing the folded state of the CTD. Experiments in the presence of an osmoticant agent suggest that channel gating involves a change in solute-inaccessible volume in the CTD of â¼1,900 Å3 This volume matches the void space inside the coiled coil according to the cryogenic electron microscopy structure of TRPM8. The results indicate that a folding-unfolding reaction of a specialized temperature-sensitive structure is coupled to TRPM8 gating.
Assuntos
Domínios Proteicos , Dobramento de Proteína , Canais de Cátion TRPM/química , Animais , Temperatura Baixa , Microscopia Crioeletrônica , Humanos , Ativação do Canal Iônico , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Oócitos , Conformação Proteica , Canais de Cátion TRPM/metabolismo , Termodinâmica , Xenopus laevisRESUMO
2,4,6-Trinitrotoluene (TNT) is a nitroaromatic explosive, highly toxic and mutagenic for organisms. In this study, we report for the first time the screening and isolation of TNT-degrading bacteria from Antarctic environmental samples with potential use as bioremediation agents. Ten TNT-degrading bacterial strains were isolated from Deception Island. Among them, Pseudomonas sp. TNT3 was selected as the best candidate since it showed the highest tolerance, growth, and TNT biotransformation capabilities. Our results showed that TNT biotransformation involves the reduction of the nitro groups. Additionally, Pseudomonas sp. TNT3 was capable of transforming 100 mg/L TNT within 48 h at 28 °C, showing higher biotransformation capability than Pseudomonas putida KT2440, a known TNT-degrading bacterium. Functional annotation of Pseudomonas sp. TNT3 genome revealed a versatile set of molecular functions involved in xenobiotic degradation pathways. Two putative xenobiotic reductases (XenA_TNT3 and XenB_TNT3) were identified by means of homology searches and phylogenetic relationships. These enzymes were also characterized at molecular level using homology modelling and molecular dynamics simulations. Both enzymes share different levels of sequence similarity with other previously described TNT-degrading enzymes and with their closest potential homologues in databases.
Assuntos
Trinitrotolueno , Regiões Antárticas , Biodegradação Ambiental , Biotransformação , Ilhas , Filogenia , PseudomonasRESUMO
The most common denominator of many of the neurodegenerative diseases is badly folded protein accumulation, which results in the formation of insoluble protein deposits located in different parts of the organism, causing cell death and tissue degeneration. Dendritic systems have turned out to be a promising new therapeutic approach for the treatment of these diseases due to their ability to modulate the folding of these proteins. With this perspective, and focused on typeâ 2 diabetes (T2D), characterized by the presence of deposits containing the amyloidogenic islet amyloid polypeptide (IAPP), we demonstrate how different topologies of cationic carbosilane dendrimers inhibit the formation of insoluble protein deposits in pancreatic islets isolated from transgenic Tg-hIAPP mice. Also, the results obtained by the modification of dendritic carbosilane wedges with the chemical chaperone 4-phenylbutyric acid (4-PBA) at the focal point confirmed their potential as anti-amyloid agents with a concentration efficiency in their therapeutic action five orders of magnitude lower than that observed for free 4-PBA. Computational studies, which determined the main interaction between IAPP and dendrimers at the atomic level, support the experimental work.
Assuntos
Amiloidose/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/química , Fenilbutiratos/química , Silanos/química , Animais , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos TransgênicosRESUMO
Amphiphilic Janus dendrimers (JDs) are repetitively branched molecules with hydrophilic and hydrophobic components that self-assemble in water to form a variety of morphologies, including vesicles analogous to liposomes with potential pharmaceutical and medical application. To date, the self-assembly of JDs has not been fully investigated thus it is important to gain insight into its mechanism and dependence on JDs' molecular structure. In this study, the aggregation behavior in water of a second-generation bis-MPA JD was evaluated using experimental and computational methods. Dispersions of JDs in water were carried out using the thin-film hydration and ethanol injection methods. Resulting assemblies were characterized by dynamic light scattering, confocal microscopy, and atomic force microscopy. Furthermore, a coarse-grained molecular dynamics (CG-MD) simulation was performed to study the mechanism of JDs aggregation. The obtaining of assemblies in water with no interdigitated bilayers was confirmed by the experimental characterization and CG-MD simulation. Assemblies with dendrimersome characteristics were obtained using the ethanol injection method. The results of this study establish a relationship between the molecular structure of the JD and the properties of its aggregates in water. Thus, our findings could be relevant for the design of novel JDs with tailored assemblies suitable for drug delivery systems.
Assuntos
Dendrímeros/química , Simulação de Dinâmica Molecular , Água/química , Interações Hidrofóbicas e Hidrofílicas , Estrutura MolecularRESUMO
BACKGROUND: Antifreeze proteins (AFPs) production is a survival strategy of psychrophiles in ice. These proteins have potential in frozen food industry avoiding the damage in the structure of animal or vegetal foods. Moreover, there is not much information regarding the interaction of Antarctic bacterial AFPs with ice, and new determinations are needed to understand the behaviour of these proteins at the water/ice interface. RESULTS: Different Antarctic places were screened for antifreeze activity and microorganisms were selected for the presence of thermal hysteresis in their crude extracts. Isolates GU1.7.1, GU3.1.1, and AFP5.1 showed higher thermal hysteresis and were characterized using a polyphasic approach. Studies using cucumber and zucchini samples showed cellular protection when samples were treated with partially purified AFPs or a commercial AFP as was determined using toluidine blue O and neutral red staining. Additionally, genome analysis of these isolates revealed the presence of genes that encode for putative AFPs. Deduced amino acids sequences from GU3.1.1 (gu3A and gu3B) and AFP5.1 (afp5A) showed high similarity to reported AFPs which crystal structures are solved, allowing then generating homology models. Modelled proteins showed a triangular prism form similar to ß-helix AFPs with a linear distribution of threonine residues at one side of the prism that could correspond to the putative ice binding side. The statistically best models were used to build a protein-water system. Molecular dynamics simulations were then performed to compare the antifreezing behaviour of these AFPs at the ice/water interface. Docking and molecular dynamics simulations revealed that gu3B could have the most efficient antifreezing behavior, but gu3A could have a higher affinity for ice. CONCLUSIONS: AFPs from Antarctic microorganisms GU1.7.1, GU3.1.1 and AFP5.1 protect cellular structures of frozen food showing a potential for frozen food industry. Modeled proteins possess a ß-helix structure, and molecular docking analysis revealed the AFP gu3B could be the most efficient AFPs in order to avoid the formation of ice crystals, even when gu3A has a higher affinity for ice. By determining the interaction of AFPs at the ice/water interface, it will be possible to understand the process of adaptation of psychrophilic bacteria to Antarctic ice.
Assuntos
Proteínas Anticongelantes/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Aminoácidos , Regiões Antárticas , Proteínas Anticongelantes/química , Proteínas Anticongelantes/genética , Bactérias/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Cucurbita/metabolismo , Cucurbitaceae/metabolismo , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , Análise de Sequência de DNA , Sequenciamento Completo do GenomaRESUMO
Dendrimers functionalized with folic acid (FA) are drug delivery systems that can selectively target cancer cells with folate receptors (FR-α) overexpression. Incorporation of polyethylene glycol (PEG) can enhance dendrimers solubility and pharmacokinetics, but ligand-receptor binding must not be affected. In this work we characterized, at atomic level, the binding functionality of conventional site-specific dendrimers conjugated with FA with PEG 750 or PEG 3350 as a linker. After Molecular Dynamics simulation, we observed that both PEG's did not interfere over ligand-receptor binding functionality. Although binding kinetics could be notably affected, the folate fragment from both dendrimers remained exposed to the solvent before approaching selectively to FR-α. PEG 3350 provided better solubility and protection from enzymatic degradation to the dendrimer than PEG 750. Also, FA-PEG3350 dendrimer showed a slightly better interaction with FR-α than FA-PEG750 dendrimer. Therefore, theoretical evidence supports that both dendrimers are suitable as drug delivery systems for cancer therapies.
Assuntos
Dendrímeros/química , Receptor 1 de Folato/química , Receptor 1 de Folato/metabolismo , Ácido Fólico/química , Polietilenoglicóis/química , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Solventes/químicaRESUMO
BACKGROUND: Nanotechnology is a science that involves imaging, measurement, modeling and a manipulation of matter at the nanometric scale. One application of this technology is drug delivery systems based on nanoparticles obtained from natural or synthetic sources. An example of these systems is synthetized from poly(3-hydroxybutyrate-co-3-hydroxyvalerate), which is a biodegradable, biocompatible and a low production cost polymer. The aim of this work was to investigate the uptake mechanism of PHBV nanoparticles in two different epithelial cell lines (HeLa and SKOV-3). RESULTS: As a first step, we characterized size, shape and surface charge of nanoparticles using dynamic light scattering and transmission electron microscopy. Intracellular incorporation was evaluated through flow cytometry and fluorescence microscopy using intracellular markers. We concluded that cellular uptake mechanism is carried out in a time, concentration and energy dependent way. Our results showed that nanoparticle uptake displays a cell-specific pattern, since we have observed different colocalization in two different cell lines. In HeLa (Cervical cancer cells) this process may occur via classical endocytosis pathway and some internalization via caveolin-dependent was also observed, whereas in SKOV-3 (Ovarian cancer cells) these patterns were not observed. Rearrangement of actin filaments showed differential nanoparticle internalization patterns for HeLa and SKOV-3. Additionally, final fate of nanoparticles was also determined, showing that in both cell lines, nanoparticles ended up in lysosomes but at different times, where they are finally degraded, thereby releasing their contents. CONCLUSIONS: Our results, provide novel insight about PHBV nanoparticles internalization suggesting that for develop a proper drug delivery system is critical understand the uptake mechanism.
Assuntos
Portadores de Fármacos/metabolismo , Sistemas de Liberação de Medicamentos , Nanopartículas/metabolismo , Neoplasias/tratamento farmacológico , Poliésteres/metabolismo , Transporte Biológico , Linhagem Celular Tumoral , Endocitose , Células HeLa , Humanos , Nanopartículas/ultraestruturaRESUMO
The exopolyphosphatase of Escherichia coli processively and completely hydrolyses long polyphosphate chains to ortho-phosphate. Genetic surveys, based on the analysis of single ppx(-) or ppk(-) mutants and on the double mutant, demonstrate a relationship between these genes and the survival capacity. The exopolyphosphatase belongs to the ASKHA protein superfamily, hence, its active site is well known; however, the knowledge of the way in which this enzyme binds polyP remains incomplete. Here we present different computational approaches, site-direct mutagenesis and kinetic data to understand the relationship between structure and function of exopolyphosphatase. We propose H(378) as a fundamental gatekeeper for the recognition of long chain polyphosphate.
Assuntos
Hidrolases Anidrido Ácido/química , Proteínas de Bactérias/química , Escherichia coli/metabolismo , Sítios de Ligação , Domínio Catalítico , Hidrogênio/química , Cinética , Conformação Molecular , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mutação , Polifosfatos/química , Ligação Proteica , Eletricidade Estática , TermodinâmicaRESUMO
Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels are involved in a large variety of physiological processes. Regulatory ß-subunits are one of the mechanisms responsible for creating BK channel diversity fundamental to the adequate function of many tissues. However, little is known about the structure of its voltage sensor domain. Here, we present the external architectural details of BK channels using lanthanide-based resonance energy transfer (LRET). We used a genetically encoded lanthanide-binding tag (LBT) to bind terbium as a LRET donor and a fluorophore-labeled iberiotoxin as the LRET acceptor for measurements of distances within the BK channel structure in a living cell. By introducing LBTs in the extracellular region of the α- or ß1-subunit, we determined (i) a basic extracellular map of the BK channel, (ii) ß1-subunit-induced rearrangements of the voltage sensor in α-subunits, and (iii) the relative position of the ß1-subunit within the α/ß1-subunit complex.
Assuntos
Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Animais , Transferência de Energia , Feminino , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Modelos Moleculares , Oócitos , Conformação Proteica , Domínios Proteicos , Xenopus laevisRESUMO
Poly(amidoamine) dendrimers are the most recognized class of dendrimer. Amino-terminated (PAMAM-NH2) and hydroxyl-terminated (PAMAM-OH) dendrimers of generation 4 are widely used, since they are commercially available. Both have different properties, mainly based on their different overall charges at physiological pH. Currently, an important function of dendrimers as carriers of short single-stranded DNA has been applied. These molecules, known as antisense oligonucleotides (asODNs), are able to inhibit the expression of a target mRNA. Whereas PAMAM-NH2 dendrimers have shown to be able to transfect plasmid DNA, PAMAM-OH dendrimers have not shown the same successful results. However, little is known about their interaction with shorter and more flexible molecules such as asODNs. Due to several initiatives, the use of these neutral dendrimers as a scaffold to introduce other functional groups has been proposed. Because of its low cytotoxicity, it is relevant to understand the molecular phenomena involving these types of dendrimers. In this work, we studied the behavior of an antisense oligonucleotide in presence of both types of dendrimers using molecular dynamics simulations, in order to elucidate if they are able to form stable complexes. In this manner, we demonstrated at atomic level that PAMAM-NH2, unlike PAMAM-OH, could form a well-compacted complex with asODN, albeit PAMAM-OH can also establish stable interactions with the oligonucleotide. The biological activity of asODN in complex with PAMAM-NH2 dendrimer was also shown. Finally, we revealed that in contact with PAMAM-OH, asODN remains outside the cells as TIRF microscopy results showed, due to its poor interaction with this dendrimer and cell membranes.
RESUMO
Polyvinylpolypyrrolidone (PVPP) is a fining agent, widely used in winemaking and brewing, whose mode of action in removing phenolic compounds has not been fully characterised. The aim of this study was to evaluate the experimental and theoretical binding affinity of PVPP towards six phenolic compounds representing different types of phenolic species. The interaction between PVPP and phenolics was evaluated in model solutions, where hydroxyl groups, hydrophobic bonding and steric hindrance were characterised. The results of the study indicated that PVPP exhibits high affinity for quercetin and catechin, moderate affinity for epicatechin, gallic acid and lower affinity for 4-methylcatechol and caffeic acid. The affinity has a direct correlation with the hydroxylation degree of each compound. The results show that the affinity of PVPP towards phenols is related with frontier orbitals. This work demonstrates a direct correlation between the experimental affinity and the interaction energy calculations obtained through computational chemistry methods.
Assuntos
Alimentos , Modelos Teóricos , Fenóis/química , Povidona/análogos & derivados , Adsorção , Catequina/química , Catequina/isolamento & purificação , Catecóis/química , Catecóis/isolamento & purificação , Alimentos/normas , Ácido Gálico/química , Ácido Gálico/isolamento & purificação , Estrutura Molecular , Fenóis/isolamento & purificação , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Povidona/química , Quercetina/química , Quercetina/isolamento & purificação , SoluçõesRESUMO
Mycobacterium tuberculosis employs various virulence strategies to subvert host immune responses in order to persist and cause disease. Interaction of M. tuberculosis with mannose receptor on macrophages via surface-exposed lipoarabinomannan (LAM) is believed to be critical for cell entry, inhibition of phagosome-lysosome fusion, and intracellular survival, but in vivo evidence is lacking. LprG, a cell envelope lipoprotein that is essential for virulence of M. tuberculosis, has been shown to bind to the acyl groups of lipoglycans but the role of LprG in LAM biosynthesis and localization remains unknown. Using an M. tuberculosis lprG mutant, we show that LprG is essential for normal surface expression of LAM and virulence of M. tuberculosis attributed to LAM. The lprG mutant had a normal quantity of LAM in the cell envelope, but its surface was altered and showed reduced expression of surface-exposed LAM. Functionally, the lprG mutant was defective for macrophage entry and inhibition of phagosome-lysosome fusion, was attenuated in macrophages, and was killed in the mouse lung with the onset of adaptive immunity. This study identifies the role of LprG in surface-exposed LAM expression and provides in vivo evidence for the essential role surface LAM plays in M. tuberculosis virulence. Findings have translational implications for therapy and vaccine development.