RESUMEN
Calcium is one of the most prominent second messengers. It is involved in a wide range of functions at the single-cell level but also in modulating regulatory mechanisms in the entire organism. One process mediating calcium signaling involves hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) by the phospholipase-C (PLC). Thus, calcium and PtdIns(4,5)P2 are intimately intertwined two second-messenger cascades that often depend on each other. Another relevant lipid associated with calcium signaling is cholesterol. Both PtdIns(4,5)P2 and cholesterol play key roles in the formation and maintenance of specialized signaling nanodomains known as lipid rafts. Lipid rafts are particularly important in calcium signaling by concentrating and localizing calcium channels such as the Orai1 channel. Depletion of internal calcium stores is initiated by the production of inositol-1,4,5-trisphosphate (IP3). Calcium depletion from the ER induces the oligomerization of STIM1, which binds Orai1 and initiates calcium influx into the cell. In the present review, we analyzed the complex interactions between cholesterol, PtdIns(4,5)P2, and the complex formed by the Orai1 channel and the signaling molecule STIM1. We explore some of the complex mechanisms governing calcium homeostasis and phospholipid metabolism, as well as the interaction between these two apparently independent signaling cascades.
Asunto(s)
Fosfatidilinositol 4,5-Difosfato , Fosfatidilinositoles , Calcio/metabolismo , Señalización del Calcio/fisiología , Membrana Celular/metabolismo , Colesterol/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositoles/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Proteína ORAI1/metabolismoRESUMEN
Objective: Low HDL-C (high-density lipoprotein cholesterol) is the most frequent dyslipidemia in Mexicans, but few studies have examined the underlying genetic basis. Our purpose was to identify genetic variants associated with HDL-C levels and cardiovascular risk in the Mexican population. Approach and Results: A genome-wide association studies for HDL-C levels in 2335 Mexicans, identified four loci associated with genome-wide significance: CETP, ABCA1, LIPC, and SIDT2. The SIDT2 missense Val636Ile variant was associated with HDL-C levels and was replicated in 3 independent cohorts (P=5.9×10−18 in the conjoint analysis). The SIDT2/Val636Ile variant is more frequent in Native American and derived populations than in other ethnic groups. This variant was also associated with increased ApoA1 and glycerophospholipid serum levels, decreased LDL-C (low-density lipoprotein cholesterol) and ApoB levels, and a lower risk of premature CAD. Because SIDT2 was previously identified as a protein involved in sterol transport, we tested whether the SIDT2/Ile636 protein affected this function using an in vitro site-directed mutagenesis approach. The SIDT2/Ile636 protein showed increased uptake of the cholesterol analog dehydroergosterol, suggesting this variant affects function. Finally, liver transcriptome data from humans and the Hybrid Mouse Diversity Panel are consistent with the involvement of SIDT2 in lipid and lipoprotein metabolism. Conclusions: This is the first genome-wide association study for HDL-C levels seeking associations with coronary artery disease in the Mexican population. Our findings provide new insight into the genetic architecture of HDL-C and highlight SIDT2 as a new player in cholesterol and lipoprotein metabolism in humans.
Asunto(s)
HDL-Colesterol/sangre , Enfermedad de la Arteria Coronaria/genética , Hiperlipoproteinemia Tipo II/genética , Proteínas de Transporte de Nucleótidos/genética , Polimorfismo de Nucleótido Simple , Adulto , Edad de Inicio , Animales , Biomarcadores/sangre , Estudios de Casos y Controles , Niño , Enfermedad de la Arteria Coronaria/sangre , Enfermedad de la Arteria Coronaria/diagnóstico , Enfermedad de la Arteria Coronaria/epidemiología , Modelos Animales de Enfermedad , Femenino , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Células HEK293 , Factores de Riesgo de Enfermedad Cardiaca , Humanos , Hiperlipoproteinemia Tipo II/sangre , Hiperlipoproteinemia Tipo II/diagnóstico , Hiperlipoproteinemia Tipo II/epidemiología , Masculino , Análisis de la Aleatorización Mendeliana , México/epidemiología , Ratones , Persona de Mediana Edad , Proteínas de Transporte de Nucleótidos/metabolismo , Fenotipo , Medición de RiesgoRESUMEN
The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are encoded by a family of four genes (HCN1-4). All isoforms are expressed in the heart, HCN4 being the most abundant in the sinoatrial node (SAN). HCN channels are responsible for the "funny" current (If) associated with the generation and autonomic control of the diastolic depolarization phase of cardiac action potential. In this work we performed a proteomic analysis of HCN4 transfected in HEK293 cells. Most of the identified proteins in the HCN4 network belonged to mitochondria. The subcellular localization of HCN channels was predicted in plasma membrane, mitochondria and nucleus. Experimentally, HCN2 (full-length, truncated), HCN3 (full-length, truncated) and HCN4 (truncated) were detected in rat heart mitochondria by immunoblotting. If sensitive to ZD7288, was recorded by patch-clamp in mitoplasts from cardiomyocytes. Mitochondrial membrane potential (ΔΨm) assessment in H9c2 cells revealed that ZD7288 induced almost 50% higher hyperpolarization respect to control at 30 min. Furthermore, ZD7288 reduced oxygen consumption attributed to ATP synthesis in H9c2 cells. In conclusion, we identify for the first time functional HCN channels in mammalian cardiac mitochondria and demonstrate their impact on ΔΨm and respiration.
Asunto(s)
Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Potencial de la Membrana Mitocondrial , Mitocondrias Cardíacas/metabolismo , Consumo de Oxígeno , Animales , Línea Celular , Células HEK293 , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/análisis , Ratones Endogámicos BALB C , Miocitos Cardíacos/metabolismo , Ratas WistarRESUMEN
BACKGROUND: The use of biomaterials has been expanded to improve the characteristics of vaccines. Recently we have identified that the peptide PH(1-110) from polyhedrin self-aggregates and incorporates foreign proteins to form particles. We have proposed that this peptide can be used as an antigen carrying system for vaccines. However, the immune response generated by the antigen fused to the peptide has not been fully characterized. In addition, the adjuvant effect and thermostability of the particles has not been evaluated. RESULTS: In the present study we demonstrate the use of a system developed to generate nano and microparticles carrying as a fusion protein peptides or proteins of interest to be used as vaccines. These particles are purified easily by centrifugation. Immunization of animals with the particles in the absence of adjuvant result in a robust and long-lasting immune response. Proteins contained inside the particles are maintained for over 1 year at ambient temperature, preserving their immunological properties. CONCLUSION: The rapid and efficient production of the particles in addition to the robust immune response they generate position this system as an excellent method for the rapid response against emerging diseases. The thermostability conferred by the particle system facilitates the distribution of the vaccines in developing countries or areas with no electricity.
Asunto(s)
Antígenos/inmunología , Inmunoglobulinas/metabolismo , Proteínas de la Matriz de Cuerpos de Oclusión/química , Péptidos/química , Vacunas/inmunología , Animales , Antígenos/química , Estabilidad de Medicamentos , Femenino , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/inmunología , Inmunización , Ratones , Nanopartículas , Tamaño de la Partícula , Agregado de Proteínas , Proteínas Recombinantes de Fusión/inmunología , Termodinámica , Vacunas/químicaRESUMEN
Hyperpolarization-activated cationic HCN channels comprise four members (HCN1-4) that control dendritic integration, synaptic transmission and action potential firing. In the kidney, HCN1, HCN2 and HCN3 are differentially expressed and contribute to the transport of sodium, potassium (K+) and ammonium into the nephrons. HCN3 is regulated by K+ diets in the kidney. In this work we performed a proteomic analysis of HCN3 expressed in human embryonic kidney cells (HEK293 cells). More than 50% of the interacting proteins belonged to mitochondria. Therefore, we explored the presence of HCN channels in kidney mitochondria. By immunoblotting and immunogold electron microscopy HCN3 protein expression was found in rat kidney mitochondria; it was also confirmed in human kidney. Patch-clamp recordings of renal mitochondria and mitochondria from HEK293 cells overexpressing HCN1, HCN2 and HCN3 channels, stained with MitoTracker Green FM, indicated that only HCN3 could produce inwardly K+ currents that were inhibited by ZD7288, a specific blocker of HCN channels. Furthermore, ZD7288 caused inhibition of the oxygen consumption coupled to ATP synthesis and hyperpolarization of the inner mitochondrial membrane. In conclusion, we show for the first time that pacemaker HCN channels contribute to K+ transport in mitochondria facilitating the activity of the respiratory chain and ATP synthesis by controlling the inner mitochondrial membrane potential.
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Riñón/metabolismo , Mitocondrias/metabolismo , Canales de Potasio/metabolismo , Potenciales de Acción , Respiración de la Célula , Cromatografía Liquida , Activación del Canal Iónico , Mitocondrias/genética , Nucleótidos Cíclicos/metabolismo , Proteoma , Proteómica/métodos , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: Baculoviruses are widely used for the production of recombinant proteins, biopesticides and as gene delivery systems. One of the viral forms called polyhedra has been recently exploited as a scaffold system to incorporate or encapsulate foreign proteins or peptide fragments. However, an efficient strategy for foreign protein incorporation has not been thoroughly studied. RESULTS: Based on the crystal structure of polyhedrin, we conducted an in silico analysis of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) polyhedrin protein to select the minimum fragments of polyhedrin that could be incorporated into polyhedra. Using confocal and transmission electron microscopy we analyzed the expression and cellular localization of the different polyhedrin fragments fused to the green fluorescent protein (EGFP) used as reporter. The amino fragment 1-110 contains two repeats formed each of two ß sheets followed by a α helix (amino acids 1-58 and 58-110) that are important for the formation and stability of polyhedra. These fragments 1-58, 58-110 and 1-110 could be incorporated into polyhedra. However, only fragments 1-110 and 58-110 can self-aggregate. CONCLUSIONS: These results demonstrate that 58-110 is the minimum fragment that contributes to the assembly of the recombinant polyhedra via self-aggregation. This is the minimum sequence that can be used to efficiently incorporate foreign proteins into polyhedra.
Asunto(s)
Biología Computacional , Nucleopoliedrovirus/genética , Agregado de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Estructurales Virales/química , Proteínas Estructurales Virales/genética , Animales , Citoplasma/genética , Proteínas Fluorescentes Verdes/genética , Proteínas de la Matriz de Cuerpos de Oclusión , Proteínas Recombinantes de Fusión/biosíntesis , Células Sf9 , SpodopteraRESUMEN
Baculoviridae is a large family of double-stranded DNA viruses that selectively infect insects. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the best-studied baculovirus from the family. Many studies over the last several years have shown that AcMNPV can enter a wide variety of mammalian cells and deliver genetic material for foreign gene expression. While most animal viruses studied so far have developed sophisticated mechanisms to selectively infect specific cells and tissues in an organism, AcMNPV can penetrate and deliver foreign genes into most cells studied to this date. The details about the mechanisms of internalization have been partially described. In the present study, we have identified a cholesterol recognition amino acid consensus (CRAC) domain present in the AcMNPV envelope fusion protein GP64. We demonstrated the association of a CRAC domain with cholesterol, which is important to facilitate the anchoring of the virus at the mammalian cell membrane. Furthermore, this initial anchoring favors AcMNPV endocytosis via a dynamin- and clathrin-dependent mechanism. Under these conditions, efficient baculovirus-driven gene expression is obtained. In contrast, when cholesterol is reduced from the plasma membrane, AcMNPV enters the cell via a dynamin- and clathrin-independent mechanism. The result of using this alternative internalization pathway is a reduced level of baculovirus-driven gene expression. This study is the first to document the importance of a novel CRAC domain in GP64 and its role in modulating gene delivery in AcMNPV.
Asunto(s)
Secuencias de Aminoácidos , Baculoviridae/fisiología , Colesterol/metabolismo , Proteínas Virales de Fusión/metabolismo , Acoplamiento Viral , Línea Celular , Endocitosis , Humanos , Proteínas Virales de Fusión/genéticaRESUMEN
Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2n first appeared in Wuhan, China in 2019. Soon after, it was declared a pandemic by the World Health Organization. The health crisis imposed by a new virus and its rapid spread worldwide prompted the fast development of vaccines. For the first time in human history, two vaccines based on recombinant genetic material technology were approved for human use. These mRNA vaccines were applied in massive immunization programs around the world, followed by other vaccines based on more traditional approaches. Even though all vaccines were tested in clinical trials prior to their general administration, serious adverse events, usually of very low incidence, were mostly identified after application of millions of doses. Establishing a direct correlation (the cause-effect paradigm) between vaccination and the appearance of adverse effects has proven challenging. This review focuses on the main adverse effects observed after vaccination, including anaphylaxis, myocarditis, vaccine-induced thrombotic thrombocytopenia, Guillain-Barré syndrome, and transverse myelitis reported in the context of COVID-19 vaccination. We highlight the symptoms, laboratory tests required for an adequate diagnosis, and briefly outline the recommended treatments for these adverse effects. The aim of this work is to increase awareness among healthcare personnel about the serious adverse events that may arise post-vaccination. Regardless of the ongoing discussion about the safety of COVID-19 vaccination, these adverse effects must be identified promptly and treated effectively to reduce the risk of complications.
Asunto(s)
Vacunas contra la COVID-19 , COVID-19 , Humanos , Vacunas contra la COVID-19/efectos adversos , COVID-19/prevención & control , COVID-19/epidemiología , Incidencia , Vacunación/efectos adversos , Anafilaxia/inducido químicamente , Anafilaxia/etiología , SARS-CoV-2/inmunología , Síndrome de Guillain-Barré/etiología , Miocarditis/etiología , Miocarditis/inducido químicamenteRESUMEN
STIM1 and Orai1 are the central core of the Store Operated Calcium Entry (SOCE). This calcium influx mechanism is triggered after the activation of Gq protein-coupled receptors at the plasma membrane (PM) that activate phospholipase C. The phospholipase C produces Inositol triphosphate (IP3) which rapidly diffuses throughout the cytosol, resulting in the binding and activation of IP3 receptors (IP3R) and the rapid efflux of calcium from the endoplasmic reticulum (ER) to the cytosol. The calcium depletion in the ER is sensed by the stromal interaction molecule 1 (STIM1) a single-pass transmembrane protein at the ER that binds intraluminal calcium through an EF-hand domain in its amino terminal region (Fig. 1A). The cytosolic portion of STIM1 contains multiple domains. The region that interacts and activates Orai channels is known as SOAR (the STIM1 Orai activating region) [1]. For SOAR be accessible to Orai1, STIM1 must get an extended conformation that unlocks SOAR from its coiled-coil 1 (CC1) region [2]. The extended conformation is triggered by calcium depletion at the ER that oligomerizes STIM1. The oligomers of STIM1 then translocate to a close distance between two opposing membranes, forming what is known as ER-PM junctions. STIM1 accumulates at ER-PM junctions conforming the denominated STIM1 puncta.
Asunto(s)
Calcio , Fosfatos de Inositol , Calcio/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Membrana Celular/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Fosfatos de Inositol/metabolismo , Proteína ORAI1/metabolismoRESUMEN
SIDT2 is a lysosomal protein involved in the degradation of nucleic acids and the transport of cholesterol between membranes. Previous studies identified two "cholesterol recognition/interaction amino acid consensus" (CRAC) motifs in SIDT1 and SIDT2 members. We have previously shown that the first CRAC motif (CRAC-1) is essential for protein translocation to the PM upon cholesterol depletion in the cell. In the present study, we show that SIDT2 and the apolipoprotein A1 (ApoA1) form a complex which requires the second CRAC-2 motif in SIDT2 to be established. The overexpression of SIDT2 and ApoA1 results in enhanced ApoA1 secretion by HepG2 cells. This is not observed when overexpressing the SIDT2 with the CRAC-2 domain mutated to render it unfunctional. All these results provide evidence of a novel role for SIDT2 as a protein forming a complex with ApoA1 and enhancing its secretion to the extracellular space.
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Apolipoproteína A-I , Hepatocitos , Transporte de Proteínas , Hepatocitos/metabolismo , Colesterol/metabolismo , Lisosomas/metabolismoRESUMEN
We have studied Danio rerio (Zebrafish) TRPA1 channel using a method that combines single channel electrophysiological and optical recordings to evaluate lateral mobility and channel gating simultaneously in single channels. TRPA1 channel activation by two distinct chemical ligands: allyl isothiocyanate (AITC) and TRPswitch B, results in substantial reduction of channel lateral mobility at the plasma membrane. Incubation with the cholesterol sequestering agent methyl-ß-cyclodextrin (MßCD), prevents the reduction on lateral mobility induced by the two chemical agonists. This results strongly suggest that the open conformation of TRPA1 modulates channel lateral mobility probably by facilitating the insertion of the channel into cholesterol-enriched domains at the plasma membrane.
Asunto(s)
Canales de Potencial de Receptor Transitorio , Animales , Canal Catiónico TRPA1 , Canales de Potencial de Receptor Transitorio/metabolismo , Pez Cebra/metabolismo , Fenómenos Electrofisiológicos , ColesterolRESUMEN
Baculoviridae is a large family of arthropod-infective viruses. Recombinant baculoviruses have many applications, the best known is as a system for large scale protein production in combination with insect cell cultures. More recently recombinant baculoviruses have been utilized for the display of proteins of interest with applications in medicine. In the present review we analyze the different strategies for the display of proteins and peptides on the surface of recombinant baculoviruses and provide some examples of the different proteins displayed. We analyze briefly the commercially available systems for recombinant baculovirus production and display and discuss the future of this emerging and powerful technology.
Asunto(s)
Artrópodos , Baculoviridae , Animales , Baculoviridae/genética , Péptidos/genética , Técnicas de Cultivo de CélulaRESUMEN
We have developed a novel microarray system based on three technologies: 1) molecular beacons designed to interact with DNA targets at room temperature (25-27°C), 2) tridimensional silk-based microarrays containing the molecular beacons immersed in the silk hydrogel, and 3) shallow angle illumination, which uses separated optical pathways for excitation and emission. Unlike conventional microarrays that exhibit reduced signal-to-background ratio, require several stages of incubation, rinsing, and stringency control, and measure only end-point results, our microarray technology provides enhanced signal-to-background ratio (achieved by separating the optical pathways for excitation and emission, resulting in reduced stray light), performs analysis rapidly in one step without the need for labeling DNA targets, and measures the entire course of association kinetics between target DNA and the molecular beacons. To illustrate the benefits of our technology, we conducted microarray assays designed for the identification of influenza viruses. We show that in a single microarray slide, we can identify the virus subtype according to the molecular beacons designed for hemagglutinin (H1, H2, and H3) and neuraminidase (N1, N2). We also show the identification of human and swine influenza using sequence-specific molecular beacons. This microarray technology can be easily implemented for reagentless point-of-care diagnostics of several contagious diseases, including coronavirus variants responsible for the current pandemic.
RESUMEN
The use of a variety of techniques based on super-resolution (SR) microscopy unveiled a close and complex relationship between cytoskeleton reorganization and SOCE. By using SR microscopy many new proteins involved in SOCE regulation have been identified over the last few years. Many enigmas remain unsolved in this highly dynamic field, however, recent developments in SR microscopy promise new answers soon. In the present review, we describe the most relevant findings in SOCE components and SOCE modulation using different methods derived from SR microscopy.
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Calcio , Microscopía , Calcio/metabolismo , Señalización del Calcio/fisiología , Proteína ORAI1/metabolismo , Molécula de Interacción Estromal 1/metabolismoRESUMEN
Polyhedrins are viral proteins present in a large family of baculoviruses that form occlusion bodies (polyhedra). These structures protect the virus particles from the outside environment until they are ingested by susceptible insects. Occluded viruses can sustain inclement weather for long periods of time. Therefore, the polyhedra is a natural preservative that keeps the viral structure intact at ambient temperature for years. In a previous study we identified the first 110 amino acids from polyhedrin (PH(1-110)) as a good candidate to carry antigens of interest. As a proof of concept, we produced a fusion protein with PH(1-110) and the green fluorescent protein (PH(1-110)GFP). The fusion protein associates spontaneously during its synthesis resulting in the formation of nanoparticles. Nasal immunization with these nanoparticles and in the absence of any adjuvant, results in a robust immune response with the production of IgG immunoglobulins that remained elevated for months and that selectively recognize the GFP but not PH(1-110). These results indicate that PH(1-110) is poorly immunogenic but capable of enhancing the immune response to GFP.
Asunto(s)
Nanopartículas , Vacunas , Temperatura , Antígenos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismoRESUMEN
In the present work we describe a novel system for the identification of microRNAs (miRNAs) in fluids. The method is based on combined novel 3D microarray technology using silk as scaffold and total internal reflection fluorescence microscopy (TIRFM), which allows for the rapid identification of miRNAs using a portable device.
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MicroARNs/análisis , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Seda/química , Biomarcadores de Tumor/genética , Diseño de Equipo , Geles/química , Células HEK293 , Humanos , MicroARNs/genética , MicroARNs/aislamiento & purificación , Análisis de Secuencia por Matrices de Oligonucleótidos/métodosRESUMEN
Coronavirus 19 Disease (COVID-19) originating in the province of Wuhan, China in 2019, is caused by the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), whose infection in humans causes mild or severe clinical manifestations that mainly affect the respiratory system. So far, the COVID-19 has caused more than 2 million deaths worldwide. SARS-CoV-2 contains the Spike (S) glycoprotein on its surface, which is the main target for current vaccine development because antibodies directed against this protein can neutralize the infection. Companies and academic institutions have developed vaccines based on the S glycoprotein, as well as its antigenic domains and epitopes, which have been proven effective in generating neutralizing antibodies. However, the emergence of new SARS-CoV-2 variants could affect the effectiveness of vaccines. Here, we review the different types of vaccines designed and developed against SARS-CoV-2, placing emphasis on whether they are based on the complete S glycoprotein, its antigenic domains such as the receptor-binding domain (RBD) or short epitopes within the S glycoprotein. We also review and discuss the possible effectiveness of these vaccines against emerging SARS-CoV-2 variants.
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Vacunas contra la COVID-19/inmunología , COVID-19/inmunología , Epítopos Inmunodominantes/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Vacunas contra la COVID-19/genética , Humanos , Evasión Inmune , Inmunogenicidad Vacunal , Mutación , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Resultado del TratamientoRESUMEN
N-methyl-D-aspartate receptors (NMDAR) are glutamate-gated calcium channels named after their artificial agonist. NMDAR are implicated in cell proliferation under normal and pathophysiological conditions. However, the role of NMDAR during mitosis has not yet been explored in individual cells. We found that neurotransmitter-evoked calcium entry via endogenous NMDAR in cortical astrocytes was transient during mitosis. The same occurred in HEK293 cells transfected with the NR1/NR2A subunits of NMDAR. This transient calcium entry during mitosis was due to phosphorylation of the first intracellular loop of NMDAR (S584 of NR1 and S580 of NR2A) by cyclin B/CDK1. Expression of phosphomimetic mutants resulted in transient calcium influx and enhanced NMDAR inactivation independent of the cell cycle phase. Phosphomimetic mutants increased entry of calcium in interphase and generated several alterations during mitosis: increased mitotic index, increased number of cells with lagging chromosomes and fragmentation of pericentriolar material. In summary, by controlling cytosolic calcium, NMDAR modulate mitosis and probably cell differentiation/proliferation. Our results suggest that phosphorylation of NMDAR by cyclin B/CDK1 during mitosis is required to preserve mitotic fidelity. Altering the modulation of the NMDAR by cyclin B/CDK1 may conduct to aneuploidy and cancer.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Calcio/metabolismo , Ciclina B/metabolismo , Mitosis/fisiología , Receptores de N-Metil-D-Aspartato , Animales , Astrocitos/metabolismo , Células Cultivadas , Células HEK293 , Humanos , Masculino , Fosforilación , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
Porcine Circovirus Type 2 (PCV2) is one of the most important pathogens in pigs around the world. PCV2 is a non-enveloped virus and its capsid is formed by a single protein known as open reading frame 2 (ORF2). The aim of this study was to evaluate the antigenicity and immunogenicity of genetically-encoded protein nanoparticles (NPs) containing ORF2 from PCV2 fused to the first 110 amino acids of the N-terminus of polyhedrin from the insect virus Autographa californica nucleopolyhedrovirus (PH(1â¯-1â¯1â¯0)). Our group has previously described that some polyhedrin fragments self-aggregate forming polyhedra-like particles. We identified a self-aggregating signal within the first 110 amino acids from polyhedrin (PH(1â¯-1â¯1â¯0)). Fusing the ORF2 from PCV2 to the carboxyl terminus from PH(1â¯-1â¯1â¯0) results in the formation of NPs which incorporate the antigen of interest. Using this system we synthesized NPs containing PH(1â¯-1â¯1â¯0) fused to ORF2 (PH(1â¯-1â¯1â¯0)PCV2) and purify them to immunize pigs and evaluate the humoral immune response generated by these NPs comparing them to a commercially available vaccine. Pigs immunized with PH(1â¯-1â¯1â¯0)PCV2 NPs produced antibodies against ORF2 from PCV2 as indicated by western blot and ELISA analysis. Antibodies obtained with PH(1â¯-1â¯1â¯0)PCV2 NPs were comparable to those obtained using a commercial PCV2 vaccine. These antibodies neutralized the infection of a recombinant PCV2 expressing the green fluorescent protein (GFP). These results together suggest that the self-aggregating peptide PH(1â¯-1â¯1â¯0) can be used for the synthesis of subunit vaccines against PCV2.
Asunto(s)
Infecciones por Circoviridae/veterinaria , Circovirus/genética , Circovirus/inmunología , Nanopartículas , Sistemas de Lectura Abierta/inmunología , Síndrome Multisistémico de Emaciación Posdestete Porcino/prevención & control , Vacunas Virales/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Femenino , Inmunización , Masculino , Sistemas de Lectura Abierta/genética , Porcinos , Vacunas de Subunidad , Vacunas Virales/química , Vacunas Virales/genéticaRESUMEN
Store-operated calcium entry (SOCE) is an essential calcium influx mechanism in animal cells. One of the most important auto regulatory control systems involves calcium-dependent inactivation (CDI) of the Orai channel, which prevents excessive calcium influx. In the present study we analyze the role of two channels in the induction of CDI on Orai1. Here we show that calcium entering through freely diffusing TRPV1 channels induce strong CDI on Orai1 while calcium entering through P2X4 channel does not. TRPV1 can induce CDI on Orai1 because both channels were found in close proximity in the cell membrane. This was not observed with P2X4 channels. To our knowledge, this is the first study demonstrating that calcium arising from different channels may contribute to the modulation of Orai1 through CDI in freely diffusing single channels of living cells. Our results highlight the role of TRPV1-mediated CDI on Orai1 in cell migration and wound healing.