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BACKGROUND: Gene transfer into the inner ear is a promising approach for treating sensorineural hearing loss. The special electrochemical environment of the scala media raises a formidable challenge for effective gene delivery at the same time as keeping normal cochlear function intact. The present study aimed to define a suitable strategy for preserving hearing after viral inoculation directly into the scala media performed at various postnatal developmental stages. METHODS: We assessed transgene expression of green fluorescent protein (GFP) mediated by various types of adeno-associated virus (AAV) and lentivirus (LV) in the mouse cochlea. Auditory brainstem responses were measured 30 days after inoculation to assess effects on hearing. RESULTS: Patterns of GFP expression confirmed extensive exogenous gene expression in various types of cells lining the endolymphatic space. The use of different viral vectors and promoters resulted in specific cellular GFP expression patterns. AAV2/1 with cytomegalovirus promoter apparently gave the best results for GFP expression in the supporting cells. Histological examination showed normal cochlear morphology and no hair cell loss after either AAV or LV injections. We found that hearing thresholds were not significantly changed when the injections were performed in mice younger than postnatal day 5, regardless of the type of virus tested. CONCLUSIONS: Viral inoculation and expression in the inner ear for the restoration of hearing must not damage cochlear function. Using normal hearing mice as a model, we have achieved this necessary step, which is required for the treatment of many types of congenital deafness that require early intervention.
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Umbral Auditivo/efectos de los fármacos , Conducto Coclear/metabolismo , Potenciales Evocados Auditivos del Tronco Encefálico/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Terapia Genética/métodos , Vectores Genéticos/farmacología , Pérdida Auditiva Sensorineural/terapia , Animales , Umbral Auditivo/fisiología , Dependovirus , Potenciales Evocados Auditivos del Tronco Encefálico/fisiología , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Técnicas Histológicas , Lentivirus , Ratones , Transgenes/genéticaRESUMEN
Among all available antimicrobials, antibiotics hold a prime position in the treatment of infectious diseases. However, the emergence of antimicrobial resistance (AMR) has posed a serious threat to the effectiveness of antibiotics, resulting in increased morbidity, mortality, and escalation in healthcare costs causing a global health crisis. The overuse and misuse of antibiotics in global healthcare setups have accelerated the development and spread of AMR, leading to the emergence of multidrug-resistant (MDR) pathogens, which further limits treatment options. This creates a critical need to explore alternative approaches to combat bacterial infections. Phytochemicals have gained attention as a potential source of alternative medicine to address the challenge of AMR. Phytochemicals are structurally and functionally diverse and have multitarget antimicrobial effects, disrupting essential cellular activities. Given the promising results of plant-based antimicrobials, coupled with the slow discovery of novel antibiotics, it has become highly imperative to explore the vast repository of phytocompounds to overcome the looming catastrophe of AMR. This review summarizes the emergence of AMR towards existing antibiotics and potent phytochemicals having antimicrobial activities, along with a comprehensive overview of 123 Himalayan medicinal plants reported to possess antimicrobial phytocompounds, thus compiling the existing information that will help researchers in the exploration of phytochemicals to combat AMR.
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Mutations in Gjb2 and Gjb6 genes, coding for connexin26 (Cx26) and Cx30 proteins, respectively, are linked to about half of all cases of human autosomal non-syndromic prelingual deafness. Molecular mechanisms of the hearing impairments, however, are unclear. Most cochlear gap junctions (GJs) are co-assembled from Cx26 and Cx30 and deletion of either one of them causes deafness. Our previous studies have shown that normal hearing is possible in the absence of the Cx30 gene when Cx26 is over-expressed. To further test unique functional requirements for various types of connexins in the hearing, we investigated whether the hearing in the conditional Cx26 (cCx26) null mice could be rescued by genetically over-expressing Cx30. Multiple lines of control and experimental mouse models were used. Auditory brainstem response (ABR) measurements showed normal hearing in targeted gene deletion mice when the deleted Cx26 or Cx30 was transgenically expressed from integrated bacterial artificial chromosome (BAC), demonstrating the effectiveness of the BAC rescue approach. In contrast, severe hearing loss was found in cCx26 null mice in which Cx30 was over-expressed. Morphology observations were consistent with the ABR data. Cochleae of cCx26 null mice with and without the transgenic over-expression of Cx30 both showed the typical immature feature of postnatal cochlear development-the closed tunnel of Corti. Immunolabeling data and Western blot quantification indicated that the Cx26 protein expression preceded that of Cx30 during the early postnatal period in the cochlea. Null expression of Cx26 may therefore uniquely result in a transient period when a total elimination of GJs in functionally-important regions of the developing cochlea is possible. We conclude that Cx26 plays an essential role in the development of the auditory sensory epithelium and its unique developmental functions required for normal hearing is not replaceable by Cx30.
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Cóclea/embriología , Conexinas/biosíntesis , Uniones Comunicantes/metabolismo , Animales , Cromosomas Artificiales Bacterianos/genética , Cóclea/anatomía & histología , Cóclea/metabolismo , Conexina 26 , Conexina 30 , Conexinas/genética , Eliminación de Gen , Pérdida Auditiva/genética , Humanos , Ratones , Ratones TransgénicosRESUMEN
Shape of the protein stability curves changes to achieve higher melting temperature. Broadly, these changes have been classified as upward shift (increased G(s)), rightward shift (increase in T(s)) and flattening of the stability curves (decrease in C(p)). Comparative studies on homologous mesophilic-thermophilic protein pairs highlighted the differential contribution of these three strategies amongst proteins. But unambiguous way of identification of the strategies, which will be preferred for a protein, is still not achieved. We have performed comparative thermodynamic studies using differential scanning calorimeter (DSC) on thermostable variants of a lipase from Bacillus subtilis. These variants are products of 1, 2, 3 and 4 rounds of directed evolution and harbor mutations having definite contribution in thermostability unlike natural thermophilic proteins. We have shown that upward and rightward shift in stability curves are prime strategies in this lipase. Our results along with that from the other study on laboratory evolved xylanase A suggest that optimization of suboptimal thermodynamic parameters is having a dominant influence in selection of thermodynamic strategies for higher thermostability.
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Bacillus subtilis/enzimología , Evolución Molecular Dirigida , Lipasa/química , Lipasa/genética , Mutación/genética , Bacillus subtilis/genética , Rastreo Diferencial de Calorimetría , Estabilidad de Enzimas , Calor , TermodinámicaRESUMEN
Understanding the structural basis of altered properties of proteins due to changes in temperature or pH provides useful insights in designing proteins with improved stability. Here we report the basis for the pH-dependent thermostability of the Bacillus subtilis lipase (Lip A) using spectroscopic and X-ray crystallographic studies. At pH values above 7, lipase denatures and aggregates when heated at temperatures above 45 degrees C. However, at pH below 6 lipase denatures upon heating but the activity and its native structure is completely recovered upon cooling. In order to obtain the structural basis of this unusual stability of lipase, we determined high-resolution crystal structures of the lipase in two different crystal forms at pH 4.5 and 5. These structures show linear oligomerization of lipase using only two types of dimeric associations and these inter-molecular interactions are completely absent in several crystal forms of wild-type and mutant proteins obtained at basic pH. In accordance with the crystallographic studies, spectroscopic investigations reveal an invariant secondary structure in the pH range of 4-10. Quaternary organization of lipase at low pH resulted in changes in the tryptophan environment and binding of 1-anilino-8-naphthalene sulfate (ANS) at low pH. Low pH stability of the lipase is not observed in the presence of sodium chloride (>0.2 M) indicating the importance of ionic interactions at low pH. Inter- and intra-molecular ionic interactions that occur at pH below 6.0 are proposed to trap the molecule in a conformation that allows its complete refolding upon cooling.
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Bacillus subtilis/enzimología , Lipasa/química , Lipasa/metabolismo , Bacillus subtilis/genética , Sitios de Unión , Dicroismo Circular , Cristalografía por Rayos X , Dimerización , Estabilidad de Enzimas , Calor , Concentración de Iones de Hidrógeno , Cinética , Lipasa/genética , Modelos Moleculares , Pliegue de Proteína , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , TemperaturaRESUMEN
Mutations in the genes coding for connexin 26 (Cx26) and connexin 31 (Cx31) cause non-syndromic deafness. Here, we provide evidence that mutations at these two connexin genes can interact to cause hearing loss in digenic heterozygotes in humans. We have screened 108 GJB2 heterozygous Chinese patients for mutations in GJB3 by sequencing. We have excluded the possibility that mutations in exon 1 of GJB2 and the deletion of GJB6 are the second mutant allele in these Chinese heterozygous probands. Two different GJB3 mutations (N166S and A194T) occurring in compound heterozygosity with the 235delC and 299delAT of GJB2 were identified in three unrelated families (235delC/N166S, 235delC/A194T and 299delAT/A194T). Neither of these mutations in Cx31 was detected in DNA from 200 unrelated Chinese controls. Direct physical interaction of Cx26 with Cx31 is supported by data showing that Cx26 and Cx31 have overlapping expression patterns in the cochlea. In addition, by coimmunoprecipitation of mouse cochlear membrane proteins, we identified the presence of heteromeric Cx26/Cx31 connexons. Furthermore, by cotransfection of mCherry-tagged Cx26 and GFP-tagged Cx31 in human embryonic kidney (HEK)-293 cells, we demonstrated that the two connexins were able to co-assemble in vitro in the same junction plaque. Together, our data indicate that a genetic interaction between these two connexin genes can lead to hearing loss.
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Conexinas/genética , Sordera/genética , Mutación , Animales , Western Blotting , Línea Celular , Cóclea/metabolismo , Conexina 26 , Conexinas/metabolismo , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos , Linaje , Mapeo de Interacción de ProteínasRESUMEN
The molecular mechanisms underlying polarized sorting of proteins in neurons are poorly understood. Here we report the identification of a 16 amino-acid, dileucine-containing motif that mediates dendritic targeting in a variety of neuronal cell types in slices of rat brain. This motif is present in the carboxy (C) termini of Shal-family K+ channels and is highly conserved from C. elegans to humans. It is necessary for dendritic targeting of potassium channel Kv4.2 and is sufficient to target the axonally localized channels Kv1.3 and Kv1.4 to the dendrites. It can also mediate dendritic targeting of a non-channel protein, CD8.
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Dendritas/metabolismo , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/metabolismo , Secuencias de Aminoácidos/genética , Secuencias de Aminoácidos/fisiología , Secuencia de Aminoácidos , Animales , Antígenos CD8/genética , Antígenos CD8/metabolismo , Células Cultivadas , Secuencia Conservada , Endocitosis , Técnicas In Vitro , Canal de Potasio Kv1.3 , Canal de Potasio Kv1.4 , Leucina , Datos de Secuencia Molecular , Canales de Potasio/genética , Transporte de Proteínas/fisiología , Células Piramidales/metabolismo , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Canales de Potasio ShalRESUMEN
Kinesin-13s are critical microtubule regulators which induce microtubule disassembly in an ATP dependent manner. To clarify their mechanism, we report here the crystal structure of a functional construct of the kinesin-13 Kif2C/MCAK in an ATP-like state and bound to the αß-tubulin heterodimer, a complex mimicking the species that dissociates from microtubule ends during catalytic disassembly. Our results picture how Kif2C stabilizes a curved tubulin conformation. The Kif2C α4-L12-α5 region undergoes a remarkable 25° rotation upon tubulin binding to target the αß-tubulin hinge. This movement leads the ß5a-ß5b motif to interact with the distal end of ß-tubulin, whereas the neck and the KVD motif, two specific elements of kinesin-13s, target the α-tubulin distal end. Taken together with the study of Kif2C mutants, our data suggest that stabilization of a curved tubulin is an important contribution to the Kif2C mechanism.Kinesin-13s are microtubule depolymerizing enzymes. Here the authors present the crystal structure of a DARPin fused construct comprising the short neck region and motor domain of kinesin-13 in complex with an αß-tubulin heterodimer, which shows that kinesin-13 functions by stabilizing a curved tubulin conformation.
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Cinesinas/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Cristalización , Escherichia coli , Regulación Enzimológica de la Expresión Génica , Cinesinas/química , Microtúbulos , Mutación , Conformación ProteicaRESUMEN
OBJECTIVES: Mutations in connexin (Cx) 26 are the most common cause of nonsyndromic hereditary hearing impairments. Our goal is to investigate molecular mechanisms responsible for hearing impairment caused by various types of Cx26 mutations. STUDY DESIGN: Mutant Cxs linked to deafness were expressed in HEK293 cells. The permeability of reconstituted gap junctions (GJs) and hemichannels were studies. METHODS: HEK293 cells were used to reconstitute GJs and hemichannels in vitro. Ionic as well as biochemical permeabilities of reconstituted GJs were evaluated. RESULTS: We found two-point substitution mutations located in the first extracellular loop of Cx26, E47K, and G45E affected the GJ functions in dramatically different manners. E47K mutant Cx26 formed nonfunctional GJs that lacked GJ- and hemichannel-mediated biochemical and ionic coupling. In contrast, G45E mutation resulted in apoptosis and cell death within 24 hours of transfection. Increasing concentration of extracellular calcium ([Ca2+]0) rescued the cells in a dose-dependent manner. The rescued cells formed functional G45E GJs permeable to both ions and fluorescent tracer molecules. CONCLUSIONS: The primary effect of G45E Cx26 mutation is to cause leaky GJ hemichannels when cells are bathed in normal [Ca2+]0. Our data showed that abnormally open hemichannels with resultant cell death, in addition to GJ and hemichannel uncoupling, is a novel molecular mechanism by which Cx26 mutations may result in hearing impairment. One plausible therapeutic strategy for this type of Cx mutation, therefore, is to manipulate [Ca2+]0 and/or the Ca-binding affinity of GJ hemichannels.
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Muerte Celular/fisiología , Conexinas/genética , Sordera/genética , Sordera/fisiopatología , Uniones Comunicantes/fisiología , Apoptosis/fisiología , Células Cultivadas , Conexina 26 , Humanos , Mutagénesis Sitio-Dirigida , TransfecciónRESUMEN
Affinity maturation by random mutagenesis and selection is an established technique to make binding molecules more suitable for applications in biomedical research, diagnostics and therapy. Here we identified an unexpected novel mechanism of affinity increase upon in vitro evolution of a tubulin-specific designed ankyrin repeat protein (DARPin). Structural analysis indicated that in the progenitor DARPin the C-terminal capping repeat (C-cap) undergoes a 25° rotation to avoid a clash with tubulin upon binding. Additionally, the C-cap appears to be involved in electrostatic repulsion with tubulin. Biochemical and structural characterizations demonstrated that the evolved mutants achieved a gain in affinity through destabilization of the C-cap, which relieves the need of a DARPin conformational change upon tubulin binding and removes unfavorable interactions in the complex. Therefore, this specific case of an order-to-disorder transition led to a 100-fold tighter complex with a subnanomolar equilibrium dissociation constant, remarkably associated with a 30% decrease of the binding surface.
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Repetición de Anquirina , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Tubulina (Proteína)/química , Secuencias de Aminoácidos , Ancirinas/química , Dicroismo Circular , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática , Cinética , Modelos Moleculares , Mutagénesis , Mutación , Unión Proteica , Ingeniería de Proteínas , Ribosomas/química , Espectrometría de Fluorescencia , Resonancia por Plasmón de SuperficieRESUMEN
At high temperatures, protein stability is influenced by chemical alterations; most important among them is deamidation of asparagines. Deamidation kinetics of asparagines depends on the local sequence, solvent, pH, temperature, and the tertiary structure. Suitable replacement of deamidated asparagines could be a viable strategy to improve deamidation-mediated loss in protein properties, specifically protein thermostability. In this study, we have used nano RP-HPLC coupled ESI MS/MS approach to identify residues susceptible to deamidation in a lipase (6B) on heat treatment. Out of 15 asparagines and six glutamines in 6B, only five asparagines were susceptible to deamidation at temperatures higher than 75°C. These five positions were subjected to site saturation mutagenesis followed by activity screen to identify the most suitable substitutions. Only three of the five asparagines were found to be tolerant to substitutions. Best substitutions at these positions were combined into a mutant. The resultant lipase (mutC) has near identical secondary structure and improved thermal tolerance as compared to its parent. The triple mutant has shown almost two-fold higher residual activity compared to 6B after four cycles at 90°C. MutC has retained more than 50% activity even after incubation at 100°C. Engineering asparagines susceptible to deamidation would be a potential strategy to improve proteins to withstand very high temperatures.
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Amidas/metabolismo , Asparagina/metabolismo , Bacillus subtilis/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sustitución de Aminoácidos , Bacillus subtilis/genética , Dominio Catalítico , Estabilidad de Enzimas , Pliegue de Proteína , Estructura Secundaria de Proteína , TemperaturaRESUMEN
Vitamin C (VC) is a well-known antioxidant and strong free radical scavenger. Its antioxidant activity is useful for protection of cellular macromolecules, particularly DNA, from oxidative damage induced by different agents. This study was undertaken to evaluate the optimum level of VC in attenuating the chromosome aberrations (CAs) and DNA damage after iron sulfate (FeSO4) acute administration in Wistar rats. The results exhibited that the increase of CAs and DNA damage induced by FeSO4, 200 mg Fe/kg, could be reduced significantly by VC pretreatment at the dose of 500 mg/kg (p<0.001), but not in the 100 mg/kg group. The findings provide evidence that VC at the dose of 500 mg/kg exerted a possible protective effect against FeSO4 induced CAs and DNA damage. The possible mechanisms of VC may be attributed to its property as a free radical scavenger or to its indirect action in reducing the level of reactive oxygen species (ROS).
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Improvement in protein thermostability was often found to be associated with increase in its proteolytic resistance as revealed by comparative studies of homologous proteins from extremophiles or mutational studies. Structural elements of protein responsible for this association are not firmly established although loops are implicated indirectly due to their structural role in protein stability. To get a better insight, a detailed study of protein wide mutants and their influence on stability and proteolytic resistance would be helpful. To generate such a data set, a model protein, Bacillus subtilis lipase was subjected to loop scanning site-saturation mutagenesis on 86 positions spanning all loops including termini. Upon screening of ~16,000 clones, 17 single mutants with improved thermostability were identified with increment in apparent melting temperature (Tm(app) ) by 1-6°C resulting in an increase in free energy of unfolding (ΔG(unf) ) by 0.04-1.16 kcal/mol. Proteolytic resistance of all single mutants upon incubation with nonspecific protease, Subtilisin A, was determined. Upon comparison, post-proteolysis residual activities as well as kinetics of proteolysis of mutants showed excellent correlation with ΔG(unf) , (r > 0.9), suggesting that proteolysis was strongly correlated with the global stability of this protein. This significant correlation in this set, with least possible sequence changes (single aa substitution), while covering >60% of protein surface strongly argues for the covariance of these two variables. Compared to studies from extremophiles, with large sequence heterogeneity, the observed correlation in such a narrow sequence space (ΔΔG(unf) = 1.57 kcal⻹) justifies the robustness of this relation.
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Proteínas Bacterianas/química , Estabilidad Proteica , Proteolisis , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Cinética , Lipasa/química , Lipasa/genética , Modelos Moleculares , Mutagénesis , Desplegamiento Proteico , Temperatura , TermodinámicaRESUMEN
The goal of sequencing the entire human genome for $1000 is almost in sight. However, the total costs including DNA sequencing, data management, and analysis to yield a clear data interpretation are unlikely to be lowered significantly any time soon to make studies on a population scale and daily clinical uses feasible. Alternatively, the targeted enrichment of specific groups of disease and biological pathway-focused genes and the capture of up to an entire human exome (~1% of the genome) allowing an unbiased investigation of the complete protein-coding regions in the genome are now routine. Targeted gene capture followed by sequencing with massively parallel next-generation sequencing (NGS) has the advantages of 1) significant cost saving, 2) higher sequencing accuracy because of deeper achievable coverage, 3) a significantly shorter turnaround time, and 4) a more feasible data set for a bioinformatic analysis outcome that is functionally interpretable. Gene capture combined with NGS has allowed a much greater number of samples to be examined than is currently practical with whole-genome sequencing. Such an approach promises to bring a paradigm shift to biomedical research of Mendelian disorders and their clinical diagnoses, ultimately enabling personalized medicine based on one's genetic profile. In this review, we describe major methodologies currently used for gene capture and detection of genetic variations by NGS. We will highlight applications of this technology in studies of genetic disorders and discuss issues pertaining to applications of this powerful technology in genetic screening and the discovery of genes implicated in syndromic and non-syndromic hearing loss.
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Sordera/genética , Pruebas Genéticas/métodos , Genoma Humano , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ADN/métodos , Biología Computacional , Bases de Datos Genéticas , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Reproducibilidad de los ResultadosRESUMEN
Current major barriers for using next-generation sequencing (NGS) technologies in genetic mutation screening on an epidemiological scale appear to be the high accuracy demanded by clinical applications and high per-sample cost. How to achieve high efficiency in enriching targeted disease genes while keeping a low cost/sample is a key technical hurdle to overcome. We validated a cDNA-probe-based approach for capturing exons of a group of genes known to cause deafness. Polymerase chain reaction amplicons were made from cDNA clones of the targeted genes and used as bait probes in hybridization for capturing human genomic DNA (gDNA) fragments. The cDNA library containing the clones of targeted genes provided a readily available, low-cost, and regenerable source for producing capture probes with standard molecular biology equipment. Captured gDNA fragments by our method were sequenced by the Illumina NGS platform. Results demonstrated that targeted exons captured by our approach achieved specificity, multiplexicity, uniformity, and depth of coverage suitable for accurate sequencing applications by the NGS systems. Reliable genotype calls for various homozygous and heterozygous mutations were achieved. The results were confirmed independently by conventional Sanger sequencing. The method validated here could be readily expanded to include all-known deafness genes for applications such as genetic hearing screening in newborns. The high coverage depth and cost benefits of the cDNA-probe-based exon capture approach may also facilitate widespread applications in clinical practices beyond screening mutations in deafness genes.
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Sordera/genética , Marcación de Gen , Pruebas Genéticas/economía , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ADN/métodos , Biología Computacional , Sordera/diagnóstico , Exones/genética , Predisposición Genética a la Enfermedad , Pruebas Genéticas/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/economía , Humanos , Recién Nacido , Tamizaje Masivo , Análisis de Secuencia de ADN/economíaRESUMEN
In studies on polyol-mediated protein stabilization, the polyols are the preferred variable and less importance is given to the intrinsic properties of the protein used. We investigated the stabilizing effects of glycerol on three in vitro evolved lipase mutants with varying stabilities and also in a broad pH range of 3.3-12.1. Significant linear negative correlation between increment in stability due to glycerol and prior stability suggests that stabilizing effects of glycerol depend on the prior stability of the protein. Polar/nonpolar surface area and charge do not have a bearing on the stabilizing effects of glycerol.
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Bacillus subtilis/enzimología , Glicerol/química , Lipasa/química , Bacillus subtilis/genética , Lipasa/genética , Mutación , Desnaturalización Proteica , Estabilidad ProteicaRESUMEN
Rational and in vitro evolutionary approaches to improve either protein stability or aggregation resistance were successful, but empirical rules for simultaneous improvement of both stability and aggregation resistance under denaturing conditions are still to be ascertained. We have created a robust variant of a lipase from Bacillus subtilis named "6B" using multiple rounds of in vitro evolution. T(m) and optimum activity temperature of 6B is 78 °C and 65 °C, respectively, which is ~22 °C and 30 °C higher than that of wild-type lipase. Most significantly, 6B does not aggregate upon heating. Physical basis of remarkable thermostability and non-aggregating behavior of 6B was explored using X-ray crystallography, NMR and differential scanning calorimetry. Our structural investigations highlight the importance of tightening of mobile regions of the molecule such as loops and helix termini to attain higher thermostability. Accordingly, NMR studies suggest a very rigid structure of 6B lipase. Further investigation suggested that reduction/perturbation of the large hydrophobic patches present in the wild-type protein structure, decreased propensity of amino acid sequence for aggregation and absence of aggregation-prone intermediate during thermal unfolding of 6B can account for its resistance to aggregation. Overall, our study suggest that better anchoring of the loops with the rest of the protein molecule through mutations particularly on the sites that perturb/disturb the exposed hydrophobic patches can simultaneously increase protein stability and aggregation resistance.
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Bacillus subtilis/enzimología , Lipasa/química , Lipasa/metabolismo , Proteínas Bacterianas , Sitios de Unión , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Cristalografía por Rayos X , Estabilidad de Enzimas , Lipasa/genética , Espectroscopía de Resonancia Magnética , Modelos Químicos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación/genética , Unión Proteica , Conformación Proteica , Desnaturalización Proteica , Multimerización de Proteína , Desplegamiento Proteico , TermodinámicaRESUMEN
Irreversibility of thermally denatured proteins due to aggregation limits thermodynamic characterization of proteins and also confounds the identification of thermostable mutants in protein populations. Identification of mutations that prevent the aggregation of unfolded proteins provides insights into folding pathways. In a lipase from Bacillus subtilis, evolved by directed evolution procedures, the irreversibility due to temperature-mediated aggregation was completely prevented by a single mutation, M137P. Though the parent and the mutants unfold completely on heating, mutants having substitutions M137P, along with M134E and S163P, completely or partially prevent the formation of aggregation-prone intermediate(s) at 75 degrees C. The three mutants show only a marginal increase in free energy of unfolding (DeltaG(H(2)O)), however, the profiles of the residual activity with temperature shows remarkable shift to higher temperature compared to parent. The intermediate(s) were characterized by enhanced binding of bis-ANS, a probe to titrate surface hydrophobicity, aggregation profiles and by estimation of soluble protein. Inclusion of salt in the refolding conditions prevents the reversibility of mutant having charge substitution, while the reversibility of mutant with the introduction of proline was unaffected, indicating the role of charge mediated interaction in M134E in preventing aggregation. Partial prevention of thermal aggregation in wild-type lipase with single substitution, M137P, incorporated by site-directed mutagenesis, suggests that the affect of M137P is independent of the intrinsic thermostability of lipase. Various effects of the mutations suggest their role is in prevention of the formation of aggregation prone intermediate(s). These mutations, describe yet another strategy to enhance the thermotolerance of proteins, where their influence is observed only on the denatured ensemble.
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Bacillus subtilis/enzimología , Calor , Lipasa/química , Análisis Mutacional de ADN , Lipasa/genética , Mutación/genética , Prolina/química , Desnaturalización Proteica , Pliegue de ProteínaRESUMEN
OBJECTIVE: Connexin26 (Cx26) and Cx30 are the major protein subunits forming gap junction (GJ) intercellular channels in the cochlea. Mutations in these 2 Cxs are the major cause of nonsyndromic early childhood deafness in humans. The underlying mechanism for cochlear abnormality is unclear. Here, we used targeted Cx30 gene deletion (Cx30-/-) mice to investigate molecular mechanisms responsible for Cx mutation-linked deafness. Our hypothesis is that specific loss of GJ-mediated biochemical coupling in the cochlea is sufficient to cause deafness. STUDY DESIGN: We compared: (1) expression of major cochlear GJ protein subunits, Cx26 and Cx30; and (2) biochemical coupling among cochlear supporting cells in the cochleae of wild type and Cx30-/- mice. METHODS: Immunolabeling was used to examine the expression of the remaining Cx protein expression in the cochlea of Cx30-/- mice. We also used a fluorescent dye diffusion assay performed on a novel flattened cochlear preparation to examine GJ-mediated metabolite transfer among cochlear supporting cells. RESULTS: Estimation of the residual ionic conductance indicated that considerable intercellular ionic coupling remained in the cochlea of Cx30-/- mice. Direct measurement of GJ-mediated biochemical coupling showed that the transfer of metabolites among cochlear supporting cells in Cx30-/- mice was severely reduced. CONCLUSION: Our data support that deficiency in GJ-mediated biochemical coupling is sufficient to cause Cx mutation-linked deafness.
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Conexinas/genética , Sordera/genética , Sordera/patología , Mutación/genética , Mutación/fisiología , Animales , Cóclea/anatomía & histología , Cóclea/fisiología , Conexina 26 , Conexina 30 , Difusión , Epitelio/patología , Colorantes Fluorescentes , Uniones Comunicantes/genética , Glucosa/metabolismo , Ratones , Ratones NoqueadosRESUMEN
Mutations in connexins (Cxs), the constitutive protein subunits of gap junction (GJ) intercellular channels, are one of the most common human genetic defects that cause severe prelingual non-syndromic hearing impairments. Many subtypes of Cxs (e.g., Cxs 26, 29, 30, 31, 43) and pannexins (Panxs) are expressed in the cochlea where they contribute to the formation of a GJ-based intercellular communication network. Cx26 and Cx30 are the predominant cochlear Cxs and they co-assemble in most GJ plaques to form hybrid GJs. The cellular localization of specific Cx subtypes provides a basis for understanding the molecular structure of GJs and hemichannels in the cochlea. Information about the interactions among the various co-assembled Cx partners is critical to appreciate the functional consequences of various types of genetic mutations. In vitro studies of reconstituted GJs in cell lines have yielded surprisingly heterogeneous mechanisms of dysfunction caused by various Cx mutations. Availability of multiple lines of Cx-mutant mouse models has provided some insight into the pathogenesis processes in the cochlea of deaf mice. Here we summarize recent advances in understanding the structure and function of cochlear GJs and give a critical review of current findings obtained from both in vitro studies and mouse models on the mechanisms of Cx mutations that lead to cell death in the cochlea and hearing loss.