RESUMEN
In drug discovery, efficient screening of protein-drug interactions (PDIs) is hampered by the limitations of current biophysical approaches. Here, we develop a biological nanopore sensor for single-molecule detection of proteins and PDIs using the pore-forming toxin YaxAB. Using this YaxAB nanopore, we demonstrate label-free, single-molecule detection of interactions between the anticancer Bcl-xL protein and small-molecule drugs as well as the Bak-BH3 peptide. The long funnel-shaped structure and nanofluidic characteristics of the YaxAB nanopore enable the electro-osmotic trapping of diverse folded proteins and high-resolution monitoring of PDIs. Distinctive nanopore event distributions observed in the two-dimensional (ΔI/Io-versus-IN) plot illustrate the ability of the YaxAB nanopore to discriminate individual small-molecule drugs bound to Bcl-xL from non-binders. Taken together, our results present the YaxAB nanopore as a robust platform for label-free, ultrasensitive, single-molecule detection of PDIs, opening up a possibility for low-cost, highly efficient drug discovery against diverse drug targets.
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Nanoporos , Nanotecnología/métodos , Interacciones FarmacológicasRESUMEN
The sustained growth of the market for ophthalmic medical devices has increased the demand for alternatives to animal testing for the evaluation of eye irritation. The International Organization for Standardization has acknowledged the need to develop novel in vitro tests to replace animal testing. Here, we evaluated the applicability of an alternative method based on a human corneal model to test the safety of ophthalmic medical devices. 2-Hydroxyethyl methacrylate (HEMA) and Polymethyl methacrylate (PMMA), which are used to fabricate contact lenses, were used as base materials. These materials were blended with eye irritant and non-irritant chemicals specified in the OECD Test Guideline (TG) 492 and Globally Harmonized System (GHS) classification. Then, three GLP-certified laboratories performed three replicates using the developed method using 3D reconstructed human cornea epithelium, MCTT HCETM. OECD TG 492 describes the procedure used to evaluate the eye hazard potential of the test chemical based on its ability to induce cytotoxicity in a reconstructed human cornea-like epithelium (RhCE) tissue. Results: The within-laboratory reproducibility (WLR) and between-laboratory reproducibility (BLR) were both 100%. When a polar extraction solvent was used, the sensitivity, specificity, and accuracy were all 100% in each laboratory. When a non-polar extraction solvent was used, the sensitivity was 80%, the specificity was 100%, and the accuracy was 90%. The proposed method exhibited excellent reproducibility and predictive capacity within and between laboratories. Therefore, the proposed method using the MCTT HCETM model could be used to evaluate eye irritation caused by ophthalmic medical devices.
RESUMEN
Biomolecular interactions, such as protein-protein, protein-nucleic acid, and protein/nucleic acid-ligand interactions, play crucial roles in various cellular signaling and biological processes, and offer attractive therapeutic targets in numerous human diseases. Currently, drug discovery is limited by the low efficiency and high cost of conventional ensemble-averaging-based techniques for biomolecular interaction analysis and high-throughput drug screening. Nanopores are an emerging technology for single-molecule sensing of biomolecules. Owing to the notable merits of single-molecule sensing, nanopore sensors have contributed tremendously to nucleic acid sequencing and disease diagnostics. In this minireview, we summarize the recent developments and outlooks in single-molecule sensing of various biomolecular interactions for drug discovery applications using biological and solid-state nanopore sensors.
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Nanoporos , Ácidos Nucleicos , Descubrimiento de Drogas , Humanos , Ligandos , Nanotecnología/métodosRESUMEN
Nanopore sensors are a highly attractive platform for single-molecule sensing for sequencing, disease diagnostics, and drug screening. Outer membrane protein G (OmpG) nanopores have advantages for single-molecule sensing owing to their rigid monomeric structure, which comprises seven flexible loops, providing distinct gating patterns upon analyte binding. Blocking of the protein-protein interaction between B-cell lymphoma-extra-large (Bcl-xL) and the BH3 domain of Bcl-2 homologous antagonist/killer (Bak-BH3) has been reported as a promising strategy for anticancer therapy. Here, we characterized the interaction between Bcl-xL and Bak-BH3 as well as its inhibition by a small-molecule inhibitor using click chemistry-based Bak-BH3 peptide-conjugated OmpG nanopores. The binding of Bcl-xL to Bak-BH3 generated characteristic gating signals involving significant changes in the amplitudes of noise and gating parameters such as gating frequency, open probability, and durations of open and closed states. Notably, specific inhibition of Bcl-xL by the small-molecule antagonist, ABT-737, led to the recovery of the noise and gating parameters. Collectively, these results revealed that the chemically modified OmpG nanopore can serve as a valuable sensor platform for ultrasensitive, rapid, and single-molecule-based drug screening against protein-protein interactions, which are therapeutic targets for various diseases.
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Proteínas de Escherichia coli , Nanoporos , Apoptosis , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de Escherichia coli/metabolismo , Nanotecnología , Porinas/química , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína bcl-X/metabolismoRESUMEN
Apoptosis plays an essential role in maintaining cellular homeostasis and preventing cancer progression. Bcl-xL, an anti-apoptotic protein, is an important modulator of the mitochondrial apoptosis pathway and is a promising target for anticancer therapy. In this study, we identified octenidine as a novel Bcl-xL inhibitor through structural feature-based deep learning and molecular docking from a library of approved drugs. The NMR experiments demonstrated that octenidine binds to the Bcl-2 homology 3 (BH3) domain-binding hydrophobic region that consists of the BH1, BH2, and BH3 domains in Bcl-xL. A structural model of the Bcl-xL/octenidine complex revealed that octenidine binds to Bcl-xL in a similar manner to that of the well-known Bcl-2 family protein antagonist ABT-737. Using the NanoBiT protein-protein interaction system, we confirmed that the interaction between Bcl-xL and Bak-BH3 domains within cells was inhibited by octenidine. Furthermore, octenidine inhibited the proliferation of MCF-7 breast and H1299 lung cancer cells by promoting apoptosis. Taken together, our results shed light on a novel mechanism in which octenidine directly targets anti-apoptotic Bcl-xL to trigger mitochondrial apoptosis in cancer cells.
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Inteligencia Artificial , Iminas/farmacología , Piridinas/farmacología , Proteína bcl-X/antagonistas & inhibidores , Antineoplásicos/química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Humanos , Iminas/química , Simulación del Acoplamiento Molecular , Neoplasias/patología , Unión Proteica/efectos de los fármacos , Piridinas/química , Proteína Destructora del Antagonista Homólogo bcl-2/química , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína bcl-X/químicaRESUMEN
There is growing evidence that the accumulation of DNA damage induced by fine particulate matter (PM2.5) exposure is an underlying mechanism of pulmonary disease onset and progression. However, there is a lack of experimental evidence on whether common factors (age, gender) affect PM2.5 induced genomic damage. Here, we assessed the DNA damage potency of PM2.5 using conventional genotoxicity testing in old male and female mice aged 8 and 40 weeks. Mice were intratracheally instilled with diesel exhaust PM2.5 (DEP, NIST SRM 1650b), twice a week for 4 weeks. Exposure to DEP was not associated with an increase in the frequency of micronucleated polychromatic erythrocytes and did not induce a systemic genotoxic effect in the bone marrow. Meanwhile, the results from the comet assay showed a significant increase in DNA damage in DEP exposed mouse lung specimens. The positive relationship between DEP exposure and DNA damage is stronger in the older than in the younger group. Statistical analysis showed that there was a modifying effect of age on the association between PM2.5 exposure and DNA damage. Our results suggest that the age factor should be considered to better understand the cellular adverse effects of PM2.5.
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Envejecimiento/fisiología , Mutágenos/toxicidad , Emisiones de Vehículos/toxicidad , Animales , Peso Corporal/efectos de los fármacos , Médula Ósea/efectos de los fármacos , Médula Ósea/metabolismo , Ensayo Cometa , Daño del ADN , Femenino , Pulmón/patología , Masculino , Ratones Endogámicos C57BL , Pruebas de MicronúcleosRESUMEN
Several epidemiological studies concluded that inhalation of diesel exhaust particles (DEP) is associated with an increase in the relative risk of lung cancer. In vitro research evaluating the genetic damage and/or changes in gene expression have been attempted to explain the relationship between DEP exposure and carcinogenicity. However, to date, investigations have been largely confined to studies in immortalized or tumorigenic epithelial cell models. Few studies have investigated damage at the chromosomal level to DEP exposure in normal cell lines. Here, we present the genotoxic effects of DEP in normal cells (embryonic human lung fibroblasts) by conventional genotoxicity testing (micronuclei (MN) and comet assay). We show the differentially expressed genes and enriched pathways in DEP-exposed WI-38 cells using RNA sequencing data. We observed a significant increase in single-strand DNA breaks and the frequency of MN in DEP-exposed cells in a dose-dependent manner. The differentially expressed genes following DEP exposure were significantly enriched in the pathway for responding to xenobiotics and DNA damage. Taken together, these results show that DEP exposure induced DNA damage at the chromosomal level in normal human lung cells and provide information on the expression of genes associated with genotoxic stress.
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Células Epiteliales/efectos de los fármacos , Pulmón/efectos de los fármacos , Pulmón/embriología , Estrés Oxidativo/efectos de los fármacos , Material Particulado/metabolismo , Emisiones de Vehículos , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ensayo Cometa , Daño del ADN , Expresión Génica , Humanos , Mutágenos/farmacología , Óxido Nítrico/metabolismo , RNA-Seq , Especies Reactivas de OxígenoRESUMEN
The performance of an air cleaner is evaluated by the clean air delivery rate (CADR), which is defined as the measure of the delivery of contaminant-free air. Herein, we conducted comparative analyses of various particulate air filters with various collection efficiencies. We installed each filter in identical commercial air cleaners to determine the effects of the collection efficiency on the CADR. Three different filters (E11, E12, and H13 classes) were prepared to determine the effects of the filter collection efficiency and pressure drop on the air cleaner performance (ie, the CADR). Based on experimental data, filters E11 and E12 had similar CADRs and flow rates. However, filter H13, which had the highest collection efficiency and the lowest flow rate, had the lowest CADR. This indicates that even if a filter with higher collection efficiency is installed in an air cleaner, the larger pressure drop causes a reduction in the air flow rate. The CADR value is widely distributed for a flow rate range for commercially available models; however, the collection efficiencies for most air cleaners on the market lie in a narrow range. Therefore, the flow rate has the most direct impact on the performance of a commercial air cleaner.
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Filtros de Aire , Contaminación del Aire Interior , Aire , Aire Acondicionado , Polvo , Filtración , Tamaño de la PartículaRESUMEN
Neuraminidase (NA), one of the major surface glycoproteins of influenza A virus (IAV), is an important diagnostic biomarker and antiviral therapeutic target. Cytolysin A (ClyA) is a nanopore sensor with an internal constriction of 3.3 nm, enabling the detection of protein conformations at the single-molecule level. In this study, a nanopore-based approach is developed for analysis of the enzymatic activity of NA, which facilitates rapid and highly sensitive diagnosis of IAV. Current blockade analysis of the d-glucose/d-galactose-binding protein (GBP) trapped within a type I ClyA-AS (ClyA mutant) nanopore reveals that galactose cleaved from sialyl-galactose by NA of the influenza virus can be detected in real time and at the single-molecule level. Our results show that this nanopore sensor can quantitatively measure the activity of NA with 40-80-fold higher sensitivity than those previously reported. Furthermore, the inhibition of NA is monitored using small-molecule antiviral drugs, such as zanamivir. Taken together, our results reveal that the ClyA protein nanopore can be a valuable platform for the rapid and sensitive point-of-care diagnosis of influenza and for drug screening against the NA target.
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Citotoxinas/metabolismo , Pruebas de Enzimas/métodos , Virus de la Influenza A/enzimología , Nanoporos , Neuraminidasa/metabolismo , Citotoxinas/química , Modelos Moleculares , Neuraminidasa/química , Conformación ProteicaRESUMEN
We performed general toxicity studies of Gryllus bimaculatus (two-spotted cricket) glycosaminoglycan (GbG), including a single, 4-week repeated oral dose toxicity test in ICR mice, and short-term genotoxicity tests. The mutagenic potential of the purified GbG was non-genotoxic when it was evaluated using short-term genotoxicity tests, namely Ames, chromosome aberration (CA), and micronuclei (MN) tests. In Salmonella typhimurium and Escherichia coli assays, GbG did not produce any mutagenic response in the absence or presence of S9 mix with five bacterial strains (TA98, TA100, TA1535, TA1537, and WP2uvrA). Chromosome aberration test showed that GbG had no significant effect on Chinese hamster ovary (CHO) cells. In mouse micronuclei tests after twice oral treatments per day for two days, no significant alteration in the occurrence of micronucleated polychromatic erythrocytes was observed in ICR male mice intraperitoneally administered with GbG at doses of 15.63, 31.25, or 62.50 mg/kg. These results indicate that GbG has no mutagenic potential in these in vitro and in vivo systems. After GbG was orally administered at doses of 20, 40, 80, and 160 mg/kg for a single oral dose toxicity study and at 0, 40, 80, and 160 mg/kg bw/day for 4-week oral dose toxicity study, there were no observed clinical signs or deaths related to treatment in any group tested. Therefore, the approximate lethal oral dose of GbG was considered to be higher than 160 mg/kg in mice. Throughout the administration period, no significant changes in diet consumption, ophthalmologic findings, organ weight, clinical pathology (hematology, clinical chemistry, coagulation, and urinalysis), or gross pathology were detected. Microscopic examination did not identify any treatment-related histopathologic changes in organs of GbG-treated mice in the high dose group. These results indicate that the no-observed adverse effect level (NOAEL) of GbG is higher than 160 mg/kg bw/day in mice.
RESUMEN
We conducted a me-too validation study to confirm the reproducibility, reliability, and predictive capacity of KeraSkin™ skin irritation test (SIT) as a me-too method of OECD TG 439. With 20 reference chemicals, within-laboratory reproducibility (WLR) of KeraSkin™ SIT in the decision of irritant or non-irritant was 100%, 100%, and 95% while between-laboratory reproducibility (BLR) was 100%, which met the criteria of performance standard (PS, WLR≥90%, BLR≥80%). WLR and BLR were further confirmed with intra-class correlation (ICC, coefficients >0.950). WLR and BLR in raw data (viability) were also shown with a scatter plot and Bland-Altman plot. Comparison with existing VRMs with Bland-Altman plot, ICC and kappa statistics confirmed the compatibility of KeraSkin™ SIT with OECD TG 439. The predictive capacity of KeraSkin™ SIT was estimated with 20 reference chemicals (the sensitivity of 98.9%, the specificity of 70%, and the accuracy of 84.4%) and additional 46 chemicals (for 66 chemicals [20 + 46 chemicals, the sensitivity, specificity and accuracy: 95.2%, 82.2% and 86.4%]). The receiver operating characteristic (ROC) analysis suggested a potential improvement of the predictive capacity, especially sensitivity, when changing cut-off (50% â 60-75%). Collectively, the me-too validation study demonstrated that KeraSkin™ SIT can be a new me-too method for OECD TG 439.
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Epidermis/efectos de los fármacos , Adhesión a Directriz/normas , Irritantes/toxicidad , Modelos Biológicos , Organización para la Cooperación y el Desarrollo Económico/normas , Pruebas de Irritación de la Piel/normas , Epidermis/metabolismo , Epidermis/patología , Humanos , Irritantes/metabolismo , Pruebas de Irritación de la Piel/métodosRESUMEN
Legionella pneumophila causes a potentially fatal form of pneumonia by replicating within macrophages in the Legionella-containing vacuole (LCV). Bacterial survival and proliferation within the LCV rely on hundreds of secreted effector proteins comprising high functional redundancy. The vacuolar membrane-localized MavN, hypothesized to support iron transport, is unique among effectors because loss-of-function mutations result in severe intracellular growth defects. We show here an iron starvation response by L. pneumophila after infection of macrophages that was prematurely induced in the absence of MavN, consistent with MavN granting access to limiting cellular iron stores. MavN cysteine accessibilities to a membrane-impermeant label were determined during macrophage infections, revealing a topological pattern supporting multipass membrane transporter models. Mutations to several highly conserved residues that can take part in metal recognition and transport resulted in defective intracellular growth. Purified MavN and mutant derivatives were directly tested for transporter activity after heterologous purification and liposome reconstitution. Proteoliposomes harboring MavN exhibited robust transport of Fe2+, with the severity of defect of most mutants closely mimicking the magnitude of defects during intracellular growth. Surprisingly, MavN was equivalently proficient at transporting Fe2+, Mn2+, Co2+, or Zn2+ Consequently, flooding infected cells with either Mn2+ or Zn2+ allowed collaboration with iron to enhance intracellular growth of L. pneumophila ΔmavN strains, indicating a clear role for MavN in transporting each of these ions. These findings reveal that MavN is a transition-metal-ion transporter that plays a critical role in response to iron limitation during Legionella infection.
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Proteínas Bacterianas , Proteínas de Transporte de Catión , Legionella pneumophila , Metales/metabolismo , Vacuolas , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Humanos , Legionella pneumophila/genética , Legionella pneumophila/metabolismo , Enfermedad de los Legionarios/genética , Enfermedad de los Legionarios/metabolismo , Enfermedad de los Legionarios/patología , Macrófagos/metabolismo , Macrófagos/microbiología , Macrófagos/patología , Células U937 , Vacuolas/genética , Vacuolas/metabolismoRESUMEN
Tripartite efflux pumps and the type I secretion system of Gram-negative bacteria are large protein complexes that span the entire cell envelope. These complexes expel antibiotics and other toxic substances or transport protein toxins from bacterial cells. Elucidating the binary and ternary complex structures at an atomic resolution are crucial to understanding the assembly and working mechanism. Recent advances in cryoelectron microscopy along with the construction of chimeric proteins drastically shifted the assembly models. In this review, we describe the current assembly models from a historical perspective and emphasize the common assembly mechanism for the assembly of diverse tripartite pumps and type I secretion systems.
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Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Farmacorresistencia Bacteriana , Bacterias Gramnegativas/fisiología , Sistemas de Secreción Tipo I/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Microscopía por Crioelectrón , Genes MDR/genética , Bacterias Gramnegativas/genética , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Proteínas Periplasmáticas/genética , Proteínas Periplasmáticas/metabolismo , Multimerización de Proteína , Sistemas de Secreción Tipo I/genéticaRESUMEN
The need for in vitro eye irritation test replacing in vivo is steadily increasing. The MCTT HCE™ eye irritation test (EIT) using 3D reconstructed human cornea-like epithelium, was developed to identify ocular irritants from non-irritants those that are not requiring classification and labelling for eye irritation. Here, we report the results of me-too validation study, which was conducted to evaluate the reliability and relevance of the MCTT HCETM EIT, according to performance standards (PS) of OECD TG 492. The optimal cutoff to determine irritation in the prediction model was preliminarily established at 45% with the receiver operation characteristics (ROC) curve for 141 reference substances. To demonstrate the reproducibility of within- and between-laboratory (WLR and BLR), a set of 30 PS reference chemicals were tested in three laboratories three times. The WLR and BLR concordance with the binary decision of whether non-irritant or irritant was estimated to be 90-100% and 90%, respectively, and both met the PS requirements. The predictive capacity of the respective laboratories for the 30 reference chemicals were evaluated based on three different estimation methods, and the results were comparable, with sensitivity ranging from 89.6 to 93.3%, the specificity ranging from 62.2 to 66.7%, and the accuracy ranging from 75.9 to 80.0%. Additional test with the new set of 30 PS substances in the revised OECD GD 216 yielded a performance of sensitivity ranging from 92.6-93.3%, the specificity 62.2-66.7% and the accuracy 77.4-80.0%. 95.0% sensitivity, 67.2% specificity, and 83.0% accuracy were obtained for 141 reference substances in total. Furthermore, separate cutoffs for liquids and solids, 35% and 60%, respectively, produced better predictivity, which was established as a final prediction model. Collectively, our study demonstrated that MCTT HCETM EIT meets the reproducibility and predictivity criteria stated in OECD TG 492 PS.
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Alternativas a las Pruebas en Animales , Epitelio Corneal/efectos de los fármacos , Irritantes/toxicidad , Pruebas de Toxicidad/métodos , Humanos , Reproducibilidad de los ResultadosRESUMEN
GSK3ß is a key kinase that plays a role in cellular signaling pathways. In Alzheimer's disease (AD), GSK3ß has been implicated in hyperphosphorylation of tau proteins in the neuron, which is a hallmark of AD. Morin, a flavonoid that is abundant in nature, was found as an inhibitor of GSK3ß that can reduce tau pathology in vivo and in vitro. In this study, we determined the crystal structure of GSK3ß in complex with morin. The structure revealed that morin inhibits GSK3ß by binding to the ATP binding pocket. Our findings augment the potential of morin as a functional food to help prevent AD, as well as to provide structural information to develop new therapeutics based on the morin skeleton.
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Flavonoides/química , Glucógeno Sintasa Quinasa 3 beta/química , Animales , Cristalografía por Rayos X , Humanos , Ratones , Conformación Molecular , Unión Proteica , Resonancia por Plasmón de SuperficieRESUMEN
The MacAB-TolC tripartite efflux pump is involved in resistance to macrolide antibiotics and secretion of protein toxins in many Gram-negative bacteria. The pump spans the entire cell envelope and operates by expelling substances to extracellular space. X-ray crystal and electron microscopic structures have revealed the funnel-like MacA hexamer in the periplasmic space and the cylindrical TolC trimer. Nonetheless, the inner membrane transporter MacB still remains ambiguous in terms of its oligomeric state in the functional complex. In this study, we purified a stable binary complex using a fusion protein of MacA and MacB of Escherichia coli, and then supplemented MacA to meet the correct stoichiometry between the two proteins. The result demonstrated that MacB is a homodimer in the complex, which is consistent with results from the recent complex structure using cryo-electron microscopy single particle analysis. Structural comparison with the previously reported MacB periplasmic domain structure suggests a molecular mechanism for regulation of the activity of MacB via an interaction between the MacB periplasmic domain and MacA. Our results provide a better understanding of the tripartite pumps at the molecular level.
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Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/ultraestructura , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/ultraestructura , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/ultraestructura , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/ultraestructura , Sitios de Unión , Simulación por Computador , Modelos Químicos , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
DHHC enzymes catalyze palmitoylation, a major post-translational modification that regulates a number of key cellular processes. There are up to 24 DHHCs in mammals and hundreds of substrate proteins that get palmitoylated. However, how DHHC enzymes engage with their substrates is still poorly understood. There is currently no structural information about the interaction between any DHHC enzyme and protein substrates. In this study we have investigated the structural and thermodynamic bases of interaction between the ankyrin repeat domain of human DHHC17 (ANK17) and Snap25b. We solved a high-resolution crystal structure of the complex between ANK17 and a peptide fragment of Snap25b. Through structure-guided mutagenesis, we discovered key residues in DHHC17 that are critically important for interaction with Snap25b. We further extended our finding by showing that the same residues are also crucial for the interaction of DHHC17 with Huntingtin, one of its most physiologically relevant substrates.
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Aciltransferasas/química , Aciltransferasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteína Huntingtina/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Proteína 25 Asociada a Sinaptosomas/metabolismo , Aciltransferasas/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencias de Aminoácidos , Repetición de Anquirina , Sitios de Unión , Cristalografía por Rayos X , Células HEK293 , Humanos , Modelos Moleculares , Mutación , Proteínas del Tejido Nervioso/genética , Palmitatos/metabolismo , Dominios Proteicos , Especificidad por SustratoRESUMEN
The protein toxin HlyA of Escherichia coli is exported without a periplasmic intermediate by the type I secretion system (T1SS). The T1SS is composed of an inner membrane ABC transporter HlyB, an outer-membrane channel protein TolC, and a membrane fusion protein HlyD. However, the assembly of the T1SS remains to be elucidated. In this study, we determine the crystal structure of a part of the C-terminal periplasmic domain of HlyD. The long α-helical domain consisting of three α helices and a lipoyl domain was identified in the crystal structure. Based on the HlyD structure, we modeled the hexameric assembly of HlyD with a long α-helical barrel, which formed a complex with TolC in an intermeshing cogwheel-to-cogwheel manner, as observed in tripartite RND-type drug efflux pumps. These observations provide a structural blueprint for understanding the type I secretion system in pathogenic Gram-negative bacteria.
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Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Cristalografía por Rayos X , Escherichia coli/química , Modelos Moleculares , Dominios Proteicos , Estructura Secundaria de Proteína , Sistemas de Secreción Tipo I/química , Sistemas de Secreción Tipo I/metabolismoRESUMEN
BACKGROUND: The universal stress proteins (USP) family member UspE is a tandem-type USP that consists of two Usp domains. The UspE expression levels of the Escherichia coli (E. coli) become elevated in response to oxidative stress and DNA damaging agents, including exposure to mitomycin C, cadmium, and hydrogen peroxide. It has been shown that UspA family members are survival factors during cellular growth arrest. The structures and functions of the UspA family members control the growth of E. coli in animal hosts. While several UspA family members have known structures, the structure of E. coli UspE remains to be elucidated. RESULTS: To understand the biochemical function of UspE, we have determined the crystal structure of E. coli UspE at 3.2 Å resolution. The asymmetric unit contains two protomers related by a non-crystallographic symmetry, and each protomer contains two tandem Usp domains. The crystal structure shows that UspE is folded into a fan-shaped structure similar to that of the tandem-type Usp protein PMI1202 from Proteus mirabilis, and it has a hydrophobic cavity that binds its ligand. Structural analysis revealed that E. coli UspE has two metal ion binding sites, and isothermal titration calorimetry suggested the presence of two Cd(2+) binding sites with a Kd value of 38.3-242.7 µM. Structural analysis suggested that E. coli UspE has two Cd(2+) binding sites (Site I: His117, His 119; Site II: His193, His244). CONCLUSION: The results show that the UspE structure has a hydrophobic pocket. This pocket is strongly bound to an unidentified ligand. Combined with a previous study, the ligand is probably related to an intermediate in lipid A biosynthesis. Subsequently, sequence analysis found that UspE has an ATP binding motif (Gly(269)- X2-Gly(272)-X9-Gly(282)-Asn) in its C-terminal domain, which was confirmed by in vitro ATPase activity monitored using Kinase-Glo® Luminescent Kinase Assay. However, the residues constituting this motif were disordered in the crystal structure, reflecting their intrinsic flexibility. ITC experiments revealed that the UspE probably has two Cd(2+) binding sites. The His117, His 119, His193, and His244 residues within the ß-barrel domain are necessary for Cd(2+) binding to UspE protein. As mentioned above, USPs are associated with several functions, such as cadmium binding, ATPase function, and involvement in lipid A biosynthesis by some unknown way.
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Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas de Choque Térmico/química , Cadmio/química , Cristalografía por Rayos X , Escherichia coli/fisiología , Proteínas de Escherichia coli/fisiología , Proteínas de Choque Térmico/fisiología , Ligandos , Conformación ProteicaRESUMEN
The resistance-nodulation-division type tripartite pump AcrAB-TolC and its homologs are responsible for multidrug resistance in Gram-negative bacteria by expelling a wide variety of toxic substrates. The three essential components, AcrA, AcrB, and TolC, must function in concert with each respective binding partner within the complex. In this study, we report an 8.2-Å resolution cryo-electron microscopy (cryo-EM) 3D reconstruction of the complex that consists of an AcrAB fusion protein and a chimeric TolC protein. The pseudoatomic structure derived from the cryo-EM reconstruction clearly demonstrates a model only compatible with the adaptor bridging mechanism, wherein the funnel-like AcrA hexamer forms an intermeshing cogwheel-like interaction with the α-barrel tip region of TolC. These observations provide a structural milestone for understanding multidrug resistance in pathogenic Gram-negative bacteria, and may also lead to the design of new antibacterial drugs.