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The oriented attachment of molecules in general and proteins in particular at the interface of nanoparticles is currently a challenge in (bio)nanotechnology. In this work carried out by research groups of the Institute of Physical Chemistry Rocasolano, Institute of Material Science of Madrid and Institute of Catalysis and Petroleochemistry, agarose-coated magnetic nanoparticles have been prepared and characterized. They have shown that these nanoparticles constitute an excellent experimental platform for the oriented attachment of recombinant proteins tagged with the β-trefoil lectin LSL150. Optimization of the preparation of the agarose-coated magnetic nanoparticles as followed by a survey of techniques such as dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR) and thermogravimetric studies, required the decoupling of particle formation from agarose coating. LSL150 interacted with these agarose-coated nanoparticles exclusively through the recognition of the sugars of the polymer, forming highly stable complexes. The marked topological polarity of this small lectin makes it an excellent molecular adaptor for the oriented attachment of proteins at the nanoparticle interface since they always face the bulk solvent. This fact opens up new possibilities for the design of novel and more efficient (bio)sensors.
Iván Acebrón, Amalia G. Ruiz-Estrada, Yurena Luengo, María del Puerto Morales, José Manuel Guisán, and José Miguel Mancheño. “Oriented Attachment of Recombinant Proteins to Agarose-Coated Magnetic Nanoparticles by Means of a β‑Trefoil Lectin Domain”. Bioconjugate Chemistry (2016) 27, 2734−2743.
(doi:10.1021/acs.bioconjchem.6b00504)
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Apoptin is a small protein from the chicken anemia virus which selectively induces apoptosis (programmed cell suicide) in over 80 different cancer cell lines, yet does not harm healthy cells. Apoptin is a promising cancer lead and is progressing through clinical trials. Nevertheless, Apoptin's strong tendency to oligomerize limits its ability to enter cells and thwarts studies of its structure. Therefore, we have prepared and characterized a monomeric Apoptin variant that retains most of the wild type protein's selective anti-cancer activity. Using NMR spectroscopy, this variant was shown to be intrinsically disordered and dynamic on ps-ms time scales. The conformational ensemble is not significantly affected by specific phosphorylation, addition of Mg++, pH changes or red/ox conditions. These findings support a model for Apoptin's mechanism of action in which cancer specific kinases phosphorylate Apoptin, leading to its accumulation in the nucleus and activation of p53-independent apoptosis.
Referencias:
1. "Insights into the mechanism of Apoptin's exquisitely selective anti-tumor action from atomic level characterization of its conformation and dynamics." Ruiz-Martínez S, Pantoja-Uceda D, Castro J, Vilanova M, Ribó M, Bruix M, Benito A, Laurents DV. Arch Biochem Biophys. (2017) 614:53-64.
doi:10.1016/j.abb.2016.12.010
2. "A truncated Apoptin protein variant selectively kills cancer cells." Ruiz-Martínez S, Castro J, Vilanova M, Bruix M, Laurents DV, Ribó M, Benito A. Invest New Drugs (2017).
doi:10.1007/s10637-017-0431-6
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The protein complex formed by the Ca2+ sensor NCS-1 and the guanine exchange factor Ric8a co-regulates in a antagonistic manner synapse number and probability of neurotransmitter release, emerging as a potential therapeutic target for diseases affecting synapses such as Fragile X syndrome (FXS), the most common heritable autism disorder. By combining crystallographic and chemoinformatic methodologies, a new and small phenothiazine derivative has been found to inhibit this protein complex, whose contact surface is big and complex. The administration of the compound reduces the aberrant excess of synapse number to normal levels and improves associative learning in a Drosophila FXS model. Finally, the structure-function studies have demonstrated the mechanism of action of this new molecule. This work opens the path to the generation of new drugs to treat neuronal diseases affecting synapse function, such as Autism or Alzheimer.
This work has been carried out by researchers from three CSIC Institutes (Instituto de Química-Física “Rocasolano”, Instituto Cajal and Centro de Investigaciones Biológicas) and the BSRC “Alexander Fleming” in Greece.
Alicia Mansilla, Antonio Chaves-Sanjuan, Nuria E. Campillo, Ourania Semelidou, Loreto Martínez-González, Lourdes Infantes, Juana María González-Rubio, Carmen Gil, Santiago Conde, Efthimio M. C. Skoulaki, Alberto Ferrús, Ana Martínez, María José Sánchez-Barrena. “Interference of the complex between NCS-1 and Ric8a with phenothiazines regulates synaptic function and is an approach for fragile X syndrome”. Proc. Nat. Acad. Sci., PNAS (2017).
doi:10.1073/pnas.1611089114
EFE press release
CSIC press release
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A complex link exists between cell-wall recycling/repair and the manifestation of resistance to β-lactam antibiotics in many Enterobacteriaceae and Pseudomonas aeruginosa. This process is mediated by specific cell-wall-derived muropeptide products. These muropeptides are internalized into the cytoplasm and bind to the transcriptional regulator AmpR, which controls the cytoplasmic events that lead to expression of β-lactamase, an antibiotic-resistance determinant. By a combination of X-ray crystallography, mass spectrometry and molecular dynamics techniques we have characterized the effector-binding domain (EBD) of AmpR. Our results provide insights on the muropeptides triggering antibiotics resistance and revises the dogma in the field.
This is part of a collaborative effort between the IQFR and the Univ. of Notre Dame (Indiana, USA).
Dik, D.A.; Domínguez-Gil, T.; Lee, M.; Hesek, D.; Byun, B.; Fishovitz, J.; Boggess, B.; Hellman, L.M.; Fisher, J. F.; Hermoso, J.A.; Mobashery, S. “Muropeptide Binding and the X-Ray Structure of the Effector Domain of the Transcriptional Regulator AmpR of Pseudomonas aeruginosa”. J. Am. Chem. Soc. (2017).
doi:10.1021/jacs.6b12819
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We study thermochemical [1] and electronic properties [2] of halogen-containing species with relevance to several atmospherical processes (e.g. catalytic ozone destruction and air quality).
On the one hand, we found that Gn (Gaussian-n, n = 3,4) ab initio computations are accurate theoretical methods to provide reliable heat of formation and carbon-halogen bond-energy values of a wide variety of chlorinated and brominated organic species [1]. These data will be implemented in climate models in order to evaluate the atmospheric-impact of these compounds.
On the other hand, we have shown that the CASPT2 methodology ("Complete Active Self Consistent Field Perturbation Theory”) is also an excellent method for providing reliable values of absorption optical parameters (within the UV-Vis range) of representative species such as IBr and HgBr2 which have particular connotation in photochemical atmospheric processes [2].
[1] J.Z. Dávalos, R. Notario, C.A. Cuevas, J.M. Oliva, A. Saiz-Lopez: “Thermochemistry of halogen-containing organic compounds with influence on atmospheric chemistry”. Comp. Theor. Chem. 1099 (2017) 36-44. DOI:10.1016/j.comptc.2016.11.009
[2] S.P. Sitkiewicz, J.M. Oliva, J.Z. Dávalos, R. Notario, A. Saiz-Lopez, D.R. Alcoba, O.B. Oña, D. Roca-Sanjuán; “Ab initio quantum-chemical computations of the electronic states in HgBr2 and IBr: Molecules of interest on the Earth's atmosphere”. J. Chem. Phys. 145 (2016) 244304, 1-14. DOI:10.1063/1.4971856
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Alejandro Manjavacas Arévalo, a former PhD student at the IQFR, has been distinguished with the Young Investigator 2016 prize, granted jointly by the Spanish Physical Royal Society and the BBVA Foundation.
Dr Manjavaca, now at the New Mexico University (USA) as Associated Investigator, carried out his PhD research on “Light-matter interactions at nano-level” at this Institute, under the supervision of Prof. J. García Abajo. His doctoral dissertation, together with that of Dr. Luis Cerdán also from the IQFR, was distinguished with the “Premio Extraordinario 2012-2013” by the Complutense University of Madrid.
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Alfonso Saiz-Lopez, Research Scientist at CSIC and Head of the Link to the ERC press release in this Institute, has obtained an ERC Consolidator Grant 2016 funded with 2 M euros by the European Research Council for the 5-year project “Climate dimension of natural halogens in the Earth system: Past, present, future (CLIMAHAL)”.
Through an extremely competitive selection process, the ERC Consolidator Grants will fund 314 projects (24 in Spain) out of 2300 applications. The CLIMAHAL project will use a multidisciplinary approach including spectroscopic and kinetic methods, and theoretical modelling to determine for the first time how natural halogen molecules affect the climate of our planet in past, present and future scenarios.
The ERC Consolidator Grants open every year to support research of consolidated and exceptional scientists of any nationality and age. The ERC selects pioneering and high risk projects with ground-breaking ideas within their fields of research.
