Suchergebnisse

This is the peer reviewed version of the following article: Pazos, E. and Vázquez, M. E. (2014), Advances in lanthanide‐based luminescent peptide probes for monitoring the activity of kinase and phosphatase. Biotechnology Journal, 9: 241-252. doi:10.1002/biot.201300203, which has been published in final form at https://doi.org/10.1002/biot.201300203. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions ; Signaling pathways based on protein phosphorylation and dephosphorylation play critical roles in the orchestration of complex biochemical events and form the core of most signaling pathways in cells (i.e. cell cycle regulation, cell motility, apoptosis, etc.). The understanding of these complex signaling networks is based largely on the biochemical study of their components, i.e. kinases and phosphatases. The development of luminescent sensors for monitoring kinase and phosphatase activity is therefore an active field of research. Examples in the literature usually rely on the modulation of the fluorescence emission of organic fluorophores. However, given the exceptional photophysical properties of lanthanide ions, there is an increased interest in their application as emissive species for monitoring kinase and phosphatase activity. This review summarizes the advances in the development of lanthanide‐based luminescent peptide sensors as tools for the study of kinases and phosphatases and provides a critical description of current examples and synthetic approaches to understand these lanthanide‐based luminescent peptide sensors ; We thank the support of the Spanish government's grants SAF2010-20822-C02, CTQ2012-31341, CTQ2009-14431,SAF2007-61015 and the Xunta de Galicia INCITE09 209 084PR, GRC2010/12, PGIDIT08CSA-047209PR. E.P.thanks the Fundación Barrié de la Maza for her postdoc-toral fellowship ; SI

We describe a lanthanide biosensor that responds to CDK4 kinase activity in melanoma cell extracts through a significant and dose dependent increase in luminescence, thanks to sensitization of a DOTA[Tb3+] complex incorporated into a CDK4 substrate peptide by a unique tryptophan residue in an adjacent phosphoaminoacid binding moiety ; This work was funded by the CNRS (Centre National de la Recherche Scientifique) and a Marie-Curie fellowship EC-FP7 Framework (PIEF-GA-2013-623151) supporting JAGV. CB was funded by the INCA (PRTK-2014). Financial support from the Spanish MINECO (CTQ2015-70698-R), the Xunta de Galicia (Centro singular de investigacio´n de Galicia accreditation 2016–2019), and the European Union (European Regional Development Fund – ERDF), are gratefully acknowledged ; SI

The nickel(II)‐mediated self‐assembly of a multimeric DNA binder is described. The binder is composed of two metal‐chelating peptides derived from a bZIP transcription factor (brHis2) and one short AT‐hook domain equipped with two bipyridine ligands (HkBpy2). These peptides reversibly assemble in the presence of NiII ions at selected DNA sequences of 13 base pairs ; Financial support from the Spanish grants SAF2016‐76689‐R, RTI2018‐099877‐B‐I00, Orfeo‐cinqa network CTQ2016‐81797‐REDC, the Xunta de Galicia (2015‐CP082, ED431C‐2017/19 and Centro Singular de Investigación de Galicia accreditation 019–2022, ED431G 2019/03), the European Union (European Regional Development Fund—ERDF), and the European Research Council (Advanced Grant No. 340055) are gratefully acknowledged. S.L.‐A. thanks the Spanish MINECO for her FPI fellowship (BES‐2017‐080555); J.R. thanks the Xunta de Galicia for her PhD fellowship ; SI

A fragment of the DNA basic region (br) of the GCN4 bZIP transcription factor has been modified to include two His residues at designed i and i+4 positions of its N-terminus. The resulting monomeric peptide (brHis2) does not bind to its consensus target DNA site (5′-GTCAT-3′). However, addition of Pd(en)Cl2 (en, ethylenediamine) promotes a high-affinity interaction with exquisite selectivity for this sequence. The peptide–DNA complex is disassembled by addition of a slight excess of a palladium chelator, and the interaction can be reversibly switched multiple times by playing with controlled amounts of either the metal complex or the chelator. Importantly, while the peptide brHis2 fails to translocate across cell membranes on its own, addition of the palladium reagent induces an efficient cell internalization of this peptide. In short, we report (1) a designed, short peptide that displays highly selective, major groove DNA binding, (2) a reversible, metal-dependent DNA interaction, and (3) a metal-promoted cell internalization of this basic peptide ; This work has received financial support from the MINECO (SAF2013-41943-R, SAF2016-76689-R, and CTQ2015-70698-R), the Xunta de Galicia (2015-CP082, ED431C 2017/19, and Centro Singular de Investigación de Galicia Accreditation 2016–2019, ED431G/09), the European Union (European Regional Development Fund, ERDF), and the European Research Council (Advanced Grant No. 340055). Support of COST CM1306 and the orfeo-cinqa network are also acknowledged. J.R. thanks the Xunta de Galicia for a Ph.D. fellowship. We also wish to acknowledge the generous support by the Fundación AECC (IDEAS197VAZQ grant) ; SI

This is the peer reviewed version of the following article: Calo-Lapido, R., Penas, C., Jiménez-Balsa, A., Vázquez, M. E., Mascareñas, J. L. (2019), A chemical approach for the synthesis of the DNA-binding domain of the oncoprotein MYC. Org. Biomol. Chem., 17: 6748-6752, which has been published in final form at https://doi.org/10.1039/C9OB01209E. This article may be used for non-commercial purposes in accordance with RSC Terms and Conditions for Use of Self-Archived Versions ; We describe the first chemical synthesis of a functional mutant of the DNA binding domain of the oncoprotein MYC, using two alternative strategies which involve either one or two Native Chemical Ligations (NCLs). Both routes allowed the efficient synthesis of a miniprotein which is capable of heterodimerizing with MAX, and replicate the DNA binding of the native protein. The versatility of the reported synthetic approach enabled the straightforward preparation of MYC and Omomyc analogues, as well as fluorescently labeled derivatives ; Financial support from the MINECO (CTQ2015-70698-R and SAF2016-76689-R, orfeo-cinqa CTQ2016-81797-REDC), Xunta de Galicia (2015-CP082, ED431C 2017/19, ED431B 2018/04, Centro singular de investigación de Galicia accreditation 2016–2019, ED431G/09), the European Union (European Regional Development Fund - ERDF), Fundación AECC (IDEAS197VAZQ), and the European Research Council (Advanced Grant No. 340055) is acknowledged. R. C. L. thanks Xunta de Galicia and the European Social Fund for her fellowship. A. J. B. thanks Xunta de Galicia for his post-doctoral fellowship ; SI

We describe a ds-oligonucleotide–peptide conjugate that is able to efficiently dismount preformed DNA complexes of the bZIP regions of oncoproteins c-Fos and c-Jun (AP-1), and therefore might be useful as disrupters of AP-1-mediated gene expression pathways ; We thank the financial support provided by the Spanish grants SAF2010-20822-C02, SAF2013-41943-R and CTQ2012-31341, the Xunta de Galicia GRC2013-041 and the ERDF. E.P. thanks the Xunta de Galicia for her postdoctoral contract; C.P. and C.P. thank the Spanish Government for their Ph.D. fellowship ; SI

We propose that peptides are highly versatile platforms for the precise design of supramolecular metal architectures, and particularly, for the controlled assembly of helicates. In this context, we show that the bacteriophage T4 Fibritin foldon (T4Ff) can been engineered on its N-terminus with metal-chelating 2,2′-bipyridine units that stereoselectively assemble in the presence of Fe(II) into parallel, three-stranded peptide helicates with preferred helical orientation. Modeling studies support the proposed self-assembly and the stability of the final helicate. Furthermore, we show that these designed mini-metalloproteins selectively recognize three-way DNA junctions over double-stranded DNA ; Financial support from the Spanish grants CTQ2015-70698-R, CTQ2017-87889-P, the Xunta de Galicia (Centro singular de investigación de Galicia accreditation 2016–2019, ED431G/09) and the European Union (European Regional Development Fund - ERDF), is gratefully acknowledged. JG-G, thanks the Spanish MINECO for his FPI fellowship, GB thanks the ERC for her EU METALIC-II 2013-2442/001-001-EMA2 mobility scheme fellowship, and GS. thanks the Universitat Autònoma de Barcelona for its support to his PhD. J-DM and GS are thankful for the support given by the Generalitat de Catalunya 2017SGR1323. Support of COST Action CM1306 is kindly acknowledged. MEV, also wish to acknowledge the generous support by the Fundación Asociación Española Contra el Cáncer AECC (IDEAS197VAZQ grant) ; SI

Obtaining artificial proteins that mimic the DNA binding properties of natural transcription factors could open new ways of manipulating gene expression at will. In this context it is particularly interesting to develop simple synthetic systems. Inspired by the modularity of natural transcription factors, we have designed synthetic miniproteins that combine the zinc finger module of the transcription factor GAGA and AT-hook peptide domains. These constructs are capable of binding to composite DNA sequences of up to 14 base pairs with high affinity and good selectivity. In particular, we have synthesized three different chimeras and characterized their DNA binding properties by electrophoresis and fluorescence anisotropy. We have also used, for the first time in the study of peptide-based DNA binders, nanopore force spectroscopy to obtain further data on the DNA interaction. ; This work received support from the MINECO (SAF2013-41943- R, BFU2016-81754-ERC, CTQ2015-70698-R, SAF2014-58398-R, and SAF2016-76689-R), the Xunta de Galicia (2015-CP082, ED431C 2017/19 and Centro singular de investigación de Galicia accreditation 2016–2019, ED431G/09), the Fundación AECC (IDEAS197VAZQ), the European Union (European Regional Development Fund – ERDF), and the European Research Council (Advanced Grant No. 340055). Support of the orfeocinqa network is also acknowledged. J. R. thanks the Xunta de Galicia for a PhD fellowship; D. R.-L. is a recipient of a Ramón y Cajal Fellowship (RYC-2013-12799) ; SI

MitoBlue is a fluorescent bisamidine that can be used to easily monitor the changes in mitochondrial degradation processes in different cells and cellular conditions. MitoBlue staining pattern is exceptional among mitochondrial dyes and recombinant fluorescent probes, allowing the dynamic study of mitochondrial recycling in a variety of situations in living cells. MitoBlue is a unique tool for the study of these processes that will allow the detailed characterization of communication between mitochondria and lysosomes ; We are thankful for the support given by the Spanish grants BFU2013-43513-R, SAF2016-76689-R, CTQ2015-70698-R, RTI2018-099877-B-I00, CTQ2013-49317-EXP, CTQ2013-41339-P, CTQ2016-75870-P, CTQ2015-71896-REDT (Red de Fotoquímica Biológica) and Instituto de Salud Carlos III (ISCIII; RETIC ARADYALRD16/0006/0012), Junta de Andalucía (PI0250-2016 and UMA18-FEDERJA-007), Orfeo-cinqa network CTQ2016-81797-REDC; the Xunta de Galicia (2015-CP082, ED431C 2017/19, Centro Singular de investigación de Galicia accreditation 2019–2022, ED431G 2019/03), the European Union (European Regional Development Fund - ERDF), and the European Research Council (Advanced Grant No. 340055) are gratefully acknowledged. M.E.V. also acknowledges the support provided by the Fundación AECC (IDEAS197VAZQ grant) ; SI

Connexins (Cxs) are integral membrane proteins that form high-conductance plasma membrane channels, allowing communication from cell to cell (via gap junctions) and from cells to the extracellular environment (via hemichannels). Initially described for their role in joining excitable cells (nerve and muscle), gap junctions (GJs) are found between virtually all cells in solid tissues and are essential for functional coordination by enabling the direct transfer of small signalling molecules, metabolites, ions, and electrical signals from cell to cell. Several studies have revealed diverse channel-independent functions of Cxs, which include the control of cell growth and tumourigenicity. Connexin43 (Cx43) is the most widespread Cx in the human body. The myriad roles of Cx43 and its implication in the development of disorders such as cancer, inflammation, osteoarthritis and Alzheimer's disease have given rise to many novel questions. Several RNA- and DNA-binding motifs were predicted in the Cx43 and Cx26 sequences using different computational methods. This review provides insights into new, ground-breaking functions of Cxs, highlighting important areas for future work such as transfer of genetic information through extracellular vesicles. We discuss the implication of potential RNA- and DNA-binding domains in the Cx43 and Cx26 sequences in the cellular communication and control of signalling pathways ; This work was supported in part through funding from the Society for Research on Bone and Mineral Metabolism - Grant number FEIOMM2016 (to M.D.M.), by grant PRECIPITA-2015-000139 from the FECYT-Ministry of Economy and Competitiveness (to M.D.M), by grants PI13/00591 and PI16/00035 from the Health Institute "Carlos III" (ISCIII, Spain) and co-financed by the European Regional Development Fund, "A way of making Europe" from the European Union (to M.D.M.), by a grant from Xunta de Galicia (pre-doctoral fellowship) to M.V.-E., and by a grant from the Ministry of Education, Culture and Sports, Spain (FPU grant to ...

We report the first Ru(II) coordination compounds that interact with DNA through a canonical minor groove insertion mode and with selectivity for A/T rich sites. This was made possible by integrating a bis-benzamidine minor groove DNA-binding agent with a ruthenium(II) complex. Importantly, one of the enantiomers (Δ-[Ru(bpy)2b4bpy]2+, Δ-4Ru) shows a considerably higher DNA affinity than the parent organic ligand and the other enantiomer, particularly for the AATT sequence, while the other enantiomer preferentially targets long AAATTT sites with overall lower affinity. Finally, we demonstrate that the photophysical properties of these new binders can be exploited for DNA cleavage using visible light ; Financial support from the Spanish grants CTQ2015-70698-R, SAF2016-76689-R and Orfeo-cinqa network CTQ2016-81797-REDC; the Ministerio de Ciencia, Innovación y Universidades of Spain, RTI2018-099877-B-I00; the Xunta de Galicia (2015-CP082, ED431C 2017/19, Centro singular de investigación de Galicia accreditation 2016–2019, ED431G/09), the European Union (European Regional Development Fund – ERDF), and the European Research Council (Advanced Grant No. 340055) are gratefully acknowledged. We also acknowledge the support from the Portuguese Foundation for Science and Technology, FCT, (IF/00894/2015) and CICECO – Aveiro Institute of Materials, FCT Ref. UID/CTM/50011/2019, financed by national funds through the FCT/MCTES. M. E. V. also acknowledges the support of the Fundación Asociación Española Contra el Cáncer (AECC) (IDEAS197VAZQ grant) ; SI