Suchergebnisse

Aggregation, nuclear location, and nucleic acid interaction are common features shared by a number of proteins related to neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, transmissible spongiform encephalopathy, Huntington's disease, spinobulbar muscular atrophy, dentatorubro-pallidoluysian atrophy, and several spinocerebellar ataxias. β-Amyloid peptides, tau protein, α-synuclein, superoxide dismutase1, prion protein, huntingtin, atrophin1, androgen receptor, and several ataxins are proteins prone to becoming aggregated, to translocate inside cell nucleus, and to bind DNA. In this chapter, we review those common features suggesting that neurological diseases too may share a transcriptional disorder, making it an important contribution to the origin of the disease. © 2013 Elsevier Inc. ; SAF2006-02424 y P2009/TIC-1476 from the Spanish Government and Comunidad de Madrid. ; Peer Reviewed

17 páginas, 7 figuras, 1 tabla ; The RcsCDB phosphorelay system controls an extremely large regulon in Enterobacteriaceae that involves processes such as biofilm formation, flagella production, synthesis of extracellular capsules and cell division. Therefore, fine-tuning of this system is essential for virulence in pathogenic microorganisms of this group. The final master effector of the RcsCDB system is the response regulator (RR) RcsB, which activates or represses multiple genes by binding to different promoter regions. This regulatory activity of RcsB can be done alone or in combination with additional transcriptional factors in phosphorylated or dephosphorylated states. The capacity of RcsB to interact with multiple promoters and partners, either dephosphorylated or phosphorylated, suggests an extremely conformational dynamism for this RR. To shed light on the activation mechanism of RcsB and its implication on promoter recognition, we solved the crystal structure of full-length RcsB from Salmonella enterica serovar Typhimurium in the presence and absence of a phosphomimetic molecule BeF3-. These two novel structures have guided an extensive site-directed mutagenesis study at the structural and functional level that confirms RcsB conformational plasticity and dynamism. Our data allowed us to propose a β5-T switch mechanism where phosphorylation is coupled to alternative DNA binding ways and which highlights the conformational dynamism of RcsB to be so pleiotropic. ; Spanish Government (Ministry of Economy and Competitiveness) Grants [BIO2013–42619-P, BIO2016–78571-P to A. M, BFU2016–78606-P to P.C, BIO2016–77639-P to F.G.dP]; Valencian Government Grant Prometeo [II/2014/029 to A.M.]; Ministry of Economy and Competitiveness Ramón y Cajal contract (to P.C.); Diamond Light Source block allocation group (BAG) Proposal [MX10121]; Spanish Synchrotron Radiation Facility ALBA Proposal [2015071314]. Funding for open access charge: Spanish Government (Ministry of Economy and Competitiveness) Grants [BIO2013-42619-P, BIO2016-78571-P to A. M, BIO2016-77639-P to F.G.dP, BFU2016-78606-P to P.C.]; Valencian Government grant Prometeo [II/2014/029 to A.M.]. ; Peer reviewed

16 pags., 5 figs.-- Open Access funded by Creative Commons Atribution Licence 4.0 ; BACKGROUND: MeCP2-a chromatin-binding protein associated with Rett syndrome-has two main isoforms, MeCP2-E1 and MeCP2-E2, differing in a few N-terminal amino acid residues. Previous studies have shown brain region-specific expression of these isoforms which, in addition to their different cellular localization and differential expression during brain development, suggest that they may also have non-overlapping molecular mechanisms. However, differential functions of MeCP2-E1 and E2 remain largely unexplored. RESULTS: Here, we show that the N-terminal domains (NTD) of MeCP2-E1 and E2 modulate the ability of the methyl-binding domain (MBD) to interact with DNA as well as influencing the turn-over rates, binding dynamics, response to neuronal depolarization, and circadian oscillations of the two isoforms. Our proteomics data indicate that both isoforms exhibit unique interacting protein partners. Moreover, genome-wide analysis using ChIP-seq provide evidence for a shared as well as a specific regulation of different sets of genes. CONCLUSIONS: Our study supports the idea that Rett syndrome might arise from simultaneous impairment of cellular processes involving non-overlapping functions of MECP2 isoforms. For instance, MeCP2-E1 mutations might impact stimuli-dependent chromatin regulation, while MeCP2-E2 mutations could result in aberrant ribosomal expression. Overall, our findings provide insight into the functional complexity of MeCP2 by dissecting differential aspects of its two isoforms. ; This work was supported by the Canadian Institutes of Health Research [MOP-130417 to JA and MOP-102758 to JBV]. Genome Canada and Genome British Columbia for fnancial support for the University of Victoria-Genome BC Proteomics Centre [204PRO and 214PRO], Leading Edge Endowment Fund (University of Victoria), the Segal McGill Chair in Molecular Oncology at McGill University, the Warren Y. Soper Charitable Trust and the Alvin Segal Family Foundation to the Jewish General Hospital to CHB. Max Planck Society and DFG CRC 1080; DFG CRC 902 to EMS. European Research Council under the European Union's Horizon 2020 research and innovation program [743216 to EMS and 727264 (EPIPHARM) to ME]. Spanish Ministerio de Economia y Competitividad [BFU2013-47064-P and BFU2016-78232-P to AVC and SAF201455000-R to ME]. Instituto de Salud Carlos III and co-funded by European Union (ERDF/ESF, "Investing in your future") [PI15/00663, and Miguel Servet contract CPII13/0017] to OA. Spanish Ministerio de Educacion Cultura y Deporte [FPU 13/3870] to RCG. Terry Fox Research Institute Program Project [TFRI #1039] and Canadian Cancer Society Research Institute [CCSRI #703489] to MH. European Community's Seventh Framework Programme (FP7/2007–2013)/ERC [268626/ EPINORC project]; Integrated Project of Excellence [PIE13/00022] (ONCOPRO‑FILE); CIBER 2016 CB16/12/00312 (CIBERONC); the Spanish Cancer Research Network [RD12/0036/0039]; the Cellex Foundation; Obra Social "La Caixa"; and the Health and Science Departments of the Catalan Government (Generalitat de Catalunya) AGAUR [2014SGR633] to ME. ME is an ICREA Research Professor. French Association of Rett Syndrome funded research conducted by CEM, HM and LK, genotoul bioinformatics platform Toulouse Midi-Pyrenees (Bioinfo Genotoul) provided computing and storage resources. ; Peer reviewed

Curaxins and especially the second-generation derivative curaxin CBL0137 have important antitumor activities in multiple cancers such as glioblastoma, melanoma and others. Although most of the authors suggest that their mechanism of action comes from the activation of p53 and inactivation of NF-kB by targeting FACT, there is evidence supporting the involvement of DNA binding in their antitumor activity. In this work, the DNA binding properties of curaxin CBL0137 with model quadruplex DNA oligomers were studied by 1H NMR, CD, fluorescence and molecular modeling. We provided molecular details of the interaction of curaxin with two G-quadruplex structures, the single repeat of human telomere d(TTAGGGT)4 and the c-myc promoter Pu22 sequence. We also performed 1H and 31P NMR experiments were also performed in order to investigate the interaction with duplex DNA models. Our data support the hypothesis that the interaction of curaxin with G-quadruplex may provide a novel insight into the DNA-binding properties of CBL0137, and it will be helpful for the design of novel selective DNA-targeting curaxin analogues. ; This research was funded by PIANO DI SOSTEGNO ALLA RICERCA 2020—Linea 2 azione B (DEFENS) and by the Spanish Ministerio de Ciencia e Innnvacion (PID2019-107158GB-I00) and recognition from the Autonomous Catalan government (2017SGR114). ; Peer reviewed

Tau hyperphosphorylation can be considered as one of the hallmarks of Alzheimer's disease and other tauophaties. Besides its well-known role as a microtubule associated protein, Tau displays a key function as a protector of genomic integrity in stress situations. Phosphorylation has been proven to regulate multiple processes including nuclear translocation of Tau. In this contribution, we are addressing the physicochemical nature of DNA-Tau interaction including the plausible influence of phosphorylation. By means of surface plasmon resonance (SPR) we measured the equilibrium constant and the free energy, enthalpy and entropy changes associated to the Tau-DNA complex formation. Our results show that unphosphorylated Tau binding to DNA is reversible. This fact is in agreement with the protective role attributed to nuclear Tau, which stops binding to DNA once the insult is over. According to our thermodynamic data, oscillations in the concentration of dephosphorylated Tau available to DNA must be the variable determining the extent of Tau binding and DNA protection. In addition, thermodynamics of the interaction suggest that hydrophobicity must represent an important contribution to the stability of the Tau-DNA complex. SPR results together with those from Tau expression in HEK cells show that phosphorylation induces changes in Tau protein which prevent it from binding to DNA. The phosphorylation-dependent regulation of DNA binding is analogous to the Tau-microtubules binding inhibition induced by phosphorylation. Our results suggest that hydrophobicity may control Tau location and DNA interaction and that impairment of this Tau-DNA interaction, due to Tau hyperphosphorylation, could contribute to Alzheimer's pathogenesis. © 2014 Camero et al. ; Spanish Government and Comunidad de Madrid. CIBERNED (Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas ; Peer Reviewed

In a previous paper (Syrjänen et al., 2014), we reported the first structural characterisation of a synaptonemal complex (SC) protein, SYCP3, which led us to propose a model for its role in chromosome compaction during meiosis. As a component of the SC lateral element, SYCP3 has a critical role in defining the specific chromosome architecture required for correct meiotic progression. In the model, the reported compaction of chromosomal DNA caused by SYCP3 would result from its ability to bridge distant sites on a DNA molecule with the DNA-binding domains located at each end of its strut-like structure. Here, we describe a single-molecule assay based on optical tweezers, fluorescence microscopy and microfluidics that, in combination with bulk biochemical data, provides direct visual evidence for our proposed mechanism of SYCP3-mediated chromosome organisation. ; This work was supported by the European Union's Horizon 2020 research and innovation programme under grant agreement no 654148 Laserlab-Europe, by VICI grants (GJLW and EJGP), a European Research Council starting grant (GJLW) and a H2020 FETopen grant (GJLW and EJGP), by Medical Research Council project grant MR/N000161/1 and Wellcome Trust investigator award 104641/Z/14/Z (LP), and a BBSRC DTP studentship (JLS). ORD is a Sir Henry Dale Fellow jointly funded by the Wellcome Trust and Royal Society (award number 104158/Z/14/Z).

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

Tau protein has been proposed as a trigger of Alzheimer's disease once it is hyperphosphorylated. However, the role that native tau forms play inside the neuronal nucleus remains unclear. In this work we present results concerning the interaction of tau protein with double-stranded DNA, single-stranded DNA, and also with a histone-DNA complex. The tau-DNA interaction results in a structure resembling the beads-on-a-string form produced by the binding of histone to DNA. DNA retardation assays show that tau and histone induce similar DNA retardation. A surface plasmon resonance study of tau-DNA interaction also confirms the minor groove of DNA as a binding site for tau, similarly to the histone binding. A residual binding of tau to DNA in the presence of Distamycin A, a minor groove binder, suggests the possibility that additional structural domains on DNA may be involved in tau interaction. Finally, DNA melting experiments show that, according to the Zipper model of helix-coil transition, the binding of tau increases the possibility of opening the DNA double helix in isolated points along the chain, upon increasing temperature. This behavior is analogous to histones and supports the previously reported idea that tau could play a protective role in stress situations. Taken together, these results show a similar behavior of tau and histone concerning DNA binding, suggesting that post-translational modifications on tau might impair the role that, by modulating the DNA function, might be attributable to the DNA-tau interaction. © 2014-IOS Press and the authors. All rights reserved. ; Spanish Government (SAF2006-02424 and SAF2010-15525) and Comunidad de Madrid (P2009/TIC-1476).

Silver nanoclusters (AgNCs) prepared by the reduction of silver ions in the presence of DNA oligonucleotides have attracted great interest as potential diagnostic tools for their tunable and high fluorescent properties. In this work, three DNA sequences that consist of a 12-nucleotide long probe sequence at the 5′-end linked to the complementary sequence to three miRNAs are studied. First, the interaction of these sequences with Ag(I) was characterized by means of circular dichroism spectroscopy. By applying multivariate methods to the analysis of spectroscopic data, two complexes with different Ag(I) : DNA ratios were resolved. Secondly, the impact of several experimental variables, such as temperature, borohydride concentration and reaction time, on the formation of AgNCs templated by these three sequences was studied. Finally, the fluorescence properties of the duplexes formed by DNA probes with complementary DNA or miRNA sequences were studied. The results presented here highlight the role of the secondary structure adopted by the DNA probe on the fluorescence properties of DNA-stabilized AgNCs which, in turn, affect the development of methods for miRNA detection. ; We thank the contribution of Gerard Pastor and Sara Guardia (University of Barcelona). Funding from the Spanish Ministerio de Ciencia e Innovación (PID2019-107158GB-I00) and recognition from the Autonomous Catalan government (2017SGR114) are acknowledged. ; Peer reviewed

Electrostatic interactions between DNA molecules have been extensively studied experimentally and theoretically, but several aspects (e.g. its role in determining the pitch of the cholesteric DNA phase) still remain unclear. Here, we performed large-scale all-atom molecular dynamics simulations in explicit water and 150 mM sodium chloride, to reconstruct the potential of mean force (PMF) of two DNA oligomers 24 base pairs long as a function of their interaxial angle and intermolecular distance. We find that the potential of mean force is dominated by total DNA charge, and not by the helical geometry of its charged groups. The theory of homogeneously charged cylinders fits well all our simulation data, and the fit yields the optimal value of the total compensated charge on DNA to ≈65% of its total fixed charge (arising from the phosphorous atoms), close to the value expected from Manning's theory of ion condensation. The PMF calculated from our simulations does not show a significant dependence on the handedness of the angle between the two DNA molecules, or its size is on the order of [Formula: see text]. Thermal noise for molecules of the studied length seems to mask the effect of detailed helical charge patterns of DNA. The fact that in monovalent salt the effective interaction between two DNA molecules is independent on the handedness of the tilt may suggest that alternative mechanisms are required to understand the cholesteric phase of DNA. ; This research used resources of the National Energy Research Scientific Computing Center, a DOE Office of Science User Facility supported by the Office of Science of the US Department of Energy under Contract No. DE-AC02-05CH11231. RC was supported by the United Kingdom Engineering and Physical Sciences Research Council (grant No. EP/H004319/1), the French Institut National du Cancer, Grant No. INCa 5960, by the French Agence Nationale de la Recherche, Grant No. ANR-13-BSV5-0010-03, and the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme (FP7/2007-2013) under REA grant agreement n 608959. We acknowledge support of the Spanish Ministry of Economy and Competitiveness, 'Centro de Excelencia Severo Ochoa 2013-2017'. We acknowledge the support of the CERCA Programme/Generalitat de Catalunya.