BOOK OF ABSTRACTS
VIII National Conference BIFI 2017
ORGANIZING COMMITTEE 3 PROGRAM ...... 4 TALKS ...... 5 Tuesday 31st January ...... 6 Wednesday, 1st February ...... 14 Thursday, 2nd February ...... 22 SHORT ORAL COMMUNICATIONS ...... 27 POSTERS...... 38
ORGANIZING COMMITTEE
Patricia Ferreira (Institute BIFI and Department of Biochemistry and Molecular and Cell Biology, U. Zaragoza) Jesús Gómez Gardeñes (Institute BIFI and Department of Theoretical Physics, U. Zaragoza) Maria Luisa Peleato (Institute BIFI and Department of Biochemistry and Molecular and Cell Biology, U. Zaragoza) Javier Sancho (Institute BIFI and Department of Biochemistry and Molecular and Cell Biology, U. Zaragoza) Francisco Sanz (Institute BIFI, U. Zaragoza) Emma Sevilla Miguel (Institute BIFI and Department of Biochemistry and Molecular and Cell Biology, U. Zaragoza)
Support
Elisa Cauhé (Institute BIFI, U. Zaragoza) Yolanda Vergara (Institute BIFI, U. Zaragoza)
Conference Secretariat
Beatriz Antolí (Institute BIFI, U. Zaragoza) Isabel Vidal (Institute BIFI, U. Zaragoza)
PROGRAM
TALKS
Tuesday 31st January
Mechanical properties of viruses: structural determinants and biological relevance
Mauricio G. Mateu
Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Universidad Autonoma de Madrid, Campus de Cantoblanco, 28049 Madrid, Spain
In our group we use different model systems to study the structural determinants and biological relevance of self-assembly, conformational stability and dynamics and mechanical properties of virus particles. In this presentation I will provide a summary of our ongoing studies on virus biophysics using atomic force microscopy and a combination of other biophysical, biochemical or virological techniques: i) analysis of self-assembly of the capsid lattice of the human immunodeficiency virus (HIV) onto a negatively charged substrate, and of mechanical properties of the assembled HIV lattice; ii) analysis of the reversible self-assembly pathway in solution of the capsid of a structurally simple icosahedral virus, the minute virus of mice (MVM), and quantitation of a succession of transient intermediates; iii) a dissection of the structural determinants and biological relevance of local and global mechanical stiffness of viral particles of MVM. These and other studies are contributing to establish intimate relationships in virus particles between molecular structure, mechanical properties, propensity to specific conformational rearrangements and viral biology.
3D Puzzles for integrative structural biology Pablo Chacon IQFR-CSIC
Modeling 3D-EM reconstructions with computational tools currently enables the interpretation at near-atomic resolution of different functional states of macromolecules, thereby deciphering the functional mechanism of biologically relevant complexes. Recent advances in cryo-electron microscopy (cryo-EM), such as direct electron detectors, specimen preparation, image processing, and data automation, are increasing the number of determined structures, particularly at high resolutions. Here, we present several new integrative approaches to retrieve structural information from these accurate reconstructions by incorporating modeling constraints from any complementary biophysical techniques. First, using a two-step integrative approach we unravel the topology of a key unassigned proteasome helical bundle using cryo-EM data, distance restraints, and secondary structure predictions information. This method unambiguously localized all helices and provided a topologically correct model later confirmed by crystallography. Second, we model at near atomic resolution the structure of human of the transcription TFIID in complex with TFIIA and core promoter DNA, determined by single-particle cryo-EM using different hybrid methods developed by us.
Co-creating the citizen science agenda in Spain
Jesús Clemente1,2, Eduardo Lostal1 , MariCarmen Ibáñez1,2, Francisco Sanz1,2, Fermín Serrano1,2
1Fundación Ibercivis, C/ Mariano Esquillor s/n Edificio I+D, Zaragoza, Spain 2Institute for Biocomputation and Complex Physics Systems of the University of Zaragoza, Spain
Citizen science refers to the participation of the general public, amateurs and volunteers in research activities. Participants, even those with limited skills or lower performance, are still valid knowledge generators. The combination of openness with digital technologies make the practices more relevant because of its scalability in number and range of collaborations. These bidirectional contributions occur all along the research cycle, not only for experimental data acquisition or basic analysis tasks of scientific data, transforming the way research is done in diverse areas of knowledge such as astronomy, environmental monitoring, biotechnology and cultural heritage. The term citizen science is gaining relevance worldwide in the last five years with direct cross- roads with Open Science and Responsible Research and Innovation. One of the key documents used to create the vision of citizen science in Europe is the White Paper on Citizen Science for Europe delivered by the Socientize project in 2014. This project was initiated by the European Commission under the Directorate General for Communications Networks, Content & Technology (DG CONNECT) in 2011 under the coordination of the University of Zaragoza and the Ibercivis Foundation. This document has been referenced by several countries that have created their own policy agendas. Since 2015, the Ibercivis Foundation leads the Observatory of Citizen Science in Spain. In 2017, in collaboration with the Cotec Foundation, Ibercivis is leading the process of co-creating the citizen science agenda for Spain. This capacity building process is oriented to strengthen capabilities at institutional and individual levels and involves tens of experts, facilitators and participants from different communities and organisations. Participants are brought together to share a vision of citizen science in Spain, to assess major trends and to identify main areas in need of policy action, and to determine best strategies and implementable actions to provoke these changes. In this session we will present an holistic vision of citizen science through a number of inspirational research projects and the ongoing policy making process itself.
NMR structures help to consider native flexibility in structure-based models for protein folding
Antonio Rey Deparamento de. Química Física I, Universidad Complutense de Madrid & BIFI.
Simulation has become a fundamental tool to complement the experimental biophysical techniques devoted to the investigation of the protein folding problem. To tackle this process, and taking into account its very long time scale and the huge number of degrees of freedom involved, researchers have introduced coarse-grained models, which simplify the geometrical representation of the system and its energetic interactions. Among them, native structure-based models have emerged as a powerful technique to analyze the folding process. They define the interactions in the system from the contacts present in the folded state, whose structure has to be experimentally known. Nuclear magnetic resonance (NMR) is able to obtain the structure in solution, where thermal fluctuations allow observing its structural flexibility. Moreover, this flexibility is of paramount importance to explain the function of proteins, either in their natural environment or in an increasing number of biotechnological applications. Each NMR determined structure is therefore recorded into the Protein Data Bank as a set of conformations, termed “models”, which taken together partially mirror this flexibility. However, in present structure- based simulation models the flexibility is ignored, defining the native contacts just from one single “model”, usually the first one. Here we try to change this situation, by including in the simulation Hamiltonian information from the full set of NMR “models” present in the experimental structure, i.e., trying to consider the experimentally determined flexibility of the native state. The most challenging task in this project is to define an interaction potential which does not eliminate, by definition, the information already present in the experimental data. This novel way to employ structure-based potentials has not been seriously tackled before. The results we have obtained with this new simulation model and those computed from a single structure are compared in order to assess the novelties our approach can provide.
Two-dimensional Turbulence: the footprint of coherent events in the complex phase of Fourier space.
José Miguel Reynolds Universidad Complutense de Madrid
While studying turbulence, it is common to analyze the Fourier transform of the evolved fields. However, most of these studies focus only on the amplitude of the Fourier transform, completely ignoring the complex phase. From the time of Kolmogorov, the slopes of the power spectrum have been extensively investigated. In contrast, studies of the phase are scarce, mainly due to the difficulties of its interpretation. Here it is shown,for the case of 2D incompressible Navier-Stokes equations, that clear coherent patterns do appear in the complex phase of the Fourier spectrum, mainly within the dissipation range. These events are shown to be associated with intermittent structures in real space.
The physics of multilayer networks
Manlio De Domenico1 , Clara Granell2, Mason A. Porter3, and Alex Arenas1 ∗ ∗ 1Departament d’Enginyeria Informatica i Matematiques, Universitat Rovira i Virgili, 43007 Tarragona, Spain 2Carolina Center for Interdisciplinary Applied Mathematics, Department of Mathematics, University of North Carolina, Chapel Hill, NC 27599-3250, USA 3Oxford Centre for Industrial and Applied Mathematics, Mathematical Institute, University of Oxford, OX2 6GG, UK; and CABDyN Complexity Centre, University of Oxford, Oxford OX1 1HP, UK
The study of networks plays a crucial role in investigating the structure, dynamics, and function of a wide variety of complex systems in myriad disciplines. Despite the success of traditional network analysis, standard networks provide a limited representation of these systems, which often includes different types of relationships (i.e., “multiplexity”) among their constituent components and/or multiple interacting subsystems. Such structural complexity has a significant effect on both dynamics and function. Throwing away or aggregating available structural information can generate misleading results and provide a major obstacle towards attempts to understand the system under analysis. The recent “multilayer” approach for modeling networked systems explicitly allows the incorporation of multiplexity and other features of realistic networked systems. On one hand, it allows one to couple different structural relationships by encoding them in a convenient mathematical object. On the other hand, it also allows one to couple different dynamical processes on top of such interconnected structures. The resulting framework plays a crucial role in helping to achieve a thorough, accurate understanding of complex systems. The study of multilayer networks has also revealed new physical phenomena that remained hidden when using the traditional network representation of graphs. Here we survey progress towards a deeper understand- ing of dynamical processes on multilayer networks, and we highlight some of the physical phenomena that emerge from multilayer structure and dynamics.
On nucleic acids and their information ratchets
J. Ricardo Arias-Gonzalez1,2
1. Instituto Madrileño de Estudios Avanzados en Nanociencia, C/Faraday 9, Cantoblanco, 28049 Madrid, Spain 2. CNB-CSIC-IMDEA Nanociencia Associated Unit “Unidad de Nanobiotecnología”, Cantoblanco, 28049 Madrid, Spain
Nucleic acids are information macromolecular platforms that develop multiple biological roles in the cell. Associated to them, there exist motor proteins that process this nanoscale software. In the first part of this talk, we present single-molecule experiments on non-canonical nucleic acid structures, known as G-quadruplexes, capable of forming four-stranded conformations. We compare the mechanical stability of DNA and RNA human sequences that are important for telomere regulation and unveil their distinct folding dynamics. In the second part, we develop an information theory framework to characterize the enzyme-mediated information transfer involved in DNA replication under the real, non-equilibrium conditions. Using this formalism, we reproduce measured error rates and explain the typical 100-fold increase in fidelity that is experimentally found when proofreading and edition take place. Our model reveals the energy consumed by DNA polymerases to achieve real error rates in replication.
I+D+i de barrio / Citizen R&D&i
Luis Martín Nuez1, Esther Borao1, Jorge Mata1,, JoseLuis Berrocal1, Ruben Martín1, Fergus Reig1 , Borja Latorre1
1 Makeroni
At the gates of an imminent fourth industrial revolution, many people in the world have already flown their own drone, monitor their pulse and oxygen in blood with a simple smarthwatch and have been able to print a keychain on a 3D printer. All thanks to a simple “equation” with false mathematical appearance: R&D+i. But unlike the elitist proposal of the innovation that was posed in the 90s for the future, in which only the big companies would create great inventions, the time has given the necessary tools to the citizenship to change the world in a decentralized way. Now, big companies are afraid of what a group of young people can do with a couple of computers in a garage. The time of citizen R & D + i has arrived. Or as we call it in Zaragoza “I+D+i de barrio”.
Wednesday, 1st February
The evolutionary puzzle of viruses with multipartite genomes
Susanna Manrubia National Biotechnology Centre (CSIC)
Viruses count amongst the most amazing organisms on Earth regarding their evolutionary and adaptive abilities. They resort to several different forms of coding information in their genomes; together with an array of different mutational mechanisms, they have succeeded in infecting all cellular organisms and in escaping any antiviral strategy (natural or artificial). We will present and discuss a puzzling example of viral adaptive strategy: viruses with multipartite genomes. Multipartite viruses possess fragmented genomes with fragments encapsidated in independent viral particles. This demands co-infection of cells to complete the viral cycle, a condition that poses severe restrictions on the number of viral particles infecting single cells. This kind of viruses infects mostly plants and represents about 16% of all viral species described. As of today, the adaptive advantage of multipartition in front of complete or fragmented genomes encapsidated in a single particle remains undisclosed.
MANGO: Deploying an Heterogeneous Infrastructure for High Performance Computing.
José Flich1,, Universitat Politècnica de València, Spain
The performance/power efficiency wall poses the major challenge faced nowadays by HPC. Looking straight at the heart of the problem, the hurdle to the full exploitation of today computing technologies ultimately lies in the gap between the applications’ demand and the underlying computing architecture: the closer the computing system matches the structure of the application, the most efficiently the available computing power is exploited. Consequently, enabling a deeper customization of architectures to applications is the main pathway towards computation power efficiency. The MANGO project will be presented, which aims to build on this consideration and will set inherent architecture-level support for application-based customization as one of its underlying pillars. In addition to mere performance and power-efficiency, it is of paramount importance to meet new non-functional requirements posed by emerging classes of applications. In particular, a growing number of HPC applications demand some form of time- predictability, or more generally Quality-of-Service (QoS), particularly in those scenarios where correctness depends on both performance and timing requirements and the failure to meet either of them is critical. Examples of such time-critical application include: - online video transcoding - the server-side on-the-fly conversion of video contents, which involves very computation-intensive operations on huge amounts of data to be performed within near real-time deadlines. - medical imaging - characterized by both stringent low-latency requirements and massive computational demand. Time predictability and QoS, unfortunately, are a relatively unexplored area in HPC. While traditional HPC systems are based on a “the faster, the better” principle, real time is a feature typically found in systems used for mission-critical applications, where timing constraints usually prevail over performance requirements. In such scenarios, the most straightforward way of ensuring isolation and time-predictability is through resource overprovisioning, which is in striking contrast to power/performance optimization. In fact, predictability, power, and performance appear to be three inherently diverging perspectives on HPC. We collectively refer to this range of trade-offs as the PPP (Power, Performance, Predictability) space. In this talk we will show how MANGO will address those trade-offs and how the PPP space can be optimized.
Neuronal cultures: exploring complex phenomena in a dish Jordi Soriano
Dept. Física de la Matèria Condensada, Av. Diagonal 645, E-08028 Barcelona, Spain Universitat de Barcelona Institute of Complex Systems, Barcelona, Spain
In neuronal cultures, an ensemble of dissociated neurons self-organize and shape a network with rich spontaneous activity within days. These in vitro systems provide a simple yet versatile experimental platform to monitor the behavior of a living neuronal network, and model it through Physics toolboxes such as dynamical systems or network theory. The spatial arrangement of the neurons over the substrate where they grow can be tuned to give rise to contrasting architectures, for instance a homogeneous and an aggregated one. In this talk I will present different examples of the potential of these two configurations to address important questions in neuroscience. For homogeneous cultures, on the one hand, I will show how the combination of spontaneous activity measurements with numerical simulations helps understanding the mechanisms behind the initiation and propagation of spontaneous activity. For aggregated cultures, on the other hand, I will show their appropriateness as “network theory lab” to understand pivotal aspects such as the relation within the structural and functional connectivity or the resilience of networks to damage.
Energy efficiency best practices applied to CESAR's data center
Guillermo Losilla Anadón1, Patricia Santos Marco1 1 Instituto de Biocomputación y Física de Sistemas Complejos (BIFI)
The Supercomputing Laboratory of the Aragón Supercomputing Center (CESAR-LSC, [1]) is a medium-sized data center located in the "Edificio I+D" building of UNIZAR's Río Ebro campus. It hosts most BIFI's computing infrastructure, currently equivalent to 10.000 cores and 200 TeraFLOPs: 3 HPC supercomputers, several cloud & grid computing platforms, 2 special purpose systems, Ibercivis volunteer computing central services... In this talk we will review the evolution of the data center in terms of energy efficiency (hardware layout, UPS & cooling systems involved, monitoring, automation...), from the beginning to its current design. We will see some lessons learnt and all the measures and improvements that have been progressively applied, always from a “low-cost” perspective.
References
[1] http://cesar.unizar.es
Pan-genomes: estimating the true genomic diversity of species
Bruno Contreras-Moreira1,2,3, Carlos P. Cantalapiedra1, María J García-Pereira1, Inmaculada Yruela1,3, Sean P. Gordon4, John P. Vogel4, Pilar Catalán3,5, Ernesto Igartua1, Ana M. Casas1, Pablo Vinuesa6
1 Estación Experimental de Aula Dei-CSIC), Zaragoza, Spain. 2 Fundación ARAID, Zaragoza, Spain. 3 Grupo de Bioquímica, Biofísica y Biología Computacional (BIFI, UNIZAR), Unidad Asociada al CSIC 4 DOE Joint Genome Institute, Walnut Creek, CA, USA. 5 Universidad de Zaragoza-EPS, Huesca, Spain. 6Centro de Ciencias Genómicas-UNAM, Cuernavaca, Morelos, Mexico.
A pan-genome is defined as the union of all the genes and non-coding DNA found in all individuals of a species or broader taxon. While pan-genomes have been extensively studied in bacteria, and related to their ecological and pathogenic properties, our knowledge of pan-genomes in wild plants and crops is still limited. In this talk I will review how genome and RNA sequencing of both monocot and dicot models (Brachypodium distachyon and Arabidopsis thaliana) and crops (Hordeum vulgare) show that plants, as microorganisms, contain core genes, detected in all accessions, and also accessory sequences, which are present only in some of them. Core genes are more likely to be involved in essential biological functions, whereas accessory loci evolve faster and are conditionally expressed with roles in defense and development. Comparison of de- novo genome assemblies of B. distachyon accessions indicates that accessory genes accumulate in pericentromeric regions, close to transposable elements, in agreement with expression and enrichment analyses in barley. Our results demonstrate that pan-genomes are useful to explore germplasm diversity and show that mobile genetic elements play a key role in shaping plant genomes.
Lessons from Staphylococcus. aureus transcriptome analysis Iñigo Lasa
Universidad Pública de Navarra
In my talk, I will present new insights in bacterial transcription deduced from the characterization of Staphylococcus aureus transcriptome through deep sequencing of both short and long RNA fractions. The study showed the existence of a genome-wide RNase III-dependent processing of overlapping transcripts into short, 19-22 nt RNAs, that affects to three-quarters of sense RNAs from annotated genes. Aiming to find a biological function for this process, we have been investigating a particular gene transcription architecture named as “overlapping operons”. This term refers to genes that being located in the middle of an operon are transcribed in opposite direction to the rest of the operon. I will present results indicating that overlapping operons provide a mechanisms for coordinating gene expression between neighbor genes. Finally, evidences showing that bacterial 3’-UTRs can play post-transcriptional regulatory functions will be presented.
Innovative drug discovery and development strategies for antibacterial therapy: a focus on neglected diseases Santiago Ramón-García1, 2, 3
1 University of British Columbia; 2 ARAID, Research Agency of Aragon; 3 University of Zaragoza
Drug discovery and development is a long and costly process that can take hundreds of millions of euros of investment and more than 15 years from the time a drug is identified in the lab to the moment it reaches the patient. These figure are not an option when it comes to neglected diseases, such as tuberculosis or buruli ulcer, for which there is little or none interests in developing new therapeutic interventions due to the established perception of low or null return on capital investment. To overcome these limitations and speed up the development process we are applying two innovative concepts: drug repurposing and synergistic drug interactions. In this talk I will describe how we are implementing these concepts to bring much needed new therapies to the most in need.
A mechanism for protein recycling
Aitor Hierro 1,2 ,
1 Structural Biology Unit, CIC bioGUNE, Bizkaia Technology Park, 48160 Derio, Spain. 2 IKERBASQUE, Basque Foundation for Science, 48011 Bilbao, Spain.
Cells constantly recycle proteins and lipids, with a direct impact on nutrient uptake, resensitisation to environmental signals, immune surveillance and waste management. Retromer is a multiprotein complex that recycles cargo proteins from endosomes to the trans- Golgi network and the plasma membrane. Defects in retromer impair various cellular processes and underlie some forms of Alzheimer’s and Parkinson’s diseases. The mechanisms for the recruitment of retromer to endosomal membranes and cargo recognition have remained elusive. Our latest results demonstrate how signal-recognition is mechanistically coupled to membrane recruitment [1]. Thus, retromer is simultaneously both the adaptor and the scaffolding complex for sorting cargo into tubular-vesicular carriers.
References [1] Lucas M, Gershlick DC, Vidaurrazaga A, Rojas AL, Bonifacino JS, Hierro A. (2016) Cell.167(6):1623-1635.
Thursday, 2nd February
The role of matrix stiffness on cell migration
JM García-Aznar1, J Escribano1, C Borau1, C Valero1, N Movilla1, C del Amo1
1 Multiscale in Mechanical and Biological Engineering (M2BE), Aragón Institute of Engineering Research (I3A), Dept Mechanical Engineering, Universidad de Zaragoza.
Cell migration is crucial in most of morphogenetic and regenerative processes, also contributing to the development of numerous diseases, including cancer. Different extracellular factors can regulate this cell migration, being the stiffness of the matrix one of the most relevant properties. Actually, cells have more difficulties to deform the matrix and subsequently to move over or through it, depending on matrix stiffness and whether this migration is 2D or 3D. To advance in the understanding of how matrix stiffness guides cell migration, we propose to combine cell migration experiments and numerical simulations. First experimental work [1] is based on the study of how cells by means of contraction are able to sense the stiffness of the surrounding medium and how we simulate this contraction using a particle-based approach [2]. Secondly, we simulate how cells are able to sense a stiffness gradient guiding the collective migration of cells over flat surfaces [3]. Finally, we develop novel 3D microfluidic-based cell cultures combined with collagen-based matrices to recreate migration in 3D [4]. This migration has been simulated by means of the extension of a previous model where cell movement is guided by the dynamics of dendritic protrusions [5].
References
[1] Mitrossilis D., Fouchard J., Pereira D., Postic F., Richert A., Saint-Jean M., & Asnacios A. (2010). Proceedings of the National Academy of Sciences, 107(38), 16518-16523. [2] Borau C., Kim T., Bidone T., García-Aznar J.M., & Kamm R.D. (2012). PLoS One, 7(11), e49174. [3] Sunyer R., Conte V., Escribano J., Elosegui-Artola A., Labernadie A., Valon L., Navajas D., García-Aznar J.M., Muñoz J.J., Roca-Cusachs P., Trepat X. (2016). Science, 353(6304), 1157-1161. [4] Moreno-Arotzena O., Borau C., Movilla N., Vicente-Manzanares M., & García-Aznar J.M. (2015). Annals of biomedical engineering, 43(12), 3025-3039. [5] Ribeiro F.O., Gómez-Benito M.J., Folgado J., Fernandes, P.R., & García-Aznar, J.M. (2017). Computer Methods in Biomechanics and Biomedical Engineering, 20(1), 59-74.
From antiapoptosis to detOXIfication: stumblin' in!
Sonia Vega, Guillermina Goñi, Myriam Alías, Pedro Latorre, Alan Vigueras, Raquel Moreno- Loshuertos, Patricio Fernández-Silva, Isabel Usón, Ramón Hurtado-Guerrero, Adrián Velázquez-Campoy, Milagros Medina and José A. Carrodeguas
Bcl-XL is an antiapoptotic member of the Bcl-2 family of apoptosis regulators. It resides mainly at the outer mitochondrial membrane, where it prevents proapoptotic members from oligomerizing forming pores to release apoptotic factors from mitochondria in which is known as the point of no return in apoptosis commitment. Most in vitro studies with this protein have used a mutant lacking its carboxyl terminal transmembrane domain that is experimentally problematic due to its hydrophobicity, but nevertheless very important for the protein's function. In an attempt to improve knowledge about the function and structure of Bcl-XL we have expressed the full- length protein in bacteria, purified it and analyzed it by several techniques. Our results indicated the presence of a likely tetramer of the protein that was apparently able to bind flavin, a so far unreported role for Bcl-XL. Several in vitro assays were carried out to characterize this new activity and the protein was cristalized and its structure solved. But, was it really Bcl-XL?.
Mitochondrial and nuclear DNA matching shapes metabolism and healthy ageing. Raquel Moreno
Human mitochondrial DNA (mtDNA) shows extensive within population sequence variability. Many studies suggest that mtDNA variants may be associated with ageing or diseases, although mechanistic evidence at the molecular level is lacking. Mitochondrial replacement has the potential to prevent transmission of disease-causing oocyte mtDNA. However, extension of this technology requires a comprehensive understanding of the physiological relevance of mtDNA sequence variability and its match with the nuclear-encoded mitochondrial genes. Studies in conplastic animals allow comparison of individuals with the same nuclear genome but different mtDNA variants, and have provided both supporting and refuting evidence that mtDNA variation influences organismal physiology. However, most of these studies did not confirm the conplastic status, focused on younger animals, and did not investigate the full range of physiological and phenotypic variability likely to be influenced by mitochondria. Here we systematically characterized conplastic mice throughout their lifespan using transcriptomic, proteomic,metabolomic, biochemical, physiological and phenotyping studies. We show that mtDNA haplotype profoundly influences mitochondrial proteostasis and reactive oxygen species generation,insulin signalling, obesity, and ageing parameters including telomere shortening and mitochondrial dysfunction, resulting in profound differences in health longevity between conplastic strains.
Hypercooperative Hydrogen Bonds May Stabilize an Amyloid-Like Pathological Conformation of TDP-43
Miguel Mompeán1, Marco Baralle2, Emanuele Buratti2, Douglas V. Laurents1
1 Instituto de Química Física “Rocasolano”, Consejo Superior de Investigaciones Científicas, Serrano 119, E-28006, Madrid, Spain 2 International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, I-34149, Trieste Italy
As people around the world are living longer and longer, a large increase in dementia is forecast for the coming decades. TDP-43, an essential RNA-binding protein, forms aggregates in 96% of cases of sporadic ALS and is linked to other neurodegenerative diseases. Our goal is to study the structural basis of harmful TDP-43 aggregates using NMR and other techniques. TDP-43 contains a folded N-terminal domain and two RRM RNA-binding domains. TDP-43 also possesses a chiefly disordered C-terminal region (residues 260 - 414) which plays roles in functional RNA regulatory assemblies as well as pathologically relevant aggregation. Microscopy studies and the ability of TDP-43 aggregates to resist harsh treatment and to seed new pathological aggregates capable of cell to cell spreading [1] strongly suggest that they have a self-templating, amyloid- like structure. The Gln/Asn-rich 341–367 residue segment is key for efficient aggregation of endogenous TDP-43 [2]. Based on extensive spectroscopic and computational experiments, we proposed in 2015 that this segment adopts a beta-hairpin structure that assembles to form an amyloid-like structure [3]. More recently, we have shown that Asn and Gln side chains H-bonds in amyloids may be stabilized by a remarkably strong class of hypercooperativity [4], which could decisively stabilize TDP-43 aggregates. The clinical existence of this conformer is supported by the LC-MS/MS characterization of TDP-43 from ex-vivo aggregates, which show that residues 341-367 were protected in vivo from Ser phosphorylation, Gln/Asn deamidation and Met oxidation [6]. Hyperstable Asn/Gln side chain H-bonds may also contribute to the conformational stability of other pathological amyloids, such as the polyQ stretches in huntingtin, as well as functional amyloids like Sup35 (GNNQQNY), and CPEB (QQQQRQQQQ), implicated in memory consolidation.
References
[1] Nonaka, T., Masuda-Suzukake, M., Arai, T., et al. (2013) Cell Reports 4, 124-134. [2] Budini, M., Romano, V., Baralle F., et al. (2015) Hum. Mol. Gene. 24, 9-20. [3] Mompeán, M., Hervás R., Xu, T. et al., (2015) J. Phys. Chem. Lett. 6: 2608-2615. [4] Mompeán, M., Nogales, A., Ezquerra T., and Laurents D. (2016) J. Phys. Chem. Lett. 7: 1859-1864. [5] Mompeán, M., Baralle, M., Buratti, E. and Laurents D. (2016) Front. Mol. Neurosci. 9: 125. [6] Kametani, F., Obi, T., Shishido, T. et al. (2016) Sci. Reports 6: 23281.
The rotating motor protein ATP synthase induces mechanical softening in lipid bilayers: implications for membrane remodeling
Paolo Natale
Dto. Química Física I, Universidad Complutense de Madrid. Avenida Complutense s/n 28040 Madrid, Spain
ATP synthase is a rotating membrane protein that synthesizes adenosine triphosphate (ATP), the chemical energy source of the cell. To unveil the mechanical impact of this molecular motor protein on the bending properties of its lipid environment, we have functionally reconstituted the ATP synthase in giant unilamellar vesicles and tracked the membrane fluctuations by means of flickering spectroscopy. We find that ATP synthase rotates at a frequency of about 20 Hz, promoting large out-of-equilibrium deformations at discrete hot- spots in lipid vesicles and thus inducing an overall membrane softening. Therefore, the rotation of ATP synthases promote mechanically adapted membranes with a high bending compliance and able to support high local curvatures. Our results evidence a mechanical functionality of the ATP synthase for biomembrane re-structuring and shaping.
SHORT ORAL COMMUNICATIONS
Colossus: Cloud computing CESAR infrastructure at the service of companies and researchers
Ruben Valles1, Carlos Gimeno1
1 Institute for Biocomputation and Physics of Complex Systems (BIFI), University of Zaragoza, Zaragoza, Spain.
Cloud technology has evolved a lot, especially in the recent years. It allows the usage of virtualized computing, storage, network resources in a simple and effective way. The resources are very flexible and thanks to virtualization technologies, different operating systems like Windows and Linux can be executed equally. For this reasons, among others, cloud computing is growing in adoption by companies and researchers. CESAR is the Supercomputing Centre of Aragon, which among its powerful supercomputing facilities has a new generation OpenStack cloud infrastructure called Colossus. Almost 2000 computing cores, 20TB RAM and 600TB storage are the presentation card of this versatile cloud computing tool, which will be presented and will serve as training platform on how to access and use it.
Accessible Biochemistry:
The arrival of Biohacking in you Urban Laboratory
Cristina Hernández Ruiz1
1 Institute for Biocomputation and Physics of Complex Systems (BIFI), University of Zaragoza, Zaragoza, Spain.
Cesar-Etopia hereby presents its WetLab, the first urban biochemistry laboratory at BIFI. Added to the biohacking trend and Bio Do-It-Yourself (DIYbio) our WetLab offers a place where biochemical techniques and equipment can be accessible to everyone. New methodologies and ways of workings make this space be a generator and developer of interdisciplinary projects where science, art, technology and education can walk together. Through courses, scientific workshops and collaborative projects, we use the potential of applied biotechnology in research going across the barrier between the professional science world and people. Our goal is to make visible the invisible, to foster scientific knowledge and improve channels of communication allowing anyone interested to have direct, practical experience, even if they lack of a scientific background. “Bioarte”, “Kombucha”, “Ciencia Do it Yourself” and “Vigilantes del cierzo” are some initial projects by means of which our WetLab is taking its first steps forward. These projects are producing biomaterials such as bacterial cellulose, developing new textiles to be applied in fashion and cinema; PCR techniques are being used, as is α-complementation in artistic works or exhibitions in galleries and museums; strawberry plants are being handed out to members of the public for mapping environmental environmental pollution. Experiments are being conducted with basic scientific concepts in workshops, with the involvement of the entire family. Accessories and devices are being made in our maker space with the latest 3D printer and Arduino technologies, which enable scientific activity while significantly reducing costs. The biohacking age is here.
References
[1] http://cesaretopia.com/ [2] http://genspace.org/ [3] http://waag.org/en
JANUS II in action: statics-dynamic equivalence through the fluctuation-dissipation ratio.
Sergio Perez-Gaviro1,2 for the Janus Collaboration
1 Centro Universitario de la Defensa (CUD-Zaragoza), Academia General Militar de Zaragoza, Spain. 2 Institute for Biocomputation and Physics of Complex Systems (BIFI), University of Zaragoza, Zaragoza, Spain.
Our custom made supercomputer JANUS II entered into full operation some time ago. Its first challenge has been to study the non-equilibrium Fluctuation-Dissipation Theorem (FDT) violations for the Edwards-Anderson model in 3 dimensions, as a way to investigate equilibrium properties of spin glasses [1]. Janus II has given us the opportunity to access to unexplored dynamic regimes where unexpected results have been observed. Thanks to the special purpose computers JANUS [2] and JANUS II [3], we carried on simulations for very large systems, avoiding in this way the always bothering finite size scaling effects, and for very long simulation times, permitting us to investigate unexplored dynamical regimes from huge to small t/tw ratios. Our main result is a quantitative statics- dynamics dictionary, which could allow the experimental exploration of important features of the spin-glass phase without requiring uncontrollable extrapolations to infinite times or system sizes.
References
[1] The Janus Collaboration, Proceedings of the National Academy of Sciences (accepted for publication in December 2016). [2] The Janus Collaboration, Comp. Phys. Comm. 178, 208-216 (2008). [3] The Janus Collaboration, Comp. Phys. Comm. 185, 550-559 (2014).
Janus II: the importance of simulation in spin glasses
J.Moreno-Gordo on behalf of the Janus Collaboration
The Janus project is a collaboration of BIFI members with researchers from Universities of Spain (Extremadura and Complutense of Madrid) and Italy (Ferrara and Rome-La Sapienza). Within the framework of this project, dedicated computers have been built for the efficient study of disordered systems in Physics. The new generation computer Janus II has recently entered in production. The talk will include a brief introduction to the Janus II computer and the systems that Janus II studies (spin glasses), as well as the possibilities that this new computer offers compared to its predecessors. It will describe the work currently in production and will discuss the advance that will involve in the knowledge of the physics of spin glasses.
The interplay between disordered regions and water molecules strongly influence the interaction between methyl-CpG binding protein 2 and DNA
Rafael Claveria-Gimeno1,2,3, Pilar M. Lanuza1,3,4, Ignacio Morales-Chueca1,2, Olga C. Jorge5, Sonia Vega1, Olga Abian1,2,3,4,6*, Manel Esteller5,7,8*, Adrian Velazquez-Campoy1,3,4,9*
1Institute of Biocomputation and Physics of Complex Systems (BIFI), Joint Unit IQFR-CSIC- BIFI, Universidad de Zaragoza, Zaragoza, Spain 2Instituto Aragonés de Ciencias de la Salud (IACS), Zaragoza, Spain 3Aragon Institute for Health Research (IIS Aragon), Zaragoza, Spain 4Department of Biochemistry and Molecular and Cell Biology, Universidad de Zaragoza, Zaragoza, Spain 5Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), 08908 Hospitalet de Llobregat, Barcelona, Spain 6Centro de Investigación Biomédica en Red en el Área Temática de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain 7Department of Physiological Sciences II, School of Medicine, University of Barcelona, L'Hospitalet de Llobregat, Barcelona, Spain 8Institucio Catalana de Recerca i Estudis Avançats, Barcelona, Spain 9Fundacion ARAID, Government of Aragon, 50018 Zaragoza, Spain
Methyl CpG binding protein 2 (MeCP2) is a DNA binding protein involved in epigenetic regulation and chromatin remodeling that preferentially interacts with methylated DNA regions, though it also interacts with multiple protein partners. Mutations in MeCP2 are linked to Rett syndrome, a rare neurodevelopmental disease causing mental, motor and growth impairment, the leading cause of intellectual retardation in girls. MeCP2 is intrinsically disordered; most of its polypeptide chain remains unstructured under physiological conditions. Unstructured regions are important because they provide the required structural plasticity for establishing multiple interactions with different binding partners through processes where partial folding and binding are coupled, resulting in allosteric regulation. We present a biophysical characterization of the methyl binding domain (MBD) from MeCP2 reporting the contribution of the flanking domains and different environmental variables to its structural stability and dsDNA interaction employing spectroscopic and calorimetric techniques. Strikingly, while isolated MBD showed an entropically-driven moderate affinity binding, the flanking disordered domains significantly increased the structural stability of MBD, completely modified the binding profile to an enthalpically-driven high affinity binding, and provided an additional independent dsDNA binding site. The interaction with dsDNA is characterized by a considerable deprotonation and an unusually large negative heat capacity change. A cluster of constrained, hydrogen-bonding water molecules trapped within the binding interface seems to be responsible for the large binding heat capacity. Therefore, the dynamics and mobility of disordered regions together with extrinsic factors (ionic concentration and water molecules) are key determinants of MeCP2 global structural properties and functional capabilities.
Fast sampling coarse-grained molecular dynamics simulations of binding and aggregating disordered proteins
Agustí Emperador1, Modesto Orozco1
1 Institute for Research in Biomedicine Barcelona
Molecular dynamics simulations are limited to the ms timescale due to the small size of the time step, usually 2 fs. We have simulated systems of interacting intrinsically disordered proteins (IDP) using a coarse-grained implicit solvent model with a very low viscosity [1] that produces a very fast sampling of the conformational space of the system. The extensive sampling obtained in our µs long simulations allows us to observe phenomena that happen in a timescale far beyond the ms, like induced fit and refolding upon binding of IDPs, or the onset of aggregation in protein solutions. We have studied the association of ACTR to its binding partner NCBD and the aggregation of the amyloid-β peptide. Our force field produces completely reversible protein- protein binding, resulting in a fraction of monomers in agreement with that found at the concentrations used in experiments. This solves the problem of fast global aggregation at low concentrations found in standard explicit solvent atomistic simulations [2]
References [1] Emperador A., Sfriso P., Villarreal M. A., Gelpi J. L., and Orozco M. (2015) J. Chem. Theory Comput. 11, 5929-5938 [2] Abriata L. A., and Dal Peraro M. (2015) Sci. Rep. 5, 11549
Development of a structural and energetic model of the "unfolded state" of proteins. Juan José Galano-Frutos1,, Javier Sancho2.
1 Institute for Biocomputation and Physics of Complex System / Department of Biochemistry and Molecular and Cellular Biology of the University of Zaragoza. 2 Institute for Biocomputation and Physics of Complex System / Department of Biochemistry and Molecular and Cellular Biology of the University of Zaragoza.
The traditional structural and functional representation of proteins, limited to their native folded conformations, has been challenged by a new description where partially or completely unfolded conformations are becoming increasingly important. About 30% of eukaryotic proteins contain intrinsically unfolded regions under physiological conditions, and they perform crucial roles within interaction networks and signalling cascades [1]. Furthermore, most of non-infectious diseases are associated to destabilizing mutations favouring alternative conformations. A deep understanding of the principles governing the stability of proteins is required for applying quantitative methods to the description and control of biological and technological phenomena in which that stability plays a key role. The poor knowledge about the non-native conformations, such as the partially unfolded intermediate states, the intrinsically unfolded domains, and the ensemble of conformations known as the “unfolded state”, constitutes a pressing need. In this work we are developing a realistic structural and energetic model for the “unfolded state” in proteins that allows calculating one thermodynamic property, the unfolding ⵠH (ⵠHu), as a first step towards the prediction of the stability (ⵠGu). Here, from the solved wild type structure of Barnasa (a good model in this case by several reasons: 1) small, simple protein (110 residues), 2) X-ray high resolution (1.5 Å, PDB:1A2P [2]), 3) reversible, complete and two-states unfolding, 4) no presence of disulfide bonds, and last but maybe the most important thing 5) high experimental ⵠHu value [3]) which has been taken as the “folded state”, and from a randomly, sufficient selected subset from the ~2200 unfolded structures released from the ProtSa server [4,5], which would represent the “unfolded state”, we performed MD simulations on each of these states. We have taken into account the solvent effect on the calculation of the theoretical ⵠHu by differentiating between the ⵠHu contributions of the first water shells (“biological waters”) and the bulk. Despite huge absolute Enthalpies are obtained from both simulated states, i.e. folded and unfolded, the ⵠHu calculation ( ⵠHu = Hu – Hf - ⵠHsolv-eff) seems to be accurate and reproducible. Moreover, we are also estimating another relevant property related to the unfolding process, the unfolding ⵠCp, whose experimental value is also known for Barnasa [3]. This second prediction will provide our methodology a greater reliability and relevance and allow us to propose a feasible set of structures for the “unfolded state” of a protein. Very important statistical and physical aspects, such as whether the selected “unfolded state” fits a Boltzmann distribution are also analyzed in this work. References [1] Tompa P. (2012) TIBS 37: 509-516. [2] Martin C. et al. (1999) Acta Crystallogr D Biol Crystallogr. 55(Pt 2): 386-398. [3] Martínez J. C. et al. (1994) Biochemistry 33: 3919-3926. [4] Bernadó P. et al. (2006) Biophysical J. 91: 4536-4543. [5] Estrada J. et al. (2009) BMC Bioinformatics 10, 104.
A kinetic and thermodynamic view of the protein-ligand interactions during the RFK activity of the FAD synthetase from Corynebacterium ammoniagenes.
María Sebastiána, Ana Serranoa,1, Adrián Velázquez-Campoya,b, and Milagros Medinaa,b
aDepartamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, and Institute of Biocomputation and Physics of Complex Systems, Universidad de Zaragoza, Spain bFundación ARAID, Diputación General de Aragón, Spain 1Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, Madrid, Spain
Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are biomolecules produced from riboflavin (RF) or vitamin B2 through two sequential reactions. In prokaryotes, both reactions are catalyzed by a single bifunctional enzyme, named FAD synthetase (FADS). This protein is organized in two modules, the N-terminal one, that carries out a FMN adenylyltransferase activity, and the C-terminal one, which transforms RF into FMN through a riboflavin kinase (RFK) activity [1]. The RFK activity of the FADS from Corynebacterium ammoniagenes (CaFADS) exhibits a strong inhibition by excess of its RF substrate [2]. Inhibition of key enzymes is a common tool to regulate metabolic pathways. Therefore, the RFK activity of CaFADS is worthy of study, since FMN and FAD play a key role in the cellular metabolism, where they act as cofactors of a plethora of flavoproteins and flavoenzymes. In the present study we use a truncated CaFADS variant -which only contains the completely active RFK domain- to propose a detailed explanation of the inhibition mechanism suffered by this module. For that purpose, we integrate kinetics –both in the pre-steady and in the steady state− thermodynamic data and previous structural information [3]. Our kinetic study in the steady state reveals that, additionally to the inhibition performed by the RF substrate, both of the reaction products –FMN and ADP− inhibit the RFK activity. Additionally, thermodynamic data show that all of the substrates and products compete for binding the module, being this binding in some occasions faster than the substrates one, as the pre-steady state kinetics data reveal. Altogether, our study highlights the importance of a regulated synthesis of FMN and FAD.
[1] Frago, S., et al., Structural analysis of FAD synthetase from Corynebacterium ammoniagenes. BMC Microbiol, 2008. 8: p. 160. [2] Serrano, A., et al., Key residues at the riboflavin kinase catalytic site of the bifunctional riboflavin kinase/FMN adenylyltransferase from Corynebacterium ammoniagenes. Cell Biochem Biophys, 2013. 65(1): p. 57-68. [3] Herguedas, B., et al., Structural insights into the synthesis of FMN in prokaryotic organisms. Acta Crystallogr D Biol Crystallogr, 2015. 71(Pt 12): p. 2526-42
Deciphering the action mechanism of Fur homolog from the pathogen Clostridium difficile Angela Fernández-Otal1, M. Luisa Peleato1, María F. Fillat1, Angel Lanas2and M. Teresa Bes1.
1Department of Biochemistry and Molecular and Cell Biology, and Institute for Biocomputation and Physics of Complex Systems (BIFI), University of Zaragoza, Zaragoza, Spain. 2Instituto de Investigación Sanitaria Aragón (IIS).
Clostridium difficile is an anaerobic, Gram-positive, spore-forming opportunistic bacterium that maybe part of the normal intestinal microbiota in healthy individuals. However, C. difficile has been recognized as a toxin-producing pathogen and is the most common cause of antibiotic- associated colitis. It has gained notoriety linked to the emergence of hypervirulent strains that display resistance to antibiotics [1]. In combination with other strategies, the identification of bioactive compounds that may specifically modulate essential molecules of this microorganism and thereby inhibit or eradicate the infection could lead to the rational design of new antimicrobials. Iron acquisition in the host is vital for survival of bacterial pathogens, but high levels of intracellular iron can increase oxidative damage. Therefore control of iron acquisition and utilization must be tightly regulated in these organisms. Mechanisms of iron homeostasis in bacteria are controlled by the metal-dependent transcriptional regulator Fur (Ferric Uptake Regulator). Moreover, Fur mediates the expression of several virulence genes as response to environmental iron-deficient conditions and oxidative stress, both of them situations that occur during the infection process [2]. Recent studies link resistance to some antibiotics with altering both the expression of redox proteins and iron metabolism in C. difficile [3]. Therefore Fur emerges as an attractive target for the development of new antimicrobial compounds against this pathogen since a part of its well-known role in control of iron homeostasis it has been described as a redox responsive protein in other microorganisms. The aim of this work was to study the redox characteristics of C. difficile Fur (Cd Fur) and their implication in the mechanism of action of this protein. For this purpose, we analyzed the biochemical features of the native protein and its seven single cysteine mutants. The Cd Fur protein showed two forms: a reduced form with DNA-binding activity capable of interacting with its own promoter, and an oxidized inactive form. The change between them was dependent on the protein redox state. Moreover, a single cysteine residue was essential for the binding of the protein to the corepressor metal.
References [1] Chong PM, Lynch T, McCorrister S, Kibsey P, Miller M, Gravel D, et al. (2014). PLoS ONE 9(1): e82622. [2] Fillat M.F. (2014) Arch BiochemBiophys.Vol 546, 41-52. [3] Ho T.D. and Ellermeier C.D. (2015)J Bacteriol. Vol 197(18):2930-2940.
Construction and characterization of TB vaccines based on MTBVAC in modern lineages of Mycobacterium tuberculosis. Irene Pérez1, Carlos Martín1,2, Jesús Gonzalo-Asensio1,2.
1Grupo de Genética de Micobacterias, Universidad de Zaragoza, Zaragoza, Spain 2CIBER enfermedades respiratorias. Instituto de Salud Carlos III, Madrid, Spain
Tuberculosis (TB) is caused by Mycobacterium tuberculosis (MTB) and is one of the infectious diseases that cause more mortality [1]. BCG is the only actual vaccine against TB, but its efficacy against pulmonary diseases in adults is variable. To overcome this problem of BCG, it has been constructed a new live attenuated vaccine candidate, MTBVAC, which is in clinical trials (phase Ib, NCT02013245). MTBVAC is based on the deletion in phoP and fadD26 genes, both related with virulence factors [2]. MTBVAC belongs to lineage 4 (Euro-American-Asian) of Mycobacterium tuberculosis (Mtb). The principal objective of this work is to construct the same deletions of MTBVAC in two strains of linages 2 (Asian Beijing) and 3 (African-Indian). These are modern lineages, which together with lineage 4, are highly distributed and responsible for the current transmission of MTB in humans. We have selected clinical strains from lineages 2 and 3 using RFLP and spoligotyping techniques. Vaccines will be constructed in these lineages using the same method that was used to construct MTBVAC. Suicide plasmids which contains the gene (phoP or fadD26) disrupted by an antibiotic resistance gene and a counter-selectable (negative) marker will be used [2]. Mutants will be constructed through a two-step process involving a positive/negative double selection. The antibiotic marker will be eliminated by using a γδ-resolvase which acts on res sites flanking the resistance marker since these constructions will be used as potential vaccine candidates. MTBVAC has been extensively characterized. It has been studied the transcriptome of the transcription factor PhoP, its lipidomics profile by thin-layer chromatography in which it was observed the devoid of cell-wall lipids phthiocerol dimycocerosates (DIM), diacyltrehaloses and polyacyltrehaloses (DAT/PAT) as direct consequence of fadD26 and phoP deletions, respectively [2]. It has also been studied the expression profile of the principal PhoP-regulated genes (pks2, pks3, espA…) by qRT-PCR [3, 4] and the inability to secrete ESAT-6 [5] by Western blot. To study whether these newly constructed vaccines show the same properties as MTBVAC, we plan to deeply characterize these phenotypes prior to conduct to preclinical studies.
References [1] WHO (2015). Global Tuberculosis Report 2015. [2] Arbues, A., et al., (2013) Vaccine, 31(42): p. 4867-73. [3] Gonzalo-Asensio, J., et al., (2008) PLoS One, 3(10): p. e3496. [4] Solans, L., et al., (2014) PLoS Pathog, 10(5): p. e1004183. [5] Gonzalo-Asensio, J., et al., (2014) Proc Natl Acad Sci U S A, 111(31): p. 11491-6. Funded by: Diputación General de Aragón (DGA). European Project TBVAC2020.
POSTERS
P1. Tissue and cell pattern formation through the Delta-Notch mechanism of cellular differentiation. Pablo J. Blasco-Hernández1, Pierpaolo Bruscolini1, Fernando Falo1.
1 Instituto de Biocomputación y Física de Sistemas Complejos (BIFI)
In this work, we propose a model for tissue growth combined with the process of cell differentiation given by the Delta-Notch mechanism [1]. A simple tissue growth model has been implemented where equations that control the concentrations of delta and notch proteins in each cell, have been incorporated [2]. From these concentrations the fate of each cell is determined. We analyze the different patterns that arise in the tissue according to the different temporal scales of the problem: growth, cell division and cell differentiation [3].
References [1]Bray, S. J.: Notch signalling: a simple pathway becomes complex. Nature Reviews in Molecular Cell Biology. , 7:678 2006. [2] Collier, J. R., N. A. Monk, P. Maini et al.: Pattern formation by lateral inhibition with feedback: a mathematical model of delta-notch intercellular signalling. J Theor Biol, 429- 446, 1996. [3] Blasco-Hernández PJ, Trabajo Fin de Grado de Físicas (2016). Universidad de Zaragoza
P2. Molecular motors modeling: helicase model Guillermo Díez-Señorans1, Rafael Tapia-Rojo2, Fernando Falo1
1 Instituto de Biocomputación y Física de Sistemas Complejos (BIFI). 2 Department of Biological Science, Columbia University.
Molecular motors are the proteins which perform mechanical work at the scale of the cell. The modeling of this system has been a challenge to physics in the last twenty years [1]. In this work we review some of the most used models, and we propose other ones suitable to helicase and RNA polymerase biology [2]. Such models are based on a mesoscopic approach to DNA dynamics (Peyrard-Bishop model) [3], as well as DNA-protein interaction [4].
References [1] R. Dean Astumian, Thermodynamics and Kinetics of a Brownian Motor, Science 276, 917- 922, (1997) [2] Alberts, Bray, Hopkin, Johnson, Lewis, Raff, Roberts, Walter, Essential cell biology, Garland Science, (2010) [3] Michel Peyrard, Nonlinear dynamics and statistical physic of ADN, Nonlinearity 17, R1- R14, (2004) [4] R. Tapia-Rojo, D. Prada-Gracia, J.J Mazo y F. Falo, Mesoscopic model for free-energy- landscape analysis of ADN sequences, Phys. Rev. E 86, 021908, (2012)
P3. FurC controls key genes involved in cyanobacterial nitrogen metabolism and affects heterocyst differentiation
Cristina Sarasa, Emma Sevilla,, Esther Broset, María Luisa Peleato, María F. Fillat.
Department of Biochemistry and Molecular and Cell Biology and Institute for Biocomputation and Physics of Complex Systems. Faculty of Sciences. University of Zaragoza, Pedro Cerbuna, 12. Zaragoza, Spain.
FUR (ferric uptake regulator) proteins constitute a large family of transcriptional regulators that exhibit a wide range of functions. FurC from Anabaena sp. PCC 7120 is the least studied regulator and the one with less similarities with FUR paralogues. Apart from other functions, such as modulation of other fur palalogues, and its clear implication in the oxidative stress processes. [1,2]. It has been reported that FurC was likely related with nitrogen metabolism, since an overexpression of NtcA (Global Regulator of Nitrogen Metabolism) leads to an increase of transcriptions levels of FurC. [3]. NtcA also regulates several genes, such as nblA gene responsible for the degradation of phycobilisomes, an early response to a nitrogen deficiency. The main goal of this study was focused on the study of the implication of FurC in nitrogen metabolism. To this aim we have compared the transcription levels of several key genes involved in nitrogen metabolism in the Anabaena sp. PCC 7120 wild type strain and the FurC- overexpressing mutant EB2770. Transcriptional analysis showed that under nitrogen starvation, the wild type strain showed an increase in the furC expression levels. Moreover, transcriptional analysis under nitrogen sufficient conditions using a furC-overexpressing mutant showed that both ntcA and nblA genes displayed increased transcription levels. Subsequently, these results were combined with DNA-protein binding assays (EMSA) to confirm a direct interaction of FurC with the novel targets ntcA and nblA. To this aim, FurC was obtained by heterologous overexpression in Escherichia coli and subsequent purification by ion-exchange chromatography. No binding was observed between FurC and nblA promoter, whereas when targeting ntcA, FurC specifically bound to the promoter region displaying a complex that evidenced the interaction. In addition, bright-field microscopy showed that the furC-overexpressing mutant was unable to differenciate heterocysts under nitrogen deficiency. These results suggest the existence of a cross-regulation between NtcA and FurC. Besides the involvement of the level of FurC in heterocysts formation evidence the implication in nitrogen metabolism in cyanobacteria.
References [1] Hernandez, J., Hernández, J. A., López-Gomollón, S., Bes, M. T., Fillat, M. F., & Peleato, M. L. (2004). FEMS Microbiology Letters, 236(2), 275–282. [2] Yingping, F., Lemeille, S., Talla, E., Janicki, A., Denis, Y., Zhang, C. C., & Latifi, A. (2014). Environmental Microbiology Reports, 6(5), 468–475. [3] Picossi, S., Flores, E., & Herrero, A. (2014). BMC Genomics, 15, 22.
P4. A new approach for Helicobacter pylori eradication: In vivo assessment of novel flavodoxin inhibitors efficacy
1,2 1,2 4 4 5 5 S. Salillas , M. Alías , V. Michel3, J. Bueno , J. Arribas , A. Lucía , L. Rodrigues , A. 4 5 4 6 4 3 Mahía1,2,6, C. Sostres , J. A. Aínsa , J. Castillo , M.D. Díaz-de-Villegas , A. Lanas , E. Touati , 1,2 and J. Sancho .
1Universityof Zaragoza. Department of Biochemistry and Molecular and Cellular Biology. Zaragoza, Spain. 2Universitiy of Zaragoza. Biocomputation and Physics of Complex Systems Institute (BIFI). BIFI- IQFR(CSIC)-Joint Unit. Zaragoza, Spain. 3Pasteur Institute. Unit of Helicobacter Pathogenesis, Department of Microbiology. Paris, France. 4University Hospital Lozano Blesa. Service of Digestive Diseases. Zaragoza, Spain. 5University of Zaragoza. Department of Microbiology, Preventive Medicine and Public Health. Zaragoza, Spain. 6University of Zaragoza-CSIC. Department of Catalysis and Catalytic Processes. Institute of Chemical Synthesis and Homogeneous Catalysis (ISQCH). Zaragoza, Spain.
The highprevalence of antimicrobial resistance is a current challenge associated with high morbidity and mortality, which appears to be increased over time. One of the most common antibiotic resistant pathogens is Helicobacter pylori, Hp, which colonises the gastric mucosa of more than half of the human population. The majority of Hp-infected patientsare clinically asymptomatic individuals, but some of them develop gastritis, peptic ulcers and even gastric cancer, as well as extragastric diseases such as diabetes, anaemia or hepatic, neurodegenerative and cardiovascular disorders. As previously mentioned, increasing H. pylori resistance to antibiotics used in conventional treatments constitutes an important health problem, which requires the development of new anti-Hp drugs, without disrupting the gastrointestinal microflora. That is why some essential proteins of H. pylori, such as its flavodoxin (Hp-Fld), have been proposed as specific targets. Under the hypothesis that small molecules could bind to a distinct pocket created in the Hp-Fld and thus inhibit this vital enzyme, we have recently synthesized new potential antimicrobials by derivatization of three previously identified flavodoxin inhibitors. Their bactericidal effects, which were tested by MIC determination, indicated the good in vitro activity of some of these new compounds. According to its MCC values, most of them also exhibited a decreased toxicity toward HeLa cells when comparing with lead inhibitors. What is more, efficacy testing in mice showed promising effects for some of these new compounds, which reached improved eradication rates. In accordance with all these results, it is remarkable the anti- Hp potential of these new analogues. In any case, further research is needed in order to establish the mechanism of action of these new compounds and improve its pharmacokinetic properties.
[1] Franceschi F. et al. (2015). Helicobacter. 20 (Suppl S1), 40-46. [2] Sugano K. et al. (2015). Gut. 64, 1353-1367. [3]Galano J. J. et al. (2013). J. Med. Chem. 56, 6248−6258. [4] Cremades N. et al. (2009). ACS Chem Biol. 4 (11), 928-938.
P5. Study of the catalytic mechanism of the adenylylation of FMN in the bifunctional FAD synthetase from Corynebacterium ammoniagenes
Ricardo Burbano1, Juan Seco1, Martha C. Daza1, Markus Doerr1, Milagros Medina2, and Isaías Lans1.
1 Grupo de Bioquímica Teórica, Universidad Industrial de Santander, Bucaramanga, Colombia 2 Instituto de Biocomputación y Física de Sistemas Complejos (BIFI), Universidad de Zaragoza, Zaragoza, Spain
In organisms from all kingdoms, the FMN and FAD cofactors of flavoproteins are synthetized from riboflavin (RF). Initial phosphorylation of RF produces FMN and the subsequent adenylylation of FMN yields FAD. In eukaryotes, the FMN and FAD synthesis are catalyzed by two different enzymes, riboflavin kinases (RFK) and FMN adenylyltransferase (FMNAT) respectively, while in prokaryotes these two processes are catalyzed by a bifunctional enzyme, FAD synthetase (FADS) [1-2]. The RFK activity in the bifunctional FADS takes place at the C- terminal module, while the FMNAT activity takes place at the N-terminal module. The FMNAT module of prokaryotic FADS does not present neither structural nor sequence homology with FMNATs from eukarya [3]. Such divergence has identified prokaryotic bifunctional FADSs as potential drug targets for the design of inhibitors which could fight resistant pathogen microorganisms. In this study, we aim to propose the catalytically competent FMNAT-substrates complex, as well as the mechanism the FMN adenylylation during catalysis based on the crystal structure of the bifunctional FAD synthetase from Corynebacterium ammoniagenes [3]. Hybrid molecular dynamic simulations (QM/MM;PM3/CHARMM) coupled to Umbrella Sampling, finite-temperature string method and the WHAM method, have provided the potential of mean force (PMF) of the adenylylation of FMN processes. The results confirm the relevant role of the residues H31, S164, T165 and R168 [4], which are involved in the orientation of interacting substrates and in the stabilization of the catalytically competent complex. In addition we were able to observe at atomistic level, several interactions which can contribute to FMN adenylylation catalysis. These interactions facilitate the approach between the α-phosphate of ATP and the phosphate of FMN.
References
[1] Fischer M., Bacher A. (2005) Nat. Prod. Rep. 22, 324-50. [2] Brizio C., Galluccio M., Wait R., Torchetti, E.M., Bafunno, V., Accardi R., Gianazza E., Indiveri C., Barile M. (2006) Biochem. Biophys. Res. Commun. 344, 1008-16. [3] Herguedas B., Martinez-Julvez M., Frago S., Medina M., Hermoso J.A. (2010) J. Mol. Biol. 400, 218-230. [4] Serrano A., Frago S., Velazquez-Campoy A., Medina M. (2012) Int. J. Mol. Sci. 13, 14492- 14517.
P6. Contribution of transcription factor protein ductility to organismic complexity
Inmaculada Yrue1a1,2, Christopher J. Oldfield3, Karl J. Niklas4 and A. Keith Dunker3
1 Estación Experimental de Aula Dei, Consejo Superior de Investigaciones Científicas (EEAD- CSIC), Avda. Montañana, 1005, 50059 Zaragoza, Spain. 2 Grupo de Bioquímica, Biofísica y Biología Computacional (BIFI, UNIZAR), Unidad Asociada al CSIC, Spain. 3 Center for Computational Biology and Bioinformatics, Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA. 4 School of Integrative Plant Science, Cornell University, Ithaca, NY 14853, USA.
Studies of diverse phylogenetic lineages reveal that protein disorder increases in concert with organismic complexity but that differences nevertheless exist among lineages [1,2]. To gain insight into the correlation between protein disorder and organismic complexity, we focused on the transcription factor (TF) families of 17 species spanning bacteria, yeast, algae, land plants, and animals. The data show that organismic complexity as gauged by the number of different cell types is positively and strongly correlated with the total number of TFs, the number of their spliced variants, and their total disordered residue content (r2 > 0.8). Although the fraction of disordered residues in TF sequences is often poorly correlated with organismic complexity (r2 < 0.5), the correlation becomes strong (r2 >0.8) when analyses are focused on families of TFs involved in essential developmental processes common to all organisms, or when focused on specific TFs (i.e., orthologues) involved in cell division, differentiation or proliferation. We interpret these data to indicate that the increasing fraction of disordered residues in specific TFs likely had an important factor contributing to the evolution of organismic complexity and not merely a concurrence unrelated to cause and effect.
References [1] Niklas K.J., Dunker A.K. (2016). 2 Alternative splicing, intrinsically disordered. In Multicellularity: origins and evolution. Vienna Series in Theoretical Biology. MIT Press, pp. 17-39. ISBN:0262333759, 9780262333757. [2] Dunker A.K., Bondos S.E., Huang F., Oldfield C.J. (2015). Intrinsically disordered proteins and multicellular organisms. Semin Cell Dev Biol. 37,44-55.
P7. The bifunctional FAD synthetase of the human pathogen Streptococcus pneumoniae
María Sebastián1,2, Erandi Lira-Navarrete3, Ana Serrano4, Carlos Marcuello5, Adrián Velázquez-Campoy2,6, Anabel Lostao6,7, Ramón Hurtado-Guerrero2,6, Milagros Medina1,2, and Marta Martínez-Júlvez1,2 1Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Spain 2 Instituto de Biocomputación y Física de Sistemas Complejos (BIFI) and GBsC-CSIC and BIFI Joint Units, Universidad de Zaragoza, Spain 3 Copenhagen Center for Glycomics, University of Copenhagen, DK-2200, Denmark 4 CIB, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain 5 Departamento de Física. Faculdade de Ciências da Universidade de Lisboa. 6 Fundación ARAID, Diputación General de Aragón, Spain 7 Laboratorio de Microscopías Avanzadas (LMA), Instituto de Nanociencia de Aragón (INA), Universidad de Zaragoza, Spain
Prokaryotic bifunctional FAD synthetases (FADSs) catalyze the biosynthesis of the FMN and FAD, key cofactors to maintain the flavoproteome homeostasis in all type of organisms. Here we shed light to the properties of the hitherto unstudied bacterial FADS from the human pathogen Streptococcus pneumoniae (SpnFADS) [1]. We show that, as other members of the family [2], SpnFADS catalyzes the three typical activities: riboflavin kinase (RFK), ATP:FMN adenylyltransferase (FMNAT), and pyrophosphorylase (FADpp) activities. However, several biophysical properties differ from others FADSs. In particular, i) the RFK activity is not inhibited by the RF substrate, ii) the FMNAT activity requires the FMN substrate in its reduced state, iii) the prior presence of adenine nucleotide ligands is required for the binding of oxidized flavinic substrates/products, and iv) the monomer is the predominant state. Collectively, our results add interesting mechanistic differences among the few prokaryotic bifunctional FADSs characterized so far, which might reflect the adaptation of the enzyme to relatively different environments. In addition, these differences can be exploited for designing selective compounds targeting FADSs for the treatment of diverse infectious diseases.
References
[1] Mitchell A.M. and Mitchell T.J. (2010). Clin Microbiol Infect 16, 411-418 [2] Herguedas B., Martínez-Júlvez M., Frago S. Medina M., and Hermoso J.A. (2010). J Mol Biol 400, 218-230.
P8. High chlorophyll does not interfere in protein-phosphatase inhibition microcystin assay in field samples. Cristina Sarasa1, Emma Sevilla1, Luis Mata2, Pedro Razquin2, María Luisa Peleato1
1 Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Zaragoza. 2 Zeulab. Calle Bari, 25 Duplicado, 50197 Zaragoza
Microcystins are hepatotoxins produced by some cyanobacteria that are a serious environmental and health risk. Several methods have been developed to allow the quantification of microcystins, which are mainly endotoxins. Among those methods, the protein phosphatase inhibition assay is a good candidate as a screening method because of its sensitivity, simplicity and specificity. Among the different methods of microcystin screening for public health purposes, the protein- phosphatase assay presents the great advantage of the absence of false-negative results. The protein phospatase inhibition methods are based on spectrophotometric measures so that the presence of chlorophyll in the extracts potentially may constitute a problem in microcystin determination. Previously we found that methanol 80% is able to reduce chlorophyll recovery1 and avoid any chlorophyll interference. In order to complete this study, a comparative study has been performed and a commercial kit based in protein-phosphatese inhibition (MicrocysTest®) was assayed in the presence of samples from a high chlorophyl cyanobacterial culture. Anabaena PCC 7120, Microcystis aeruginosa PCC 7806 and Microcystis aeruginosa PCC7005 were grown in BG11 and cells were extracted using freeze and thaw procedures, usually indicated for ELISA test assays2. Different dilutions of this highly concentrated extracts were used in order to obtain a wide range of chlorophyll concentrations. These samples were doped with 1µg/L of microcystin- LR and assayed either with MicrocysTest® and ELISA test (Abraxis®). The absorbance at 665nm and at 405nm in the final conditions of the protein phosphatase inhibition assay, was determined and equivalents of microcystin-LR quantified. Whatever the chlorophyll concentration in the original sample is in the range of natural scums during the toxic bloom, once the extracts were performed, no interferences were observed in the assay. It is important to notice that in fresh water is very unusual to find so high chlorophyll concentrations unless a cyanobacterial bloom has taken place. The quantification of microcystin by the ELISA test gave similar values. When a culture of Microcystis aeruginosa PCC7806 cells with high chlorophyll content were extracted using the freeze/thaw procedure usually recommended for ELISA test we found that microcystin levels of microcystin detected are much more low than using our previously described procedure using 80% methanol. Conclusions: - Chlorophyll content, even at very high levels corresponding to a toxic bloom, does not interfere with Protein-phosphatase assay (MicrocysTest®) - Both test, Microcystest and Elisa (Abraxis®) quantify very similarly microcystin - The microcystin extraction procedure is critical. Freeze and thaw procedures are not able to extract all the toxin in the sample. References [1] Sevilla et al. (2009). Water Science & Technology 60: 1903-1909 (2009). [2] Loftin, K. A., Meyer, M. T., Rubio, F., Kamp, L., Humphries, E., & Whereat, E. (2008). Comparison of two cell lysis procedures for recovery of microcystins in water samples. USGS Open-File Report 2008-1341. http://pubs.usgs.gov/of/2008/1341 P9. Potential use of the Twin Arginine Translocation System to secrete heterologous antigens in a recombinant MTBVAC tuberculosis vaccine. Juan Calvet, Jesús Gonzalo-Asensio, Carlos Martín y Esther Broset Grupo de Genética de Micobacterias, Departamento de Microbiología y Medicina preventive. Facultad de Medicina, Universidad de Zaragoza, 50009 Zaragoza
New vaccine candidate against tuberculosis (TB), MTBVAC, is a live-attenuated bacteria derived from a Mycobacterium tuberculosis strain. This vaccine is characterized by inactivation of two genes known to be involved in M. tuberculosis virulence. (Arbues et al. Vaccine 2013) Among others phenotypes, MTBVAC is characterized by enhanced secretion of substrates from the TAT pathway (Twin Arginine Translocation). A non-coding RNA named mcr7 is transcribed by a PhoP-regulated mechanism. Mcr7 post-transcriptionally inhibits translation of TatC, an essential constituyent of the TAT secretion system. Inactivation of phoP in MTBVAC leads to an increased transla-tion of TatC and an increased secretion of TAT substrates (Solans et al. PLoS Patogens 2014). In this study, considering the adjuvanticity of mycobacteria, it is intended to use this TAT phenotype in MTBVAC to express and secrete heterologous antigens. Several signals sequences (ss) from TAT substrates (ssAg85A, ssAg85B, ssAg85C, ssBlaC, ssRv0203 and ssRv1987) were cloned in phase with the GFP (Green Flourescence Protein) gene. All these constructions were placed under the control of a strong promotor derived from L5 mycobacteriophage. The correct transcription of previous constructions was confirmed with qRT-PCR and GFP expression was verified by Flow Cytometry. Fluorescence evaluation of the secreted fraction and whole cell extracts identifies ssAG85A and ssRv1987 as the ss with the highest secretion efficiency. This study provides a proof-of-concept of potential use of these ss to develop multivalent recombinant vaccines based in MTBVAC.
Funding: Esther Broset is granted by BES-2012-052937 fellowship from Ministerio de Economía y Competitividad de España. This study is funded by BIO2014-5258P and TBVAC2020 projects.
P10. Study of the interaction between human Apoptosis Inducing Factor (hAIF) and its nuclear partners.
Silvia Romero-Tamayo1, Adrian VelázquezCampoy1,2, Milagros Medina1 and Patricia Ferreira1
1Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, and Institute of Biocomputation and Physics of Complex Systems, Universidad de Zaragoza, Spain 2ARAID Foundation.
The Apoptosis Inducing Factor (AIF) was first discovered as a caspase-independent cell death promoter, which also plays a vital role in mitochondria, where it is normally confined [1]. In healthy mitochondria, AIF is present in monomer-dimer equilibrium regulated by NADH/NAD+ levels. AIF dimer is undergoing upon NADH reduction and stabilized by long-lived charge transfer complexes between reduced FAD cofactor and oxidized coenzime. AIF dimerization is also accompanied by conformational rearrangements of its reductase and apoptotic domains that suggest some interconnection between its mitochondrial and apoptotic activities [2].
When an apoptotic stimulus is felt, the AIF is liberated into cytosol, and then translocated to the nucleus where induces DNA degradation into 50 kb fragments (typical chromatinolysis caspase- independent pattern) [3]. The interaction between hAIF and DNA occurs in an independent manner based on electrostatic interactions [4]. The lethal activity of AIF requires its interaction with nuclease proteins such as cyclophilin A (Cyp A) [5]. In vitro studies suggest that AIF-CypA interaction is stronger in the presence of NADH [6].
In this study we characterized the in vitro interaction between hAIF and its nuclear partners (DNA and Cyp A) and the influence of hAIF redox state in modulating these interactions, by using gel- retardation and isothermal titration calorimetry techniques.
References [1]., Ortiz-Lombardía M., Boitel B., Haouz A., Tello D., and Susin SA (2002) Nature Structural & Molecular Biology 9, 442-6. [2] Ferreira P., Villanueva R., Martínez-Júlvez M., Herguedas B., Marcuello C., Fernandez-Silva P., Cabon L., Hermoso J., Lostao A., Susin SA and Medina M (2014) Biochemistry, 53 (25), 4204–4215. [3] Susin SA., Lorenzo HK., Zamzami N., Marzo I., Snow BE., Brothers GM., Mangion J., Jacotot E., Costantini P., and Kroemer G (1999) Nature 397, 441-6. [4] Vahsen N., Cande C., Dupaigne P., Giordanetto F., Kroemer RT and N Modjtahedi (2006) Oncogene 25, 1763–1774. [5] Cande C., Vahsen N., Kouranti I., Schmitt E., Daugas E., Spahr C, Luban J, Kroemer and Kroemer G (2004) Oncogene 23, 1514-1521. [6] Zhu Ying., Zhang Li., Fan Jin and Hana Shihui (2007) NeuroImage 34, 1310–1316.
P11. Screening of FDA-approved drugs library identifies potential inhibitors against Helicobacter pylori ArsR essential regulator
Andrés González1,2, Jordan Morán3, Adrián Velázquez-Campoy2, Javier Sancho2,3, María F. Fillat2,3, Ángel Lanas1,4. 1Aragon Institute for Health Research (IIS Aragon) 2Institute of Biocomputation and Physics of Complex Systems (BIFI), University of Zaragoza. 3Department of Biochemistry & Molecular and Cell Biology, University of Zaragoza. 4Digestive Diseases Service, University Clinic Hospital Lozano Blesa, CIBERehd.
Helicobacter pylori is a helical-shaped, Gram-negative bacterium that persistently colonizes the stomach in about half of the human population, causing chronic gastritis as well as gastric and duodenal ulcers [1]. H. pylori infection is the principal trigger of gastric carcinogenesis and gastric cancer, and remains the third leading cause of cancer-related death in both sexes worldwide [2]. A standard triple therapy consisting of a proton pump inhibitor and two antibiotics (clarithromycin and amoxicillin/ metronidazole) is widely used as the first-line regimen for treatment of infection by this microorganism. However, the efficacy of this standard regimen has progressively declined in recent years, mainly due to widespread development of antibiotic resistance [3]. Nowadays, an effective novel therapy is mandatory to increase eradication rates and minimize both antimicrobial resistance and side effect on normal microbiota. In this context, the search for new bioactive compounds able to inhibit the growth of pathogens by acting specifically on essential molecular targets can be a valuable route for drug discovery. The regulator ArsR appears to be a promising therapeutic target against H. pylori infection. ArsR is part of the acidic-responsive ArsS-ArsR two-component signal transduction system, comprised of the sensor histidine kinase ArsS and the response regulator ArsR [4,5]. While ArsS null mutants are viable, ArsR is essential for H. pylori viability, suggesting that a subset of essential genes into the ArsR regulon can be regulated by a non-phosphorylated form of ArsR [6]. In the present study, we screened the Prestwick Chemical Library®, a collection of 1120 approved drugs, for compounds that specifically bind to ArsR and potentially inhibit its function. Compound binding was assessed by a fluorescence-based thermal shift assay. Ten active molecules belonging to different pharmacological classes were identified as potential inhibitors.
References [1] Backert S., Neddermann M., Maubach G., and Naumann M. (2016) Helicobacter 21, 19-25. [2] Venerito M., Vasapolli R., Rokkas T., and Malfertheiner P. (2015) Helicobacter 20, 36-39. [3] Federico A., Gravina A.G., Miranda A., Loguercio C., and Romano M. (2014) World J. Gastroenterol. 20, 665-672. [4] Beier D., and Frank R. (2000) J. Bacteriol. 182, 2068-2076. [5] Pflock M., Kennard S., Finsterer N., and Beier D. (2006) J. Biotechnol. 126, 52-60. [6] Gupta S.S., Borin B.N., Cover T.L., and Krezel A.M. (2009). J. Biol. Chem. 84, 6536-6545.
P12. Targeting the ferric uptake regulator (Fur) for identification of potential novel drugs against Pseudomonas aeruginosa
Andrés González1,2, Ángela Fernández-Otal1,2,3, Adrián Velázquez-Campoy2, Javier Sancho2,3, Ángel Lanas1,4, María F. Fillat2,3. 1Aragon Institute for Health Research (IIS Aragon) 2Institute of Biocomputation and Physics of Complex Systems (BIFI), University of Zaragoza. 3Department of Biochemistry & Molecular and Cell Biology, University of Zaragoza. 4Digestive Diseases Service, University Clinic Hospital Lozano Blesa, CIBERehd.
Pseudomonas aeruginosa is a Gram-negative, rod-shaped bacterium which is seldom a member of the human normal microbiota and rarely causes disease in healthy individuals. However, this opportunistic pathogen is a leading cause of nosocomial infections worldwide. Colonization rates may exceed 50% during hospitalization, especially among patients with trauma or breach in cutaneous or mucosal barriers, causing pneumonias, urinary tract infections, bloodstream infections, skin and wound infections, etc [1,2]. P. aeruginosa is also a common cause of high morbidity/mortality in patients with defects in host defenses or cystic fibrosis [3]. The increasing frequency of multi-drug-resistant P. aeruginosa strains is concerning as efficacious antimicrobial options are severely limited [2,4]. Selection of the most appropriate antibiotic is frequently complicated by the ability of P. aeruginosa to develop resistance to multiple classes of antibacterial agents, even during the course of treating an infection. This fact can severely limit the therapeutic options for treatment of serious infections. Therefore, a vast number of approaches to develop novel anti-infectives are currently pursued [5]. With the advent of modern molecular biology methods and based on knowledge of the pathogen genomes, drug discovery has now largely changed into a target-based approach [6]. New drugs that inhibit the biological activity of essential molecular targets and retain potency against multi-drug-resistant P. aeruginosa strains, with the additional benefit of minimize side effect on normal microbiota, are urgently needed. In P. aeruginosa, the ferric uptake regulator (Fur) is an essential protein that modulates transcription of genes involved not only in iron acquisition mechanisms but also in exotoxin production, defenses against oxidative stress, aerobic growth, etc. Using a fluorescence-based thermal shift assay, we evaluated the binding capability of 1120 FDA-approved small molecules to P. aeruginosa Fur. Five of these drugs bind to Fur and could potentially inhibit its function.
References [1] Lister P.D., Wolter D.J., and Hanson N.D. (2009) Clin. Microbiol. Rev. 22, 582-610. [2] Obritsch M.D., Fish D.N., MacLaren R., and Jung R. (2005) Pharmacotherapy 25, 1353-1364. [3] Langan K.M., Kotsimbos T., and Peleg A.Y. (2015) Curr. Opin. Infect. Dis. 28, 547-556. [4] Aloush V., Navon-Venezia S., Seigman-Igra Y., Cabili S., and Carmeli Y. (2006) Antimicrob. Agents Chemother. 50, 43-48. [5] Wagner S., Sommer R., Hinsberger S., Lu C., Hartmann R.W., Empting M., and Titz A. (2016) J. Med. Chem. 59, 5929-5969. [6] Eder J., Sedrani R., and Wiesmann C. (2014) Nat. Rev. Drug Discovery 13, 577-587.
P13. 3D structure prediction and AFM morphology study of human FAD synthase (isoform 2).
Víctor Taleb Seral1, Milagros Medina1,2, Piero Leone3,Maria Barile3, Anabel Lostao4,5, Marta Martínez-Júlvez1,2. 1Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Spain 2 Instituto de Biocomputación y Física de Sistemas Complejos (BIFI) and GBsC-CSIC and BIFI JointUnits, Universidad de Zaragoza, Spain 3Dipartimento di Bioscienze, Biotecnologie e Biofarmaceutica, UniversitàdegliStudi di Bari “A. Moro”, viaOrabona 4, I-70126, Bari, Italy 4 Fundación ARAID, Zaragoza, Spain 5 Laboratorio de Microscopías Avanzadas (LMA), Instituto de Nanociencia de Aragón (INA), Universidad de Zaragoza, and Fundación INA, Spain
The human FAD-synthase (hFADS) is a bi-functional enzyme involved in the synthetic pathway of the FAD cofactor, required by many enzymes for their function. This enzyme consists of a C- terminus domain or FAD-forming catalytic domain (Aa 232 to 490) and an N-terminus domain that resembles a molybdopterin-binding (MPTb) domain (Aa 13 to 197) and catalyses the FAD hydrolysis [1]. According to its localization, several isoforms of hFADS have been detected and here we report the predicted structure for the cytosolic one. The 3D structure prediction has been achieved using two different servers, GENO3D and SwissModel that take proteins with similar sequences as templates for the structure prediction. Due to the different functionality between domains, they have been predicted separately. The templates used for the C-terminus domain were S. cerevisiae FAD-synthase (PDB: 2wsi) and C. glabrata FMN adenylyltransferase (PDB: 3fwk), and for the N-terminus domain T. acidophilum CinA (PDB: 3kbq) was used. The whole predicted 3D structure of hFADS has been obtained by the RaptorX server using as templates the proteins mentioned above together with that of the T. thermophilu s CinA (PDB: 4ct8). We used also atomic force microscopy (AFM) to direct imaging the single FAD-forming domain bound to mica surfaces, while preserving their catalytic properties. AFM allowed solving individual C domain molecules in buffer as previously described for prokaryotic FADSs [2].A majority of monomerswas found at the images exhibiting an average height of 3.5 ± 0.5 nm. About one third of the molecules appear dimerized showing two peaks of 5.0 ± 0.5 nm and 4.0 ± 0.5 nm.
References [1] Giancaspero T.A., Galluccio M., Miccolis A., Leone P., Eberini I., Iametti S., Indiveri C., Barile M. (2015). Biochemical and Biophysical Research Communications, 465, 443-449. [2] Marcuello C., Arilla-Luna S., Medina M., Lostao A. (2013). Biochimica et Biophysica Acta 1834, 665–676.
P14. Old drugs, new uses. Novel compounds targeting the prokaryotic FAD synthetases
Ernesto Anoz-Carbonell1,2,3, AinhoaLucía Quintana3, María Sebastián1,2, Sandra Salillas1,2, Javier Sancho1,2, José Antonio Aínsa3, Milagros Medina1,2
1Instituto de Biocomputación y Física de SistemasComplejos (BIFI), Universidad de Zaragoza, Spain 2Departamento de Bioquímica y Biología Molecular y Celular, Universidad de Zaragoza, Spain 3Dpto. Microbiología, MedicinaPreventiva y SaludPública, Universidad de Zaragozaand CIBER EnfermedadesRespiratorias (CIBERES), Instituto de Salud Carlos III
The prokaryotic FAD synthetases (FADS), bifunctional enzymes that catalyze the biosynthesis of the essential flavin cofactors flavinmononnucleotide (FMN) and flavin adenine dinucleotide (FAD), are key proteins in flavin and flavoproteome homeostasis [1]. This crucial role in cellular metabolism, together with the significant differences with their eukaryotic counterparts, converts the FADS in a potential drug target for the development of inhibitors endowed with antimicrobial activity [2]. In this study, we have thoroughly characterized five in vitro inhibitors of the FADSs from Corynebacterium ammoniagenes and Streptococcus pneumoniae. These compounds show antimicrobial activity against gram positive and acid-fast pathogens, and one of them against gram negative microorganisms as well. However, the antibacterial mechanism of action through the in vivo inhibition of FADS has yet to be proven. Nevertheless, their therapeutic application is limited due to their high cytotoxicity on eukaryotic cell lines at the concentrations required for the antimicrobial activity. Both antibacterial and cytotoxic effects may be associated with the unspecific alteration of other essential flavoproteins and flavoenzymes, which perform a key role in cellular metabolism. In a further step, the molecular information of the antimicrobial mechanism of action will be used for the optimization of these compounds in order to generate second-generation antimicrobials with higher efficacy and less toxicity.
References [1] Frago, S., Martínez-Júlvez, M., Serrano, A., and Medina, M. (2008). BMC Microbiol. 8, 160. [2] Serrano, A., Ferreira, P., Martínez-Júlvez, M., and Medina, M. (2013). Curr. Pharm. Des. 19, 2637–2648.
P15. Overexpression of FurC produces changes in the pigment composition and yield of photosynthetic apparatus in Anabaena PCC7120
Emma Sevilla1, Cristina Sarasa1, Esther Broset 1, Rafael Cases2, María Luisa Peleato1 and María F. Fillat1.
1Departamento de Bioquímica y Biología Molecular y Celular, Instituto de Biocomputación y Física de Sistemas Complejos. 2Instituto de Ciencia de Materiales de Aragón y Departamento de Física de la Materia Condensada. Facultad de Ciencias, Universidad de Zaragoza-CSIC, Pedro Cerbuna 12, 50009 Zaragoza, Spain.
Ferric uptake regulator (Fur) proteins are transcriptional regulators that play key roles in many prokariots. The cyanobacteria Anabaena PCC 7120 contains in its genome three paralogs of Fur proteins whose genes are called all1691 (furA), all2473 (furB) and alr0954 (furC). FurC protein has been barely studied in Anabaena. It has been described as a modulator of DNA-binding properties of the two other Fur proteins and also as a possible PerR regulator. This work was focused on the study of the role of FurC protein in Anabaena PCC7120. In order to achieve this goal, an overexpression mutant of FurC protein was constructed in Anabaena PCC7120 rendering the EB2770 strain. This mutant contained approximately a 45-fold increase in the level of furC transcript referred to wild type strain. Scanning Electron Microscopy (SEM) studies were performed comparing the EB2770 strain and the wild type strain. The data revealed changes in the size, the shape and even in the roughness of the EB2770 cells. Growth curves were carried out with the wild type and the EB2770 strain and surprisingly, the overexpression of FurC protein led to an increase in the growth rate of the EB2770 strain. On the other hand, the photosynthetic pigments (chlorophyll, phycobiliproteins and carotenes) were determined in both strains and the data showed that pigment content was notably altered in the overexpression mutant. Comparison of the fluorescence emission spectra at 77K of both strains evidences differences in the efficiency and yield of energy transfer between the phycobilisome and the photosystem I and II of Anabaena PCC7120 and the FurC overexpressing strain. Finally pull down experiments were performed with purified FurC protein in order to identify proteins in an extract of Anabaena cells that could interact with FurC protein. The results indicated that two linker proteins of phycobilisome interacted with FurC protein.
P16. Spectroscopic insights into the stability of Apoptosis Inducing Factor
Raquel Villanueva1, Juan Martínez-Oliván1, Javier Sancho1, Patricia Ferreira1, and Milagros Medina1.
1 Department of Biochemistry and Molecular and Cellular Biology. Institute for Biocomputation and Physics of Complex Systems.
Apoptosis Inducing Factor (AIF) is a mitochondrial oxidoreductase essential for cell survival, acting as a supporter of respiratory chain complexes assembly by facilitating the entry of CHCHD4 factor into mitochondria [1]. On the other hand, AIF acts as a caspase-independent cell death mediator. For that, AIF is translocated into the nucleus where it induces chromatin condensation and DNA fragmentation, involving several other proteins [2]. Binding of NADH to AIF induces its reduction and the stabilization of a long-lived charge-transfer complex (CTC) between its reduced FAD cofactor and the oxidized nicotinamide moiety of the coenzyme product [3, 4]. This process produces a dramatic change in AIF structure and induces its dimerization as well as the release of the 509-560 region contained in the C-terminal apoptotic domain. This AIF NADH-driven allosteric regulation has been recently suggested to propagate from the CTC active site through very specific molecular pathways and to be central to control the AIF participation in mitochondrial homeostasis and programmed cell death [5]. Here, we have evaluated the WT hAIF∆1-101 stability under different conditions that emulate possible different conformational situations as a consequence of its subcellular localization (variations in pH), redox state and/or binding of the coenzyme. We have evaluated the thermal stability of WT hAIF∆1-101 in its oxidized monomeric and its NADH-reduced dimeric states as a function of pH by using different spectroscopic approaches.
References
[1] Hangen, E., O. Féraud, S. Lachkar, H. Mou, N. Doti, G.M. Fimia, N.V. Lam, C. Zhu, I. Godin, K. Muller, A. Chatzi, E. Nuebel, F. Ciccosanti, S. Flamant, P. Bénit, J.L. Perfettini, A. Sauvat, A. Bennaceur-Griscelli, K. Ser-Le Roux, P. Gonin, K. Tokatlidis, P. Rustin, M. Piacentini, M. Ruvo, K. Blomgren, G. Kroemer and N. Modjtahedi. (2015) Mol Cell 58, 6: 1001-14. [2] Natarajan, S.K. and D.F. Becker. (2012) Cell Health Cytoskelet, 4: 11-27. [3] Ferreira, P., R. Villanueva, M. Martínez-Júlvez, B. Herguedas, C. Marcuello, P.Fernandez- Silva, L. Cabon, J.A. Hermoso, A. Lostao, S.A. Susin and M. Medina. (2014) Biochemistry 53, 25: 4204-15. [4] Sevrioukova, I.F. (2011) Antioxid Redox Signal 14, 12: 2545-79. [5] Brosey C., C. Ho, W.Z. Long, S. Singh, K. Burnett, G.L. Hura, J.C. Nix, G.R. Bowman, T. Ellenberger, and J.A. Tainer. (2016) Structure 24, 1-13.
P17. Bedaquiline, anti MDR-TB drug, is efficiently nanoencapsulated Laura De Matteis1,*, Dorothée Jary2,3,* , Ainhoa Lucía4,5,6, Inés Serrano7, Sonia García 1, Fabrice Navarro2,3, Jesús M. de la Fuente7 , and José Antonio Ainsa 4,5,6,8
1 Instituto de Nanociencia de Aragon (INA), Universidad de Zaragoza, 2 Univ. Grenoble Alpes, Grenoble, France 3 CEA, LETI, MINATEC Campus, Technologies for Healthcare and Biology division, Grenoble, France 4 Departamento de Microbiologia, Facultad de Medicina, Universidad de Zaragoza, 5 CIBER de Enfermedades Respiratorias (CIBERES), Instituto de Salud Carlos III 6 Instituto de Investigacion Sanitaria de Aragon (IIS-Aragon) 7 Instituto de Ciencia de Materiales de Aragon (ICMA), Universidad de Zaragoza-CSIC 8 Instituto de Biocomputacion y Fisica de Sistemas Complejos, BIFI, Universidad de Zaragoza *These authors contributed equally to this work
Antibiotic resistance in Mycobacterium tuberculosis is a declared global health emergency, and rising cases of MDR-TB, and XDR-TB are making increasingly difficult to treat tuberculosis nowadays [1]. After 50 years without any new drug for TB, in 2012 the FDA approved the use of bedaquiline, the first drug designed specifically to treat MDR-TB. It is very effective but shows serious side effects, and consequently, it can only be prescribed when no other treatment options are available [2]. Novel drug delivery systems based on nanocarriers are a promising strategy to overcome current therapeutic challenges due to their unique physicochemical properties [3, 4], like small size or high surface to volume ratio. Nanocarriers improve the aqueous solubility of poorly soluble drugs, protects the drugs, and allow a controlled release of the medication. Additionally they can be modified to control their biodistribution allowing selective transport to the sites of infection. The development of effective and safe nanotherapy methods is particularly relevant in the treatment of MDR-TB, as it requires very long treatments with highly toxic drugs. In this sense the nanoencapsulation of bedaquiline is of special interest [5]. In this work, chitosan based nanocapsules and Lipid NanoParticles, based on the Lipidots® technology, have been synthesized and optimized for the encapsulation of bedaquiline. upon quantification of drug loading efficiency, improvement of the drug payload, and nanoparticles stability amelioration in storage conditions. For the best candidates, drug release has been determined in biological media for in vitro assays. The antimycobacterial activity has finally been evaluated to confirm that the drug is still active after encapsulation, and, their cytotoxicity has been assayed in different cell lines.
References [1]. WHO, Drug resistant TB: surveillance and response. Supplement to global tuberculosis report (2014). Available in http://www.who.int/tb/publications/global_report/gtbr14_supplement_web.pdf [2]. Mingote LR, Namutamba D, Apina F, Barnabas N, Contreras C, Elnour T, Frick MW, Lee C, Seaworth B, Shelly D, Skipper N, Tavora dos Santos Filho E. Lancet. (2015), Lancet. 385: 477-479. [3]. Mishra B, Patel BB, Tiwari S. (2010), Nanomedicine: NBM 6: 9-24. [4]. Onoue S, Yamada S, Chan HK. (2014); Int J Nanomed. 9: 1025-1037. [5]. Mustafa S, Pai RS, Singh G, Devi K. (2015) J Drug Targeting 23: 287 P18. Protein Motions Promote Hydride Tunneling in the Reduction of Aryl-Alcohol Oxidase Juan Carro1, Marta Martínez-Júlvez2, Milagros Medina2, Angel T. Martínez1, Patricia Ferreira2
1 Centro de Investigaciones Biológicas, CSIC, E-28040, Madrid, Spain 2Departament of Biochemistry and Molecular and Cellular Biology, and Institute for Biocomputation and Physics of Complex Systems (BIFI), University of Zaragoza, E-50009 Zaragoza, Spain
The temperature dependence of H– transfer (HT) from the substrate to the N5 of the FAD cofactor during the reductive half-reaction of aryl-alcohol oxidase (AAO) from Pleurotus eryngii has been assessed in this work to deduce the extent of the involvement of quantum-mechanical tunneling in the process. Data from kinetic isotope effects (KIEs) on both the kinetics of pre-steady state reduction of the enzyme and its steady-state kinetics employing differently deuterated substrates suggest the involvement of tunneling in the HT, according to the environmentally-coupled tunneling model. Moreover, those kinetic data, along with the crystallographic structure of the enzyme in complex with a substrate analog, indicate that the enzyme shows a pre-organized active site that would only require the approaching of the H– donor and the acceptor for the tunneled transfer to take place. Alteration of the enzyme’s active-site structure by replacement of Y92, a residue involved in the stabilization of the catalytically competent substrate-enzyme complex, showed different behaviors toward this HT, further reinforcing the above statements.
P19. Insights into apotential chloroplast NADPH dependent flavoreductasein stroma from Arabidopsis thaliana Guillermina Goñi1,2, Minna M. Koskela3, Nina Lehtimäki3, Paula Mulo3 and Milagros Medina1,2
1 Molecular and Cellular Biology, Department of Biochemistry, University of Zaragoza, Zaragoza, Spain. 2Institute for Biocomputation and Physics of Complex Systems (BIFI), University of Zaragoza, Zaragoza, Spain. 3 Molecular Plant Biology, Department of Biochemistry, University of Turku, FI-20014 Turku, Finland
In chloroplasts, Ferredoxin:NADP(H) oxidoreductase(FNR) enzymes oxidize the final reduced product of the photosynthetic electron transport chain, ferredoxin (Fd), to reduce NADP+ providing reduction power, NADPH, to different metabolic pathways. In addition, FNRs play a role in cyclic electron transport. Oppositely, in non-photosynthetic plastids FNR oxidizes NADPH to provide reduced Fd for enzymes implicated in bio assimilation and biosynthesis of metabolites. These separate plastid types predominantly contain different iso-proteins, with distinct leaf FNR (LFNR) and root FNR (RFNR) features. A locus from Arabidopsis thaliana genome encodes in addition a FAD/NAD(P)-binding oxidoreductase that is a distant homologue of FNRs and is localized mainly in stroma of chloroplasts. This protein, herein knows as FNR- like, has been cloned, heterogeneously expressed in Escherichia coli and purified in this study. Purified FNR-like binds as cofactor one molecule of FAD that can be reduced by NAD(P)H, being the process specific for NADPH versus NADH. We determined pH and ionic strength NADPH NADPH conditions for this process, as well as kcat and Km parameters. Km of FNR-like is in the range to those reported for canonical plant-type FNRs, but turnover is dramatically lower. The pre-steady-state analysis of the process indicated us that NADPH is able to fully reduce FNR-like through a hydride transfer process with the stabilization of traces of intermediate charge transfer complexes between the flavin and nicotinamide rings and with slow hydride transfer rates (∼2 s- 1). Altogether our data indicate that FNR-like is less efficient in accepting electrons from NADPH than FNRs, stabilizes less amount of spectroscopically observable charge transfers complexes and, contrary to the LFNR, can get fully reduced by the coenzyme. All these data envisage a different and specific function for FNR-like regarding LFNRs and RFNRs.
P20. Structural Study of Amyloid Aggregates
José D. Camino1, Serene W. Chen2, Jesús Sot3, Igor de la Arada 3, José L. Arrondo3, Félix M. Goñi3, Christopher M. Dobson2, Nunilo Cremades1
1 Institute for Biocomputation and Physics of Complex Systems (BIFI), University of Zaragoza, Zaragoza, Spain. 2 Department of Chemistry, University of Cambridge, Cambridge, United Kingdom. 3 Department of Biochemistry, University of Basque Country, Leioa, Spain.
α-Synuclein (α-Syn) is a presynaptic intrinsically disordered neuronalprotein whose misfolding and aggregation in the form of amyloid fibrils is the hallmark of Parkinson’s disease and other neurodegenerative disorders collectively referred to synucleinopathies. Recent evidence suggest that different types of fibrillar strains or polymorphs of α-Syn have different seeding capabilities and present different degrees of infectivity, inducing variable neuronal vulnerability and pathology, suggesting a potential link between the structure and pathology of different α-Syn fibrillar strains. With the aim of understanding the origins of the multiplicity of α-Syn fibril polymorphism and its relation with neuronal toxicity, we have developed different strategies to purify different α-Syn fibril polymorphs and have characterized their structure by means of Fourier Transform Infrared.
References [1] Cremades N., Cohen S. I., Deas E., Abramov A. Y., Chen A. Y., Orte A., Sandal M., Clarke R. W., Dunne P., Aprile F. A., Bertoncini C. W., Wood N. W., Knowles T. P., Dobson C. M., and Klenerman D. (2012) Direct observation of the interconversion of normal and toxic forms of alpha-synuclein. Cell 149(5): 1048-59. [2] Campioni S., Carret G., Jordens S., Nicoud L., Mezzenga R., and Riek R. (2014) The presence of an Air−Water interface affects formation and elongation of α-Synuclein fibrils. JACS 136: 2866−2875. [3] Chen S. W., Drakulic S., Deas E., Ouberai M., Aprile F. A., Arranz R., Ness S., Roodveldt C. Guilliams T., De-Genst E. J., Klenerman D., Wood N. W., Knowles T. P., Alfonso C. Rivas G., Abramov A. Y., Valpuesta J. M., Dobson C. M., and Cremades N. (2015) Structural characterization of toxic oligomers that are kinetically trapped during alpha- synuclein fibril formation. PNAS 112(16):E1994-2003.
P21. High throughput prediction of hot spots of interaction for large scale mapping of mutations in the human interactome Didier Barradas Bautista1, Juan Fernández Recio1,
1 Barcelona Supercomputing Center.
Proteins are the embodiment of the message encoded in the genes and they act as the building blocks and effector part of the cell. From gene regulation to cell signalling, as well as cell recognition and movement, protein-protein interactions (PPIs) drive many important cellular events by forming intricate interaction networks. The number of all non-redundant human binary interactions, forming the so-called interactome, ranges from 130,000 to 650,000 interactions as estimated by different studies [1,2]. In some diseases, like cancer, these PPIs are altered by the presence of mutations in individual proteins, which can change the interaction networks of the cell resulting in a pathological state [3]. To understand how these mutations can alter the PPIs, we need to look at the three-dimensional structure of the protein complexes at the atomic level. However, there are available structures for less than 10% of the estimated human interactome [4]. Computational approaches such as protein-protein docking can help to extend the structural coverage of known PPIs [5]. In this work, we used docking calculations to predict interaction hotspots, i.e. residues that contribute the most to the binding energy, and interface patches by including neighbour residues to the predictions. The method can predict the location of a nsSNPs with a precision of 51.5% and a sensitivity of 80,1%. This methodology was applied to predict the location of 14,551 nsSNPs in 4,254 proteins, for more than 12,000 interactions without 3D structure. We found that 34% of the disease-associated nsSNPs were located at a protein-protein interface. This opens future opportunities for the high-throughput characterization of pathological mutations at the atomic level resolution, and can help to design novel therapeutic strategies to re- stabilize the affected PPIs by disease-associated nsSNPs.
References [1] Venkatesan K.(2009); Nat Methods. 6:83–90. [2] Yu H (2008). Science. ;322:104–10. [3] Lage K (2014). Biochim Biophys Acta BBA - Mol Basis Dis. Oct;1842(10):1971–80. [4] Mosca R, Céol A, Aloy P (2013). Nat Methods. Jan;10(1):47–53. [5] Mosca R, Pons C, Fernández-Recio J, Aloy P(2009). PLoS Comput Biol. 28;5(8):e1000490. P22. Expanding template-based protein-protein complex prediction using ab-initio docking S. Mares-Sámano, L. Ángel Rodríguez-Lumbreras and J. Fernández-Recio
1 Universidad Nacional Autónoma de México-ICF. 2,3 Barcelona Supercomputing Center
Structural characterization of protein-protein interaction (PPI) networks is crucial for understanding the underlying molecular mechanisms whereby life processes and disease arise. However, due to inherent limitations of experimental techniques, such characterization only covers an extremely reduced fraction of the human PPI network (interactome) [1]. Recent studies have shown that although available structural templates may suffice to model a significant proportion of the interactome [2, 3], model accuracy and binding specificity remain unsolved problems. Consequently, improving the ability to predict PPIs structurally will help to provide a better 3D profile of the known interactome, which may ultimately lead to the development of new therapeutic applications. Here we show a novel approach that combines template-based modeling[4] with protein-protein computational docking to the structure-based prediction of PPIs. Our approach samples different protein-protein structural models derived from docking simulations. Models are subsequently ranked using a function that incorporates an energy-based scoring term and a structural template similarity score. The energy-based scoring function includes electrostatics, van der Waals and desolvation calculations[5], whilst the template similarity score accounts for the degree of structural similarity of models against a high-resolution and diverse dataset of structural templates. Our approach highly improved the predictive success rate over individual ab-initio docking and template-based techniques across a large benchmark dataset, including 176 protein-protein complexes. When compared to the performance of the ab-initio docking algorithm, we found that the approach increased consistently the success rate, by approximately 30%, for the top 1, top 5 and top 10 solutions. The success rate improvement was even more notorious when the comparison was performed against the predictions from the traditional template-based docking. Though incorporating ab-initio docking expands considerably the scope of the template-based docking method, challenges remain for interacting proteins in which high conformational changes occur upon binding and the size and diversity of the repertoire of structural templates needs to be increased.
References [1] Mosca, R., A. Céol, and P. Aloy (2013), Nat. Methods, 10(1): p. 47-53. [2] Kundrotas, P.J., et al. (2012), Proceedings of the National Academy of Sciences, 109(24): p. 9438-9441. [3] Negroni, J., R. Mosca, and P. Aloy (2014), Structure (London, England : 1993), 22(9): p. 1356-62. [4] Kundrotas, P.J., M.F. Lensink, and E. Alexov (2008), International Journal of Biological Macromolecules, 43(2): p. 198-208. [5] Cheng, T.M.-K., T.L. Blundell, and J. Fernandez-Recio (2007), Proteins, 68(2): p. 503-515.
P23. Design, synthesis and testing of a novel and efficient fluorescent compound for the staining of amyloid beta fibrillar aggregates.
A. Mahía1, 3, M. D. Díaz-de-Villegas3, J. A. Gálvez2, 3 and J. Sancho1
1 Department of Biochemistry and Molecular and Cellular Biology. Faculty of Sciences. University of Zaragoza, Spain; Biocomputation and Physics of Complex Systems Institute (BIFI). BIFI-IQFR(CSIC)-Joint Unit. University of Zaragoza, Spain. 2 Department of Organic Chemistry. Faculty of Sciences. University of Zaragoza, Spain 3 Department of Catalysis and Catalytic Processes. Institute of Chemical Synthesis and Homogeneous Catalysis (ISQCH). Faculty of Sciences. University of Zaragoza-CSIC, Spain.
More than 46 million people are affected by Alzheimer’s disease (AD), a neurodegenerative disorder that, since it was discovered in 1906, has become the major cause of dementia in humans. AD is characterized, in its early phase, by an inability to create new memories and a failure to retrieve ancient ones and, in more advanced stages, by the loss of important human skills such as reasoning, abstraction and language. Histologically, the two classic lesions that characterize AD are senile plaques, made of extracellular fibrillar deposits of amyloid beta peptide (Aβ) aggregates, and intraneuronal neurofibrillary tangles composed of hyperphosphorilated tau protein [1]. According to the amyloid cascade hypothesis, aggregation of Aβ triggers the development of AD. In a previous study, we reported the discovery of four chemical compounds that inhibit the aggregation of Aβ17-40 and Aβ1-42 in vitro and in fungi models [2]. As these compounds interact with Aβ aggregating species, suitable fluorescent derivatives could be used to stain and detect Aβ fibrils by means of fluorescence microscopy. Therefore, we have design and synthesized four fluorescent compounds derived from one of the previously identified inhibitors. All of them interfere with Aβ17-40 aggregation (as it has been demonstrated by turbidimetry, dynamic light scattering and transmission electron microscopy) and one of them is an efficient marker for Aβ1-42 fibrils via fluorescence microscopy and exhibits a greatest sensitivity than thioflavin T (ThT) staining.
References
[1] Selkoe D.J. (2011) Cold Spring Harb Perspect Biol. 3, a004457. [2] López L. C., Dos-Reis S., Espargaró A., Carrodeguas J. A., Maddelein M., Ventura S., and Sancho J. (2012) J. Med. Chem. 55, 9521-9530.
P24. Optimal control with nonadiabatic molecular dynamics: Application to the Coulomb explosion of sodium clusters Gómez Pueyo, Adrián1, Budagosky M., Jorge A.1, and Castro, Alberto12
1 Institute for Biocomputation and Physics of Complex Systems, University of Zaragoza, Calle Mariano Esquillor, 50018 Zaragoza, Spain 2ARAID Foundation, Calle María Luna, 50018 Zaragoza, Spain
In Ref. [1] we presented an implementation of optimal control theory (OCT) for the Ehrenfest model, a non-adiabatic ab initio model where the nuclei are treated as classical particles, while the electrons receive a quantum approach based on the time-dependent density functional theory. As an application of this implementation we have optimized the Coulomb explosion of sodium clusters irradiated by laser pulses for a given fluence, frequency and duration of the pulse, considering both moving and fixed nuclei during the action of the pulse. We chose the Coulomb explosion of the sodium clusters because it is a violent, complex and far- from-equilibrium process which requires a non-perturbative approach, qualities that makes it an ideal candidate for the Ehrenfest model. The OCT implementation in this model implies the propagation of the system both forward and backwards in time, so we had to be especially careful about the precision and stability of the numerical propagator. Our results show that the frequency of the pulses that maximize the Coulomb explosion of the clusters don’t correspond with the resonance frequency of the cluster’s bonds as could be expected, which complicates the search for the initial conditions of the optimization process. We also found that for our initial 16fs pulses the nuclear movement wasn’t relevant for the optimization of the pulses, but with pulses of 32 and 64fs the pulses obtained with the static nuclei method performed sensibly worse than its moving nuclei counterpart, even being unable to find pulses that produce the Coulomb explosion of the cluster in some cases.
References [1] Gómez Pueyo A., Budagosky M. J. A., and Castro A. (2016) Phys. Rev. A 94, 063421.
P25. Activity of circular bacteriocin AS-48 against mycobacteria Clara Aguilar1,2,3, *, Begoña Gracia1,2,3,Asunción Vitoria1,2,3, Rubén Cebrián4, Mercedes Maqueda4, José Antonio Aínsa1,2,3*
1Departamento de Microbiología, Facultad de Medicina, and Instituto de Biocomputación y Física de Sistemas Complejos, BIFI, Universidad de Zaragoza, Zaragoza, Spain 2CIBER de Enfermedades Respiratorias (CIBERES), Instituto de Salud Carlos III, Spain 3Instituto de Investigación Sanitaria de Aragón (IIS-Aragón), Zaragoza, Spain 4Departamento de Microbiología, Facultad de Ciencias, Universidad de Granada, Granada, Spain.
*Corresponding author: [email protected]; [email protected]
There are very few drugs available for treatment of tuberculosis and the incidence of multi-drug resistant strains of Mycobacterium tuberculosisis increasing worldwide, so this is promoting the development of a new line of drugs that try to solve these problems. We have investigated the antibacterial peptide AS-48, produced by E. faecalis, which is targeting the bacterial membrane, and it is active against several gram-positive bacteria. Targeting the membrane is an advantage considering that there are a lot biosynthetic pathways involved, so it is less likely to select a resistance mutation. This study has assessed the bactericidal effect of AS-48 in M.tuberculosis strains, includingH37Rv and other clinical and reference strains, and also against some non-tuberculous clinical isolates of mycobacterial species. The combination of AS-48 with either lysozyme or first line drugs (commonly used in the treatment of tuberculosis)increases bactericidal action of AS- 48, showing a synergic interaction. Under these conditions, AS-48 kills M.tuberculosis at a lower dose, and exhibits a MIC (Minimal Inhibitory Concentration) close to some of the first line anti- TB agents. In addition, we assayed cytotoxicity of AS-48 against THP-1 and MHS macrophagecell lines, and found that at concentrations close to the MIC of AS-48 we could not detect any cytotoxic effect. In summary, we consider that bacteriocin AS-48 has an interesting potential in the treatment of infectious diseases, including tuberculosis therapy because of its antimicrobial activity and its low cytotoxicity against cell lines.
KEY WORDS: AS-48, antimicrobial peptide, antituberculosis activity, synergy.
P26. Studying the mechanism of action of novel stabilizing compounds of phenylalanine hydroxylase
María Conde-Giménez1,2, Sandra Salillas1,2, Juan J. Galano-Frutos1,2, María Galiana1,2, Mº Dolores Díaz-de-Villegas3, Javier Sancho1,2
1 Departamento de Bioquímica y Biología Molecular y Celular. Facultad de Ciencias. Universidad de Zaragoza (Spain) 2 Biocomputation and Physics of Complex Systems Institute (BIFI). BIFI-IQFR(CSIC)-Joint Unit. Universidad de Zaragoza (Spain) 3 Departamento de Catálisis y Procesos Catalíticos. Instituto de Síntesis Química y Catálisis Homogénea (ISQCH). Universidad de Zaragoza-CSIC (Spain)
Phenylalanine hydroxylase (PAH) is a human enzyme that catalyzes the conversion of phenylalanine into tyrosine. Mutations on the gene that codifies this protein cause phenylketonuria (PKU), a rare disorder characterized with mental retardation and development problems if it is not treated [1]. The mainstay of therapy to prevent the major manifestations of this disease is not entirely effective so that new therapeutic strategies need to be developed [2]. Some of these approaches involve the use of compounds that act displacing the PAH folding equilibrium towards the native state and thereby rescuing its enzymatic function [3,4,5]. Therefore, both identifying new promising stabilizing compounds and understanding the unfolding and refolding of the physiologically active enzyme are crucial to develop new useful therapies for different phenotypes of PKU. Here, we present some biophysical results focused on both directions. We have identified a group of promising compounds characterized by the stabilizing effect exerted on thermal PAH unfolding. Moreover, we have studied the molecular events leading to the chemical unfolding of WT PAH and we are trying to propose a tentative model for the distribution of the oligomeric forms in the denaturation pathway. Further assays need to be done to test the efficacy and toxicity of these compounds in vivo and to ascribe the stabilizing effect exhibited in vitro to a specific unfolding event.
References
[1] Blau N. et al (2010) Lancet 376: 1417-27 [2] VanSpronsen F. J.et al (2010). Molec Genetics and Metab; 99, S90-95 [3] Heintz C. et al. (2013) Hum Mut 20, 34(7):927-36 [4] Pey A.L. et al. (2008) J Clin Invest, 118(8): 2858-2867 [5] Torreblanca R. et al. (2012) ChemBioChem, 13(9):1266-1269
P27. Identifying small compounds inhibiting protein-protein interactions involving intrinsically disordered proteins: a multiple challenge Jose L. Neira1,2, Jennifer Bintz3, Maria Arruebo4,5, Bruno Rizzuti6, Thomas Bonacci3, Sonia Vega2, Angel Lanas5,7,8,9, Adrian Velazquez-Campoy2,5,10, Juan L. Iovanna3, and Olga Abian2,4,5,7
1 Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Elche, Alicante, Spain 2 Instituto de Biocomputación y Física de Sistemas Complejos (BIFI), Unidad Asociada IQFR- CSIC-BIFI, Universidad de Zaragoza, Zaragoza, Spain 3 Centre de Recherche en Cancérologie de Marseille (CRCM), INSERM U1068, CNRS UMR 7258, Aix-Marseille Université and Institut Paoli-Calmettes, Parc Scientifique et Technologique de Luminy, Marseille, France 4 Instituto Aragonés de Ciencias de la Salud (IACS), Zaragoza, Spain 5 Instituto de Investigaciones Sanitarias (IIS) Aragón, Zaragoza, Spain 6 CNR-NANOTEC, Licryl-UOS Cosenza and CEMIF.Cal, Department of Physics, University of Calabria, Cosenza, Italy 7 Centro de Investigación Biomédica en Red en el Área Temática de Enfermedades Hepáticas y Digestivas (CIBERehd), Spain 8 Servicio de Aparato Digestivo, Hospital Clínico Universitario “Lozano Blesa”, Zaragoza, Spain 9 Department of Medicine, University of Zaragoza, Zaragoza, Spain 10 Fundación ARAID, Diputación General de Aragón, Zaragoza, Spain
Intrinsically disordered proteins (IDPs) are prevalent in eukaryotes, performing signaling and regulatory functions. Often associated with human diseases, they constitute drug-development targets. NUPR1 (nuclear protein 1) is a multifunctional IDP, over-expressed and involved in pancreatic ductal adenocarcinoma (PDAC) development. Targeting protein-protein interactions involving IDPs using low molecular weight compounds is challenging because of several reasons: 1) absence of well-defined protein structure, 2) complex conformational landscape, and 3) extended protein binding interface. By screening 1120 FDA-approved compounds, fifteen candidates were selected, and their interactions with NUPR1 were characterized by experimental and simulation techniques. The protein remained disordered upon binding to all fifteen candidates. These compounds were tested in PDAC-derived cell-based assays, and all induced cell-growth arrest and senescence, reduced cell migration, and decreased chemoresistance, mimicking NUPR1-deficiency. The most effective compound completely arrested tumor development in vivo on xenografted PDAC-derived cells in mice [1]. Besides reporting the discovery of a compound targeting an intact IDP and specifically active against PDAC, our study proves the possibility to target the ‘fuzzy’ interface of a protein that remains disordered upon binding to its natural biological partners or to selected drugs.
References [1] Neira J.L., Bintz J., Arruebo M., Rizzuti B., Bonacci T., Vega S., Lanas A., Velazquez- Campoy A., Iovanna J.L., and Abian O. (2017) Identification of a drug targeting an intrinsically disordered protein involved in pancreatic adenocarcinoma. Sci. Rep. 7, 39732