Ensamblaje in vitro de la cápsida del virus de la inmunodeficiencia humana y su inhibición por péptidos diseñados racionalmente

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 22-07-201

Diseño Estructural del Pavimento en el Sector 1 de la Urb. Los Huertos - Distrito de Huanchaco - Trujillo - La Libertad

Esta investigación titulada ‘‘DISEÑO ESTRUCTURAL DEL PAVIMENTO EN EL SECTOR 1 DE LA URB. LOS HUERTOS - DISTRITO DE HUANCHACO - TRUJILLO - LA LIBERTAD’’ busca diseñar distintos tipos de pavimentos como son del tipo flexible, rígido y articulado mediante el uso de los procedimientos del método AASHTO 93 y aplicando las guías del manual de carreteras proporcionado por el MTC, los cuales harán posible el mejor planteamiento de diseño de pavimento durante el desarrollo de la tesis. La unidad de análisis en cuestión pertenece al sector 1 de la urbanización Los Huertos, la cual no cuenta con una infraestructura de pavimentos y padece de condiciones precarias en cuanto a la transitabilidad y niveles de servicio de sus vías, estas afectan el desarrollo urbanístico de la zona y por este motivo provoca un inadecuado tránsito vehicular y peatonal. Para hacer frente a la desfavorable situación que enfrenta esta población, se propone más de una opción de pavimento los cuales se comparan a través de un estudio con el fin de determinar el más adecuado para las necesidades de la población. Este proceso se llevó a cabo mediante estudios previos como el aforo vehicular en la zona en cuestión, análisis de comportamiento de suelos y la determinación de las variables de diseño para aplicar el método de diseño de AASHTO 93 para pavimento, seguidos de la elaboración de las propuestas económicas correspondientes a los diseños los cuales fueron realizados empleando softwares específicos. Esta tesis brinda una apropiada opción de pavimento para resolver los problemas que enfrenta la unidad de análisis.This research entitled ''STRUCTURAL DESIGN OF THE PAVEMENT IN SECTOR 1 OF THE URB. LOS HUERTOS - DISTRICT OF HUANCHACO - TRUJILLO - LA LIBERTAD '' seeks to design different types of pavements such as flexible, rigid and articulated by using the procedures of the AASHTO 93 method and applying the guidelines of the road manual provided by the MTC , which will make possible the best pavement design approach during the development of the thesis. The analysis unit in question belongs to sector 1 of the Los Huertos urbanization, which does not have a pavement infrastructure and suffers from precarious conditions in terms of the transitability and service levels of its roads, these affect the urban development of the area. area and for this reason causes inadequate vehicular and pedestrian traffic. To deal with the unfavorable situation faced by this population, more than one pavement option is proposed, which are compared through a study in order to determine the most suitable for the needs of the population. This process was carried out through previous studies such as the traffic capacity in the area in question, soil behavior analysis and the determination of the design variables to apply the AAHSTO 93 design method for pavement, followed by the elaboration of the economic proposals corresponding to the designs which were made using specific software. This thesis provides an appropriate pavement option to solve the problems faced by the analysis unit.Tesi

Biophysical analysis of the MHR motif in folding and domain swapping of the HIV capsid protein C-terminal domain

© 2015 Biophysical Society. Infection by human immunodeficiency virus (HIV) depends on the function, in virion morphogenesis and other stages of the viral cycle, of a highly conserved structural element, the major homology region (MHR), within the carboxyterminal domain (CTD) of the capsid protein. In a modified CTD dimer, MHR is swapped between monomers. While no evidence for MHR swapping has been provided by structural models of retroviral capsids, it is unknown whether it may occur transiently along the virus assembly pathway. Whatever the case, the MHR-swapped dimer does provide a novel target for the development of anti-HIV drugs based on the concept of trapping a nonnative capsid protein conformation. We have carried out a thermodynamic and kinetic characterization of the domain-swapped CTD dimer in solution. The analysis includes a dissection of the role of conserved MHR residues and other amino acids at the dimerization interface in CTD folding, stability, and dimerization by domain swapping. The results revealed some energetic hotspots at the domain-swapped interface. In addition, many MHR residues that are not in the protein hydrophobic core were nevertheless found to be critical for folding and stability of the CTD monomer, which may dramatically slow down the swapping reaction. Conservation of MHR residues in retroviruses did not correlate with their contribution to domain swapping, but it did correlate with their importance for stable CTD folding. Because folding is required for capsid protein function, this remarkable MHR-mediated conformational stabilization of CTD may help to explain the functional roles of MHR not only during immature capsid assembly but in other processes associated with retrovirus infection. This energetic dissection of the dimerization interface in MHR-swapped CTD may also facilitate the design of anti-HIV compounds that inhibit capsid assembly by conformational trapping of swapped CTD dimers.Spanish Government (BIO2012-37649) and Comunidad de Madrid (S-2009/MAT/1467) and by an institutional grant from Fundación Ramón Areces.Peer Reviewe

Dynamic constriction andfission of endoplasmicreticulum membranes by reticulon

The endoplasmic reticulum (ER) is a continuous cell-wide membrane network. Network formation has been associated with proteins producing membrane curvature and fusion, such as reticulons and atlastin. Regulated network fragmentation, occurring in different physiological contexts, is less understood. Here we find that the ER has an embedded fragmentation mechanism based upon the ability of reticulon to produce fission of elongating network branches. In Drosophila, Rtnl1-facilitated fission is counterbalanced by atlastin-driven fusion, with the prevalence of Rtnl1 leading to ER fragmentation. Ectopic expression of Drosophila reticulon in COS-7 cells reveals individual fission events in dynamic ER tubules. Consistently, in vitro analyses show that reticulon produces velocity-dependent constriction of lipid nanotubes leading to stochastic fission via a hemifission mechanism. Fission occurs at elongation rates and pulling force ranges intrinsic to the ER, thus suggesting a principle whereby the dynamic balance between fusion and fission controlling organelle morphology depends on membrane motility.This work was partially supported by NIH R01GM121725 to V.A.F., a 5x1000 grant from the Italian Ministry of Health and Telethon GGP11189 to A.D., Spanish Ministry of Science, Innovation and Universities grants BFU2015-70552-P to V.A.F. and A.V.S., and BFU2015-63714-R and PGC2018-099341-B-I00 to B.I., Basque Government grant IT1196-19, Russian Science Foundation Grant No. 17-75-30064 and Ministry of Science and Higher Education of the Russian Federation

Mechanical unfolding of long human telomeric RNA (TERRA)

[EN] We report the first single molecule investigation of TERRA molecules. By using optical-tweezers and other biophysical techniques, we have found that long RNA constructions of up to 25 GGGUUA repeats form higher order structures comprised of single parallel G-quadruplex blocks, which unfold at lower forces than their DNA counterparts.This work was supported by grants from the Spanish Ministry of Science and Innovation (grants RYC2007-01765 to JRA-G, BFU2011-30295-C02-01 to AV, and CTQ2010-21567-C02-02 to CG). MG was supported by the FPI fellowship BES-2009-027909. RB and EH-G were supported by Comunidad de Madrid, grant CAM-S2009MAT-1507. AV acknowledges an institutional grant from the Fundacion Ramon Areces to the CBMSO. JRA-G wants to thank Prof. J. L. Carrascosa and Prof. J. M. Valpuesta (CNB-CSIC) for their continuous support and encouragement in this research. We also acknowledge the excellent technical assistance of Beatriz de Pablos (CBMSO).Garavís, M.; Bocanegra, R.; Herrero-Galán, E.; González, C.; Villasante, A.; Arias-Gonzalez, JR. (2013). Mechanical unfolding of long human telomeric RNA (TERRA). Chemical Communications. 49(57):6397-6399. https://doi.org/10.1039/c3cc42981dS639763994957De Lange, T. (2005). Shelterin: the protein complex that shapes and safeguards human telomeres. Genes & Development, 19(18), 2100-2110. doi:10.1101/gad.1346005Blackburn, E. H. (1991). Structure and function of telomeres. Nature, 350(6319), 569-573. doi:10.1038/350569a0Biffi, G., Tannahill, D., McCafferty, J., & Balasubramanian, S. (2013). Quantitative visualization of DNA G-quadruplex structures in human cells. Nature Chemistry, 5(3), 182-186. doi:10.1038/nchem.1548Paeschke, K., Simonsson, T., Postberg, J., Rhodes, D., & Lipps, H. J. (2005). Telomere end-binding proteins control the formation of G-quadruplex DNA structures in vivo. 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G-Quadruplex Formation by Human Telomeric Repeats-Containing RNA in Na+Solution. Journal of the American Chemical Society, 130(33), 11179-11184. doi:10.1021/ja8031532Collie, G. W., Haider, S. M., Neidle, S., & Parkinson, G. N. (2010). A crystallographic and modelling study of a human telomeric RNA (TERRA) quadruplex. Nucleic Acids Research, 38(16), 5569-5580. doi:10.1093/nar/gkq259Collie, G. W., Parkinson, G. N., Neidle, S., Rosu, F., De Pauw, E., & Gabelica, V. (2010). Electrospray Mass Spectrometry of Telomeric RNA (TERRA) Reveals the Formation of Stable Multimeric G-Quadruplex Structures. Journal of the American Chemical Society, 132(27), 9328-9334. doi:10.1021/ja100345zMartadinata, H., Heddi, B., Lim, K. W., & Phan, A. T. (2011). Structure of Long Human Telomeric RNA (TERRA): G-Quadruplexes Formed by Four and Eight UUAGGG Repeats Are Stable Building Blocks. Biochemistry, 50(29), 6455-6461. doi:10.1021/bi200569fArora, A., & Maiti, S. (2009). 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Rationally Designed Interfacial Peptides Are Efficient In Vitro Inhibitors of HIV-1 Capsid Assembly with Antiviral Activity

Virus capsid assembly constitutes an attractive target for the development of antiviral therapies; a few experimental inhibitors of this process for HIV-1 and other viruses have been identified by screening compounds or by selection from chemical libraries. As a different, novel approach we have undertaken the rational design of peptides that could act as competitive assembly inhibitors by mimicking capsid structural elements involved in intersubunit interfaces. Several discrete interfaces involved in formation of the mature HIV-1 capsid through polymerization of the capsid protein CA were targeted. We had previously designed a peptide, CAC1, that represents CA helix 9 (a major part of the dimerization interface) and binds the CA C-terminal domain in solution. Here we have mapped the binding site of CAC1, and shown that it substantially overlaps with the CA dimerization interface. We have also rationally modified CAC1 to increase its solubility and CA-binding affinity, and designed four additional peptides that represent CA helical segments involved in other CA interfaces. We found that peptides CAC1, its derivative CAC1M, and H8 (representing CA helix 8) were able to efficiently inhibit the in vitro assembly of the mature HIV-1 capsid. Cocktails of several peptides, including CAC1 or CAC1M plus H8 or CAI (a previously discovered inhibitor of CA polymerization), or CAC1M+H8+CAI, also abolished capsid assembly, even when every peptide was used at lower, sub-inhibitory doses. To provide a preliminary proof that these designed capsid assembly inhibitors could eventually serve as lead compounds for development of anti-HIV-1 agents, they were transported into cultured cells using a cell-penetrating peptide, and tested for antiviral activity. Peptide cocktails that drastically inhibited capsid assembly in vitro were also able to efficiently inhibit HIV-1 infection ex vivo. This study validates a novel, entirely rational approach for the design of capsid assembly interfacial inhibitors that show antiviral activity

Association equilibrium of the HIV-1 capsid protein in a crowded medium reveals that hexamerization during capsid assembly requires a functional C-domain dimerization interface

Polymerization of the intact capsid protein (CA) of HIV-1 into mature capsidlike particles at physiological ionic strength in vitro requires macromolecularly crowded conditions that approach those inside the virion, where the mature capsid is assembled in vivo. The capsid is organized as a hexameric lattice. CA subunits in each hexamer are connected through interfaces that involve the CA N-terminal domain (NTD); pairs of CA subunits belonging to different hexamers are connected through a different interface that involves the C-terminal domain (CTD). At physiological ionic strength in noncrowded conditions, CA subunits homodimerize through this CTD-CTD interface, but do not hexamerize through the other interfaces (those involving the NTD). Here we have investigated whether macromolecular crowding conditions are able to promote hexamerization of the isolated NTD and/or full-length CA (with an inactive CTD-CTD interface to prevent polymerization). The oligomerization state of the proteins was determined using analytical ultracentrifugation in the absence or presence of high concentrations of an inert macromolecular crowding agent. Under the same conditions that promoted efficient assembly of intact CA dimers, neither NTD nor CA with an inactive CTD-CTD interface showed any tendency to form hexamers or any other oligomer. This inability to hexamerize was observed even in macromolecularly crowded conditions. The results indicate that a functional CTD-CTD interface is strictly required for hexamerization of HIV-1 CA through the other interfaces. Together with previous results, these observations suggest that establishment of NTD-CTD interactions involved in CA hexamerization during mature HIV-1 capsid assembly requires a homodimerization-dependent conformational switching of CTD.Spanish Government (BIO2009-10092, BIO2012-37649, BIO2011-28941-C03); Comunidad de Madrid (S-2009/MAT/1467); Fundación Ramón ArecesPeer Reviewe

Optical Tweezers to Force Information out of Biological and Synthetic Systems One Molecule at a Time

Over the last few decades, in vitro single-molecule manipulation techniques have enabled the use of force and displacement as controlled variables in biochemistry. Measuring the effect of mechanical force on the real-time kinetics of a biological process gives us access to the rates, equilibrium constants and free-energy landscapes of the mechanical steps of the reaction; this information is not accessible by ensemble assays. Optical tweezers are the current method of choice in single-molecule manipulation due to their versatility, high force and spatial and temporal resolutions. The aim of this review is to describe the contributions of our lab in the single-molecule manipulation field. We present here several optical tweezers assays refined in our laboratory to probe the dynamics and mechano-chemical properties of biological molecular motors and synthetic molecular devices at the single-molecule level

Molecular recognition in the human immunodeficiency virus capsid and antiviral design

Many compounds able to interfere with HIV-1 infection have been identified; some 25 of them have been approved for clinical use. Current anti-HIV-1 therapy involves the use of drug cocktails, which reduces the probability of virus escape. However, many issues remain, including drug toxicity and the emergence of drug-resistant mutant viruses, even in treated patients. Therefore, there is a constant need for the development of new anti-HIV-1 agents targeting other molecules in the viral cycle. The capsid protein CA plays a key role in many molecular recognition events during HIV-1 morphogenesis and uncoating, and is eliciting increased interest as a promising target for antiviral intervention. This article provides a structure-based, integrated review on the CA-binding small molecules and peptides identified to date, and their effects on virus capsid assembly and stability, with emphasis on recent results not previously reviewed. As a complement, we present novel experimental results on the development and proof-of-concept application of a combinatorial approach to study molecular recognition in CA and its inhibition by peptide compounds. © 2012 Elsevier B.V.FIPSE (No. 36557/06); the Spanish Ministerio de Ciencia e Innovación (BIO2009-10092); Comunidad de Madrid (CM) (S-2009/MAT/1467); Fundación Ramón Areces; Institute for Biocomputation and Physics of Complex Systems, Zaragoza, SpainPeer Reviewe

Understanding the paradoxical mechanical response of in-phase A-tracts at different force regimes

A-tracts are A:T rich DNA sequences that exhibit unique structural and mechanical properties associated with several functions i n v i vo . The crystallographic structure of A-tracts has been well characterized. However, the mechanical properties of these sequences is controversial and their response to force remains unexplored. Here, we rationalize the mechanical properties of in-phase A-tracts present in the C a e n o r h a b d i t i s e l e g a n s genome over a wide range of external forces, using single-molecule experiments and theoretical polymer models. Atomic Force Microscopy imaging shows that A-tracts induce long-range (∼200 nm) bending, which originates from an intrinsically bent structure rather than from larger bending flexibility. These data are well described with a theoretical model based on the worm-like chain model that includes intrinsic bending. Magnetic tweezers experiments show that the mechanical response of A-tracts and arbitrary DNA sequences have a similar dependence with monovalent salt supporting that the observed A-tract bend is intrinsic to the sequence. Optical tweezers experiments reveal a high stretch modulus of the A-tract sequences in the enthalpic regime. Our work rationalizes the complex multiscale flexibility of A-tracts, providing a physical basis for the versatile character of these sequences inside the cell.The authors acknowledge the computer resources, technical expertise and assistance provided by the Red Española de Supercomputacion at the Minotauro Supercomputer (BSC, Barcelona). We thank Andrew Fire (Stanford University, USA) and Ralf Seidel (University of Leipzig, Germany) for providing us biological material required for the fabrication of the DNA molecules.Peer reviewe