Modulación del TLR4 : estudios de reconocimiento molecular y diseño de fármacos por modelado molecular

Tesis de la Universidad Complutense de Madrid, Facultad de Farmacia, Departamento de Química Orgánica y Farmacéutica, leída el 04/05/2018The heterodimeric complex, formed by Toll-Like Receptor 4 (TLR4) and its accessory protein Myeloid Differentiation factor 2 (MD-2) is responsible of activating the innate immune system when sensing the presence of particular pathogen-associated molecular patterns (PAMPs) from bacteria. The outer membrane of Gram-negative bacteria is primarily populated by lipopolysaccharides (LPS) which are essential for their growth and survival. These LPSs are specifically recognized by the TLR4/MD-2 complex as follows: an LPS binds to MD-2 inside a deep molecular hydrophobic pocket causing molecular rearrangements of the receptorial complex resulting in the dimerization of another TLR4/MD-2 unit. TLR4 ectodomains dimerization event brings together the TLR4 intercellular domains initiating the activation of innate immune system signaling pathways. Interestingly, this activation is not only modulated by naturally occurring LPSs from many different Gram-negative bacteria but also by non-naturally occurring glycolipids and other non-LPS like molecules...El complejo heterodimérico, formado por el receptor Toll-like 4 (Toll-like receptor 4, TLR4) y su proteína accesoria, el Myeloid Differentiation factor 2 (MD-2), es responsable de activar la respuesta del sistema inmune innato cuando detecta la presencia de patrones moleculares asociados a patógenos (pathogen associated molecular patterns, PAMPs), que provienen de bacterias y virus. En concreto, la membrana externa de bacterias Gram-negativas está poblada principalmente por lipopolisacáridos (lipopolisaccharides, LPS), compuestos que son esenciales para su crecimiento y supervivencia. Estos LPS son reconocidos de forma específica por el complejo TLR4/MD-2 de la siguiente manera: una molécula de LPS se une a la proteína MD-2 dentro de un profundo bolsillo hidrofóbico dando lugar al reordenamiento molecular del complejo resultando en la dimerización de otra unidad de TLR4/MD-2. El evento de dimerización de los ectodominios del TLR4 hace que se acercan los dominios intracelulares que inician la activación de las vías de señalización del sistema inmune innato. Curiosamente, esta activación no sólo está modulada por LPS naturales de muchas bacterias Gram-negativas distintas, sino también por glicolípidos no naturales y otras moléculas de estructura química diferente a los LPS...Fac. de FarmaciaTRUEunpu

Novel carboxylate-based glycolipids: TLR4 antagonism, MD-2 binding and self-assembly properties

New monosaccharide-based lipid A analogues were rationally designed through MD-2 docking studies. A panel of compounds with two carboxylate groups as phosphates bioisosteres, was synthesized with the same glucosamine-bis-succinyl core linked to different unsaturated and saturated fatty acid chains. The binding of the synthetic compounds to purified, functional recombinant human MD-2 was studied by four independent methods. All compounds bound to MD-2 with similar affinities and inhibited in a concentration-dependent manner the LPS-stimulated TLR4 signaling in human and murine cells, while being inactive as TLR4 agonists when provided alone. A compound of the panel was tested in vivo and was not able to inhibit the production of proinflammatory cytokines in animals. This lack of activity is probably due to strong binding to serum albumin, as suggested by cell experiments in the presence of the serum. The interesting self-assembly property in solution of this type of compounds was investigated by computational methods and microscopy, and formation of large vesicles was observed by cryo-TEM microscopy.TOLLerant project (H2020-MSC-ETN-642157), the Italian Ministry for Foreign Affairs and International Cooperation (MAECI) and Spanish MINECO (CTQ2014-57141-R and CTQ2017-88353-R grants) are acknowledged. Vesna Hodnik of University of Ljubljana for the help with SPR, Sandra Delgado of CIC BioGUNE for the Cryo-TEM images. RJ was partially funded by the research program P4-0176 by the Slovenian Research Agency

Protein kinase A controls yeast growth in visible light

Background: A wide variety of photosynthetic and non-photosynthetic species sense and respond to light, having developed protective mechanisms to adapt to damaging effects on DNA and proteins. While the biology of UV light-induced damage has been well studied, cellular responses to stress from visible light (400–700 nm) remain poorly understood despite being a regular part of the life cycle of many organisms. Here, we developed a high-throughput method for measuring growth under visible light stress and used it to screen for light sensitivity in the yeast gene deletion collection. Results: We found genes involved in HOG pathway signaling, RNA polymerase II transcription, translation, diphthamide modifications of the translational elongation factor eEF2, and the oxidative stress response to be required for light resistance. Reduced nuclear localization of the transcription factor Msn2 and lower glycogen accumulation indicated higher protein kinase A (cAMP-dependent protein kinase, PKA) activity in many light-sensitive gene deletion strains. We therefore used an ectopic fluorescent PKA reporter and mutants with constitutively altered PKA activity to show that repression of PKA is essential for resistance to visible light. Conclusion: We conclude that yeast photobiology is multifaceted and that protein kinase A plays a key role in the ability of cells to grow upon visible light exposure. We propose that visible light impacts on the biology and evolution of many non-photosynthetic organisms and have practical implications for how organisms are studied in the laboratory, with or without illumination

Insights into the key determinants of membrane protein topology enable the identification of new monotopic folds

25 p.-9 fig.-1 tab.Monotopic membrane proteins integrate into the lipid bilayer via reentrant hydrophobic domains that enter and exit on a single face of the membrane. Whereas many membrane spanning proteins have been structurally characterized and transmembrane topologies can be predicted computationally, relatively little is known about the determinants of membrane topology in monotopic proteins. Recently, we reported the X ray structure determination of PglC, a full length monotopic membrane protein with phosphoglycosyl transferase (PGT) activity. The definition of this unique structure has prompted in vivo, biochemical, and computational analyses to understand and define two key motifs that contribute to the membrane topology and to provide insight into the dynamics of the enzyme in a lipid bilayer environment. Using the new information gained from studies on the PGT superfamily we demonstrate that the two motifs exemplify principles of topology determination that can be applied to the identification of reentrant domains among diverse monotopic proteins of interest.The Biophysical Instrumentation Facility for the Study of Complex Macromolecular Systems (NSF-0070319) is gratefully acknowledged for assistance with CD experiments. We thank Prof. Karen N. Allen for many valuable discussions, and Theresa Hwang and Hannah Bernstein for technical assistance with SCAM and activity analyses.Peer reviewe

Computational approaches to toll-like receptor 4 modulation

24 p.-10 fig.Toll-like receptor 4 (TLR4), along with its accessory protein myeloid differentiation factor 2 (MD-2), builds a heterodimeric complex that specifically recognizes lipopolysaccharides (LPS),which are present on the cell wall of Gram-negative bacteria, activating the innate immune response.Some TLR4 modulators are undergoing preclinical and clinical evaluation for the treatment of sepsis,inflammatory diseases, cancer and rheumatoid arthritis. Since the relatively recent elucidation of the X-ray crystallographic structure of the extracellular domain of TLR4, research around this fascinating receptor has risen to a new level, and thus, new perspectives have been opened. In particular,diverse computational techniques have been applied to decipher some of the basis at the atomic level regarding the mechanism of functioning and the ligand recognition processes involving the TLR4/MD-2 system at the atomic level. This review summarizes the reported molecular modeling and computational studies that have recently provided insights into the mechanism regulating the activation/inactivation of the TLR4/MD-2 system receptor and the key interactions modulating the molecular recognition process by agonist and antagonist ligands. These studies have contributed to the design and the discovery of novel small molecules with promising activity as TLR4 modulators.This work was supported by the Spanish Ministry for Economy and Competitiveness (MINECO) Grant CTQ2014-57141-R and PhD fellowship BES-2015-071588 and by the European Commission granted GLYCOPHARM Marie Curie ITN PITN-GA-2012-317297 (www.glycopharm.eu) and TOLLerant H2020-MSC-ETN-642157 (www.tollerant.eu) projects.Peer reviewe

Structures, Properties, and Dynamics of Intermediates in eEF2-Diphthamide Biosynthesis

The eukaryotic translation Elongation Factor 2 (eEF2) is an essential enzyme in protein synthesis. eEF2 contains a unique modification of a histidine (His699 in yeast; HIS) into diphthamide (DTA), obtained via 3-amino-3-carboxypropyl (ACP) and diphthine (DTI) intermediates in the biosynthetic pathway. This essential and unique modification is also vulnerable, in that it can be efficiently targeted by NAD⁺-dependent ADP-ribosylase toxins, such as diphtheria toxin (DT). However, none of the intermediates in the biosynthesis path is equally vulnerable against the toxins. This study aims to address the different susceptibility of DTA and its precursors against bacterial toxins. We have herein undertaken a detailed <i>in silico</i> study of the structural features and dynamic motion of different His699 intermediates along the diphthamide synthesis pathway (HIS, ACP, DTI, DTA). The study points out that DTA forms a strong hydrogen bond with an asparagine which might explain the ADP-ribosylation mechanism caused by the diphtheria toxin (DT). Finally, <i>in silico</i> mutagenesis studies were performed on the DTA modified protein, in order to hamper the formation of such a hydrogen bond. The results indicate that the mutant structure might in fact be less susceptible to attack by DT and thereby behave similarly to DTI in this respect

Full-atom model of the agonist LPS-bound toll-like receptor 4 dimer in a membrane environment

21 p.-14 fig. In Memoriam Prof. Kilian Muñiz (R.I.P.). Excellent person, great heart.The innate immunity TLR4/MD-2 system is a membrane receptor of paramount importance as therapeutic target. Its assembly, upon binding of Gram-negative bacteria lipopolysaccharide (LPS), and also dependent on the membrane composition, finally triggers the immune response cascade. We here combine ab-initio calculations, molecular docking, all-atom molecular dynamics simulations, and thermodynamics calculations to provide the most realistic and complete 3D models of the active full TLR4 complex embedded into a realistic membrane to date. Our studies reveal functional and structural insights into the transmembrane domain behavior in different membrane environments, the ectodomain bouncing movement, and the dimerization patterns of the intracellular TIR domain. Our work provides TLR4 models as reasonable 3D structures for the (TLR4/MD-2/LPS) 2 architecture accounting for the active (agonist) state of the TLR4, and pointing to a signal transduction mechanism across cell membrane. These observations unveil relevant molecular aspects involved in the TLR4 innate immune pathways and will promote the discovery of new TLR4 modulators.This work was financially supported by the Spanish Ministry for Science and Innovation (grants CTQ2014-57141-R, CTQ2017-88353-R, and PID2020-113588RB-I00; grants BES-2012-053653 for L.P.R., BES-2015-071588 for J.G.C. and PRE2018-086249 for A.M.R.), and the European Commission Marie Sklodowska-Curie actions (H2020-MSCA-ITN 642157 “TOLLerant”).Peer reviewe

Identification of three bacterial species associated with increased appendicular lean mass : the HUNT study.

Appendicular lean mass (ALM) associates with mobility and bone mineral density (BMD). While associations between gut microbiota composition and ALM have been reported, previous studies rely on relatively small sample sizes. Here, we determine the associations between prevalent gut microbes and ALM in large discovery and replication cohorts with information on relevant confounders within the population-based Norwegian HUNT cohort (n = 5196, including women and men). We show that the presence of three bacterial species - Coprococcus comes, Dorea longicatena, and Eubacterium ventriosum - are reproducibly associated with higher ALM. When combined into an anabolic species count, participants with all three anabolic species have 0.80 kg higher ALM than those without any. In an exploratory analysis, the anabolic species count is positively associated with femoral neck and total hip BMD. We conclude that the anabolic species count may be used as a marker of ALM and BMD. The therapeutic potential of these anabolic species to prevent sarcopenia and osteoporosis needs to be determined

Amphiphilic Guanidino­calixarenes Inhibit Lipopolysaccharide (LPS)- and Lectin-Stimulated Toll-like Receptor 4 (TLR4) Signaling

We recently reported on the activity of cationic amphiphiles in inhibiting TLR4 activation and subsequent production of inflammatory cytokines in cells and in animal models. Starting from the assumption that opportunely designed cationic amphiphiles can behave as CD14/MD-2 ligands and therefore modulate the TLR4 signaling, we present here a panel of amphiphilic guanidino­calixarenes whose structure was computationally optimized to dock into MD-2 and CD14 binding sites. Some of these calixarenes were active in inhibiting, in a dose-dependent way, the LPS-stimulated TLR4 activation and TLR4-dependent cytokine production in human and mouse cells. Moreover, guanidino­calixarenes also inhibited TLR4 signaling when TLR4 was activated by a non-LPS stimulus, the plant lectin PHA. While the activity of guanidino­calixarenes in inhibiting LPS toxic action has previously been related to their capacity to bind LPS, we suggest a direct antagonist effect of calixarenes on TLR4/MD-2 dimerization, pointing at the calixarene moiety as a potential scaffold for the development of new TLR4-directed therapeutics