Search

Selenophosphate synthetases use selenium and ATP to synthesize selenophosphate. This is required for biological utilization of selenium, most notably for the synthesis of the non-canonical amino acid selenocysteine (Sec). Therefore, selenophosphate synthetases underlie all functions of selenoproteins, which include redox homeostasis, protein quality control, hormone regulation, metabolism, and many others. This protein family comprises two groups, SelD/SPS2 and SPS1. The SelD/SPS2 group represent true selenophosphate synthetases, enzymes central to selenium metabolism which are present in all Sec-utilizing organisms across the tree of life. Notably, many SelD/SPS2 proteins contain Sec as catalytic residue in their N-terminal flexible selenium-binding loop, while others replace it with cysteine (Cys). The SPS1 group comprises proteins originated through gene duplications of SelD/SPS2 in metazoa in which the Sec/Cys-dependent catalysis was disrupted. SPS1 proteins do not synthesize selenophosphate and are not required for Sec synthesis. They have essential regulatory functions related to redox homeostasis and pyridoxal phosphate, which affect signaling pathways for growth and differentiation. In this review, we summarize the knowledge about the selenophosphate synthetase family acquired through decades of research, encompassing their structure, mechanism, function, and evolution.

Tissue inhibitor of metalloproteinase-1 (TIMP-1) is an important regulator of extracellular matrix turnover that has been traditionally regarded as a potential tumor suppressor owing to its inhibitory effects of matrix metalloproteinases. Intriguingly, this interpretation has been challenged by the consistent observation that increased expression of TIMP-1 is associated with poor prognosis in virtually all cancer types including lung cancer, supporting a tumor-promoting function. However, how TIMP-1 is dysregulated within the tumor microenvironment and how it drives tumor progression in lung cancer is poorly understood. We analyzed the expression of TIMP-1 and its cell surface receptor CD63 in two major lung cancer subtypes: lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC), and defined the tumor-promoting effects of their interaction. We found that TIMP-1 is aberrantly overexpressed in tumor-associated fibroblasts (TAFs) in ADC compared to SCC. Mechanistically, TIMP-1 overexpression was mediated by the selective hyperactivity of the pro-fibrotic TGF-β1/SMAD3 pathway in ADC-TAFs. Likewise, CD63 was upregulated in ADC compared to SCC cells. Genetic analyses revealed that TIMP-1 secreted by TGF-β1-activated ADC-TAFs is both necessary and sufficient to enhance growth and invasion of ADC cancer cells in culture, and that tumor cell expression of CD63 was required for these effects. Consistently, in vivo analyses revealed that ADC cells co-injected with fibroblasts with reduced SMAD3 or TIMP-1 expression into immunocompromised mice attenuated tumor aggressiveness compared to tumors bearing parental fibroblasts. We also found that high TIMP1 and CD63 mRNA levels combined define a stronger prognostic biomarker than TIMP1 alone. Our results identify an excessive stromal TIMP-1 within the tumor microenvironment selectively in lung ADC, and implicate it in a novel tumor-promoting TAF-carcinoma crosstalk, thereby pointing to TIMP-1/CD63 interaction as a novel therapeutic target in lung cancer.

Treballs Finals de Grau d'Enginyeria Biomèdica. Facultat de Medicina i Ciències de la Salut. Universitat de Barcelona. Curs: 2022-2023. Tutor/Director: Sala Llonch, Roser, Orozco López, Modesto, The human brain is organized in a hierarchical structure of distinct but closely connected levels. Although our knowledge about each individual level is extensive, what remains unknown is a comprehensive understanding of how events in low levels propagate through higher levels. This is a challenge for neuroscience. This project focuses on low-level protein events. Its aim is to simulate the dynamics of a subset of central nervous system proteins as their mobility and flexibility are essential for neurological signal transduction. In particular, it is centred on the subset of CNS proteins related to Parkinson’s disease. By reproducing their dynamics, the process of signal transduction in the brain can be better understood, as well as helping in the design of neuro-active drugs for Parkinson’s disease. This document provides a detailed description of the development of the project to obtain the dynamics of the proteins of study. The scope of the project goes from generating a list of proteins of interest, running and validating their dynamics and uploading the data to an open access database. A total of 49 simulations were successfully obtained and uploaded. These simulations can be further studied to understand protein functions in neurological pathways as well as study possible drug binding interactions.

Depuis maintenant de très longues années, les problèmes de poids sont devenus monnaie courante en France. D’autant plus inquiétants que les courbes de poids de la population française ne cessent d’augmenter, année après année. Si l’obésité est reconnue comme une maladie et nécessite un accompagnement personnalisé afin de maximiser les chances de guérison du patient, le surpoids a tendance à être mis de côté. Dans ce travail, nous nous pencherons sur les régimes hyperprotéinés, longtemps en vogue pour la perte de poids.Dans un premier temps, nous étudierons les fondamentaux concernant la nutrition avant de nous focaliser sur le surpoids et l’obésité.Nous verrons ensuite dans un second temps, les principes et les déroulements des régimes hyperprotéinés les plus connus.Enfin, nous envisagerons les éventuels effets indésirables de ces régimes et donc l’importance de les suivre correctement.

The type-1 cannabinoid receptor (CB1R) is widely expressed in excitatory and inhibitory nerve terminals, and by suppressing neurotransmitter release, its activation modulates neural circuits and brain function. While the interaction of CB1R with various intracellular proteins is thought to alter receptor signaling, the identity and role of these proteins are poorly understood. Using a high-throughput proteomic analysis complemented with an array of in vitro and in vivo approaches in the mouse brain, we report that the C-terminal, intracellular domain of CB1R interacts specifically with growth-associated protein of 43 kDa (GAP43). The CB1R-GAP43 interaction occurs selectively at mossy cell axon boutons, which establish excitatory synapses with dentate granule cells in the hippocampus. This interaction impairs CB1R-mediated suppression of mossy cell to granule cell transmission, thereby inhibiting cannabinoid-mediated anti-convulsant activity in mice. Thus, GAP43 acts as a synapse type-specific regulatory partner of CB1R that hampers CB1R-mediated effects on hippocampal circuit function.

[eng] Intrinsically disordered proteins (IDPs) landed on the molecular biology framework at the turn of the 20th century to challenge the established protein function- structure paradigm. Due to their inherent flexibility and disorder-to-order transitions, IDPs play an important role in the adaptive regulation and mediation of biological responses within cells. However, the intrinsic disorder makes IDPs difficult to characterise by experimental techniques, hindering the elucidation of their mechanisms of action in biological functions. Molecular dynamics simulations can capture the conformational ensembles of macromolecules, but several issues need to be when simulating IDPs, such as proper parameterisation to reproduce the intrinsic disorder, improvement of the conformational sampling, or factors related to the cellular environment such as ionic strength, pH, molecular crowding, etc. With the recent introduction of these proteins in the scientific landscape, this thesis is presented as a contribution to provide further insight into simulations of IDPs, especially on the effect of pH. Due to the high abundance of ionisable amino acids in IDPs, the incorporation of charge-conformation coupling into in-silico modelling is critical. Therefore, the effect of the dynamic change of protonation states depending on the solvent pH to generate conformational ensembles of IDPs is investigated using the constant pH Molecular Dynamics method. During the study, some shortcomings of this method were identified, which led to a detailed assessment of this approach implemented in AMBER. On the other hand, new force fields and water models designed for IDP simulation, as well as coarse-grained models or sampling techniques, are evaluated on the model IDP peptide, histatin-5, with one of the most extensive simulations of this peptide. Finally, we focus on the IDP α-synuclein (αS), which is implicated in Parkinson's disease through its fibrillation and oligomerisation leading to deposition in Lewis bodies. Using the IDP-specific force field ff14IDPSFF, β-sheet-rich intermediates are detected in a fragment of αS. In addition, we provide a first insight into the effect of pH on αS and β- synuclein, and plan to continue this study in the future, using the knowledge gained in this thesis to unravel the mechanism of fibrillogenesis of these proteins., [cat] Les proteïnes intrínsecament desordenades (IDPs) es van popularitzar en el marc de la biologia molecular a principis del segle XX per a qüestionar el paradigma de funció- estructura de les proteïnes. A causa de la seva flexibilitat intrínseca i les transicions de desordre-a-ordre, les IDPs exerceixen un paper clau en la regulació adaptativa i la mediació de respostes biològiques en les cèl·lules. El desordre intrínsec provoca que siguin difícils de caracteritzar mitjançant tècniques experimentals, la qual cosa impedeix l'elucidació dels seus mecanismes d'acció. Les simulacions de Dinàmica Molecular poden captar els conjunts de conformacions de les macromolècules, però en les simulacions de IDPs és necessari abordar prèviament diverses qüestions, com ara la parametrització, el mostreig conformacional o factors relacionats amb l'entorn cel·lular com la força iònica, el pH, etc. Amb la recent introducció d'aquestes proteïnes en el panorama científic, aquesta tesi es presenta com una contribució per a millorar les simulacions de IDPs, especialment sobre l'efecte del pH. A causa de la gran abundància d'aminoàcids ionitzables en les IDPs, la incorporació de l'acoblament carrega-conformació en les simulacions és fonamental. Per tant, investiguem l'efecte del canvi dinàmic dels estats de protonació en funció del pH sobre la generació de conjunts de conformacions de les IDPs utilitzant el mètode de Dinàmica Molecular a pH constant. Durant l'estudi, s’identifiquen algunes deficiències en el mètode, impulsant-nos així a avaluar aquesta tècnica en profunditat. D'altra banda, també posem a prova nous camps de força o models d'aigua dissenyats per a la simulació de IDP, així com models de gra gruixut o tècniques de mostreig, en la IDP model, histatin- 5, amb una de les simulacions més exhaustives del pèptid. Finalment, ens centrem en la IDP α-sinucleïna (αS), implicada en la malaltia de Parkinson a través de la fibril·lació i oligomerització que condueixen al seu dipòsit en els cossos de Lewis. Utilitzant el camp de força específic per IDPs, ff14IDPSFF, es detecten intermedis rics en fulles β en un fragment de αS. A més a més, proporcionem una primera pinzellada de l'efecte del pH sobre αS i β-sinucleïna per desentranyar el mecanisme de fibril·logènesi d'aquestes proteïnes.

Micro- and nanoparticles, and especially plastic particles, are an important current topic. The ever-growing increase of the amount of plastic used in a wide variety of fields causes a sharp increase in the associated pollution. When these particles enter a biological medium they surround themselves with a corona, which can have a complex impact on organisms. It can give the particle a “biological identity” influencing its interactions in the living medium.This thesis studies the interactions of polyethylene and polypropylene microparticles (MP) and microstructured polypropylene filters with proteins and model peptides. We characterized the MPs with several spectroscopic methods.Unaged and aged MPs were brought into contact with proteins and model peptides.We measured protein adsorption isotherms on aged and unaged MPs. Raman imaging and fluorescence measurements allowed us to directly visualize the hemoglobin and bovine serum albumin corona on the MPs. The hemoglobin function, in solution and adsorbed, was assessed by tonometry.To understand the interactions between peptides and nanoparticles at the atomic scale, we used an in silico molecular dynamics approach. Meanwhile, we have measured adsorption isotherms of fluorescent peptides to validate the simulation results.; Les micro- et nanoparticules, et en particulier celles de plastique, constituent un sujet d'actualité important. L'augmentation toujours croissante des quantités de plastiques utilisées dans des domaines très variés est à l'origine d'une forte augmentation de la pollution associée. Lorsque ces particules entrent dans un milieu biologique, elles s'entourent d'une corona dont l'impact sur les organismes peut être complexe. Elle donne à la particule une "identité biologique" influençant ses interactions dans le milieu vivant.Cette thèse étudie les interactions de microparticules (MP) de polyéthylène et de polypropylène, et de filtres microstructurés en polypropylène avec des protéines et des peptides modèles. Nous avons caractérisés les MP par différentes méthodes spectroscopiques.Des MP non-vieillies et vieillies par différentes méthodes ont été mises en contact avec des protéines et peptides modèles. Nous avons mesuré les isothermes d'adsorption des protéines sur les MP vieillis et non vieillies. Des mesures d'imagerie Raman et de fluorescence nous ont permis de visualiser directement la corona de l'hémoglobine et de l'albumine du sérum bovin sur les MP. La fonction de l'hémoglobine, en solution et adsorbée, a été évaluée par tonométrie.Pour comprendre les interactions entre peptides et nanoparticules de polyéthylène à l'échelle atomique, nous avons utilisé une approche in silico de dynamique moléculaire. En parallèle, nous avons mesuré les isothermes d'adsorption de peptides fluorescents pour pour valider les résultats de simulation.

Abstract Bacillus thuringiensis Cry1I insecticidal proteins are structurally similar to other three-domain Cry proteins, although their size, activity spectrum, and expression at the stationary phase are unique among other members of the Cry1 family. The mode of action of Cry1 proteins is not completely understood but the existence of an activation step prior to specific binding is widely accepted. In this study, we attempted to characterize and determine the importance of the activation process in the mode of action of Cry1I, as Cry1Ia protoxin or its partially processed form showed significantly higher toxicity to Ostrinia nubilalis than the fully processed protein either activated with trypsin or with O. nubilalis midgut juice. Oligomerization studies showed that Cry1Ia protoxin, in solution, formed dimers spontaneously, and the incubation of Cry1Ia protoxin with O. nubilalis brush border membrane vesicles (BBMV) promoted the formation of dimers of the partially processed form. While no oligomerization of fully activated proteins after incubation with BBMV was detected. The results of the in vitro competition assays showed that both the Cry1Ia protoxin and the approx. 50 kDa activated proteins bind specifically to the O. nubilalis BBMV and compete for the same binding sites. Accordingly, the in vivo binding competition assays show a decrease in toxicity following the addition of an excess of 50 kDa activated protein. Consequently, as full activation of Cry1I protein diminishes its toxicity against lepidopterans, preventing or decelerating proteolysis might increase the efficacy of this protein in Bt-based products. Key points • Processing Cry1I to a 50 kDa stable core impairs its full toxicity to O. nubilalis • Partially processed Cry1Ia protoxin retains the toxicity of protoxin vs O. nubilalis • Protoxin and its final processed forms compete for the same functional binding sites

In the eighteenth century, meat became strongly associated with English national identity and was often used in discourses about national differences. This article seeks to analyze how, from the 1840s to WWI, the development of food chemistry—a science then primarily concerned with the importance of proteins—contributed to strengthening this discourse. The article thus shows how science gave a renewed legitimacy to discourses establishing strong fault lines and hierarchies between different peoples or ‘races’, depending on their diets. The second part of the article shows, however, the evolutions and complex uses of this scientific discipline which eventually contributed to a relative weakening of ideas linking a meat diet and national or racial supremacy. Depuis le dix-huitième siècle, au moins, la viande est fortement associée à l’identité nationale des Anglais et à leur supériorité supposée. Le présent article se propose d’analyser la façon dont le développement de la chimie alimentaire dans la seconde moitié du dix-neuvième siècle a contribué à renforcer cet imaginaire, en raison de sa focalisation particulière sur l’apport en protéines. L’article montre ainsi comment la science a pu donner une nouvelle légitimité aux discours instituant des lignes de fracture et hiérarchies fortes entre différents peuples ou « races » en fonction de leurs régimes alimentaires. Dans un second temps, l’article vise néanmoins à montrer les évolutions et usages complexes de cette discipline scientifique, qui a également contribué à déstabiliser les discours liant régime carné et suprématie nationale ou raciale.

[eng] This thesis develops, validates, and explores applications of the Franzese-Stanley (FS) coarse-grained model for bulk water, ultimately intended to represent the solvent in large-scale simulations of biological systems. The model coarse-grains the positions of the water molecules through a discrete density field but keeps a detailed account of the hydrogen bond (HB) contributions to local density heterogeneity. It was initially proposed for hydrophobically confined water monolayers and is suitable for analytic calculations and Monte Carlo simulations. The FS monolayer displays dynamic and thermodynamic anomalies qualitatively consistent with experiments and atomistic calculations. Moreover, its extension to hydration water in protein solutions unveils the water contribution to protein folding, protein design, and protein aggregation, including effects arising from changes in temperature, pressure, salinity, and pH. Here, we extend to bulk the FS model. Although going from monolayer to bulk could drastically change the thermodynamic behavior of the model, we show that the bulk FS reproduces well the experimental data of water. Furthermore, we prove that the model is suitable for simulations of large biological systems in explicit solvent. On the one hand, we account for water interactions at the molecular level, at variance with commonly employed coarse-grained models. On the other hand, we equilibrate systems at length scales and times so large that it would be unaffordable for current atomistic simulations. First, we developed efficient parallel distributed algorithms for Metropolis and Swendsen-Wang Monte Carlo simulations. We benchmarked accessible size systems of 260³ ∼ 10⁷ water molecules using parallel Metropolis and 128³ ∼ 10⁶ with Swendsen-Wang. Second, we calibrated the model parameters according to ab initio calculations and experimental results. We found that the model fits the experimental data in a wide range of temperatures T and pressures P around ambient conditions. Third, we transferred the model to deep supercooled conditions and compared the model's predictions to those obtained from atomistic models. We found that the FS model displays a liquid-liquid critical point (LLCP) at TC = (192 ± 6) K and PC = (176 ± 2) MPa, consistent with estimates from the most accepted atomistic models. Finally, we investigated applications of the bulk FS model to hydrate large biological systems. More specifically, we developed a simulation approach to estimate the contribution of the solvent to the free energy of hydrated systems previously calculated with implicit solvent models. We considered two cases of application. First, we studied the unfolding upon increasing hydrodynamic stress of the globular domains A1, A2, and A3 of the von Willebrand factor (vWf). Here, we identify four characteristic states of the system and estimate the free energy barriers that separate them as a function of the shear rate. Second, we considered the SOD1 protein sequestration into FUS biocondensates and the anti-sequestration action of the BSA crowder, mimicking the high cytoplasm concentration. BSA are globular proteins, while FUS are intrinsically disordered proteins. Our results show that the decrease of the SOD1 partitioning coefficient into FUS biocondensates when BSA is present is a consequence of the increase in water's entropy. In both the vWf and SOD1 examples, we show that the explicit calculation of water contribution to the free energy is essential to interpret the phenomena' physics correctly. Within this approach, we overcome the intrinsic limitation of implicit solvent models that, by construction, average over the solvent degrees of freedom., [spa] A lo largo de la presente tesis, extendemos a 3D el modelo coarse-grain Franzese-Stanley (FS) que, en su formulación original para monocapas líquidas confinadas (2D), reproduce de forma cualitativa las anomalías dinámicas y termodinámicas del agua. Aquí, lo validamos en 3D, demostrando que se ajusta en un amplio rango de presiones y temperaturas a los resultados experimentales, y lo aplicamos para calcular la contribución del agua a la energía libre de sistemas biológicos de gran extensión. El objetivo principal es demostrar que el modelo en 3D es aplicable a simulaciones de gran escala, calculando explícitamente las contribuciones del solvente. Para ello, el modelo debe reproducir las propiedades del agua a condiciones ambiente, y en otras condiciones relevantes para los procesos biológicos, y debe ser posible, mediante simulaciones, equilibrar sistemas de tamaño muy grande. Para reducir el coste de las simulaciones y aumentar el rango de tamaños accesibles, desarrollamos algoritmos Monte Carlo paralelizados. Con ello, logramos equilibrar sistemas del orden de 10⁷ moléculas. A continuación, calibramos los parámetros del modelo de manera que el modelo reproduzca cuantitativamente la ecuación de estado del agua experimental en una amplia región del diagrama de fases alrededor de las condiciones ambiente. En tercer lugar, estudiamos el comportamiento del modelo en la región sobreenfriada. Nuestros resultados muestran que el modelo reproduce una transición de fase líquido-líquido entre las fases líquido-de-alta-densidad y líquido-de-baja-densidad que termina en un punto crítico. Mediante un análisis de tamaño finito, encontramos que el punto crítico localizado en el modelo FS es consistente con las estimaciones de modelos atomísticos. A pesar que estas condiciones extremas sean menos relevantes para los procesos biológicos, la consistencia de los resultados de nuestro modelo con modelos atomísticos aceptados refuerza su validez. Finalmente, desarrollamos un método de simulación que permite calcular la contribución del agua a la energía libre de sistemas biológicos. El método toma la trayectoria del sistema (previamente calculada mediante un modelo de agua implícita) y mapea las configuraciones a su representación correspondiente en el modelo FS (agua explícita). Así, damos cuenta de la evolución de las configuraciones del agua a lo largo de la trayectoria del sistema. Mediante dos casos de aplicación, concluimos que es necesario incluir los grados de libertad del solvente para describir de forma adecuada la física del sistema y, en consecuencia, para calcular las barreras de energía libre que separan los estados característicos de los procesos examinados.

Thousands of genetic variants in protein-coding genes have been linked to disease. However, the functional impact of most variants is unknown as they occur within intrinsically disordered protein regions that have poorly defined functions1–3. Intrinsically disordered regions can mediate phase separation and the formation of biomolecular condensates, such as the nucleolus4,5. This suggests that mutations in disordered proteins may alter condensate properties and function6–8. Here we show that a subset of disease-associated variants in disordered regions alter phase separation, cause mispartitioning into the nucleolus and disrupt nucleolar function. We discover de novo frameshift variants in HMGB1 that cause brachyphalangy, polydactyly and tibial aplasia syndrome, a rare complex malformation syndrome. The frameshifts replace the intrinsically disordered acidic tail of HMGB1 with an arginine-rich basic tail. The mutant tail alters HMGB1 phase separation, enhances its partitioning into the nucleolus and causes nucleolar dysfunction. We built a catalogue of more than 200,000 variants in disordered carboxy-terminal tails and identified more than 600 frameshifts that create arginine-rich basic tails in transcription factors and other proteins. For 12 out of the 13 disease-associated variants tested, the mutation enhanced partitioning into the nucleolus, and several variants altered rRNA biogenesis. These data identify the cause of a rare complex syndrome and suggest that a large number of genetic variants may dysregulate nucleoli and other biomolecular condensates in humans.

Over the last decades, non-cereal crops have been displaced in European cropping systems leading to a significant dependency on imported soybean. Continuous maize cropping under Mediterranean irrigated conditions can lead to agronomic and environmental problems. The objective of this work was to assess diversified Mediterranean irrigated cropping systems to maximize protein production while reducing synthetic N fertilizer use. A field experiment was carried out from 2019 to 2021 in an irrigated area in NE Spain. Four cropping systems, (i) continuous maize (MM), (ii) soybean in a rotation one out of three years (MSrt), (iii) barley-maize double cropping system (BM), and (iv) barley-soybean double cropping system (BS) were assessed at the crop, pre-crop and cropping system level. Productivity in terms of grain, energy and protein yield was measured at the crop and calculated for the cropping system level. As well, synthetic N fertilizer use efficiency was calculated for each cropping system. At the pre-crop level, soybean introduction led to a 28% yield increase in the following cereal (maize or barley) mainly due to the residual N effect. At the cropping system level, soybean in rotation (MSrt) did not lead to a significant increase in total protein production compared to MM (from 895 to 947 kg ha−1 yr−1), but it mildly increased synthetic N fertilizer use efficiency. Protein production in the BS system (1778 kg protein ha−1 yr−1) was significantly higher than in all other cropping systems (990 kg protein ha−1 yr−1 on average). As well, BS was the cropping system with the highest synthetic N fertilizer use efficiency compared to the other cropping systems (251 and 88 kg grain kg synthetic N fertilizer−1). Our results demonstrate that introducing soybean as a double crop following barley is a successful strategy to reduce environmental impacts resulting from N fertilizer use and increase protein production, contributing to plant protein self-sufficiency and cropping systems diversification. This research was developed in the framework of the SusCrop-ERA-NET LegumeGap project, PCI2019-103597 funded by MCIN/AEI/10.13039/501100011033 and co-funded by the European Union, and the ECO-TRACE Research project, TED2021-131895A-I00 funded by MCIN/AEI /10.13039/501100011033 and the European Union NextGenerationEU/ PRTR. Daniel Plaza-Bonilla is Ramón y Cajal fellow (RYC-2018–024536-I) co-funded by MICIN/AEI/10.13039/501100011033 and European Social Fund. Genís Simon-Miquel thanks Universitat de Lleida for the funding granted to do an international research stay at ZALF where this work was conceptualized. Moritz Reckling was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – 420661662. The authors of this manuscript kindly thank Ramon Pujol Chambert and Ramon Pujol Figueras from Jolbertal SL for their goodwill to carry out this research in their fields and with their machinery and for providing precious feedback. This acknowledgement also extends to Ariadna Marquilles, Josep Maria Perera, Gerard Piñol, Pablo Redondo, Louise Blanc, Andreu Dago, and several bachelor students from Universitat de Lleida for their field, lab and technical support.

Chronic low-dose exposure to organophosphorus (OP) toxicants is correlated with an increase in the risk of impaired cognition and neurodegenerative diseases. A mechanism to explain this relationship is needed. We suggest that the formation of organophosphate-induced high-molecular-weight protein aggregates that disrupt cell function may be the missing link. It has been demonstrated that such aggregation can be promoted by OP-labeled lysine. Alternatively, OP-labeled glutamate may be the initiator. To test this hypothesis, we treated MAP-rich tubulin Sus scrofa and human transglutaminase with chlorpyrifos oxon. Trypsin-digested proteins were subjected to liquid chromatography-tandem mass spectrometry followed by Protein Prospector searches to identify diethyl phosphate adducts and cross-linked peptides. We report the presence of diethyl phosphate adducts on the side chains of glutamate, lysine, and tyrosine, as well as cross-links between glutamate and lysine. Glutamate-lysine cross-linking could be initiated either by diethyl phosphate-activated glutamate or by diethyl phosphate-activated lysine to form stable isopeptide bonds between and within proteins. It was concluded that organophosphate-induced high-molecular-weight protein aggregates could promote brain dysfunction., This work was supported in part by the Fred & Pamela Buffet Cancer Center Support Grant [grant number P30CA036727] from the National Institutes of Health and by Ministerio de Ciencia e Innovacioń and Agencia Estatal de Investigacioń MCIN/AEI/10.13039/501100011033 [grant number PID2020-118127RB-I00].

Els sistemes alimentaris comprenen tots els elements i totes les activitats relacionades amb la producció, processament, distribució, preparació i consum d'aliments, i també els resultats d'aquestes activitats, incloent-hi també les de tipus socioeconòmic i ambiental. Aquests sistemes estan in tegrats per diverses cadenes de valor i també inclouen activitats que no es tan basades en relacions de mercat, sinó en la cultura, els hàbits socials o l'educació. La principal funció dels sistemes alimentaris és contribuir a la nutrició i la seguretat alimentària de la població, però cada vegada es posa més en relleu el seu paper clau en relació amb la gestió de recursos naturals, la resiliència dels ecosistemes o la mitigació del canvi climàtic, així com en la creació de llocs de treball i la fixació de població al territori. En cap altre àmbit tots aquests elements convergeixen de forma tan explícita i directa.

[eng] Tumour progression largely depends on the signalling networks that direct cell viability, growth and dissemination (metastasis). Transforming growth factor beta (TGF-β) and Bone Morphogenetic Protein (BMP) are cytokines that induce a plethora of physiological functions in all vertebrates, by activating or repressing various signal transducers. One of these is the Smad family of transcription factor proteins, which play an essential role in early embryogenesis, development, cell immunity, homeostasis, tissue repair and many other essential processes during human life. Mutations in the TGF-β signalling components (including Smads) inactivate the cell’s tumour suppression functions, facilitating the survival of cancer cells. Upon activation by the receptor, Smads form active homo- and hetero- trimeric complexes to interact with DNA sequences (promoters, enhancers) proteins (other transcription factors, co-activators or co-repressors), and also with RNAs. Smad-RNA complexes were reported in the scientific and medical literature; however, this research was largely carried out on a cellular level and the exact details of these interactions remain poorly understood. I have been working on the characterization of Smad-RNA complex formation. I have also determined the structure of a C2 domain present in NEDD4-L, one of the ubiquitin ligases that degrade Smad proteins In a different line of research, which is aimed at defining new targets for drug screening, I determined the structure of (among others) the protein Deadhead, belonging to the lethal(3)malignant brain tumour signature genes and responsible for the development and sex determination in D. melanogaster and several structures of a p38α (MAPK14) kinase, bound to compounds that regulate its function in cells. This work will pave the way for the future optimisation of these compounds to improve their pharmacological properties.

[eng] L-type amino acid transporters play key roles in human physiology and are involved in several human pathologies such as primary inherited aminoacidurias (Feliubadaló et al., 1999; Torrents et al., 1999), autism spectrum disease (Tărlungeanu et al., 2016) and age-related hearing loss (Espino Guarch et al., 2018) among others. However, their complete and detailed transport mechanism is still unknown as the structures solved until now are most of them in the same conformational state, open-to-in, with an exception of LAT1 bound to inhibitors that showed for the first time an inward occluded conformation (Yan et al., 2021). To decipher LATs transport mechanism we have studied the bacterial LAT alanine- serine-cysteine exchanger BasC as a LAT model. With the objective to use BasC to translate from the already well described transport mechanism of the neurotransmitter sodium symporter (NSS) family, which shares the same APC fold from the APC superfamily, to the current LAT transport mechanism in study here. BasC has been already characterized (Bartoccioni et al., 2019) and 3D structurally solved in complex with the specific nanobody 74 (Errasti-Murugarren et al., 2019). Here, the 29 nanobodies have been characterised for the following parameters. Firstly, nanobodies have been characterised by their binding to sort them by affinity through Surface Plasmon Resonance. Then, by their inhibition profile to unveil their side of interaction through inhibition screenings of BasC transport activity. Also, by their recognised conformation compatibilities between them through the feasibility of tertiary complex between BasC and nanobody pairs. Finally, for their possible conformational effect on BasC through single-molecule Fluorescence Resonance Energy Transfer (smFRET) measurements on TM1a on the cytoplasmic gate. In this regard, selected nanobodies could eventually force BasC to adopt these new conformations currently unavailable and solve them in complex through structural techniques such as X-ray crystallography and cryo-EM. Here we present two low-resolution structures of BasC-Nb71 and BasC-Nb53-58 complexes. Furthermore, the dynamics of this cytoplasmic gate were also studied under the effect of substrates (L- alanine and L-serine) and inhibitors (L-glutamine). It was detected an inward closing of the TM1a due to L-alanine and L-Serine but not due to the addition of L-glutamine, that did not provoke any closing effect by the cytosolic side. Even more, nanobodies became here a powerful tool to study the blocking effect of these substrate-induced effects. It was identified nanobody 71 as a strong blocker of it while nanobody 53 was not impeding this TM1a bend. These substrate-induced dynamic effects being blocked or not by nanobodies in combination with the two structures solved, sustains the designed smFRET approach, as the structure interactions between the nanobodies 71 and 53 towards BasC structurally explain the blocking effects of the TM1a dynamic results obtained by smFRET. Regarding the mechanism of transport of LATs, the seen substrate-induced closing effect of the cytoplasmic gate for the BasC WT variant has been studied on lysinuric protein intolerance (LPI) related variant (K154A). This Lys residue is highly conserved among all hLATs, located in the TM5 in a homologue region of the key Na2 site for the NSS family of the APC superfamily. It has been speculated the role of this conserved Lys to be one of the key factors for the sidedness of LATs and mainly responsible for the occlusion and opening of the thick gate in the cytosol. Here we obtained the first experimental evidence of the molecular mechanism deficiency when there lacks this Lys as the LPI related variant was unable to close the cytosolic gate. Overall, we present a useful approach that can be used to study the transport mechanism of membrane proteins and the miss functions of disease related variants., [cat] Els transportadors d’L-aminoàcids (LATs) tenen un paper capital en la fisiologia humana i estan involucrats en diverses patologies com les aminoacidúries primàries heretades (Feliubadaló et al., 1999; Torrents et al., 1999), l’espectre autista (Tărlungeanu et al., 2016) o la pèrdua d’oïda relacionada amb l’edat (Espino Guarch et al., 2018). Així i tot, el seu mecanisme de transport complet i en detall encara resta desconegut, atès que la gran majoria d’estructures resoltes fins a l’actualitat es troben en el mateix estat obert al citosol, amb la única excepció de LAT1 unit a inhibidors resolt tancat pel citosol (Yan et al., 2021). Amb la finalitat de revelar el mecanisme de transport dels LATs ens proposem emprar com a model l’intercanviador obligat bacterià d’alanina-serina-cisteïna (BasC). Aquest intercanviador ja ha estat prèviament caracteritzat funcionalment (Bartoccioni et al., 2019) i la seva estructura es va resoldre en complex amb el nanocòs específic 74 (Errasti-Murugarren et al., 2019). Els 29 nanocossos han sigut caracteritzats pels seus paràmetres d’unió, el seu costat d’interacció amb BasC i l’efecte conformacional que efectuen sobre BasC per assajos de transferència d’energia de ressonància fluorescent per molècules individuals (smFRET) sobre el TM1a de la porta citosòlica de BasC. Els estudis de smFRET ens han permès estudiar la dinàmica de tancament de la porta citosòlica induïda per l’adició de substrats, L-Ala i L-Ser. A més, s’ha comprovat que els nanocossos poden esdevenir una eina molt útil a l’hora de bloquejar certs moviments de la proteïna i, eventualment, bloquejar el transportador en un estat conformacional diferent del ja resolt, seleccionant els nanocossos prometedors per resoldre l’estructura de BasC en complex amb ells per tècniques estructurals com la cristal·lografia de raigs X o la crio-microscopia electrònica. Finalment, aquests efectes de tancament de la porta citosòlica induïts pels substrats van ser assajats sobre la variant relacionada amb la malaltia d’intolerància lisinúrica proteica de BasC (K154A). Aquesta variant ha resultat ser incapaç de tancar la porta citosòlica, esdevenint la primera evidència experimental sobre el rol clau d’aquest residu, K154, en el mecanisme de transport dels LATs, més concretament el tancament de la porta citosòlica. Al cap i a la fi, aquest projecte pretén presentar i posar a punt una tècnica que pot ser d’alta utilitat per estudiar el mecanisme de transport de proteïnes de membranes, així com els funcionament aberrants de les variants relacionades amb les malalties.

Protein domains are independent structural and functional modules that can rearrange to create new proteins. While the evolution of multidomain proteins through the shuffling of different preexisting domains has been well documented, the evolution of domain repeat proteins and the origin of new domains are less understood. Metallothioneins (MTs) provide a good case study considering that they consist of metal-binding domain repeats, some of them with a likely de novo origin. In mollusks, for instance, most MTs are bidomain proteins that arose by lineage-specific rearrangements between six putative domains: α, β1, β2, β3, γ and δ. Some domains have been characterized in bivalves and gastropods, but nothing is known about the MTs and their domains of other Mollusca classes. To fill this gap, we investigated the metal-binding features of NpoMT1 of Nautilus pompilius (Cephalopoda class) and FcaMT1 of Falcidens caudatus (Caudofoveata class). Interestingly, whereas NpoMT1 consists of α and β1 domains and has a prototypical Cd2+ preference, FcaMT1 has a singular preference for Zn2+ ions and a distinct domain composition, including a new Caudofoveata-specific δ domain. Overall, our results suggest that the modular architecture of MTs has contributed to MT evolution during mollusk diversification, and exemplify how modularity increases MT evolvability.

Amb l'objectiu de dissenyar un aliment que pugui ser utilitzat com a suplementació esportiva per a practicants d'exercici físic intens, que aporti energia, que suposi una font alta de proteïnes (>20%), i que es pugui consumir i transportar fàcilment. S'ha realitzat una investigació dels productes d'aquest tipus ja existents al mercat, estudiant-ne les característiques nutricionals i mancances, on s'ha determinat que no compleixen amb un percentatge de proteïnes adequat. A continuació, s'ha realitzat un càlcul per determinar la qualitat de les proteïnes de certs ingredients que es troben de manera habitual a les barres així com alguns de nous mitjançant diferents mètodes. Pel disseny de les fórmules s'ha dut a terme una selecció i valoració dels ingredients a fer servir, per tal d'assolir els requisits nutricionals i fisicoquímics desitjats en les barretes. S'han dissenyat cinc fórmules variant les proporcions d'alguns ingredients i afegint-ne de nous per tal d'arribar al 20% de proteïnes. S'ha fet una anàlisi nutricional, de la qualitat proteica i econòmic i per finalitzar s'ha comparat les formulacions amb una seleccionada de les barres comercials. S'ha seleccionat la barreta F5 que aporta 387,75 kcal/100 g i 24,19 g/100 g, d'energia i proteïnes, respectivament, l'índex d'aminoàcids de la qual és d'1,37 i té un cost de 14,82 €/kg, donant com a resultat una millora important en alguns aspectes respecte de la barra comercial. With the aim of designing a food that can be used as a sport supplement for practitioners of intense physical exercise, which provides energy, which means a high source of proteins (>20%), and which can be easily consumed and transported. Research has been carried out into products of this type already in existence on the market, studying their nutritional characteristics and deficiencies, where it has been determined that they do not meet an appropriate percentage of proteins. Below, a calculation has been made to determine the quality of proteins of certain ingredients commonly found in bars as well as some new ones using different methods. For the design of formulas, a selection and evaluation of the ingredients to be used has been carried out, in order to meet the desired nutritional and physicochemical requirements in the bars. Five formulas have been designed by varying the ratios of some ingredients and adding new ones to reach 20% proteins. Nutritional, econòmic and protein quality analysis has been done and formulations have been compared to a selected one of the commercial bars. The F5 bar has been selected, providing 387.75 kcal/100 g and 24.19 g/100 g, of energy and proteins respectively, whose amino acid index is 1.37 and has a cost of 14.82 €/kg, resulting in a major improvement in some aspects of the commercial bar. Con el objetivo de diseñar un alimento que pueda ser utilizado como suplementación deportiva para practicantes de ejercicio físico intenso, que aporte energía, que suponga una fuente alta de proteínas (>20%), y que se pueda consumir y transportar fácilmente. Se ha realizado una investigación de los productos de este tipo ya existentes al mercado, estudiando las características nutricionales y carencias, donde se ha determinado que no cumplen con un porcentaje de proteínas adecuado. A continuación, se ha realizado un cálculo para determinar la calidad de las proteínas de ciertos ingredientes que se encuentran de manera habitual en las barras, así como algunos de nuevos, mediante diferentes métodos. Para el diseño de las fórmulas se ha llevado a cabo una selección y valoración de los ingredientes a usar, para lograr los requisitos nutricionales y fisicoquímicos deseados en las barritas. Se han diseñado cinco fórmulas variando las proporciones de algunos ingredientes y añadiendo otros para llegar al 20% de proteínas. Se ha hecho un análisis nutricional, de la calidad proteica y económico, y para finalizar se ha comparado las formulaciones con una seleccionada de entre las barras comerciales. Se ha seleccionado la barrita F5 que aporta 387,75 kcal/100 g y 24,19 g/100 g de energía y proteínas, respectivamente, el índice de aminoácidos de la cual es de 1,37 y tiene un coste de 14,82€ /kg, dando como resultado una mejora importante en algunos aspectos respecto de la barra comercial. Objectius de Desenvolupament Sostenible::3 - Salut i Benestar

Male germ cells undergo an extreme but fascinating process of chromatin remodeling that begins in the testis during the last phase of spermatogenesis and continues through epididymal sperm maturation. Most of the histones are replaced by small proteins named protamines, whose high basicity leads to a tight genomic compaction. This process is epigenetically regulated at many levels, not only by posttranslational modifications, but also by readers, writers, and erasers, in a context of a highly coordinated postmeiotic gene expression program. Protamines are key proteins for acquiring this highly specialized chromatin conformation, needed for sperm functionality. Interestingly, and contrary to what could be inferred from its very specific DNA-packaging function across protamine-containing species, human sperm chromatin contains a wide spectrum of protamine proteoforms, including truncated and posttranslationally modified proteoforms. The generation of protamine knock-out models revealed not only chromatin compaction defects, but also collateral sperm alterations contributing to infertile phenotypes, evidencing the importance of sperm chromatin protamination toward the generation of a new individual. The unique features of sperm chromatin have motivated its study, applying from conventional to the most ground-breaking techniques to disentangle its peculiarities and the cellular mechanisms governing its successful conferment, especially relevant from the protein point of view due to the important epigenetic role of sperm nuclear proteins. Gathering and contextualizing the most striking discoveries will provide a global understanding of the importance and complexity of achieving a proper chromatin compaction and exploring its implications on postfertilization events and beyond. This article is categorized under: Reproductive System Diseases > Genetics/Genomics/Epigenetics Reproductive System Diseases > Molecular and Cellular Physiology.