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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.

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.

[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].

[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.

X-ray crystallography is a powerful method that has significantly contributed to our understanding of the biological function of proteins and other molecules. This method relies on the production of crystals that, however, are usually a bottleneck in the process. For some molecules, no crystallization has been achieved or insufficient crystals were obtained. Some other systems do not crystallize at all, such as nanoparticles which, because of their dimensions, cannot be treated by the usual crystallographic methods. To solve this, whole pair distribution function has been proposed to bridge the gap between Bragg and Debye scattering theories. To execute a fitting, the spectra of several different constructs, composed of millions of particles each, should be computed using a particle-pair or particle-particle (pp) distance algorithm. Using this computation as a test bench for current field-programmable gate array (FPGA) technology, we evaluate how the parallel computation capability of FPGAs can be exploited to reduce the computation time. We present two different solutions to the problem using two state-of-the-art FPGA technologies. In the first one, the main C program uses OmpSs (a high-level programming model developed at the Barcelona Supercomputing Center, that enables task offload to different high-performance computing devices) for task invocation, and kernels are built with OpenCL using reduced data sizes to save transmission time. The second approach uses task and data parallelism to operate on data locally and update data globally in a decoupled task. Benchmarks have been evaluated over an Intel D5005 Programmable Acceleration Card, computing a model of 2 million particles in 81.57 s - 24.5 billion atom pairs per second (bapps)- and over a ZU102 in 115.31 s. In our last test, over an up-to-date Alveo U200 board, the computation lasted for 34.68 s (57.67 bapps). In this study, we analyze the results in relation to the classic terms of speed-up and efficiency and give hints for future improvements focused on reducing the global job time., This work was partially supported by the Spanish Government (AEI) and European Union (FEDER) (projects CTQ2017-88948-P, PID2021-124848OB-I00, RTI2018-095232-B-C21, SEV-2015-0493 and TIN2015-65316-P, grant BES-2016-078046) and by the Generalitat de Catalunya (contracts 2017-SGR-1742, 2017-SGR-1414, 2017-SGR-670 and 2017-SGR-1328).

Several factors present in the extracellular environment regulate epithelial cell adhesion and dynamics. Among them, growth factors such as EGF, upon binding to their receptors at the cell surface, get internalized and directly activate the acto-myosin machinery. In this study we present the effects of EGF on the contractility of epithelial cancer cell colonies in confined geometry of different sizes. We show that the extent to which EGF triggers contractility scales with the cluster size and thus the number of cells. Moreover, the collective contractility results in a radial distribution of traction forces, which are dependent on integrin β1 peripheral adhesions and transmitted to neighboring cells through adherens junctions. Taken together, EGF-induced contractility acts on the mechanical crosstalk and linkage between the cell-cell and cell-matrix compartments, regulating collective responses.

A prime concern of young patients with Polycystic Ovary Syndrome (PCOS) is the control of body adiposity, given their tendency to gain weight and/or their difficulty to lose weight. Circulating growth-and-differentiation factor-15 (GDF15) facilitates the control of body weight via receptors in the brainstem. C-reactive protein (CRP) and insulin are endogenous GDF15 secretagogues. We hypothesised that PCOS in non-obese adolescents is characterised by low concentrations of circulating GDF15, when judged by the degree of CRP and insulin drive. GDF15 was added as a post-hoc endpoint of two previously reported, randomised studies in non-obese adolescent girls with PCOS (N = 58; 60% normal weight; 40% overweight) who received either an oral oestroprogestogen contraceptive (OC), or a low-dose combination of spironolactone-pioglitazone-metformin (SPIOMET) for 1 year; subsequently, all girls remained untreated for 1 year. Adolescent girls with regular menses (N = 20) served as healthy controls. Circulating GDF15, CRP and fasting insulin were assessed prior to treatment, and halfway the on- and post-treatment years. Pre-treatment, the absolute GDF15 concentrations were normal in PCOS girls, but their relative levels were markedly low, in view of the augmented CRP and insulin drives. OC treatment was accompanied by a near-doubling of circulating GDF15 (on average, from 296 to 507 pg/mL) and CRP, so that the relative GDF15 levels remained low. SPIOMET treatment was accompanied by a 3.4-fold rise of circulating GDF15 (on average, from 308 to 1045 pg/mL) and by a concomitant lowering of CRP and insulin concentrations towards normal, so that the relative GDF15 levels became markedly abundant. Post-OC, the relatively low GDF15 levels persisted; post-SPIOMET, the circulating concentrations of GDF15, CRP and insulin were all normal. BMI remained stable in both treatment groups. Only SPIOMET was accompanied by a reduction of hepato-visceral fat (by MRI) towards normal. In conclusion, early PCOS was found to be characterised by a relative GDF15 deficit that may partly explain the difficulties that young patients experience to control their body adiposity. This relative GDF15 deficit persisted during and after OC treatment. In contrast, SPIOMET treatment was accompanied by an absolute and a relative abundance of GDF15, and followed by normal GDF15, CRP and insulin concentrations. The present findings strengthen the rationale to raise the concentrations of circulating GDF15 in early PCOS, for example with a SPIOMET-like intervention that attenuates low-grade inflammation, insulin resistance and ectopic adiposity, without necessarily lowering body weight.Clinical trial registries: ISRCTN29234515 and ISRCTN11062950.

Graphical abstract, We present a method for efficiently and effectively assessing whether and where two proteins can interact with each other to form a complex. This is still largely an open problem, even for those relatively few cases where the 3D structure of both proteins is known. In fact, even if much of the information about the interaction is encoded in the chemical and geometric features of the structures, the set of possible contact patches and of their relative orientations are too large to be computationally affordable in a reasonable time, thus preventing the compilation of reliable interactome. Our method is able to rapidly and quantitatively measure the geometrical shape complementarity between interacting proteins, comparing their molecular iso-electron density surfaces expanding the surface patches in term of 2D Zernike polynomials. We first test the method against the real binding region of a large dataset of known protein complexes, reaching a success rate of 0.72. We then apply the method for the blind recognition of binding sites, identifying the real region of interaction in about 60% of the analyzed cases. Finally, we investigate how the efficiency in finding the right binding region depends on the surface roughness as a function of the expansion order.

Graphical abstract, Long non-coding RNAs (lncRNAs) can perform a variety of key cellular functions by interacting with proteins and other RNAs. Recent studies have shown that the functions of lncRNAS are largely mediated by their structures. However, our structural knowledge for most lncRNAS is limited to sequence-based computational predictions. Non-coding RNA activated by DNA damage (NORAD) is an atypical lncRNA due to its abundant expression and high sequence conservation. NORAD regulates genomic stability by interacting with proteins and microRNAs. Previous sequence-based characterization has identified a modular organization of NORAD composed of several NORAD repeat units (NRUs). These units comprise the protein-binding elements and are separated by regular spacers. Here, we experimentally determine for the first time the secondary structure of NORAD using the nextPARS approach. Our results suggest that the spacer regions provide structural stability to NRUs. Furthermore, we uncover two previously unreported NRUs, and determine the core structural motifs conserved across NRUs. Overall, these findings will help to elucidate the function and evolution of NORAD.

Protein-protein interactions (PPIs) govern all biological processes. Some small molecules modulate PPIs through induced protein proximity. In particular, molecular glue degraders are monovalent compounds that orchestrate interactions between a target protein and an E3 ubiquitin ligase, prompting the proteasomal degradation of the former. This and other pharmacological strategies of targeted protein degradation (e.g. proteolysis-targeting chimeras - PROTACs) overcome some limitations of traditional occupancy-based therapeutics. Here, we provide an overview of the "molecular glue" concept, with a special focus on natural and synthetic inducers of proximity to E3s. We then briefly highlight the serendipitous discoveries of some clinical and preclinical molecular glue degraders, and discuss the first examples of intentional discoveries. Specifically, we outline the different screening strategies reported in this rapidly evolving arena and our thoughts on future perspectives. By mastering the ability to influence PPIs, molecular glue degraders can induce the degradation of unligandable proteins, thus providing an exciting path forward to broaden the targetable proteome.

Mechanical force controls fundamental cellular processes in health and disease, and increasing evidence shows that the nucleus both experiences and senses applied forces. Such forces can lead to the nuclear translocation of proteins, but whether force controls nucleocytoplasmic transport, and how, remains unknown. Here we show that nuclear forces differentially control passive and facilitated nucleocytoplasmic transport, setting the rules for the mechanosensitivity of shuttling proteins. We demonstrate that nuclear force increases permeability across nuclear pore complexes, with a dependence on molecular weight that is stronger for passive than for facilitated diffusion. Owing to this differential effect, force leads to the translocation of cargoes into or out of the nucleus within a given range of molecular weight and affinity for nuclear transport receptors. Further, we show that the mechanosensitivity of several transcriptional regulators can be both explained by this mechanism and engineered exogenously by introducing appropriate nuclear localization signals. Our work unveils a mechanism of mechanically induced signalling, probably operating in parallel with others, with potential applicability across signalling pathways.

Altered protein phosphorylation is a major pathologic modification in tauopathies and Alzheimer’s disease (AD) linked to abnormal tau fibrillar deposits in neurofibrillary tangles (NFTs) and pre-tangles and β-amyloid deposits in AD. hTau transgenic mice, which express 3R and less 4R human tau with no mutations in a murine knock-out background, show increased tau deposition in neurons but not NFTs and pre-tangles at the age of nine months. Label-free (phospho)proteomics and SWATH-MS identified 2065 proteins in hTau and wild-type (WT) mice. Only six proteins showed increased levels in hTau; no proteins were down-regulated. Increased tau phosphorylation in hTau was detected at Ser199, Ser202, Ser214, Ser396, Ser400, Thr403, Ser404, Ser413, Ser416, Ser422, Ser491, and Ser494, in addition to Thr181, Thr231, Ser396/Ser404, but not at Ser202/Thr205. In addition, 4578 phosphopeptides (corresponding to 1622 phosphoproteins) were identified in hTau and WT mice; 64 proteins were differentially phosphorylated in hTau. Sixty proteins were grouped into components of membranes, membrane signaling, synapses, vesicles, cytoskeleton, DNA/RNA/protein metabolism, ubiquitin/proteasome system, cholesterol and lipid metabolism, and cell signaling. These results showed that over-expression of human tau without pre-tangle and NFT formation preferentially triggers an imbalance in the phosphorylation profile of specific proteins involved in the cytoskeletal–membrane-signaling axis.

[eng] Intrinsically disordered (ID) proteins or regions are protein sequences that lack stable secondary and tertiary structures. ID proteins are particularly prone to undergo liquid- liquid phase separation (LLPS). The structures arising from this physical process are usually termed liquid droplets in vitro or biomolecular condensates in cells. The importance of LLPS in biological processes has been recognized in the last years, as the formation of biomolecular condensates allows for the efficient regulation of key cellular processes such as transcription or translation regulation. Additionally, mis- regulation of LLPS has been proposed as a disease mechanism. Generally, disease mutations can also cause liquid-to-solid phase transitions, a process named maturation. The general aim of this thesis was to characterize from a physicochemical point of view the LLPS of different protein systems involved in highly relevant biological processes: in transcription and translation regulation as well as in protein sorting. Additionally, we also assessed the effect of disease-related point mutations or alternative splicing on the LLPS process of proteins related to muscular dystrophy and autism spectrum disorder. We used a wide set of physical techniques such as nuclear magnetic resonance spectroscopy and apparent absorbance measurements, as well as optical and fluorescence microscopy experiments. We determined that the androgen and progesterone nuclear hormone receptors undergo LLPS, but the resulting liquid droplets are stabilized by different types of intermolecular interactions. Also, a protein that carries out its translational regulator function through LLPS, CPEB4, forms liquid droplets cooperatively which are stabilized by π-π and cation-π interactions. LLPS is also a possible mechanism for protein sorting at the Golgi apparatus, through the formation of biomolecular condensates by the TGN46 transmembrane protein. Disease mutations of ANXA11 increase the aggregation propensity of the protein leading to the formation of protein inclusions. Finally, CPEB4 mis-splicing related to autism spectrum disorder decreases the reversibility of the liquid droplets by promoting a liquid-to-solid phase transition. In summary, LLPS is a highly relevant phenomenon in biology because of its involvement in key processes such as transcription or translation regulation and protein sorting, and defects on LLPS can lead to disease, generally due to a liquid-to-solid maturation process. Understanding of the intermolecular interactions stabilizing the phase separated states can be used to design potential drugs to target ID proteins in their biomolecular condensates for treatment of diseases., [cat] Les proteïnes o regions intrínsecament desordenades (ID) són seqüències de proteïnes que manquen una estructura secundària i terciària estables. Les proteïnes ID són especialment propenses a experimentar separació de fases líquid-líquid (LLPS). Les estructures que es formen degut a aquest procés físic s’anomenen normalment gotes líquides in vitro o condensats biomoleculars en cèl·lules. La importància de la LLPS en processos biològics s’ha reconegut ens els últims anys, ja que s’ha vist que la formació de condensats biomoleculars permet l’eficient regulació de processos cel·lulars clau. En aquesta tesi s’ha caracteritzat des d’un punt de vista fisicoquímic la LLPS de diferents sistemes proteics involucrats en processos de gran rellevància biològica: en la regulació de la transcripció i traducció, així com en classificació i secreció de proteïnes. A més a més, també s’ha estudiat l’efecte de mutacions relacionades amb malalties i del splicing alternatiu sobre el procés de LLPS de proteïnes involucrades en malalties, que en general promouen una maduració de les gotes líquides, és a dir, una transició de líquid a sòlid. S’han utilitzat un ampli espectre de tècniques físiques per caracteritzar les interaccions intermoleculars i les propietats de les gotes líquides, incloent per exemple espectroscòpia de ressonància magnètica nuclear, mesures d’absorbància aparent i experiments de microscòpia òptica i de fluorescència. Poder entendre les interaccions intermoleculars que tenen lloc entre proteïnes per estabilitzar l’estat de separació de fases permet dissenyar fàrmacs dirigits a proteïnes ID en els condensats biomoleculars que formen per tractar malalties.

Genetic variants in YWHAZ contribute to psychiatric disorders such as autism spectrum disorder and schizophrenia, and have been related to an impaired neurodevelopment in humans and mice. Here, we have used zebrafish to investigate the mechanisms by which YWHAZ contributes to neurodevelopmental disorders. We observed that ywhaz expression was pan-neuronal during developmental stages and restricted to Purkinje cells in the adult cerebellum, cells that are described to be reduced in number and size in autistic patients. GCaMP6s albino zebrafish embryos were generated by the National Institute of Genetics (Japan) and obtained from Dr. Matt Parker from the University of Portsmouth, UK. The Tg(aldoca:gap43-Venus) line was obtained from Masahiko Hibi from the Bioscience and Biotechnology Center of Nagoya University, Japan. Tg(olig2:egfp)vu12 brains were obtained from the Center for Developmental Biology, UMR 5547 CNRS, Toulouse, France. Major financial support for this research was received by BC from the Spanish ‘Ministerio de Ciencia, Innovación y Universidades’ (RTI2018-100968-B-100, PID2021-1277760B-I100), the ‘Ministerio de Sanidad, Servicios Sociales e Igualdad/Plan Nacional Sobre Drogas’ (PNSD-2017I050 and PNSD-2020I042), ‘Generalitat de Catalunya/AGAUR’ (2017-SGR-738), ICREA Academia 2021, and the European Union H2020 Program [H2020/2014-2020] under grant agreements n° 667302 (CoCA) and Eat2beNICE (728018). E.A-G was supported by the Ministerio de Economía y Competitividad (Spanish Government) and the EU H2020 program (Eat2beNICE-728018). G.C., E.G. and P.L-A acknowledge financial support from the Spanish Ministerio de Economía y Competitividad (MINECO) through the “Severo Ochoa” program for Centres of Excellence in R&D CEX2019-000910-S), MINECO/FEDER Ramon y Cajal program (RYC-2015-17935); Laserlab-Europe EU-H2020 GA no. 871124, Fundació Privada Cellex, Fundación Mig-Puig and from the Generalitat de Catalunya through the CERCA program. F.A. acknowledges financial support from the Spanish ‘Ministerio de Ciencia, Innovación y Universidades’ (PID2019-107738RB-I00, MICINN/FEDER) and SGR (2017SGR1255).

This study analyses the relationship between the climate of the countries where wheat landraces are specifically adapted, and their agronomic and grain quality characteristics. Regions with similar climate within the Medi-terranean Basin were identified based on long-term climatic data of the 23 countries origin of a collection of 153 bread wheat landraces. The panel was genotyped with 13177 SNP markers and was grown on field experiments for two years under rainfed conditions in north-east Spain and 14 agronomic and 11 grain-quality traits were assessed. Great phenotypic variability was found in the collection. The agronomic performance of the landraces varied according to the climate of the four climatic regions identified within the Mediterranean Basin (south-east, south-west, north coast and north-Balkan), which gradually varies from warm and dry to wet and cold. Cycle length, grain-filling rate and yield increased, but grain-filling duration decreased from south-eastern landraces to north Balkan ones. Grain weight accounted for 25% of yield variations and was lowest in landraces from the south-eastern region. Grain quality showed no geographical pattern related to the climatic region. Landraces from Bosnia-Herzegovina, Bulgaria and Romania differed from the rest in their high gluten strength (W), loaf volume (LV), mixing time (MT) and grain hardness, while opposite attributes were found in accessions from Jordan, Lebanon and Cyprus. Landraces from south-western Mediterranean countries had low MT, alveograph-peak, W and LV. Genotypes suitable for use in breeding programmes may be identified. Molecular analyses revealed that genetic structure was mostly influenced by high temperatures before anthesis and rainfall, solar radiation and sunshine after anthesis. This study was partially funded by the research project AGL201565351-R and PID2019-109089RB-C31 of the Spanish Ministry of Science and Innovation (https://www.ciencia.gob.es/), the CERCA Programme/Generalitat de Catalunya (http://cerca.cat/), and CGIAR Research Program on Wheat lead by CIMMYT. Thanks are given to IPK, ISC, Suceava GenBank, VIR, CRF-INIA, CGN-WUR and NSGC-USDA for providing accessions for this study. The skilled technical assistance of the staff of the Sustainable Field Crops Programme of IRTA and the Wheat Quality and Chemistry Laboratory of CIMMYT are gratefully acknowledged. Carlos Guzm ' an gratefully acknowledges the European Social Fund and the Spanish State Research Agency (Ministry of Science and Innovation) for financial funding through the Ramon y Cajal Program (RYC-201721891).

Due to the fast increase in world population, many developing countries are facing malnutrition problems (famine, food insecurity, etc.). Particularly, the deficiency of protein sources is becoming a serious problem for both human and animals’ nutrition. Thus, the demand for protein sources has increased. In this context, Single Cell Proteins (SCP) produced by microorganisms could be exploited as an alternative and non-conventional protein source. The aim of this work was to study growth dynamics and SCP production and composition by Cupriavidus necator, under various environmental conditions stress and using different carbon (glucose, formic acid and oleic acid) and nitrogen (ammonium sulfate and urea) sources types. A mono-factorial approach under well-controlled conditions was used in order to investigate the impact of temperature, pH, carbon and nitrogen sources in bioreactor cultures. Results were compared in terms of bacterial growth, biomass composition (proteins and nucleic acids) and via the elemental biomass composition. Complementary analyses were also done using flow cytometry in order to study cell morphology, membrane permeability and presence of PHB production, a biopolymer naturally produced by C. necator. The temperature evaluation results showed that this parameter had an impact on growth and SCP production and composition. At 40°C, the growth was lower and low amounts of proteins, and nucleic acids were obtained. The same conclusion was obtained at pH5. Regarding the nitrogen source, the ammonium sulfate provided better results than urea as a nitrogen source. Added to that, the formic acid was found as a promising carbon source for protein production compared to glucose and oleic acid with a high total protein percentage of 90%DW (obtained with the Dumas’s method).; En raison de l'augmentation rapide de la population mondiale, de nombreux pays en cours de développement sont concernés par des problèmes de malnutrition (famine, insécurité alimentaire, etc.). En particulier, l’insuffisance en sources de protéines devient un problème majeur pour la nutrition humaine et animale et leur demande augmente de plus en plus. Dans ce contexte, les Protéines d’Organismes Unicellulaires (POU) produites par des micro-organismes peuvent exploitées comme une source de protéines alternative et non conventionnelle. L'objectif de ces travaux était d'étudier les dynamiques de croissance et de production de POU ainsi que leur composition par Cupriavidus necator, en réponse à des conditions environnementales différentes et en utilisant différents types de sources de carbone (glucose, acide formique et acide oléique) et d'azote (sulfate d’ammonium et urée). Une approche mono-factorielle en condition d’environnement contrôlé a été utilisée afin d'étudier l'impact de la température, du pH, des sources de carbone et d'azote dans des cultures en bioréacteurs. Les résultats ont été comparés en termes de croissance bactérienne, de composition de la biomasse (en protéines et en acides nucléiques) et via la composition élémentaire de la biomasse. Des analyses complémentaires ont également été effectuées par cytomètrie en flux afin de suivre la morphologie cellulaire, la perméabilité membranaire, et la présence potentielle de PHB, bio polymère naturellement produit par C. necator. Les résultats concernant la température ont montré que ce paramètre avait un impact sur la croissance ainsi que sur la production et la composition des POU. A 40°C, la croissance était plus faible et de faibles quantités de protéines et d'acides nucléiques ont été obtenues. La même conclusion a été obtenue à pH5. Concernant la nature de la source d’azote, le sulfate d'ammonium a donné les meilleurs résultats, comparé à l'urée, comme source d'azote. De plus, l'acide formique s'est avéré être une source de carbone prometteuse pour la production de protéines comparé au glucose et à l'acide oléique avec un pourcentage en protéines totales élevé autour de 90%DW (mesuré par la méthode de Dumas).

Protein-protein-interaction networks (PPINs) organize fundamental biological processes, but how oncogenic mutations impact these interactions and their functions at a network-level scale is poorly understood. Here, we analyze how a common oncogenic KRAS mutation (KRASG13D) affects PPIN structure and function of the Epidermal Growth Factor Receptor (EGFR) network in colorectal cancer (CRC) cells. Mapping >6000 PPIs shows that this network is extensively rewired in cells expressing transforming levels of KRASG13D (mtKRAS). The factors driving PPIN rewiring are multifactorial including changes in protein expression and phosphorylation. Mathematical modelling also suggests that the binding dynamics of low and high affinity KRAS interactors contribute to rewiring. PPIN rewiring substantially alters the composition of protein complexes, signal flow, transcriptional regulation, and cellular phenotype. These changes are validated by targeted and global experimental analysis. Importantly, genetic alterations in the most extensively rewired PPIN nodes occur frequently in CRC and are prognostic of poor patient outcomes. This work was supported by European Union FP7 Grant No. 278568 “PRIMES” and Science Foundation Ireland Investigator Program Grant 14/IA/2395 to W.K. B.K. is supported by SmartNanoTox (Grant no. 686098), NanoCommons (Grant no. 731032), O.R. by MSCA-IF-2016 SAMNets (Grant no. 750688). D.M. is supported by Science Foundation Ireland Career Development award 15-CDA-3495. I.J. is supported by the Canada Research Chair Program (CRC #225404), Krembil Foundation, Ontario Research Fund (GL2-01-030 and #34876), Natural Sciences Research Council (NSERC #203475), Canada Foundation for Innovation (CFI #225404, #30865), and IBM. O.S. is supported by ERC investigator Award ColonCan 311301 and CRUK. I.S. is supported by the Canadian Cancer Society Research Institute (#703889), Genome Canada via Ontario Genomics (#9427 & #9428), Ontario Research fund (ORF/ DIG-501411 & RE08-009), Consortium Québécois sur la Découverte du Médicament (CQDM Quantum Leap) & Brain Canada (Quantum Leap), and CQDM Explore and OCE (#23929). T.C. was supported by a Teagasc Walsh Fellowship

More women are diagnosed with breast cancer (BC) than any other type of cancer. Although large-scale efforts have completely redefined cancer, a cure remains unattainable. In that respect, new molecular functions of the cell should be investigated, such as post-transcriptional regulation. RNA-binding proteins (RBPs) are emerging as critical post-transcriptional modulators of tumorigenesis, but only a few have clear roles in BC. To recognize new putative breast cancer RNA-binding proteins, we performed integrated in silico analyses of all human RBPs (n = 1392) in three major cancer databases and identified five putative BC RBPs (PUF60, TFRC, KPNB1, NSF, and SF3A3), which showed robust oncogenic features related to their genomic alterations, immunohistochemical changes, high interconnectivity with cancer driver genes (CDGs), and tumor vulnerabilities. Interestingly, some of these RBPs have never been studied in BC, but their oncogenic functions have been described in other cancer types. Subsequent analyses revealed PUF60 and SF3A3 as central elements of a spliceosome-related cluster involving RBPs and CDGs. Further research should focus on the mechanisms by which these proteins could promote breast tumorigenesis, with the potential to reveal new therapeutic pathways along with novel drug-development strategies. This research received no external funding

Vaccination against SARS-CoV-2 just started in most of the countries. However, the development of specific vaccines against SARS-CoV-2 is not the only approach to control the virus and monoclonal antibodies (mAbs) start to merit special attention as a therapeutic option to treat COVID-19 disease. Here, the main conformations and interactions between the receptor-binding domain (RBD) of spike glycoprotein of SARS-CoV-2 (S protein) with two mAbs (CR3022 and S309) and the ACE2 cell receptor are studied as the main representatives of three different epitopes on the RBD of S protein. The combined approach of 1 μs accelerated molecular dynamics (aMD) and ab-initio hybrid molecular dynamics is used to identify the most predominant interactions under physiological conditions. Results allow to determine the main receptor-binding mapping, hydrogen bonding network and salt bridges in the most populated antigen-antibody interface conformations. The deep knowledge on the protein-protein interactions involving mAbs and ACE2 receptor with the spike glycoprotein of SARS-CoV-2 increases background knowledge to speed up the development of new vaccines and therapeutic drugs., Graphical abstract Image 1

The Protein Data Bank in Europe – Knowledge Base (PDBe-KB, https://pdbe-kb.org) is an open collaboration between world-leading specialist data resources contributing functional and biophysical annotations derived from or relevant to the Protein Data Bank (PDB). The goal of PDBe-KB is to place macromolecular structure data in their biological context by developing standardised data exchange formats and integrating functional annotations from the contributing partner resources into a knowledge graph that can provide valuable biological insights. Since we described PDBe-KB in 2019, there have been significant improvements in the variety of available annotation data sets and user functionality. Here, we provide an overview of the consortium, highlighting the addition of annotations such as predicted covalent binders, phosphorylation sites, effects of mutations on the protein structure and energetic local frustration. In addition, we describe a library of reusable web-based visualisation components and introduce new features such as a bulk download data service and a novel superposition service that generates clusters of superposed protein chains weekly for the whole PDB archive., ELIXIR [IDP implementation study]; Biotechnology and Biological Sciences Research Council via the 3D-Gateway [BB/T01959X/1]; FunPDBe [BB/P024351/1]; European Molecular Biology Laboratory-European Bioinformatics Institute who supported this work; J.D. acknowledges support from the Ministry of Education, Youth and Sport of the Czech Republic [INBIO CZ.02.1.01/0.0/0.0/16_026/0008451]; R.S., K.B. and J.D. also acknowledge support from the Ministry of Education, Youth and Sport of the Czech Republic [ELIXIR-CZ LM2018131]; L.M. acknowledges support from the European Union's Horizon 2020 Programme (H2020-INFRAIA-2018-1) [823839]; Research Foundation Flanders (FWO) [G032816N, G042518N, G028821N]; W.V. acknowledges support from the Research Foundation Flanders (FWO) [G032816N, G028821N]; A.R. acknowledges support from the Fondazione Cassa Di Risparmio di Firenze [24316]; European Commission [101017567]; M.H.C. acknowledges the AIRC project to MHC [IG 23539]; J.F.-R. acknowledges support from the Spanish Ministry of Science and Innovation [PID2019-110167RB-I00]; N.R. acknowledges support from the Norwegian Research Council (Norges Forskningsråd) [288008]; E.D.L. acknowledges support from the European Union's Horizon 2020 research and innovation programme [819318]; M.J.E.S. acknowledges support from the Wellcome Trust [104955/Z/14/Z, 218242/Z/19/Z]. Funding for open access charge: Biotechnology and Biological Sciences Research Council grant [BB/T01959X/1]; Wellcome Trust [104955/Z/14/Z and 218242/Z/19/Z].

Allosteric communication between distant sites in proteins is central to biological regulation but still poorly characterized, limiting understanding, engineering and drug development1-6. An important reason for this is the lack of methods to comprehensively quantify allostery in diverse proteins. Here we address this shortcoming and present a method that uses deep mutational scanning to globally map allostery. The approach uses an efficient experimental design to infer en masse the causal biophysical effects of mutations by quantifying multiple molecular phenotypes-here we examine binding and protein abundance-in multiple genetic backgrounds and fitting thermodynamic models using neural networks. We apply the approach to two of the most common protein interaction domains found in humans, an SH3 domain and a PDZ domain, to produce comprehensive atlases of allosteric communication. Allosteric mutations are abundant, with a large mutational target space of network-altering 'edgetic' variants. Mutations are more likely to be allosteric closer to binding interfaces, at glycine residues and at specific residues connecting to an opposite surface within the PDZ domain. This general approach of quantifying mutational effects for multiple molecular phenotypes and in multiple genetic backgrounds should enable the energetic and allosteric landscapes of many proteins to be rapidly and comprehensively mapped. This work was funded by European Research Council (ERC) Advanced (883742) and Consolidator (616434) grants, the Spanish Ministry of Science and Innovation (PID2020-118723GB-I00, BFU2017-89488-P, EMBL Partnership, Severo Ochoa Centre of Excellence), the Bettencourt Schueller Foundation, the AXA Research Fund, Agencia de Gestió d’Ajuts Universitaris i de Recerca (AGAUR, 2017 SGR 1322), and the CERCA Program/Generalitat de Catalunya. J.M.S. was supported by an EMBO Long-Term Fellowship (ALTF 857-2016) and a Marie Skłodowska-Curie Fellowship (752809, EU Commission Horizon 2020)

Ankyrin (ANK) repeat proteins are coded by tandem occurrences of patterns with around 33 amino acids. They often mediate protein–protein interactions in a diversity of biological systems. These proteins have an elongated non-globular shape and often display complex folding mechanisms. This work investigates the energy landscape of representative proteins of this class made up of 3, 4 and 6 ANK repeats using the energy-landscape visualisation method (ELViM). By combining biased and unbiased coarse-grained molecular dynamics AWSEM simulations that sample conformations along the folding trajectories with the ELViM structure-based phase space, one finds a three-dimensional representation of the globally funnelled energy surface. In this representation, it is possible to delineate distinct folding pathways. We show that ELViMs can project, in a natural way, the intricacies of the highly dimensional energy landscapes encoded by the highly symmetric ankyrin repeat proteins into useful low-dimensional representations. These projections can discriminate between multiplicities of specific parallel folding mechanisms that otherwise can be hidden in oversimplified depictions. M.N.S. was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; Grant 130147/2020-6). This research was supported by the Center for Theoretical Biological Physics sponsored by the NSF (Grant PHY-2019745). A.B.O. acknowledges the Robert A. Welch Postdoctoral Fellow program. P.G.W. is also supported by the D.R. Bullard-Welch Chair at Rice University (Grant C-0016). V.B.P.L. was supported by CNPq (Grant 310017/2020-3) and FAPESP (Grants 2019/22540-3 and 2018/18668-1). D.U.F. is a CONICET researcher and is supported by Grant PICT2016/1467, UBACYT, and NASA Astrobiology Institute-Enigma (Grant 80NSSC18M0093).

Many neurodegenerative disorders display protein aggregation as a hallmark, Huntingtin and TDP-43 aggregates being characteristic of Huntington disease and amyotrophic lateral sclerosis, respectively. However, whether these aggregates cause the diseases, are secondary by-products, or even have protective effects, is a matter of debate. Mutations in both human proteins can modulate the structure, number and type of aggregates, as well as their toxicity. To study the role of protein aggregates in cellular fitness, we have expressed in a highly tractable unicellular model different variants of Huntingtin and TDP-43. They each display specific patterns of aggregation and toxicity, even though in both cases proteins have to be very highly expressed to affect cell fitness. The aggregation properties of Huntingtin, but not of TDP-43, are affected by chaperones such as Hsp104 and the Hsp40 couple Mas5, suggesting that the TDP-43, but not Huntingtin, derivatives have intrinsic aggregation propensity. Importantly, expression of the aggregating form of Huntingtin causes a significant extension of fission yeast lifespan, probably as a consequence of kidnapping chaperones required for maintaining stress responses off. Our study demonstrates that in general these prion-like proteins do not cause toxicity under normal conditions, and in fact they can protect cells through indirect mechanisms which up-regulate cellular defense pathways. This work is supported by grant PGC2018-093920-B-I00 to E.H, funded by MCINN/AEI/ 10.13039/501100011033 and by “ERDF A way of making Europe”, by the “European Union”. The Oxidative Stress and Cell Cycle group is also supported by Generalitat de Catalunya (Spain) (2017-SGR-539) and by Excellence Unit «María de Maeztu» Grant CEX2018-000792-M funded by MCIN/AEI/10.13039/501100011033. E.H. is recipient of an ICREA Academia Award (Generalitat de Catalunya, Spain).