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The proper arrangement of protein components within the respiratory electron transport chain is nowadays a matter of intense debate, since altering it leads to cell aging and other related pathologies. Here, we discuss three current views-the so-called solid, fluid and plasticity models-which describe the organization of the main membrane-embedded mitochondrial protein complexes and the key elements that regulate and/or facilitate supercomplex assembly. The soluble electron carrier cytochrome c has recently emerged as an essential factor in the assembly and function of respiratory supercomplexes. In fact, a 'restricted diffusion pathway' mechanism for electron transfer between complexes III and IV has been proposed based on the secondary, distal binding sites for cytochrome c at its two membrane partners recently discovered. This channeling pathway facilitates the surfing of cytochrome c on both respiratory complexes, thereby tuning the efficiency of oxidative phosphorylation and diminishing the production of reactive oxygen species. The well-documented post-translational modifications of cytochrome c could further contribute to the rapid adjustment of electron flow in response to changing cellular conditions. Spanish Ministry of Economy and Competitiveness (BFU2015-71017/BMC MINECO/FEDER and PGC2018-096049-B-I00 BIO/BMC MICINN/FEDER, EU)

The IUBMB Focused Meeting/FEBS Workshop titled 'Crosstalk between Nucleus and Mitochondria in Human Disease'(CrossMitoNus) will take place on September 7-10, 2021 in Seville (Spain), with the support of both the International Union of Biochemistry and Molecular Biology (IUBMB) and the Federation of European Biochemical Societies (FEBS). Mitochondria are key organelles that act as a hub for vital metabolic processes, for example, energy transduction by oxidative phosphorylation, intermediary metabolism, redox signaling, calcium and iron homeostasis, heme and steroid biosynthesis, metal homeostasis, programmed cell death, and innate immunity. Consequently, a wide assortment of diseases-including neurodegenerative disorders, diabetes, cancer, rare syndromes, and many others-relate to mitochondrial dysfunction. The high relevance of mitochondria in metabolism centers on the core of cell signaling pathways, including those involved in cell-fate decisions. Critical metabolites synthesized in mitochondria are, for instance, key modulators of the sirtuin, AMPK, mTOR, and Hypoxia-inducible Factor 1A pathways. Mitochondria are indeed the major source of reactive oxygen species, which in turn mediate several regulatory routes. Interestingly, multiple nuclear-encoded factors control essential processes in mitochondrial dynamics, namely fusion (for instance, OPA1), fission (DNM1L), transport (RHOT1), and mitophagy (PINK1). The release of mitochondrial factors like cytochrome c to the cytoplasm is indeed key for the rapid onset of the intrinsic apoptotic pathway. The CrossMitoNus meeting aims to join efforts from diverse disciplines to unveil the mitochondrial and nuclear factors that are emerging as essential elements in mitochondria-nucleus communication. Needless to say, the mechanisms regulating mitochondrial protein trafficking into and out of the nucleus and the role of these proteins in the nucleus remain to be elucidated.

Due to their capability to transport chemicals or proteins into target cells, cell-penetrating peptides (CPPs) are being developed as therapy delivery tools. However, and despite their interesting properties, arginine-rich CPPs often show toxicity for reasons that remain poorly understood. Using a (PR)n dipeptide repeat that has been linked to amyotrophic lateral sclerosis (ALS) as a model of an arginine-rich CPP, we here show that the presence of (PR)n leads to a generalized displacement of RNA- and DNA-binding proteins from chromatin and mRNA. Accordingly, any reaction involving nucleic acids, such as RNA transcription, translation, splicing and degradation, or DNA replication and repair, is impaired by the presence of the CPPs. Interestingly, the effects of (PR)n are fully mimicked by protamine, a small arginine-rich protein that displaces histones from chromatin during spermatogenesis. We propose that widespread coating of nucleic acids and consequent displacement of RNA- and DNA-binding factors from chromatin and mRNA accounts for the toxicity of arginine-rich CPPs, including those that have been recently associated with the onset of ALS., Fundación Botín, by Banco Santander through its Santander Universities Global Division and by grants from the Spanish Ministry of Science, Innovation and Universities (RTI2018-102204-B-I00, co-financed with European FEDER funds) and the European Research Council (ERC-617840) to OF; DKFZ NCT3.0 Integrative Project in Cancer Research grant (NCT3.0_2015.54 DysregPT) and SFB 1036/TP07 from the Deutsche Forschungsgemeinschaft to G.S.

Despite mitochondria being key for the control of cell homeostasis and fate, their role in DNA damage response is usually just regarded as an apoptotic trigger. However, growing evidence points to mitochondrial factors modulating nuclear functions. Remarkably, after DNA damage, cytochrome c (Cc) interacts in the cell nucleus with a variety of well‐known histone chaperones, whose activity is competitively inhibited by the haem protein. As nuclear Cc inhibits the nucleosome assembly/disassembly activity of histone chaperones, it might indeed affect chromatin dynamics and histone deposition on DNA. Several histone chaperones actually interact with Cc Lys residues through their acidic regions, which are also involved in heterotypic interactions leading to liquid–liquid phase transitions responsible for the assembly of nuclear condensates, including heterochromatin. This relies on dynamic histone–DNA interactions that can be modulated by acetylation of specific histone Lys residues. Thus, Cc may have a major regulatory role in DNA repair by fine‐tuning nucleosome assembly activity and likely nuclear condensate formation., DNA damage induces liquid–liquid phase separation within DNA foci, thereby allowing repair mechanisms to access damaged sites. DNA damage also makes mitochondrial cytochrome c translocate to the nucleus to sequester histone chaperones, for example SET/TAF‐Iβ, and impair their functions. We thus hypothesize that the INHAT activity in chromatin remodelling might be inhibited, as SET/TAF‐Iβ is a component of the INHAT complex.

Post‐translational modifications frequently modulate protein functions. Lysine acetylation in particular plays a key role in interactions between respiratory cytochrome c and its metabolic partners. To date, in vivo acetylation of lysines at positions 8 and 53 has specifically been identified in mammalian cytochrome c, but little is known about the structural basis of acetylation‐induced functional changes. Here, we independently replaced these two residues in recombinant human cytochrome c with glutamine to mimic lysine acetylation and then characterized the structure and function of the resulting K8Q and K53Q mutants. We found that the physicochemical features were mostly unchanged in the two acetyl‐mimetic mutants, but their thermal stability was significantly altered. NMR chemical shift perturbations of the backbone amide resonances revealed local structural changes, and the thermodynamics and kinetics of electron transfer in mutants immobilized on gold electrodes showed an increase in both protein dynamics and solvent involvement in the redox process. We also observed that the K8Q (but not the K53Q) mutation slightly increased the binding affinity of cytochrome c to its physiological electron donor, cytochrome c 1—which is a component of mitochondrial complex III, or cytochrome bc 1—thus suggesting that Lys8 (but not Lys53) is located in the interaction area. Finally, the K8Q and K53Q mutants exhibited reduced efficiency as electron donors to complex IV, or cytochrome c oxidase., Acetylation is the main post‐translational modification of proteins in eukaryotic cells, affecting more than 60% of mitochondrial proteins. Mammalian cytochrome c, in particular, is found acetylated at lysines 8 and 53. Here, we characterize two acetyl‐mimetic mutants (Lys‐to‐Gln mutation) of human cytochrome c to gain insight into the structural and functional changes that could further explain the acetylation role in cancer and other diseases.

17 pags., 9 figs., 1 tab., Repair of injured DNA relies on nucleosome dismantling by histone chaperones and de-phosphorylation events carried out by Protein Phosphatase 2A (PP2A). Typical histone chaperones are the Acidic leucine-rich Nuclear Phosphoprotein 32 family (ANP32) members, e.g. ANP32A, which is also a well-known PP2A inhibitor (a.k.a. IPP2A). Here we report the novel interaction between the endogenous family member B—so-called ANP32B—and endogenous cytochrome c in cells undergoing camptothecin-induced DNA damage. Soon after DNA lesions but prior to caspase cascade activation, the hemeprotein translocates to the nucleus to target the Low Complexity Acidic Region (LCAR) of ANP32B; in a similar way, our group recently reported that the hemeprotein targets the acidic domain of SET/Template Activating Factor-Iβ (SET/TAF-Iβ), which is another histone chaperone and PP2A inhibitor (a.k.a. IPP2A). The nucleosome assembly activity of ANP32B is indeed unaffected by cytochrome c binding. Like ANP32A, ANP32B inhibits PP2A activity and is thus herein referred to as IPP2A. Our data demonstrates that ANP32B-dependent inhibition of PP2A is regulated by respiratory cytochrome c, which induces long-distance allosteric changes in the structured N-terminal domain of ANP32B upon binding to the C-terminal LCAR. In agreement with the reported role of PP2A in the DNA damage response, we propose a model wherein cytochrome c is translocated from the mitochondria into the nucleus upon DNA damage to modulate PP2A activity via its interaction with ANP32B., This work was supported by the Spanish Ministry of Science, Innovation and Universities (BFU2012-31670, BFU2015-71017, PGC2018-096049- BI00), Spanish Ministry of Education and Professional Training (FPU013/04373 to FRR, FPU016/01513 to AVC), Regional Government of Andalusia (BIO198, US-1254317 US/JUNTA/FEDER,UE, US- 1257019 US/JUNTA/FEDER,UE, P18-FR-3487 and P18–HO-4091), Ramon Areces Foundation, Biointeractomics Platform (cicCartuja, Seville) and the Microscopy and NMR Services at CITIUS (University of Seville). MPG is funded by a postdoctoral grant from the Spanish Association Against Cancer Scientific Foundation (FC AECC)

Diet is one of the main sources of exposure to toxic chemicals with carcinogenic potential, some of which are generated during food processing, depending on the type of food (primarily meat, fish, bread and potatoes), cooking methods and temperature. Although demonstrated in animal models at high doses, an unequivocal link between dietary exposure to these compounds with disease has not been proven in humans. A major difficulty in assessing the actual intake of these toxic compounds is the lack of standardised and harmonised protocols for collecting and analysing dietary information. The intestinal microbiota (IM) has a great influence on health and is altered in some diseases such as colorectal cancer (CRC). Diet influences the composition and activity of the IM, and the net exposure to genotoxicity of potential dietary carcinogens in the gut depends on the interaction among these compounds, IM and diet. This review analyses critically the difficulties and challenges in the study of interactions among these three actors on the onset of CRC. Machine Learning (ML) of data obtained in subclinical and precancerous stages would help to establish risk thresholds for the intake of toxic compounds generated during food processing as related to diet and IM profiles, whereas Semantic Web could improve data accessibility and usability from different studies, as well as helping to elucidate novel interactions among those chemicals, IM and diet., This work is receiving support from the project RTI2018-098288-B-I00 (MCIU/AEI/FEDER, UE) and is based on concepts developed partly funded by projects TIN2017-88877-R (AEI/FEDER, UE), TIN2017-87600-P (AEI/FEDER. UE) and IDI/2018/000176 (Asturian Government GRUPIN projects). SRS is the beneficiary of a training contract financed by project RTI2018-098288-B-I00. SA is granted by a Juan de la Cierva postdoctoral contract from the Spanish Ministry of Science and Innovation (Ref. IJCI-2017-32156) and NS is the recipient of a postdoctoral contract awarded by the Fundación para la Investigación y la Innovación Biosanitaria del Principado de Asturias (FINBA).

Cell signaling mechanisms modulate gene expression in response to internal and external stimuli. Cellular adaptation requires a precise and coordinated regulation of the transcription and translation processes. The post-transcriptional control of mRNA metabolism is mediated by the so-called RNA-binding proteins (RBPs), which assemble with specific transcripts forming messenger ribonucleoprotein particles of highly dynamic composition. RBPs constitute a class of trans-acting regulatory proteins with affinity for certain consensus elements present in mRNA molecules. However, these regulators are subjected to post-translational modifications (PTMs) that constantly adjust their activity to maintain cell homeostasis. PTMs can dramatically change the subcellular localization, the binding affinity for RNA and protein partners, and the turnover rate of RBPs. Moreover, the ability of many RBPs to undergo phase transition and/or their recruitment to previously formed membrane-less organelles, such as stress granules, is also regulated by specific PTMs. Interestingly, the dysregulation of PTMs in RBPs has been associated with the pathophysiology of many different diseases. Abnormal PTM patterns can lead to the distortion of the physiological role of RBPs due to mislocalization, loss or gain of function, and/or accelerated or disrupted degradation. This Mini Review offers a broad overview of the post-translational regulation of selected RBPs and the involvement of their dysregulation in neurodegenerative disorders, cancer and other pathologies

12 pags., 6 figs., Programmed cell death (PCD) is crucial for development and homeostasis of all multicellular organisms. In human cells, the double role of extra‐mitochondrial cytochrome c in triggering apoptosis and inhibiting survival pathways is well reported. In plants, however, the specific role of cytochrome c upon release from the mitochondria remains in part veiled yet death stimuli do trigger cytochrome c translocation as well. Here, we identify an Arabidopsis thaliana 14‐3‐3ι isoform as a cytosolic cytochrome c target and inhibitor of caspase‐like activity. This finding establishes the 14‐3‐3ι protein as a relevant factor at the onset of plant H2O2‐induced PCD. The in vivo and in vitro studies herein reported reveal that the interaction between cytochrome c and 14‐3‐3ι exhibits noticeable similarities with the complex formed by their human orthologues. Further analysis of the heterologous complexes between human and plant cytochrome c with plant 14‐3‐3ι and human 14‐3‐3ε isoforms corroborated common features. These results suggest that cytochrome c blocks p14‐3‐3ι so as to inhibit caspase‐like proteases, which in turn promote cell death upon H2O2 treatment. Besides establishing common biochemical features between human and plant PCD, this work sheds light onto the signaling networks of plant cell death., Fundación Banco Santander Spanish Ministry of Education, Culture and Sport. Grant Number: FPU17/04604 Fundación Ramón Areces Fundación Cámara – University of Seville Spanish Ministry of Economy and Competitiveness. Grant Numbers: BFU2015‐71017/BMC MINECO/FEDER, PGC2018‐096049‐B‐I00 BIO/BMC MICINN/FEDER, RTI2018‐094793‐B‐I00 MICIU Andalusian Government. Grant Numbers: BIO198, US‐1257019

Mitochondria are the powerhouses of the cell, whilst their malfunction is related to several human pathologies, including neurodegenerative diseases, cardiovascular diseases, and various types of cancer. In mitochondrial metabolism, cytochrome c is a small soluble heme protein that acts as an essential redox carrier in the respiratory electron transport chain. However, cytochrome c is likewise an essential protein in the cytoplasm acting as an activator of programmed cell death. Such a dual role of cytochrome c in cell life and death is indeed fine-regulated by a wide variety of protein post-translational modifications. In this work, we show how these modifications can alter cytochrome c structure and functionality, thus emerging as a control mechanism of cell metabolism but also as a key element in development and prevention of pathologies.

The alternative reading frame (ARF) protein is crucial in the cellular response to oncogenic stress, being likewise the second most frequently inactivated gene in a wide spectrum of human cancers. ARF is usually sequestered in the nucleolus by the well-known oncogenic nucleophosmin (NPM) protein and is liberated in response to cell damage to exhibit its tumor-suppressor ability. However, the mechanism underlying ARF activation is unknown. Here we show that mitochondria-to-nucleus translocation of cytochrome c upon DNA damage leads to the break-off of the NPM-ARF ensemble and subsequent release of ARF from the nucleoli. Our structural and subcellular data support a molecular model in which the hemeprotein triggers the extended-to-compact conformation of NPM and competes with ARF for binding to NPM.

Cardiolipin oxidation and degradation by different factors under severe cell stress serve as a trigger for genetically encoded cell death programs. In this context, the interplay between cardiolipin and another mitochondrial factor—cytochrome c—is a key process in the early stages of apoptosis, and it is a matter of intense research. Cytochrome c interacts with lipid membranes by electrostatic interactions, hydrogen bonds, and hydrophobic effects. Experimental conditions (including pH, lipid composition, and post-translational modifications) determine which specific amino acid residues are involved in the interaction and influence the heme iron coordination state. In fact, up to four binding sites (A, C, N, and L), driven by different interactions, have been reported. Nevertheless, key aspects of the mechanism for cardiolipin oxidation by the hemeprotein are well established. First, cytochrome c acts as a pseudoperoxidase, a process orchestrated by tyrosine residues which are crucial for peroxygenase activity and sensitivity towards oxidation caused by protein self-degradation. Second, flexibility of two weakest folding units of the hemeprotein correlates with its peroxidase activity and the stability of the iron coordination sphere. Third, the diversity of the mode of interaction parallels a broad diversity in the specific reaction pathway. Thus, current knowledge has already enabled the design of novel drugs designed to successfully inhibit cardiolipin oxidation.

The topology and dynamics of the scanning ribosomal 43S pre-initiation complex (PIC) bound to mRNA and initiation factors (eIFs) are probably the least understood aspects of translation initiation in eukaryotes. Recently, we described a trapping mechanism in alphavirus that stalls the PIC during scanning of viral mRNA. Using this model, we were able to snapshot for the first time the eIF4A helicase bound to mRNA in a 48S initiation complex assembled in vitro. This interaction was only detected in the presence of the natural stem loop structure (DLP) located downstream from the AUG in viral mRNA that promoted stalling of the PIC, suggesting that DLP stability was enough to jam the helicase activity of eIF4A in a fraction of assembled 48S complexes. However, a substantial proportion of DLP mRNA molecules were effectively unwound by eIF4A in vitro, an activity that alphaviruses counteract in infected cells by excluding eIF4A from viral factories. Our data indicated that eIF4A-mRNA contact occurred in (or near) the ES6S region of the 40S subunit, suggesting that incoming mRNA sequences penetrate through the ES6S region during the scanning process. We propose a topological model of the scanning PIC and how some viruses have exploited this topology to translate their mRNAs with fewer eIF requirements., Ministerio de Ciencia e Innovación [BFU2013-4005R]

The supramolecular arrangement of respiratory complexes into supercomplexes is widely accepted. The common feature observed in the supercomplex architecture from organisms of diverse phylogenetic origin is the reduction of the distance between cytochrome bc1 (complex III) and cytochrome c oxidase (complex IV). Such an arrangement reduces the dimensionality (from 3D to 2D) of the diffusional search of cytochrome c as the electron carrier that connects both complexes. In this scenario, our recent finding of additional binding sites for cytochrome c reinforces the concept of a “restrained 2D sliding pathway” onto the supercomplex surface. Herein, we analyze novel mechanistic insights into electron transfer towards cytochrome c, including modulation of the redox potential by physical contact as well as gated, long-range electron transfer through an aqueous solution. These data establish a new horizon for the understanding of electron transfer mechanisms beyond unique and well-orientated protein complexes. Multiple and dynamic long-distance conformational ensembles compatible with electron transfer could indeed contribute to the rapid adjustment of electron flow in response to changing cellular conditions.

Alphaviruses are insect-borne viruses that alternate between replication in mosquitoes and vertebrate species. Adaptation of some alphaviruses to vertebrate hosts has involved the acquisition of an RNA structure (downstream loop [DLP]) in viral subgenomic mRNAs that confers translational resistance to protein kinase (PKR)- mediated eIF2α phosphorylation. Here, we found that, in addition to promoting eIF2-independent translation of viral subgenomic mRNAs, presence of the DLP structure also increased the resistance of alphavirus to type I interferon (IFN). Aura virus (AURAV), an ecologically isolated relative of Sindbis virus (SV) that is poorly adapted to replication in vertebrate cells, displayed a nonfunctional DLP structure and dramatic sensitivity to type I IFN. Our data suggest that an increased resistance to IFN emerged during translational adaptation of alphavirus mRNA to vertebrate hosts, reinforcing the role that double-stranded RNA (dsRNA)-activated protein kinase (PKR) plays as both a constitutive and IFN-induced antiviral effector. Interestingly, a mutant SV lacking the DLP structure (SV-ΔDLP) and AURAV both showed a marked oncotropism for certain tumor cell lines that have defects in PKR expression and/or activation. AURAV selectively replicated in and killed some cell lines derived from human hepatocarcinoma (HCC) that lacked PKR response to infection or poly(I·C) transfection. The oncolytic activities of SV-ΔDLP and AURAV were also confirmed using tumor xenografts in mice, showing tumor regression activities comparable to wild-type SV. Our data show that translation of alphavirus subgenomic mRNAs plays a central role in IFN susceptibility and cell tropism, suggesting an unanticipated oncolytic potential that some naive arboviruses may have in virotherapy. IMPORTANCE Interferons (IFNs) induce the expression of a number of antiviral genes that protect the cells of vertebrates against viruses and other microbes. The susceptibility of cells to viruses greatly depends on the level and activity of these antiviral effectors but also on the ability of viruses to counteract this antiviral response. Here, we found that the level of one of the main IFN effectors in the cell, the dsRNAactivated protein kinase (PKR), greatly determines the permissiveness of cells to alphaviruses that lack mechanisms to counteract its activation. These naive viruses also showed a hypersensitivity to IFN, suggesting that acquisition of IFN resistance (even partial) has probably been involved in expanding the host range of alphaviruses in the past. Interestingly, some of these naive viruses showed a marked oncotropism for some tumor cell lines derived from human hepatocarcinoma (HCC), opening the possibility of their use in oncolytic therapy to treat human tumors.

His/Cys coordination was recently found in several c-type cytochromes, which could act as sensors, in electron transport or in regulation. Toward a better understanding of Cys function and reactivity in these cytochromes, we compare cytochrome c6 (c6wt) from the cyanobacterium Nostoc PCC 7120 with its Met58Cys mutant. We probe the axial ligands and heme properties by combining visible and mid- to far-FTIR difference spectroscopies. Cys58 determines the strong negative redox potential and pH dependence of M58C (EmM58C = -375 mV, versus Emc6wt = +339 mV). Mid-IR (notably Cys ν(SH), His ν(C5N1), heme (CmH)) and far-IR (ν(Fe(II)-His), ν(His-Fe(III)-Cys)) markers of the heme and ligands show that Cys58 remains a strong thiolate ligand of reduced Met58Cys at alkaline pH, while it is protonated at pH 7.5, is stabilized by a strong hydrogen bonding interaction, and weakly interacts with Fe(II). These data provide a benchmark for further analysis of c-type cytochromes with natural His/Cys coordination French Agence Nationale de la Recherche (ANR) PSIIFIR ANR-15-CE05-0016 French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INBS-05 Ministerio de Ciencia e Innovación PGC2018-096049-B-I00 Junta de Andalucía BIO-198, US-1257019, US-1254317

Cytochrome c (Cc) is a protein that functions as an electron carrier in the mitochondrial respiratory chain. However, Cc has moonlighting roles outside mitochondria driving the transition of apoptotic cells from life to death. When living cells are damaged, Cc escapes its natural mitochondrial environment and, once in the cytosol, it binds other proteins to form a complex named the apoptosome-a platform that triggers caspase activation and further leads to controlled cell dismantlement. Early released Cc also binds to inositol 1,4,5-triphosphate receptors on the ER membrane, which stimulates further massive Cc release from mitochondria. Besides the well-characterized binding proteins contributing to the proapoptotic functions of Cc, many novel protein targets have been recently described. Among them, histone chaperones were identified as key partners of Cc following DNA breaks, indicating that Cc might modulate chromatin dynamics through competitive binding to histone chaperones. In this article, we review the ample set of recently discovered antiapoptotic proteins-involved in DNA damage, transcription, and energetic metabolism-reported to interact with Cc in the cytoplasm and even the nucleus upon DNA breaks.

Tagetes lucida Cav. is an ancient medicinal plant used to treat different ailments involving neurological diseases and pain. However, scientific studies to validate their medicinal properties as analgesic have not been described. The aim of this study was to evaluate the T. lucida antinociceptive response using pain models. Bioactive compounds and a possible mechanism of action were also explored. Dose-response effects of an ethanol crude extract were investigated in the writhing and formalin tests in mice and rats, respectively. The extract was fractionated to isolate active fractions and bioactive compounds (quercetagetin 7‑O‑β‑ d ‑glucoside and 6,7‑dimethoxycoumarin) using the formalin test. The antinociceptive effects were compared to the reference drugs (tramadol 10 mg/kg, diclofenac 50 mg/kg, and/or ketorolac 1 mg/kg, i.p.). The ethanol extract was explored in the presence of naloxone (3 mg/kg, i.p. a non-selective opioid receptor antagonist) and WAY100635 (0.5 mg/kg, s.c., a selective 5-HT1A receptor antagonist) to screen their participation as possible inhibitory mechanisms involved in the antinociceptive response of T. lucida. The ethanol crude extract, fractions, and pure compounds caused a significant antinociceptive response resembling the effect of the reference drugs. Both opioid and 5-HT1A receptors participated in the analgesic –like activity of the extract, which did not produce gastric damage. On the contrary, the gastric damage produced as an adverse effect of the analgesic ketorolac was prevented when combined with the extract. In conclusion, these preliminary data provide evidence and give support to the properties attributed to T. lucida in the traditional medicine to alleviate pain.

In spite of the efforts devoted to understand the relationship of society with science, the results have not been satisfactory. The aim of this study is to test the PIKA model, developed to contribute to a better understanding of the perspective of citizens in the relationship of society with science. Our hypothesis is that the interaction of citizens with science generates an image that determines how they react to it. We conceive this image as a mental map, and according to contributions from neurology, we consider that it is grounded on a neural net. The PIKA model postulates that there is a section of the image of science that accounts for the interaction of Perception, Interest, Knowledge, and willingness to Act. We used Structural Equation Modelling to obtain evidence to support this model. We used data from three Spanish samples: the 2006 and 2014 editions of the Survey on Social Perception of Science and Technology by the Spanish Foundation for Science and Technology, and the answers to the PIKA Questionnaire of a sample of students from several Spanish universities. The sample of the 2006 edition of the survey of FECYT is comprised by 7.056 subjects from 18 years of age, while the 2014 edition includes 6.136 people. The sample that has completed the PIKA questionnaire includes 2.138 students from some Spanish universities. The results provide evidence in favour of the PIKA model in the three samples. We conclude that the image of science depicted as a neural net is useful to explain the interaction of citizens with science. Nevertheless, to achieve better understanding of this interaction we need better indicators of the factors that give shape to the citizens' image of science.

Aims Circadian rhythms are the approximate 24 h biological cycles that function to prepare an organism for daily environmental changes. Circadian rhythms unquestionably play critical roles in metabolic homeostasis and the exercise has emerged as a strong non-photic time cue or zeitgeber in animal models and humans. Numerous studies about the effects of exercise on the citrate synthase activity have been published. Citrate is used to assess energy production or expenditure because it is a substrate of the Krebs Cycle, a cycle for oxidative energy production. Main methods We tested the existence of a rhythmic urinary citrate excretion in a rat model that is made to exercise at six different points during the day. Key findings The data obtained by the enzyme assays were fitted to a mathematical model (Fourier series), showing for the first time, the existence of a distinct ultradian rhythm in the urinary citrate excretion. The aerobic exercise led to the increase in the period length of the ultradian rhythm and raised the acrophase value of the urinary citrate excretion. Therefore, the urinary citrate excretion pattern changed after exercise, showing a clear circadian rhythm fitted to the mathematical model. Significance The citrate urine samples could provide accurate data for ranking an individual's metabolic status. Using exercise to maintain the circadian clock at an appropriate phase and amplitude might be effective to prevent cardiometabolic disease development.

Location of the translation initiation codon generally requires scanning of the 43S ribosomal preinitiation complex (43S PIC) from the 5' of the mRNA. Associated RNA helicases can facilitate movement of the 43S PIC by removing secondary structure present in the 5' UTR of mRNA, which is required for codon inspection. The canonical RNA-dependent helicase eIF4A is directly involved in this process, as part of the eIF4F complex (eIF4G + eIF4A + eIF4E) that associates first with mRNA and then recruits the 43S PIC to initiate scanning. The topology and operational mechanism of the scanning PIC are probably the least understood aspects of the initiation step. Recent findings from translation of alphavirus mRNA, together with new biochemical and structural data of the 43S PIC, suggest a role for the ES6S region of 40S as the gateway for mRNA entry during scanning. The presence of eIF4G-eIF4A complex in this region, interacting with the incoming mRNA, supports a model where eIF4A could work ahead of the scanning complex during translation initiation. Here we present additional data supporting this model.

Protein function is frequently modulated by post-translational modifications of specific residues. Cytochrome c, in particular, is phosphorylated in vivo at threonine 28 and serine 47. However, the effect of such modifications on the physiological functions of cytochrome c – namely, the transfer of electrons in the respiratory electron transport chain and the triggering of programmed cell death – is still unknown. Here we replace each of these two residues by aspartate, in order to mimic phosphorylation, and report the structural and functional changes in the resulting cytochrome c variants. We find that the T28D mutant causes a 30-mV decrease on the midpoint redox potential and lowers the affinity for the distal site of Arabidopsis thaliana cytochrome c1 in complex III. Both the T28D and S47D variants display a higher efficiency as electron donors for the cytochrome c oxidase activity of complex IV. In both protein mutants, the peroxidase activity is significantly higher, which is related to the ability of cytochrome c to leave the mitochondria and reach the cytoplasm. We also find that both mutations at serine 47 (S47D and S47A) impair the ability of cytoplasmic cytochrome c to activate the caspases cascade, which is essential for triggering programmed cell death.

We are indebted to Luis Menendez′s lab for helping us with acrylamide gels for sequencing and Juanjo Berlanga andMiguel Angel Rodriguez Gabriel for their support and discussions. Institutional support from the Fundacion Ramon Areces is also acknowledged. Completion of this project took approximately 3 years and the estimated cost was 10000 , excluding salaries. During translation initiation, eukaryotic initiation factor 2 (eIF2) delivers the Met-tRNA to the 40S ribosomal subunit to locate the initiation codon (AUGi) of mRNA during the scanning process. Stress-induced eIF2 phosphorylation leads to a general blockade of translation initiation and represents a key antiviral pathway in mammals. However, some viral mRNAs can initiate translation in the presence of phosphorylated eIF2 via stable RNA stem-loop structures (DLP; Downstream LooP) located in their coding sequence (CDS), which promote 43S preinitiation complex stalling on the initiation codon. We show here that during the scanning process, DLPs of Alphavirus mRNA become trapped in ES6S region (680-914 nt) of 18S rRNA that are projected from the solvent side of 40S subunit. This trapping can lock the progress of the 40S subunit on the mRNA in a way that places the upstream initiator AUGi on the P site of 40S subunit, obviating the participation of eIF2. Notably, the DLP structure is released from 18S rRNA upon 60S ribosomal subunit joining, suggesting conformational changes in ES6Ss during the initiation process. These novel findings illustrate how viral mRNA is threaded into the 40S subunit during the scanning process, exploiting the topology of the 40S subunit solvent side to enhance its translation in vertebrate hosts. Sí

In oxidative phosphorylation, the transfer of electrons from reduced cofactors to molecular oxygen via the electron transport chain (ETC) sustains the electrochemical transmembrane potential needed for ATP synthesis. A key component of the ETC is complex III (CIII, cytochrome bc1), which transfers electrons from reduced ubiquinone to soluble cytochrome c (Cc) coupled to proton translocation into the mitochondrial intermembrane space. One electron from every two donated by hydroquinone at site P is transferred to Cc via the Rieske-cytochrome c1 (Cc1) pathway. According to recent structural analyses of CIII and its transitory complex with Cc, the interaction between the Rieske subunit and Cc1 switches intermittently during CIII activity. However, the electrochemical properties of Cc1 and their function as a wire between Rieske and Cc are rather unexplored. Here, temperature variable cyclic voltammetry provides novel data on the thermodynamics and kinetics of interfacial electron transfer of immobilized Cc1. Findings reveal that Cc1 displays two channels for electron exchange, with a remarkably fast heterogeneous electron transfer rate. Furthermore, the electrochemical properties are strongly modulated by the binding mode of the protein. Additionally, we show that electron transfer from Cc1 to Cc is thermodynamically favored in the immobilized Cc1-Cc complex. Nuclear Magnetic Resonance, HADDOCK, and Surface Plasmon Resonance experiments provide further structural and functional data of the Cc1-Cc complex. Our data supports the Rieske-Cc1-Cc pathway acting as a unilateral switch thyristor in which redox potential modulation through protein-protein contacts are complemented with the relay-like Rieske behavior.

Background Even though liver kinase B1 (LKB1) is usually described as a tumor suppressor in a wide variety of tissues, it has been shown that LKB1 aberrant expression is associated with bad prognosis in Hepatocellular Carcinoma (HCC). Methods Herein we have overexpressed LKB1 in human hepatoma cells and by using histidine pull-down assay we have investigated the role of the hypoxia-related post-translational modification of Small Ubiquitin-related Modifier (SUMO)ylation in the regulation of LKB1 oncogenic role. Molecular modelling between LKB1 and its interactors, involved in regulation of LKB1 nucleocytoplasmic shuttling and LKB1 activity, was performed. Finally, high affinity SUMO binding entities-based technology were used to validate our findings in a pre-clinical mouse model and in clinical HCC. Findings We found that in human hepatoma cells under hypoxic stress, LKB1 overexpression increases cell viability and aggressiveness in association with changes in LKB1 cellular localization. Moreover, by using site-directed mutagenesis, we have shown that LKB1 is SUMOylated by SUMO-2 at Lys178 hampering LKB1 nucleocytoplasmic shuttling and fueling hepatoma cell growth. Molecular modelling of SUMO modified LKB1 further confirmed steric impedance between SUMOylated LKB1 and the STe20-Related ADaptor cofactor (STRADα), involved in LKB1 export from the nucleus. Finally, we provide evidence that endogenous LKB1 is modified by SUMO in pre-clinical mouse models of HCC and clinical HCC, where LKB1 SUMOylation is higher in fast growing tumors. Interpretation Overall, SUMO-2 modification of LKB1 at Lys178 mediates LKB1 cellular localization and its oncogenic role in liver cancer. Fund This work was supported by grants from NIH (US Department of Health and Human services)-R01AR001576-11A1 (J.M.M and M.L.M-C.), Gobierno Vasco-Departamento de Salud 2013111114 (to M.L.M.-C), ELKARTEK 2016, Departamento de Industria del Gobierno Vasco (to M.L.M.-C), MINECO: SAF2017–87301-R and SAF2014–52097-R integrado en el Plan Estatal de Investigación Cientifica y Técnica y Innovación 2013–2016 cofinanciado con Fondos FEDER (to M.L.M.-C and J.M.M., respectively), BFU2015–71017/BMC MINECO/FEDER, EU (to A.D.Q. and I.D.M.), BIOEF (Basque Foundation for Innovation and Health Research): EITB Maratoia BIO15/CA/014; Instituto de Salud Carlos III:PIE14/00031, integrado en el Plan Estatal de Investigación Cientifica y Técnica y Innovacion 2013–2016 cofinanciado con Fondos FEDER (to M.L.M.-C and J.M.M), Asociación Española contra el Cáncer (T.C.D, P·F-T and M.L.M-C), Daniel Alagille award from EASL (to T.C.D), Fundación Científica de la Asociación Española Contra el Cancer (AECC Scientific Foundation) Rare Tumor Calls 2017 (to M.L.M and M.A), La Caixa Foundation Program (to M.L.M), Programma di Ricerca Regione-Università 2007–2009 and 2011–2012, Regione Emilia-Romagna (to E.V.), Ramón Areces Foundation and the Andalusian Government (BIO-198) (A.D.Q. and I.D.M.), ayudas para apoyar grupos de investigación del sistema Universitario Vasco IT971–16 (P.A.), MINECO:SAF2015–64352-R (P.A.), Institut National du Cancer, FRANCE, INCa grant PLBIO16–251 (M.S.R.), MINECO - BFU2016–76872-R to (E.B.). Work produced with the support of a 2017 Leonardo Grant for Researchers and Cultural Creators, BBVA Foundation (M.V-R). Finally, Ciberehd_ISCIII_MINECO is funded by the Instituto de Salud Carlos III. We thank MINECO for the Severo Ochoa Excellence Accreditation to CIC bioGUNE (SEV-2016-0644). Funding sources had no involvement in study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication., Highlights • Overexpression of LKB1 in human hepatoma cells during hypoxic stress induces deregulated cell growth and survival. • SUMO-2 modifications of LKB1 at Lys178 occur in human hepatoma cells hampering its nucleocytoplasmic shuttling. • LKB1 SUMOylation is augmented in pre-clinical mouse models and clinical HCC, being a hallmark of more aggressive HCC tumors.

Due to their capability to transport chemicals or proteins into target cells, cell-penetrating peptides (CPPs) are being developed as therapy delivery tools. However, and despite their interesting properties, arginine-rich CPPs often show toxicity for reasons that remain poorly understood. Using a (PR)n dipeptide repeat that has been linked to amyotrophic-lateral sclerosis (ALS) as a model of an arginine-rich CPP, we here show that the presence of (PR)n leads to a generalized displacement of RNA- and DNA-binding proteins from chromatin and mRNA. Accordingly, any reaction involving nucleic acids such as RNA transcription, translation, splicing and degradation or DNA replication and repair are impaired by the presence of the CPP. Interestingly, the effects of (PR)n are fully mimicked by PROTAMINE, a small arginine-rich protein that displaces histones from chromatin during spermatogenesis. We propose that widespread coating of nucleic acids and consequent displacement of RNA- and DNA-binding factors from chromatin and mRNA accounts for the toxicity of arginine-rich CPPs, including those that have been recently associated to the onset of ALS.

Regulation of mitochondrial activity allows cells to adapt to changing conditions and to control oxidative stress, and its dysfunction can lead to hypoxia-dependent pathologies such as ischemia and cancer. Although cytochrome c phosphorylation—in particular, at tyrosine 48—is a key modulator of mitochondrial signaling, its action and molecular basis remain unknown. Here we mimic phosphorylation of cytochrome c by replacing tyrosine 48 with p-carboxy-methyl-L-phenylalanine (pCMF). The NMR structure of the resulting mutant reveals significant conformational shifts and enhanced dynamics around pCMF that could explain changes observed in its functionality: The phosphomimetic mutation impairs cytochrome c diffusion between respiratory complexes, enhances hemeprotein peroxidase and reactive oxygen species scavenging activities, and hinders caspase-dependent apoptosis. Our findings provide a framework to further investigate the modulation of mitochondrial activity by phosphorylated cytochrome c and to develop novel therapeutic approaches based on its prosurvival effects., Financial support was provided by the Spanish Ministry of Economy and Competitiveness (Grants BFU2015-71017-P/BMC and BFU2015-19451/BMC, cofounded by FEDER EU), European Union (Bio-MR-00130 and CALIPSO-312284), Ramon Areces Foundation, and Andalusian Government (BIO198). B.M.-B. was awarded a PhD fellowship from the Spanish Ministry of Education (AP2009-4092) and a short-term traveling fellowship from the European Bio-NMR Project. A.G.-C. was awarded a PhD fellowship from the CSIC (JaePre-2011-01248).

Chromatin is pivotal for regulation of the DNA damage process insofar as it influences access to DNA and serves as a DNA repair docking site. Recent works identify histone chaperones as key regulators of damaged chromatin’s transcriptional activity. However, understanding how chaperones are modulated during DNA damage response is still challenging. This study reveals that the histone chaperone SET/TAF-Iβ interacts with cytochrome c following DNA damage. Specifically, cytochrome c is shown to be translocated into cell nuclei upon induction of DNA damage, but not upon stimulation of the death receptor or stress-induced pathways. Cytochrome c was found to competitively hinder binding of SET/TAF-Iβ to core histones, thereby locking its histone-binding domains and inhibiting its nucleosome assembly activity. In addition, we have used NMR spectroscopy, calorimetry, mutagenesis, and molecular docking to provide an insight into the structural features of the formation of the complex between cytochrome c and SET/TAF-Iβ. Overall, these findings establish a framework for understanding the molecular basis of cytochrome c-mediated blocking of SET/TAF-Iβ, which subsequently may facilitate the development of new drugs to silence the oncogenic effect of SET/TAF-Iβ’s histone chaperone activity.

In Press, Human antigen R (HuR) is a ubiquitous 32kDa protein comprising three RNA Recognition Motifs (RRMs), whose main function is to bind Adenylate and uridylate Rich Elements (AREs) in 3′ UnTranslated Regions (UTRs) of mRNAs. In addition to binding RNA molecules, the third domain (RRM3) is involved in HuR oligomerization and apoptotic signaling. The RRM3 monomer is able to dimerize, with its self-binding affinity being dependent on ionic strength. Here we provide a deeper structural insight into the nature of the encounter complexes leading to the formation of RRM3 dimers by using Brownian Dynamics and Molecular Dynamics. Our computational data show that the initial unspecific encounter follows a downhill pathway until reaching an optimum conformation stabilized by hydrophobic interactions., I.D.-M. wishes to thank the Andalusian Government (P07-CVI-02896, P11-CVI-07216 and BIO198) for financial support.

Protein production using processed cell lysates is a core technology in synthetic biology and these systems are excellent to produce difficult toxins or membrane proteins. However, the composition of the central lysate of cell-free systems is still a “black box”. Escherichia coli lysates are most productive for cell-free expression, yielding several mgs of protein per ml of reaction. Their preparation implies proteome fractionation, resulting in strongly biased and yet unknown lysate compositions. Many metabolic pathways are expected to be truncated or completely removed. The lack of knowledge of basic cell-free lysate proteomes is a major bottleneck for directed lysate engineering approaches as well as for assay design using non-purified reaction mixtures. This study is starting to close this gap by providing a blueprint of the S30 lysate proteome derived from the commonly used E. coli strain A19. S30 lysates are frequently used for cell-free protein production and represent the basis of most commercial E. coli cell-free expression systems. A fraction of 821 proteins was identified as the core proteome in S30 lysates, representing approximately a quarter of the known E. coli proteome. Its classification into functional groups relevant for transcription/translation, folding, stability and metabolic processes will build the framework for tailored cell-free reactions. As an example, we show that SOS response induction during cultivation results in tuned S30 lysate with better folding capacity, and improved solubility and activity of synthesized proteins. The presented data and protocols can serve as a platform for the generation of customized cell-free systems and product analysis.

Over the past decade, evidence has emerged suggesting a broader role for cytochrome c (Cyt c) in programmed cell death. Recently, we demonstrated the ability of Cyt c to inhibit the nucleosome assembly activity of histone chaperones SET/template-activating factor Iß and NAP1-related protein during DNA damage in humans and plants respectively. Here, we hypothesise a dual concentration-dependent function for nuclear Cyt c in response to DNA damage. We propose that low levels of highly cytotoxic DNA lesions - such as double-strand breaks - induce nuclear translocation of Cyt c, leading to the attenuation of nucleosome assembly and, thereby, increasing the time available for DNA repair. If DNA damage persists or is exacerbated, the nuclear Cyt c concentration would exceed a given threshold, causing the haem protein to block DNA remodelling altogether., This work was supported by the Spanish Ministry of Economy, Industry and Competitiveness (BFU2015‐71017/BMC), the Andalusian Government (BIO198), the Ramón Areces Foundation and the European Regional Development Fund (FEDER). AVC is recipient of an FPU Fellowship from the Spanish Ministry of Education, Culture and Sports (FPU16/01513).

Malaria is caused by Apicomplexa protozoans from the Plasmodium genus entering the bloodstream of humans and animals through the bite of the female mosquitoes. The annotation of the Plasmodium vivax genome revealed a putative RNA binding protein (apiRBP) that was predicted to be trafficked into the apicoplast, a plastid organelle unique to Apicomplexa protozoans. Although a 3D structural model of the apiRBP corresponds to a noncanonical RNA recognition motif with an additional C-terminal ¿-helix (¿3), preliminary protein production trials were nevertheless unsuccessful. Theoretical solvation analysis of the apiRBP model highlighted an exposed hydrophobic region clustering ¿3. Hence, we used a C-terminal GFP-fused chimera to stabilize the highly insoluble apiRBP and determined its ability to bind U-rich stretches of RNA. The affinity of apiRBP toward such RNAs is highly dependent on ionic strength, suggesting that the apiRBP-RNA complex is driven by electrostatic interactions. Altogether, apiRBP represents an attractive tool for apicoplast transcriptional studies and for antimalarial drug design.

Respiratory cytochrome c has been found to be phosphorylated at tyrosine 97 in the postischemic brain upon neuroprotective insulin treatment, but how such posttranslational modification affects mitochondrial metabolism is unclear. Here, we report the structural features and functional behavior of a phosphomimetic cytochrome c mutant, which was generated by site-specific incorporation at position 97 of p-carboxymethyl-l-phenylalanine using the evolved tRNA synthetase method. We found that the point mutation does not alter the overall folding and heme environment of cytochrome c, but significantly affects the entire oxidative phosphorylation process. In fact, the electron donation rate of the mutant heme protein to cytochrome c oxidase, or complex IV, within respiratory supercomplexes was higher than that of the wild-type species, in agreement with the observed decrease in reactive oxygen species production. Direct contact of cytochrome c with the respiratory supercomplex factor HIGD1A (hypoxia-inducible domain family member 1A) is reported here, with the mutant heme protein exhibiting a lower affinity than the wild-type species. Interestingly, phosphomimetic cytochrome c also exhibited a lower caspase-3 activation activity. Altogether, these findings yield a better understanding of the molecular basis for mitochondrial metabolism in acute diseases, such as brain ischemia, and thus could allow the use of phosphomimetic cytochrome c as a neuroprotector with therapeutic applications., This work was supported by Ministry of Economy and Competitiveness and European Regional Development Fund (MINECO/FEDER) Grant BFU2015-71017/BMC, European Union; the Ramón Areces Foundation, European Social Fund, Andalusian Government (BIO-198); Consejo Superior de Investigaciones Científicas Fellowship JaePre_2011_01248 (to A.G.-C.); a Fundación Cámara PhD Fellowship (to C.A.E.-R.); Biointeractomics Platform (cicCartuja, Seville); and TA Instruments.

The transient interactions of respiratory cytochrome c with complexes III and IV is herein investigated by using heterologous proteins, namely human cytochrome c, the soluble domain of plant cytochrome c1 and bovine cytochrome c oxidase. The binding molecular mechanisms of the resulting cross-complexes have been analyzed by Nuclear Magnetic Resonance and Isothermal Titration Calorimetry. Our data reveal that the two cytochrome c-involving adducts possess a 2:1 stoichiometry – that is, two cytochrome c molecules per adduct – at low ionic strength. We conclude that such extra binding sites at the surfaces of complexes III and IV can facilitate the turnover and sliding of cytochrome c molecules and, therefore, the electron transfer within respiratory supercomplexes.

HuR/ELAVL1 is an RNA-binding protein involved in differentiation and stress response that acts primarily by stabilizing messenger RNA (mRNA) targets. HuR comprises three RNA recognition motifs (RRMs) where the structure and RNA binding of RRM3 and of full-length HuR remain poorly understood. Here, we report crystal structures of RRM3 free and bound to cognate RNAs. Our structural, NMR and biochemical data show that RRM3 mediates canonical RNA interactions and reveal molecular details of a dimerization interface localized on the -helical face of RRM3. NMR and SAXS analyses indicate that the three RRMs in full-length HuR are flexibly connected in the absence of RNA, while they adopt a more compact arrangement when bound to RNA. Based on these data and crystal structures of tandem RRM1,2- RNA and our RRM3-RNA complexes, we present a structural model of RNA recognition involving all three RRM domains of full-length HuR. Mutational analysis demonstrates that RRM3 dimerization and RNA binding is required for functional activity of fulllength HuR in vitro and to regulate target mRNAs levels in human cells, thus providing a fine-tuning for HuR activity in vivo.

Apoptosis is a highly regulated form of programmed cell death, essential to the development and homeostasis of multicellular organisms. Cytochrome c is a central figure in the activation of the apoptotic intrinsic pathway, thereby activating the caspase cascade through its interaction with Apaf-1. Our recent studies have revealed 14-3-3ε (a direct inhibitor of Apaf-1) as a cytosolic cytochrome c target. Here we explore the cytochrome c / 14-3-3ε interaction and show the ability of cytochrome c to block 14-3-3ε-mediated Apaf-1 inhibition, thereby unveiling a novel function for cytochrome c as an indirect activator of caspase-9/3. We have used calorimetry, NMR spectroscopy, site mutagenesis and computational calculations to provide an insight into the structural features of the cytochrome c / 14-3-3ε complex. Overall, these findings suggest an additional cytochrome c-mediated mechanism to modulate apoptosome formation, shedding light onto the rigorous apoptotic regulation network.

There are many chronic diseases related with inflammation. The chronic inflammation can produce other problems as cancer. Therefore, it is necessary to design drugs with better anti-inflammatory activity than those in the clinic. Likewise, these could be used in chronic treatments with minimum adverse effects. The amide or ester functionality in combination with the insertion of a silyl alkyl moiety is able to improve some drug properties. In this context, the evaluation of a group of silicon containing ibuprofen derivatives (SCIDs) as antioxidants and anti-inflammatory agents is reported. Antioxidant activity was evaluated by the 2,2-Diphenyl-1-picrylhydrazyl (DPPH⨪), 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic) acid (ABTS•+) and the Fe(II) chelating ability methods. The anti-inflammatory activity was determined by using the carrageenan induced rat paw edema. The gastrotoxic profile of the SCIDs that displayed significant anti-inflammatory activity was determined by the indomethacin induced ulceration method. The SCIDs performed better than ibuprofen as chelating agents for Fe(II) and as scavengers for the free radicals DPPH• and ABTS•+. On the anti-inflammatory test, compound 4a inhibited the edema up to 87%, while 4d &10b achieved significant inflammation inhibition at a lower effective dose 50 (ED50) than ibuprofen´s. None of the SCIDs endowed with anti-inflammatory activity, showed significant gastrotoxic effects with respect to those displayed by ibuprofen. Based on the experimental results and aided by the theoretical docking approach, it was possible to rationalize how the SCIDs may bind to cyclooxygenase isoforms and helped to explain their reduced gastrotoxicity. The evaluated effects were improved in SCIDs with respect to ibuprofen.

The function of pluripotency genes in differentiation is a matter of investigation. We report here that Nanog and Oct4 are reexpressed in two mouse embryonic stem cell (mESC) lines following exposure to the differentiating agent DETA/NO. Both cell lines express a battery of both endoderm and mesoderm markers following induction of differentiation with DETA/NO-based protocols. Confocal analysis of cells undergoing directed differentiation shows that the majority of cells expressing Nanog express also endoderm genes such as Gata4 and FoxA2 (75.4% and 96.2%, resp.). Simultaneously, mRNA of mesodermal markers Flk1 and Mef2c are also regulated by the treatment. Acetylated histone H3 occupancy at the promoter of Nanog is involved in the process of reexpression. Furthermore, Nanog binding to the promoter of Brachyury leads to repression of this gene, thus disrupting mesendoderm transition, This work was supported by Consejería de Igualdad, Salud y Políticas Sociales, Junta de Andalucía (PI0022/2008, PI105/ 2010, and PI10/00964), Consejer´ıa de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía (CTS-7127/2011, P10.CTS.6505, and PAI BIO-311), Instituto de Salud Carlos III/FEDER (Red TerCel, RD06/0010/0025, RD12/0019/0028), Ministerio de Economía y Competitividad (IPT-2011-1615- 900000), Ministerio de Salud y Consumo (TRA-120), and Servicio Andaluz de Salud (SAS 11245)

The discovery of effective new antimalarial agents is urgently needed. One of the most frequently studied molecules anchored to the parasite surface is the merozoite surface protein-1 (MSP1). At red blood cell invasion MSP1 is proteolytically processed, and the 19-kDa C-terminal fragment (MSP119) remains on the surface and is taken into the red blood cell, where it is transferred to the food vacuole and persists until the end of the intracellular cycle. Because a number of specific antibodies inhibit erythrocyte invasion and parasite growth, MSP119 is therefore a promising target against malaria. Given the structural homology of cupredoxins with the Fab domain of monoclonal antibodies, an approach combining NMR and isothermal titration calorimetry (ITC) measurements with docking calculations based on BiGGER is employed on MSP119-cupredoxin complexes. Among the cupredoxins tested, rusticyanin forms a well defined complex with MSP119 at a site that overlaps with the surface recognized by the inhibitory antibodies. The addition of holo-rusticyanin to infected cells results in parasitemia inhibition, but negligible effects on parasite growth can be observed for apo-rusticyanin and other proteins of the cupredoxin family. These findings point to rusticyanin as an excellent therapeutic tool for malaria treatment and provide valuable information for drug design.

Tyrosine nitration is a common post-translational modification affecting protein structure and function. It is based on the addition of a -NO2 group at the ortho position of the phenolic hydroxyl group of tyrosine to yield 3-nitrotyrosine (3-NTyr). Understanding how tyrosine nitration affects the structure and functionality of proteins is of considerable interest, as it is associated with pathogenesis in diseases related to oxidative stress in all living organisms. There are several methods to nitrate tyrosine residues in native proteins. Among them, nitration by the chemical agent peroxynitrite stands out for its biological relevance. Recently, a genetically evolved suppressor tRNA has been developed to provide in vivo incorporation of 3-NTyr into proteins. In this minireview, we discuss the advantages and limitations of these chemical and biological methods and propose a non-damaging method to analyze the configuration and dynamics of nitrotyrosine residues in native proteins by NMR spectroscopy.

Higher-order plants and mammals use similar mechanisms to repair and tolerate oxidative DNA damage. Most studies on the DNA repair process have focused on yeast and mammals, in which histone chaperone-mediated nucleosome disassembly/reassembly is essential for DNA to be accessible to repair machinery. However, little is known about the specific role and modulation of histone chaperones in the context of DNA damage in plants. Here, the histone chaperone NRP1, which is closely related to human SET/TAF-I , was found to exhibit nucleosome assembly activity in vitro and to accumulate in the chromatin of Arabidopsis thaliana after DNA breaks. In addition, this work establishes that NRP1 binds to cytochrome c, thereby preventing the former from binding to histones. Since NRP1 interacts with cytochrome c at its earmuff domain, that is, its histone-binding domain, cytochrome c thus competes with core histones and hampers the activity of NRP1 as a histone chaperone. Altogether, the results obtained indicate that the underlying molecularmechanisms in nucleosome disassembly/reassembly are highly conserved throughout evolution, as inferred from the similar inhibition of plant NRP1 and human SET/TAF-I by cytochrome c during DNA damage response

TIA-1 (T-cell restricted intracellular antigen-1) is an RNA-binding protein involved in splicing and translational repression. It mainly interacts with RNA via its second and third RNA recognition motifs (RRMs), with specificity for U-rich sequences directed by RRM2. It has recently been shown that RRM3 also contributes to binding, with preferential binding for C-rich sequences. Here we designed UC-rich and CU-rich 10-nt sequences for engagement of both RRM2 and RRM3 and demonstrated that the TIA-1 RRM23 construct preferentially binds the UC-rich RNA ligand (5΄-UUUUUACUCC-3΄). Interestingly, this binding depends on the presence of Lys274 that is C-terminal to RRM3 and binding to equivalent DNA sequences occurs with similar affinity. Small-angle X-ray scattering was used to demonstrate that, upon complex formation with target RNA or DNA, TIA-1 RRM23 adopts a compact structure, showing that both RRMs engage with the target 10-nt sequences to form the complex. We also report the crystal structure of TIA-1 RRM2 in complex with DNA to 2.3 Å resolution providing the first atomic resolution structure of any TIA protein RRM in complex with oligonucleotide. Together our data support a specific mode of TIA-1 RRM23 interaction with target oligonucleotides consistent with the role of TIA-1 in binding RNA to regulate gene expression.

The Reactive Nitrogen and Oxygen Species (the so-called RNOS), which are well-known radicals formed in the mitochondria under nitro-oxidative cell stress, are responsible for nitration of tyrosines in a wide variety of proteins and, in particular, in cytochrome c (Cc). Only three out of the five tyrosine residues of human Cc, namely those at positions 67, 74 and 97, have been detected in vivo as nitrotyrosines. However, nitration of the two other tyrosines, namely those at positions 46 and 48, has never been detected in vivo despite they are both well-exposed to solvent. Here we investigate the changes in heme coordination and alkaline transition, along with the peroxidase activity and in cell degradation of Cc mutants in which all their tyrosine residues – with the only exception of that at position 46 or 48 – are replaced by phenylalanines. In Jurkat cell extracts devoid of proteases inhibitors, only the high-spin iron nitrated forms of these monotyrosine mutants are degraded. Altogether the resulting data suggest that nitration of tyrosines 46 and 48 makes Cc easily degradable upon turning the heme iron state to high-spin.

Cytochrome c delicately tilts the balance between cell life (respiration) and cell death (apoptosis). Whereas cell life is governed by transient electron transfer interactions of cytochrome c inside the mitochondria, the cytoplasmic adducts of cytochrome c that lead to cell death are amazingly stable. Interestingly, the contacts of cytochrome c with its counterparts shift from the area surrounding the heme crevice for the redox complexes to the opposite molecule side when the electron flow is not necessary. The cytochrome c signalosome shows a higher level of regulation by post-translational modifications-nitration and phosphorylation-of the hemeprotein. Understanding protein interfaces, as well as protein modifications, would puzzle the mitochondrial cytochrome c-controlled pathways out and enable the design of novel drugs to silence the action of pro-survival and pro-apoptotic partners of cytochrome c. © 2011 European Biophysical Societies' Association., The authors wish to thank Jonathan Martínez-Fábregas for helpful advice and critical reading of the manuscript. This work was funded by the Spanish Ministry of Science and Innovation (BFU2009-07190) and the Andalusian Government (BIO198 and P08-CVI-3876).

Transient complexes, with a lifetime ranging between microseconds and seconds, are essential for biochemical reactions requiring a fast turnover. That is the case of the interactions between proteins engaged in electron transfer reactions, which are involved in relevant physiological processes such as respiration and photosynthesis. In the latter, the copper protein plastocyanin acts as a soluble carrier transferring electrons between the two membrane-embedded complexes cytochrome b6f and photosystem I. Here we review the combination of experimental efforts in the literature to unveil the functional and structural features of the complex between cytochrome f and plastocyanin, which have widely been used as a suitable model for analyzing transient redox interactions. © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Metalloproteins play major roles in cell metabolism and signalling pathways. In many cases, they show moonlighting behaviour, acting in different processes, depending on the physiological state of the cell. To understand these multitasking proteins, we need to discover the partners with which they carry out such novel functions. Although many technological and methodological tools have recently been reported for the detection of protein interactions, specific approaches to studying the interactions involving metalloproteins are not yet well developed. The task is even more challenging for metalloproteins, because they often form short-lived complexes that are difficult to detect. In this review, we gather the different proteomic techniques and biointeractomic tools reported in the literature. All of them have shown their applicability to the study of transient and weak protein-protein interactions, and are therefore suitable for metalloprotein interactions.

15 páginas, 9 figuras, 2 tablas., Tyrosine nitration is one of the most common post-transcriptional modifications of proteins, so affecting their structure and function. Human cytochrome c, with five tyrosine residues, is an excellent case study as it is a well-known protein playing a double physiological role in different cell compartments. On one hand, it acts as electron carrier within the mitochondrial respiratory electron transport chain, and on the other hand, it serves as a cytoplasmic apoptosis-triggering agent. In a previous paper, we reported the effect of nitration on physicochemical and kinetic features of monotyrosine cytochrome c mutants. Here, we analyse the nitration-induced changes in secondary structure, thermal stability, haem environment, alkaline transition and molecular dynamics of three of such monotyrosine mutants – the so-called h-Y67, h-Y74 and h-Y97 – which have four tyrosines replaced by phenylalanines and just keep the tyrosine residue giving its number to the mutant. The resulting data, along with the functional analyses of the three mutants, indicate that it is the specific nitration of solvent-exposed Tyr74 which enhances the peroxidase activity and blocks the ability of Cc to activate caspase-9, thereby preventing the apoptosis signaling pathway., This work was supported by the Spanish Ministry of Science and Innovation (BFU2006-01361/BMC and BFU2009-07190).

5 páginas, 5 figuras, The metal cofactor determines the thermal stability in cupredoxins, but how the redox state of copper modulates their melting points remains unknown. The metal coordination environment is highly conserved in cyanobacterial plastocyanins. However, the oxidised form is more stable than the reduced one in thermophilic Phormidium, but the opposite occurs in mesophilic Synechocystis. We have performed neutral amino-acid substitutions at loops of Phormidium plastocyanin far from the copper site. Notably, mutation P49G/G50P confers a redox-dependent thermal stability similar to that of the mesophilic plastocyanin. Moreover, X-ray absorption spectroscopy reveals that P49G/G50P mutation makes the electron density distribution at the oxidised copper site shift towards that of Synechocystis plastocyanin., This work was supported by the Spanish Ministry of Science and Innovation (MICINN, BFU2006-01361, BFU2009-07190, FIS2008-03951), the Andalusian Government (BIO-198 and P06-CVI-01713), the ESRF (SC-2486). F.J.M.L. is the recipient of a FPI fellowship (BES-2005-10404) from MICINN.

Pancreatic and duodenal homeobox 1 (Pdx1) is a transcription factor that regulates the embryonic development of the pancreas and the differentiation toward ß cells. Previously, we have shown that exposure of mouse embryonic stem cells (mESCs) to high concentrations of diethylenetriamine nitric oxide adduct (DETA-NO) triggers differentiation events and promotes the expression of Pdx1. Here we report evidence that Pdx1 expression is associated with release of polycomb repressive complex 2 (PRC2) and P300 from its promoter region. These events are accompanied by epigenetic changes in bivalent markers of histones trimethylated histone H3 lysine 27 (H3K27me3) and H3K4me3, site-specific changes in DNA methylation, and no change in H3 acetylation. On the basis of these findings, we developed a protocol to differentiate mESCs toward insulin-producing cells consisting of sequential exposure to DETA-NO, valproic acid, and P300 inhibitor (C646) to enhance Pdx1 expression and a final maturation step of culture in suspension to form cell aggregates. This small molecule-based protocol succeeds in obtaining cells that express pancreatic ß-cell markers such as PDX1, INS1, GCK, and GLUT2 and respond in vitro to high glucose and KCl, Centro Anadaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Postprint