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Selenocysteine, the 21st amino acid specified by the genetic code, is a rare selenium-containing residue found in the catalytic site of selenoprotein oxidoreductases. Selenocysteine is analogous to the common cysteine amino acid, but its selenium atom offers physical-chemical properties not provided by the corresponding sulfur atom in cysteine. Catalytic sites with selenocysteine in selenoproteins of vertebrates are under strong purifying selection, but one enzyme, glutathione peroxidase 6 (GPX6), independently exchanged selenocysteine for cysteine <100 million years ago in several mammalian lineages. We reconstructed and assayed these ancient enzymes before and after selenocysteine was lost and up to today and found them to have lost their classic ability to reduce hydroperoxides using glutathione. This loss of function, however, was accompanied by additional amino acid changes in the catalytic domain, with protein sites concertedly changing under positive selection across distant lineages abandoning selenocysteine in glutathione peroxidase 6. This demonstrates a narrow evolutionary range in maintaining fitness when sulfur in cysteine impairs the catalytic activity of this protein, with pleiotropy and epistasis likely driving the observed convergent evolution. We propose that the mutations shared across distinct lineages may trigger enzymatic properties beyond those in classic glutathione peroxidases, rather than simply recovering catalytic rate. These findings are an unusual example of adaptive convergence across mammalian selenoproteins, with the evolutionary signatures possibly representing the evolution of novel oxidoreductase functions.
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Selenio , Selenocisteína , Animales , Selenocisteína/genética , Selenocisteína/química , Selenocisteína/metabolismo , Cisteína/genética , Cisteína/metabolismo , Selenio/metabolismo , Selenoproteínas/genética , Selenoproteínas/química , Selenoproteínas/metabolismo , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Aminoácidos , Glutatión , Azufre , Mamíferos/genética , Mamíferos/metabolismoRESUMEN
T cell epitopes presented on the surface of mammalian cells are subjected to a complex network of antigen processing and presentation. Among them, C-terminal antigen processing constitutes one of the main bottlenecks for the generation of epitopes, as it defines the C-terminal end of the final epitope and delimits the peptidome that will be presented downstream. Previously (Amengual-Rigo and Guallar, Sci Rep 111(11):1-8, 2021), we demonstrated that NetCleave stands out as one of the best algorithms for the prediction of C-terminal processing, which in its turn can be crucial to design peptide-based vaccination strategies. In this chapter, we provide a pipeline to exploit the full capabilities of NetCleave, an open-source and retrainable algorithm for predicting the C-terminal antigen processing for the MHC-I and MHC-II pathways.
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Presentación de Antígeno , Epítopos de Linfocito T , Animales , Algoritmos , Mamíferos/metabolismoRESUMEN
A peroxygenase-catalysed hydroxylation of organosilanes is reported. The recombinant peroxygenase from Agrocybe aegerita (AaeUPO) enabled efficient conversion of a broad range of silane starting materials in attractive productivities (up to 300â mM h-1 ), catalyst performance (up to 84â s-1 and more than 120 000 catalytic turnovers). Molecular modelling of the enzyme-substrate interaction puts a basis for the mechanistic understanding of AaeUPO selectivity.
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Cyclin-dependent kinases (CDKs), together with their cyclin partners, are the master cell cycle regulators. Remarkably, the cyclin family was extended to include atypical cyclins, characterized by distinctive structural features, but their partner CDKs remain elusive. Here, we conducted a yeast two-hybrid screen to identify new atypical cyclin-CDK complexes. We identified 10 new complexes, including a complex between CDK6 and cyclin I (CCNI), which was found to be active against retinoblastoma protein. CCNI upregulation increased the proliferation of breast cancer cells in vitro and in vivo, with a magnitude similar to that seen upon cyclin D upregulation, an effect that was abrogated by CDK6 silencing or palbociclib treatment. In line with these findings, CCNI downregulation led to a decrease in cell number and a reduction in the percentage of cells reaching S phase. Finally, CCNI upregulation correlated with the high expression of E2F target genes in large panels of cancer cell lines and tissue samples from breast cancer patients. In conclusion, we unveil CCNI as a new player in the pathways that activate CDK6, enriching the wiring of cell cycle control.
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Neoplasias de la Mama , Ciclina I , Humanos , Femenino , Ciclina I/genética , Ciclinas/genética , Ciclinas/metabolismo , Proliferación Celular/genética , Neoplasias de la Mama/genética , Expresión Génica , Proteínas de Ciclo Celular/genética , Ciclo Celular , Quinasa 6 Dependiente de la Ciclina/genéticaRESUMEN
Domain swapping is a mechanism of protein oligomerization by which two or more subunits exchange structural elements to generate an intertwined complex. Numerous studies support a diversity of swapping mechanisms in which structural elements can be exchanged at different stages of the folding pathway of a subunit. Here, we used single-molecule optical tweezers technique to analyze the swapping mechanism of the forkhead DNA-binding domain of human transcription factor FoxP1. FoxP1 populates folded monomers in equilibrium with a swapped dimer. We generated a fusion protein linking two FoxP1 domains in tandem to obtain repetitive mechanical folding and unfolding trajectories. Thus, by stretching the same molecule several times, we detected either the independent folding of each domain or the elusive swapping step between domains. We found that a swapped dimer can be formed directly from fully or mostly folded monomer. In this situation, the interaction between the monomers in route to the domain-swapped dimer is the rate-limiting step. This approach is a useful strategy to test the different proposed domain swapping mechanisms for proteins with relevant physiological functions.
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Pinzas Ópticas , Pliegue de Proteína , Factores de Transcripción Forkhead/metabolismo , Humanos , Sustancias Macromoleculares , Dominios Proteicos , Proteínas , Proteínas Represoras/metabolismoRESUMEN
IκBs exert principal functions as cytoplasmic inhibitors of NF-kB transcription factors. Additional roles for IκB homologues have been described, including chromatin association and transcriptional regulation. Phosphorylated and SUMOylated IκBα (pS-IκBα) binds to histones H2A and H4 in the stem cell and progenitor cell compartment of skin and intestine, but the mechanisms controlling its recruitment to chromatin are largely unknown. Here, we show that serine 32-36 phosphorylation of IκBα favors its binding to nucleosomes and demonstrate that p-IκBα association with H4 depends on the acetylation of specific H4 lysine residues. The N-terminal tail of H4 is removed during intestinal cell differentiation by proteolytic cleavage by trypsin or chymotrypsin at residues 17-19, which reduces p-IκBα binding. Inhibition of trypsin and chymotrypsin activity in HT29 cells increases p-IκBα chromatin binding but, paradoxically, impaired goblet cell differentiation, comparable to IκBα deletion. Taken together, our results indicate that dynamic binding of IκBα to chromatin is a requirement for intestinal cell differentiation and provide a molecular basis for the understanding of the restricted nuclear distribution of p-IκBα in specific stem cell compartments.
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Cromatina , Histonas , Acetilación , Cromatina/genética , Histonas/metabolismo , Humanos , Inhibidor NF-kappaB alfa/genética , Nucleosomas/genéticaRESUMEN
Molecular dynamics (MD) simulations have become a standard tool to correlate the structure and function of biomolecules and significant advances have been made in the study of proteins and their complexes. A major drawback of conventional MD simulations is the difficulty and cost of obtaining converged results, especially when exploring potential energy surfaces containing considerable energy barriers. This limits the wide use of MD calculations to determine the thermodynamic properties of biomolecular processes. Indeed, this is true when considering the conformational entropy of such processes, which is ultimately critical in assessing the simulations' convergence. Alternatively, a wide range of structure-based models (SBMs) has been used in the literature to unravel the basic mechanisms of biomolecular dynamics. These models introduce simplifications that focus on the relevant aspects of the physical process under study. Because of this, SBMs incorporate the need to modify the force field definition and parameters to target specific biophysical simulations. Here we introduce SBMOpenMM, a Python library to build force fields for SBMs, that uses the OpenMM framework to create and run SBM simulations. The code is flexible and user-friendly and profits from the high customizability and performance provided by the OpenMM platform.
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Simulación de Dinámica Molecular , Proteínas , Conformación Molecular , TermodinámicaRESUMEN
The original version of this article contained an error in the spelling of the author Christian A.M. Wilson, which was incorrectly given as Christian M.A. Wilson. This has now been corrected in both the PDF and HTML versions of the article.
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Knots are natural topologies of chains. Yet, little is known about spontaneous knot formation in a polypeptide chain-an event that can potentially impair its folding-and about the effect of a knot on the stability and folding kinetics of a protein. Here we used optical tweezers to show that the free energy cost to form a trefoil knot in the denatured state of a polypeptide chain of 120 residues is 5.8 ± 1 kcal mol-1. Monte Carlo dynamics of random chains predict this value, indicating that the free energy cost of knot formation is of entropic origin. This cost is predicted to remain above 3 kcal mol-1 for denatured proteins as large as 900 residues. Therefore, we conclude that naturally knotted proteins cannot attain their knot randomly in the unfolded state but must pay the cost of knotting through contacts along their folding landscape.
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Modelos Moleculares , Pliegue de Proteína , Termodinámica , Proteínas Virales/química , Bacteriófagos/metabolismo , Método de Montecarlo , Pinzas Ópticas , Conformación Proteica , Desnaturalización Proteica , Proteínas Virales/genéticaRESUMEN
The HIV-1 gp41 Membrane Proximal External Region (MPER) is recognized by broadly neutralizing antibodies and represents a promising vaccine target. However, MPER immunogenicity and antibody activity are influenced by membrane lipids. To evaluate lipid modulation of MPER immunogenicity, we generated a 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC)-based proteoliposome collection containing combinations of phosphatidylserine (PS), GM3 ganglioside, cholesterol (CHOL), sphingomyelin (SM) and the TLR4 agonist monophosphoryl lipid A (MPLA). A recombinant gp41-derived miniprotein (gp41-MinTT) exposing the MPER and a tetanus toxoid (TT) peptide that favors MHC-II presentation, was successfully incorporated into lipid mixtures (>85%). Immunization of mice with soluble gp41-MinTT exclusively induced responses against the TT peptide, while POPC proteoliposomes generated potent anti-gp41 IgG responses using lower protein doses. The combined addition of PS and GM3 or CHOL/SM to POPC liposomes greatly increased gp41 immunogenicity, which was further enhanced by the addition of MPLA. Responses generated by all proteoliposomes targeted the N-terminal moiety of MPER overlapping the 2F5 neutralizing epitope. Our data show that lipids impact both, the epitope targeted and the magnitude of the response to membrane-dependent antigens, helping to improve MPER-based lipid carriers. Moreover, the identification of immunodominant epitopes allows for the redesign of immunogens targeting MPER neutralizing determinants.