Artículos (Bioquímica Vegetal y Biología Molecular)

URI permanente para esta colecciónhttps://hdl.handle.net/11441/10824

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Mostrando 1 - 20 de 516

Why is the chloroplast redox regulatory machinery so complex?
(Cell Press, 2026-09-03) Pérez Ruiz, Juan Manuel; Cejudo Fernández, Francisco Javier; Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia, Innovación y Universidades (MICIU). España; Agencia Estatal de Investigación. España
Dithiol-disulfide exchange of cysteine residues has deep effects on protein conformation, hence on enzyme activity, which is the basis of redox regulation. The redox state of cells, including cellular compartments, is dynamically adjusted by reducing, i.e. NAD(P)H, and oxidizing signals, such as reactive oxygen species (ROS), generated during metabolic activity. Among ROS, H2O2 triggers the oxidation of cysteine thiols and the formation of disulfide bridges, thus having a relevant contribution to redox regulation. Disulfide reduction is catalyzed by thioredoxins (Trxs), small polypeptides with a characteristic structure, the Trx fold, and an active site formed by the WCGPC motif, or variants of this motif. In heterotrophic organisms, Trxs are reduced by NADPH via an NADPH-dependent Trx reductase (NTR), defining a relatively simple two-component system formed by one or two NTRs and, at most, three Trxs. In contrast, photosynthetic organisms harbor complex redox regulatory machinery. Redox regulation is remarkably complex in plant chloroplasts, organelles equipped with more than 20 Trxs, which rely on two sources of reducing power: photosynthetically reduced ferredoxin (FdxRED), via a Fdx-dependent Trx reductase (FTR); and NADPH, the electron donor to NTRC, an enzyme containing NTR and Trx domains, exclusively found in oxygenic photosynthetic organisms. Thus, the long-standing question is why chloroplasts require such complex redox regulatory machinery. Here, we propose a model of chloroplast redox regulation based on the interplay between reducing and oxidizing branches. Moreover, we propose that the complex chloroplast redox regulatory machinery allows the continuous fine-tuning of photosynthetic performance in response to the unpredictable changes of light intensity that plants face in their natural environments.
FGFR1 drives metabolic adaptation associated with temozolomide resistance in glioblastoma
(Elsevier, 2026-08-01) Zarzuela, Laura; López-Cepero, Ignacio G.; Rattigan, Kevin M.; Reina Bando, Ana; Martín-Montalvo, Jorge; Sierra-Párraga, Jesús M.; Sánchez Escabias, Elena; Morillo Huesca, Macarena; Murdoch, Piedad del Socorro; Durán, Raúl V.; Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia, Innovación y Universidades (MICIU). España; Agencia Estatal de Investigación. España; Instituto de Salud Carlos III
Therapy resistance is a major limitation in therapeutic efficacy for glioblastoma (GBM) patients, positioning GBM among the deadliest tumor types. In this work, we have dissected resistance mechanisms in GBM, which resulted in the identification of FGFR1 pathway as a major controller of the signaling and metabolic rewiring associated to temozolomide (TMZ) resistance. Hence, in FGFR1-positive, p53 WT GBM cells, FGFR1 controls a p53-mediated cell cycle arrest to allow DNA repair in response to TMZ. FGFR1 also regulates a complete metabolic rewiring promoting lipid catabolism and preventing lipid peroxidation. Indeed, FGFR1 inhibition completely abolishes this signaling and metabolic reprograming, restoring sensitivity to TMZ. Our results also indicated a correlation of FGFR1 with poor prognosis in GBM patients, and validated the dual treatment with TMZ and FGFR1 inhibitors as an efficient strategy to induce tumor cell death in FGFR1-positive, p53 WT pre-clinical animal GBM models. These data position FGFR1 as a promising candidate for future clinical evaluation to limit therapy resistance to TMZ in FGFR1-positive GBM patients.
Antisense RNA controls the degradation of phycobilisomes in heterocyst-forming cyanobacteria by a transcriptional interference mechanism
(Oxford University Press, 2026-06-22) Álvarez Escribano, Isidro; Brenes-Álvarez, Manuel; Georg, Jens; Vioque Peña, Agustín; Muro Pastor, Alicia M.; Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia e Innovación (MICIN). España; European Union (UE); Agencia Estatal de Investigación. España; Deutsche Forschungsgemeinschaft / German Research Foundation (DFG)
Transcriptional interference is rarely documented in bacteria. In cyanobacteria, phycobilisomes are the major light-harvesting antennae, and their degradation under non-optimal conditions follows a tightly regulated genetic program leading to the production of NblA, a widely conserved pro- teolytic adapter. NblA production is regulated at both the transcriptional and post-transcriptional levels. Here, we uncover an additional regulatory layer mediated by an antisense RNA conserved in heterocyst-forming cyanobacteria. In Nostoc sp. PCC 7120, transcription of the abundant an- tisense RNA ( as_nblA ) limits nblA mRNA accumulation. A strain unable to transcribe as_nblA produces an excess of NblA, ultimately so harmful that suppressor mutations of nblA expression accumulate rapidly, underscoring the essential role of as_nblA. Rifampicin time-series experi- ments and the inability of as_nblA to regulate nblA expression in trans support transcriptional interference as the primary regulatory mechanism of as_nblA . Mathematical modeling of nblA expression, supported by biological data, shows that as_nblA plays a pivotal role in preventing leaky nblA expression under non-inducing conditions. Our work highlights the importance of antisense RNA-mediated regulation, particularly transcriptional interference, in establishing thresholds that prevent spurious expression of genes encoding critical cellular functions.
Organelle-specific hydrogen sulfide metabolism governs redox homeostasis to regulate plant autophagy and cadmium stress resilience
(Elsevier, 2026-06-17) Carrillo, Reyes; Moreno, Inmaculada; Aroca Aguilar, Ángeles; Gotor, Cecilia; Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia, Innovación y Universidades (MICIU). España; European Union (UE); Junta de Andalucía
Hydrogen sulfide (H2S), traditionally regarded as a toxic threat to the environment and living organisms, is now considered an important gasotransmitter that helps regulate redox balance and autophagy in plants. However, how different sources of H2S in certain organelles help control stress responses remains unclear. In our study, how H2S is handled in various subcellular compartments of Arabidopsis thaliana was investigated in depth using null mutants defective in H2S production (des1, sir, cas-c1, str1 and str2) or consumption (oas-a1, oas-b and oas-c). Under normal physiological conditions, disruption of H2S homeostasis in any cellular compartment altered endogenous H2S levels and induced basal autophagy. These findings suggest that maintaining a specific subcellular H2S threshold is necessary to control ATG8-dependent autophagic flux. Under cadmium-induced stress, wild-type plants presented coordinated increases in cytosolic OAS-A1 levels, H2S accumulation, and protein persulfidation and decreases in sulfenylation and autophagy activation. This redox reprogramming establishes a protective redox control driven by H2S, where cadmium-induced H2O2 promotes cysteine sulfenylation followed by persulfidation, thus preventing irreversible overoxidation. Notably, this adaptive redox control was dysregulated in all the organelle-specific mutants, regardless of changes in H2S or H2O2 levels, demonstrating that the entire H2S network is required for redox protection. Cytosolic and mitochondrial mutants presented the greatest defects in Cd-induced autophagy, indicating that these compartments play a central role in stress-adaptive recycling.
Protein persulfidation: a ubiquitous modification regulating a broad spectrum of biological processes
(Oxford University Press, 2025-10-18) Romero, Luis C.; Carrillo, Reyes; Montesinos-Pereira, David; Luque Algaba, Carmen; Aroca Aguilar, Ángeles; Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia e Innovación (MICIN). España; European Union (UE); Junta de Andalucía
Hydrogen sulfide signaling occurs mainly through protein persulfidation, an important post-translational modification and a highly dynamic process in plants. Beyond enzyme activity, persulfidation affects protein localization, structure, and interactions. This review provides an overview of the mechanisms governing this modification and the pivotal role of the thioredoxin system mediating depersulfidation, which is essential for maintaining redox homeostasis. Proteomic studies in Arabidopsis thaliana and crop species reveal that protein persulfidation is a widespread posttranslational modification in plants, with over 11 700 polypeptides identified as susceptible. Functional analysis shows a high proportion of these proteins are involved in primary metabolic pathways and secondary metabolism. Gene Ontology analysis highlights additional processes regulated by persulfidation, underscoring its broad regulatory role in plant biology. These processes are mainly related to proteolysis, defense response, embryo development, protein transport, and response to cold. In addition, other processes regulated by hydrogen sulfide, which have been widely studied, also stand out, such as response to abscisic acid, response to oxidative stress, and response to water deprivation. A comprehensive description of the persulfidated proteins implicated in the processes highlighted by the Gene Ontology analysis is provided. This integrated role of H2S with other molecules offers a panoramic overview of its importance in plant biology which also helps to raise new questions and working directions for the future research.
Hands-on protocol for preparing water-soluble fractions from agri-food samples for NMR-based metabolomics analysis
(Wiley, 2026-04-01) Fernández Veloso, Andrea; Hiniesta Valero, Jaime; Guerra Castellano, Alejandra; Rosa Acosta, Miguel Ángel de la; Díaz Moreno, Irene; Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia, Innovación y Universidades (MICIU). España; Agencia Estatal de Investigación. España
The aim of this study was to address the lack of protocols for nuclear magnetic resonance (NMR)-based metabolomics in the agri-food sector by providing a reproducible workflow for the preparation and analysis of water-soluble metabolite fractions. These fractions, rich in primary metabolites such as sugars, amino acids, and organic acids, are key to assessing agri-food products’ composition, quality, as well as to monitor their manufacturing and development processes. The protocol differentiates solid and liquid matrices, optimizing extraction procedures accordingly. Representative agri-food products—strawberry leaves (solid) and wine (liquid)— were analyzed to demonstrate the method’s versatility and applicability. Key steps include tailored sample preparation, optimization of NMR acquisition, and spectral quality control, ensuring high data quality and reproducibility. The proposed workflow enhances reproducibility across agri-food metabolomics studies and facilitates integration into broader food quality, traceability, and safety frameworks.
Disruption of Asparagine Synthetase Is Associated to Increased Biomass in Lotus japonicus
(Wiley, 2026-03-24) Rosa Téllez, Sara Virginia; García Calderón, Margarita; Medeiros, David Barbosa; Márquez Cabeza, Antonio José; Fernie, Alisdair R.; Betti, Marco; Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia, Innovación y Universidades (MICIU). España
Asparagine (Asn) constitutes the major form of nitrogen translocated within Lotus japonicus plants. In this work we use knock-out (KO) LORE1 mutants-deficient in the asparagine synthetase gene (LjASN1), which is the most highly expressed ASN gene in plants grown under non-symbiotic (NS) conditions, but much less expressed under symbiotic (S) conditions. The analysis of two different Ljasn1 homozygous mutant lines grown under NS or S conditions indicated that a much higher biomass was produced in Ljasn1 mutants grown under NS conditions compared to the WT (wild-type), whereas little difference with the WT was observed in mutant plants under S conditions. Metabolomic analysis revealed that Ljasn1 mutant plants are quite distinct to WT plants when grown under NS conditions, but not under S conditions. Asn levels were considerably reduced in Ljasn1 mutant plants compared to the WT when plants were grown under NS but not under S conditions. A general decrease in amino acids and an increase in carbon compounds, such as sugars and oxo-acids, was detected in NS roots and shoots, respectively, which may explain the growth phenotypes observed. RNAseq analysis showed changes related to oxidative metabolism under NS conditions, and C/N metabolism under S conditions. The data indicate that the LjASN1 deficiency produces important changes in the C/N balance and metabolite allocation of L. japonicus plants resulting in higher biomass content and lower Asn levels, two interesting traits for biotechnological crops engineering.
Flavonoid and isoflavonoid biosynthetic gene expression is affected by MYB36 transcription factor deficiency in Lotus japonicus
(Elsevier, 2026-04-29) Monje Rueda, María Dolores; Ove-Balang, Peter Pal; Morales-Escribano, Sonia; Márquez Cabeza, Antonio José; García Calderón, Margarita; Betti, Marco; Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia, Innovación y Universidades (MICIU). España; Ministry of education, science, research and sport. Slovak Republic
MYB36 was recently identified as a transcription factor (TF) that may be involved in isoflavonoid metabolism and stress responses in the model legume Lotus japonicus. To further explore its role, we performed a comparative transcriptomic and (iso)flavonoid profiling analysis of wild-type (WT) plants and two independents homozygous Ljmyb36 mutant lines under two abiotic stress conditions: UV-B irradiation and salinity. The results obtained showed a markedly distinct and almost reciprocal global transcriptomic response of WT plants under both types of stress conditions. Under UV-B irradiation, genes related to isoflavonoid biosynthesis were induced, accompanied by increased isoflavonoid accumulation. In contrast, under salt stress, most of the genes modulated in the mutants corresponded to genes specifically involved in isoflavonoid, but not flavonol biosynthesis, highlighting candidate genes potentially regulated by MYB36. Metabolite profiling also indicated opposite changes in flavonoid and isoflavonoid metabolite in response to salt and UV-B treatments, reflecting the reciprocal patterns of gene expression found in salt and UV-B treatment conditions. These results suggest a role, for the first time, of MYB36 as a specific transcription factor in the regulation of (iso)flavonoid biosynthesis. In addition, the results obtained support the role of MYB36 as a crucial TF in the response to salt stress in legume plants.
Phenolic acid-induced phase separation and translation inhibition mediate plant interspecific competition
(Nature Portfolio, 2023-08-23) Xie, Zhouli; Zhao, Shuai; Li, Ying; Deng, Yuhua; Shi, Yabo; Chen, Xiaoyuan; Li, Yue; Li, Haiwei; Gutiérrez Beltrán, Emilio; Wang, Wei; Bioquímica Vegetal y Biología Molecular; Natural Science Foundation of China; Beijing Nova Program of Science and Technology; European Commission (EC); Natural Science Foundation of Fujian Province
Phenolic acids (PAs) secreted by donor plants suppress the growth of their susceptible plant neighbours. However, how structurally diverse ensembles of PAs are perceived by plants to mediate interspecific competition remains a mystery. Here we show that a plant stress granule (SG) marker, RNA-BINDING PROTEIN 47B (RBP47B), is a sensor of PAs in Arabidopsis. PAs, including salicylic acid, 4-hydroxybenzoic acid, protocatechuic acid and so on, directly bind RBP47B, promote its phase separation and trigger SG formation accompanied by global translation inhibition. Salicylic acid-induced global translation inhibition depends on RBP47 family members. RBP47s regulate the proteome rather than the absolute quantity of SG. The rbp47 quadruple mutant shows a reduced sensitivity to the inhibitory effect of the PA mixture as well as to that of PA-rich rice when tested in a co-culturing ecosystem. In this Article, we identified the long sought-after PA sensor as RBP47B and illustrated that PA-induced SG-mediated translational inhibition was one of the PA perception mechanisms.
Genome sequence of Stenotrophomonas indicatrix CPHE1, a powerful phenanthrene-degrading bacterium
(Springer Nature, 2023-01-18) Lara Moreno, Alba; Morillo, Esmeralda; Merchán Ignacio, Francisco; González Pimentel, José Luis; Villaverde, Jaime; Microbiología y Parasitología; Bioquímica Vegetal y Biología Molecular; Ministerio de Economía y Competitividad (MINECO). España
Environmental pollution caused by polycyclic aromatic hydrocarbons (PAHs) involves a high-risk and have received considerable attention due to their carcinogenic, teratogenic, and mutagenic properties. Phenanthrene (PHE) is a low molecular weight PAH, which has three benzene rings. It is one of the most common PAH found in contaminated environments mainly due to its low volatilization ability and hydrophobic character. A PHE degrading bacterium was isolated from an industrial contaminated soil using enrichment culture techniques. Based on macroscopic, microscopic examination and phylogenetic analysis, this bacterium was classified as Stenotrophomonas indicatrix and named strain CPHE1. Several authors have reported about bacteria stains, which can degrade PHE, but this is the first time where the ability of S. indicatrix to biodegrade and mineralize PHE has been demonstrated.
Acute Combination of Nitrogen Deprivation and High Irradiance Induces the Simultaneous Accumulation of Astaxanthin and Lutein in Continuous Cultures of the Microalga Chromochloris zofingiensis
(MDPI, 2026) Morales-Pineda, María; García González, Mercedes; García-Gómez, María Elena; Romero Campero, Francisco José; Ramos González, Marcos; Bioquímica Vegetal y Biología Molecular; Ciencias de la Computación e Inteligencia Artificial; Ministerio de Ciencia, Innovación y Universidades (MICIU). España; Agencia Estatal de Investigación. España; European Union (UE)
Carotenoids play a central role in photosynthesis and cellular protection, and microalgae represent a sustainable platform for their commercial production. Here, we optimized the accumulation of the high-value carotenoids astaxanthin and lutein in continuous photoautotrophic cultures of Chromochloris zofingiensis by modulating nitrogen supply and light intensity. Reducing nitrate availability strongly promoted astaxanthin accumulation, whereas lutein levels remained largely unaffected. For 4% N in the dry biomass, accumulation of astaxanthin was highest and that of lutein lowest, while the opposite was recorded for 9% N. Average irradiance positively affected lutein accumulation independently of nitrate, whereas that of astaxanthin only increased under nitrogen-limiting conditions. Integrated transcriptomics and carotenoid profiling analysis revealed nitrogen availability as the dominant regulatory factor, with a synergistic interaction with light that enhances their individual effects. Nitrate limitation redirected metabolic flux from lycopene toward β-carotene and its subsequent conversion to astaxanthin via BKT1 overexpression, while high irradiance induced CYP97A1 and CYP97C expression, favoring lutein biosynthesis. Together, these findings demonstrate that targeted control of nitrogen and light enables the continuous and programmable production of C. zofingiensis biomass with a specific astaxanthin-to-lutein ratio, highlighting its potential for industrial carotenoid bioprocesses.
Too dim, too bright, and just right: Systems analysis of the Chlamydomonas diurnal program under limiting and excess light
(Oxford University Press (OUP), 2025) Dupuis, Sunnyjoy; Ojeda Servián, Valle; Gallaher, Sean D.; Purvine, Samuel O.; Glaesener, Anne G.; Ponce, Raquel; Nicora, Carrie D.; Bloodsworth, Kent; Lipton, Marie S.; Merchant, Sabeeha S.; Bioquímica Vegetal y Biología Molecular; Department of Energy. United States
Photosynthetic organisms coordinate their metabolism and growth with diurnal light, which can range in intensity from limiting to excessive. Little is known about how light intensity impacts the diurnal program in Chlamydomonas reinhardtii, or how diurnal rhythms in gene expression and metabolism shape photoprotective responses at different times of day. To address these questions, we performed a systems analysis of synchronized Chlamydomonas populations acclimated to low, moderate, and high diurnal light. Transcriptomic and proteomic data revealed that the Chlamydomonas rhythmic gene expression program is resilient to limiting and excess light: genome-wide, waves of transcripts, and proteins peak at the same times in populations acclimated to stressful light intensities as in populations acclimated to moderate light. Yet, diurnal photoacclimation gives rise to hundreds of gene expression changes, even at night. Time course measurements of photosynthetic efficiency and pigments responsive to excess light showed that high light-acclimated cells partially overcome photodamage in the latter half of the day prior to cell division. Although gene expression and photodamage are dynamic over the diurnal cycle, Chlamydomonas populations acclimated to low and high diurnal light maintain altered photosystem abundance, thylakoid architecture, and non-photochemical quenching capacity through the night phase. This suggests that cells remember or anticipate the light intensities that they have typically encountered during the day. The integrated data constitute an excellent resource for understanding photoacclimation in eukaryotes under environmentally relevant conditions.
Classification and Nomenclature of Metacaspases and Paracaspases: No More Confusion with Caspases
(Elsevier, 2020-03-05) Minina, Elena A.; Staal, Jens; Alvarez, Vanina E.; Berges, John A.; Berman-Frank, Ilana; Beyaert, Rudi; Bidle, Kay D.; Bornancin, Frédéric; Gutiérrez Beltrán, Emilio; Bozhkov, Peter V.; Bioquímica Vegetal y Biología Molecular
2-Cys peroxiredoxins and the chaperone cpHSP70 act in concert in chloroplast biogenesis in Arabidopsis seedlings
(American Society of Plant Biologists, 2026-03-27) Rodríguez Marín, Fernando; Gallardo Martínez, Antonia María; Hernández Jiménez, María Luisa; González García, María de la Cruz; Cejudo Fernández, Francisco Javier; Pérez Ruiz, Juan Manuel; Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia e Innovación (MICIN). España; Agencia Estatal de Investigación. España; European Union (UE)
The redox balance between NADPH-dependent thioredoxin reductase C (NTRC) and 2-Cys peroxiredoxins (PRXs) help chloroplast photosynthetic performance acclimate rapidly to environmental cues. The Arabidopsis (Arabidopsis thaliana) 2cpab mutant, which lacks chloroplast 2-Cys PRXs A and B, shows impaired embryogenesis and cotyledon development. This phenotype indicates that these enzymes have a relevant function at early stages of plant development, although this function is poorly understood. Here, we show that the albino cotyledons of the 2cpab mutant have unstructured chloroplasts and decreased chloroplast lipid contents, revealing a key function of 2-Cys PRXs in cotyledon chloroplast differentiation. These phenotypes are mimicked by NTRC overexpression, whereas loss of NTRC function has no effect. RNA-Seq analyses showed transcriptomic changes resembling the response to proteotoxic stress in 2cpab seedlings, which was confirmed by the high sensitivity of 2cpab seedlings to heat stress. In de-etiolating seedlings 2-Cys PRXs interact with chloroplast-localized chaperone heat shock protein 70 (cpHSP70); moreover, Arabidopsis seedlings simultaneously lacking 2-Cys PRXs and cpHSP70-1 exhibit a dramatic alteration of seedling development. Based on these results, we propose that 2-Cys PRXs contribute to cotyledon chloroplast differentiation by affecting organellar proteostasis. This function is exerted in concert with chaperone cpHSP70 and is essential for seedling establishment.
Broadening the Workflow for Synchrotron-Based X-Ray Fluorescence and X-Ray Absorption Spectroscopy Imaging of Low-Abundance Cellular Metals
(Wiley, 2026-03-06) Tamargo Azpilicueta, Joaquín; Giner Arroyo, Rafael Luis; Rivero-García, Pablo; Díaz-Moreno, Sofía; Gómez González, Miguel A; Telfer, Abbey; Campanella, Michelangelo; Rosa Acosta, Miguel Ángel de la; Díaz Moreno, Irene; Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia, Innovación y Universidades (MICIU). España; Agencia Estatal de Investigación. España; Junta de Andalucía
Metals play an essential role in cellular homeostasis and are key components of several formulations currently used in the clinic. Synchrotron-based X-ray microscopy at submicron resolution is a powerful approach to map intracellular elemental distributions and to monitor how these patterns change upon genetic or pharmacological perturbations. However, existing sample-preparation protocols often rely on costly and highly specialized equipment for vitrification and dehydration, limiting their widespread adoption. Here, we present an adapted plunge-freezing and freeze-drying workflow that enables the preparation of mammalian cell samples for X-ray fluorescence (XRF) and X-ray absorption spectroscopy (XAS) studies with submicron resolution in a cost-effective and versatile manner. Furthermore, we define acquisition parameters optimized for the reliable detection of low-abundance metals, such as endogenous iron. We anticipate that this accessible protocol will facilitate the broader implementation of synchrotron-based inner-shell spectromicroscopy in cell biology.
NTRC's novel role: Bridging chloroplast redox oscillations and nuclear circadian clock
(Cell Press, 2025) Cejudo Fernández, Francisco Javier; Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia e Innovación (MICIN). España; Agencia Estatal de Investigación. España
Deciphering the role of Zn2+-binding histidines from TIA-1 on the assembly and dynamics of stress granules
(Wiley, 2024) Corrales Guerrero, Laura; Díaz Moreno, Irene; Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia e Innovación (MICIN). España; Agencia Estatal de Investigación. España
T-cell intracellular antigen-1 (TIA-1) is a key RNA-binding protein that participates in translation regulation and RNA splicing. TIA-1 undergoes Liquid-Liquid Phase Separation (LLPS) as a fundamental mechanism that enables the condensation of RNA and proteins into membraneless organelles called stress granules (SGs). However, this dynamic behavior can lead to aberrant fibril formation, implicated in neurodegenerative disorders, and must be tightly regulated. In this study, we investigated the role in the cell of histidine residues His94 and His96, responsible for Zn2+ binding. Using fluorescence microscopy, we found that the specific binding site formed by these residues is critical for SG assembly. Furthermore, it also plays a role maintaining the dynamic behavior of SG-assembled TIA-1. Collectively, our findings confirm the physiological relevance of TIA-1 His94 and His96 in the Zn2+-mediated regulatory mechanism for protection against fibril formation in SGs13 .
PharaohFUN: phylogenomic analysis for plant protein history and function elucidation
(Oxford University Press, 2026-01-22) Ramos González, Marcos; Ramos González, Víctor; Serrano Pérez, Emma; Arvanitidou, Christina; Hernández García, Jorge; García González, Mercedes; Romero Campero, Francisco José; Ciencias de la Computación e Inteligencia Artificial; Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia, Innovación y Universidades (MICIU). España; Agencia Estatal de Investigación. España
Since DNA sequencing has become commonplace, the development of efficient methods and tools to explore gene sequences has become indispensable. In particular, despite photosynthetic eukaryotes constituting the largest percentage of terrestrial biomass, computational functional characterization of gene sequences in these organisms still predominantly relies on comparisons with Arabidopsis thaliana and other angiosperms. This paper introduces PharaohFUN, a web application designed for the evolutionary and functional analysis of protein sequences in photosynthetic eukaryotes, leveraging orthology relationships between them. PharaohFUN incorporates a homogeneous representative sampling of key species in this group, bridging clades that have traditionally been studied separately, thus establishing a comprehensive evolutionary framework to draw conclusions about sequence evolution and function. For this purpose, it incorporates modules for exploring gene tree evolutionary history, expansion and contraction events, ancestral states, domain identification, multiple sequence alignments, and diverse functional annotation. It also incorporates different search modes to facilitate its use and increase its reach within the community. Tests were performed on the whole transcription factor toolbox of A. thaliana and on CCA1 protein to assess its utility for both large-scale and fine-grained phylogenetic studies. These exemplify how PharaohFUN accurately traces the corresponding evolutionary histories of these proteins by unifying results for land plants, streptophyte and chlorophyte microalgae. Thus, PharaohFUN democratices access to these kinds of analyses in photosynthetic organisms for every user, independently of their prior training in bioinformatics.
Isoform-specific regulation of PKM by acetylation
(National Academy of Sciences, 2025-11-25) Pavlenko, Dariia; Tamargo Azpilicueta, Joaquín; Nudelman, Hila; Ankri, Yuval; Shahar, Anat; Díaz Moreno, Irene; Arbely, Eyal; Bioquímica Vegetal y Biología Molecular; European Union (UE); Israel Science Foundation; Ministerio de Ciencia, Innovación y Universidades (MICIU). España
Pyruvate kinase (PK) is a crucial glycolytic protein involved in vital cellular processes ranging from cell proliferation to immune responses. The activity and functions of PK are tightly regulated by diverse mechanisms, including posttranslational N -lysine acetylation. Although previous studies have explored the impact of acetylation on selected lysine residues within the M2 isoform of PK (PKM2), a more comprehensive selection of acetylation sites and their respective effects on both PKM2 and the highly homologous PKM1 isoform is lacking. Here, we describe the structural, functional, and regulatory effects of site-specific acetylation on an expanded set of conserved lysines in PKM2 and selected lysines in PKM1. To study homogeneously acetylated proteins, we genetically encoded the incorporation of acetylated lysine into PKM variants expressed in bacteria and cultured mammalian cells. Our integrated biochemical, structural, and computational approach revealed K115 acetylation as an inhibitory modification in both PKM1 and PKM2 that stabilizes a closed active site conformation of the proteins. We also show that, in contrast to K115 acetylation, previously reported acetylation of K305 inhibits PKM2 but has no effect on the activity and oligomerization of PKM1. These findings propose the existence of both uniform and isoform-specific regulatory mechanisms of PKM, mediated by acetylation.
Multisite phosphorylation of the AML-linked C-terminal of nucleophosmin (NPM1) orchestrates protein stability, DNA binding and charge block-driven phase separation
(Oxford University Press, 2026-02-27) Rivero-García, Pablo; Giner Arroyo, Rafael Luis; Tamargo Azpilicueta, Joaquín; Telfer, Abbey; Frezza, Elisa; Velázquez-Campoy, Adrián; Díaz-Moreno, Sofía; Rosa Acosta, Miguel Ángel de la; Díaz Moreno, Irene; Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia, Innovación y Universidades (MICIU). España; Fundación Ramón Areces; Ministerio de Educación. España; Consejo Superior de Investigaciones Científicas (CSIC)
Nucleophosmin (NPM1) is a nucleolar protein commonly mutated in ~30% of newly diagnosed acute myeloid leukemia (AML) cases. These mutations occur in the terminal exon of the NPM1 gene, affecting the C-terminal DNA-binding domain of the protein and causing its delocalization to the cytoplasm—a hallmark of NPM1-mutated AML. NPM1 shuttling to the nucleoplasm is tightly regulated by posttranslational modifications, such as phosphorylation of Ser254, Ser260, and Tyr271 of the DNA-binding domain. However, the structural mechanisms underlying this process remain unclear. In this work, we show that Ser-to-Asp (S254D–S260D) and Tyr-to-pCMF (para-carboxymethyl phenylalanine) (Y271pCMF) phosphomimetic mutations induce significant structural and dynamical rearrangements, as well as drastic modifications in electrostatic surface potential. These changes compromise recognition of a G-quadruplex sequence from the c-MYC promoter by reducing DNA-binding affinity, reshape histone capturing dynamics, and fade charge segregation in the histone-binding domain. Combination of such substitutions in a triple phosphomimetic variant (S254D–S260D–Y271pCMF) further destabilizes the domain’s structure and triggers protein aggregation. Altogether, these findings suggest that phosphorylation of Ser254, Ser260, and Tyr271 of the C-end DNA-binding domain weakens both DNA affinity and charge block-driven liquid–liquid phase separation, offering a molecular explanation for the delocalization of NPM1 outside of the nucleolus.

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