Artículos (Bioquímica Vegetal y Biología Molecular)

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

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Non-Canonical, Extralysosomal Activities of Lysosomal Peptidases in Physiological and Pathological Conditions: New Clinical Opportunities for Cancer Therapy
(Multidisciplinary Digital Publishing Institute (MDPI), 2025-01-07) Conesa Bakkali, Ryan; Morillo Huesca, Macarena; Martínez Fábregas, Jonathan; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular; Junta de Andalucía; European Union (UE); Ministerio de Ciencia e Innovación (MICIN). España; Universidad de Sevilla
Lysosomes are subcellular compartments characterised by an acidic pH, containing an ample variety of acid hydrolases involved in the recycling of biopolymers. Among these hydrolases, lysosomal proteases have merely been considered as end-destination proteases responsible for the digestion of waste proteins, trafficked to the lysosomal compartment through autophagy and endocytosis. However, recent reports have started to unravel specific roles for these proteases in the regulation of initially unexpected biological processes, both under physiological and pathological conditions. Furthermore, some lysosomal proteases are no longer restricted to the lysosomal compartment, as more novel non-canonical, extralysosomal targets are being identified. Currently, lysosomal proteases are accepted to play key functions in the extracellular milieu, attached to the plasma membrane and even in the cytosolic and nuclear compartments of the cell. Under physiological conditions, lysosomal proteases, through non-canonical, extralysosomal activities, have been linked to cell differentiation, regulation of gene expression, and cell division. Under pathological conditions, these proteases have been linked to cancer, mostly through their extralysosomal activities in the cytosol and nuclei of cells. In this review, we aim to provide a comprehensive summary of our current knowledge about the extralysosomal, non-canonical functions of lysosomal proteases, both under physiological and pathological conditions, with a particular interest in cancer, that could potentially offer new opportunities for clinical intervention.
R-DeeP/TripepSVM identifies the RNA-binding OB-fold-like protein PatR as regulator of heterocyst patterning
(Oxford University Press, 2025-02-10) Brenes-Álvarez, Manuel; Ropp, Halie R.; Papagiannidis, Dimitrios; Potel, Clement M.; Stein, Frank; Scholz, Ingeborg; Steglich, Claudia; Savitski, Mikhail M.; Vioque Peña, Agustín; Muro Pastor, Alicia María; Hess, Wolfgang R.; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular; Deutsche Forschungsgemeinschaft / German Research Foundation (DFG); Ministerio de Ciencia e Innovación (MICIN). España
RNA-binding proteins (RBPs) are central components of gene regulatory networks. The differentiation of heterocysts in filamentous cyanobacteria is an example of cell differentiation in prokaryotes. Although multiple non-coding transcripts are involved in this process, no RBPs have been implicated thus far. Here we used quantitative mass spectrometry to analyze the differential fractionation of RNA–protein complexes after RNase treatment in density gradients yielding 333 RNA-associated proteins, while a bioinformatic prediction yielded 311 RBP candidates in Nostoc sp. PCC 7120. We validated in vivo the RNA-binding capacity of six RBP candidates. Some participate in essential physiological aspects, such as photosynthesis (Alr2890), thylakoid biogenesis (Vipp1) or heterocyst differentiation (PrpA, PatU3), but their association with RNA was unknown. Validated RBPs Asl3888 and Alr1700 were not previously characterized. Alr1700 is an RBP with two oligonucleotide/oligosaccharide-binding (OB)-fold-like domains that is differentially expressed in heterocysts and interacts with non-coding regulatory RNAs. Deletion of alr1700 led to complete deregulation of the cell differentiation process, a striking increase in the number of heterocyst-like cells, and was ultimately lethal in the absence of combined nitrogen. These observations characterize this RBP as a master regulator of the heterocyst patterning and differentiation process, leading us to rename Alr1700 to PatR.
Thioredoxin A regulates protein synthesis to maintain carbon and nitrogen partitioning in cyanobacteria.
(American Society of Plant Biologists, 2024-02-22) Mallén Ponce, Manuel J.; Florencio Bellido, Francisco Javier; Huertas Romera, María José; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia, Innovación y Universidades (MICINN). España; Agencia Estatal de Investigación. España; European Commission (EC). Fondo Europeo de Desarrollo Regional (FEDER); Junta de Andalucía; Universidad de Sevilla; Universidad de Sevilla. BIO284: Expresión Génica y Regulación Metabólica en Cianobacterias.
Thioredoxins play an essential role in regulating enzyme activity in response to environmental changes, especially in photosynthetic organisms. They are crucial for metabolic regulation in cyanobacteria, but the key redox-regulated central processes remain to be determined. Physiological, metabolic, and transcriptomic characterization of a conditional mutant of the essential Synechocystis sp. PCC 6803 thioredoxin trxA gene (STXA2) revealed that decreased TrxA levels alter cell morphology and induce a dormant-like state. Furthermore, TrxA depletion in the STXA2 strain inhibited protein synthesis and led to changes in amino acid pools and nitrogen/carbon reserve polymers, accompanied by oxidation of the elongation factor-Tu. Transcriptomic analysis of TrxA depletion in STXA2 revealed a robust transcriptional response. Downregulated genes formed a large cluster directly related to photosynthesis, ATP synthesis, and CO2 fixation. In contrast, upregulated genes were grouped into different clusters related to respiratory electron transport, carotenoid biosynthesis, amino acid metabolism, and protein degradation, among others. These findings highlight the complex regulatory mechanisms that govern cyanobacterial metabolism, where TrxA acts as a critical regulator that orchestrates the transition from anabolic to maintenance metabolism and regulates carbon and nitrogen balance.
Mutational analysis of the inactivating factors, IF7 and IF17 from Synechocystis sp. PCC 6803: Critical role of arginine amino acid residues for glutamine synthetase inactivation
(John Wiley & Sons, 2011-11) Saelices, Lorena; Galmozzi, Carla V.; Florencio Bellido, Francisco Javier; Muro Pastor, María Isabel; Universidad de Sevilla. Departamento de Genética; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia e Innovación (MICIN). España; Junta de Andalucía
The Synechocystis sp. PCC 6803 glutamine synthetase type I (GS) activity is controlled by a process that involves protein-protein interaction with two inactivating factors (IF7 and IF17). IF7 is a natively unfolded, 65-residue-long protein, homologous to the carboxy-terminal region of IF17. Both proteins have abundance of positively charged amino acid residues and a high isoelectric point. In this study, we analyse the IF amino acid residues involved in GS inactivation by a mutational approach, both in vitro and in vivo. The results clearly indicate that the GS-IF complex formation must be determined mainly by electrostatic interactions. We have identified three conserved arginine residues of IF7 and IF17 that are essential for the interaction of these proteins with GS. All these residues map in the homologous region of IFs. Furthermore, in vitro analysis of a truncated IF17 protein without the 82-residue-long amino-terminal part, together with the analysis of a Synechocystis strain expressing a chimeric protein, containing this amino-terminal part of IF17 fused to IF7, demonstrates that amino-terminal region of IF17 mostly confers a higher stability to this protein.
The inactivating factor of glutamine synthetase IF17 Is an intrinsically disordered protein, which folds upon binding to its target
(American Chemical Society, 2011-10-12) Saelices, Lorena; Galmozzi, Carla V.; Florencio Bellido, Francisco Javier; Muro Pastor, María Isabel; Neira, José L.; Universidad de Sevilla. Departamento de Genética; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia e Innovación (MICIN). España; Generalitat Valenciana; Junta de Andalucía
In cyanobacteria, ammonium is incorporated into carbon skeletons by the sequential action of glutamine synthetase and glutamate synthase (GOGAT). The activity of Synechocystis sp. PCC 6803 glutamine synthetase type I (GS) is controlled by a post-transcriptional process involving protein-protein interactions with two inactivating factors: the 65-residue-long protein (IF7) and the 149-residue-long one (IF17). The sequence of the C terminus of IF17 is similar to IF7; IF7 is an intrinsically disordered protein (IDP). In this work, we study the structural propensities and affinity for GS of IF17 and a chimera protein, IF17N/IF7 (constructed by fusing the first 82 residues of IF17 with the whole IF7) by fluorescence, CD, and NMR. IF17 and IF17N/IF7 are IDPs with residual non-hydrogen-bonded structure, probably formed by α-helical, turn-like, and PPII conformations; several theoretical predictions support these experimental findings. IF17 seems to fold upon binding to GS, as suggested by CD thermal denaturations and steady-state far-UV spectra. The apparent affinity of IF17 for GS, as measured by fluorescence, is slightly smaller (K D ∼1 μM) than that measured for IF7 (∼0.3 μM). The K Ds determined by CD are similar to those measured by fluorescence, but slightly larger, suggesting possible conformational rearrangements in the IFs and/or GS upon binding. Further, the results with IFN17/IF7 suggest that (i) binding of IF17 to the GS is modulated not only by its C-terminal region but also by its N-terminus and (ii) there are weakly structured (that is, "fuzzy") complexes in the ternary GS-IF system.
N and C control of ABC-type bicarbonate transporter Cmp and its LysR-type transcriptional regulator CmpR in a heterocyst-forming cyanobacterium, Anabaena sp.
(Wiley, 2012-06-06) López Igual, Rocío; Picossi, S.; López-Garrido, J.; Flores, E.; Herrero, A.; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia e Innovación (MICIN). España; European Commission (EC). Fondo Europeo de Desarrollo Regional (FEDER)
In the model, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120, gene cluster alr2877-alr2880, which encodes an ABC-type transport system, was induced under conditions of carbon limitation and its inactivation impaired the uptake of bicarbonate. Thus, this gene cluster encodes a Cmp bicarbonate transporter. ORF all0862, encoding a LysR-type transcriptional regulator, was expressed under carbon limitation and at higher levels in the absence than in the presence of combined nitrogen, with a positive effect of the N-control transcription factor NtcA. all0862 was expressed from two putative transcription start sites located 164 and 64 bp upstream from the gene respectively. The latter was induced under carbon limitation and was dependent on positive autoregulation by All0862. All0862 was required for the induction of the Cmp bicarbonate transporter, thus representing a CmpR regulator of Anabaena sp. These results show a novel mode of co-regulation by C and N availability through the concerted action of N- and C-responsive transcription factors.
Green microbes: Potential solutions for key sustainable development goals
(Wyley, 2024-07-26) Díaz Troya, Sandra; Huertas Romera, María José; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular
he latest assessment of progress towards theSustainable Development Goals (SDGs) has identi-fied major obstacles, such as climate change, globalinstability and pandemics, which threaten effortsto achieve the SDGs even by 2050. Urgent actionis needed, particularly to reduce poverty, hungerand climate change. In this context, microalgae areemerging as a promising solution, particularly in thecontext of food security and environmental sustain-ability. As versatile organisms, microalgae offer nu-tritional benefits such as high- quality proteins andessential fatty acids, and can be cultivated in non-arable areas, reducing competition for resources andimproving the sustainability of food systems. The roleof microalgae also includes other applications in aq-uaculture, where they serve as sustainable alterna-tives to animal feed, and in agriculture, where theyact as biofertilizers and biostimulants. These micro-organisms also play a key role in interventions ondegraded land, stabilizing soils, improving hydrologi-cal function and increasing nutrient and carbon avail-ability. Microalgae therefore support several SDGsby promoting sustainable agricultural practices andcontributing to land restoration and carbon seques-tration efforts. The integration of microalgae in theseareas is essential to mitigate environmental impactsand improve global food security, highlighting theneed for increased research and development, aswell as public and political support, to exploit their fullpotential to advance the SDGs
Functional dissection and evidence for intercellular transfer of the heterocyst-differentiation PatS morphogen
(John Wiley & Sons, 2013-06) Corrales Guerrero, Laura; Mariscal Romero, Vicente; Flores, Enrique; Herrero, Antonia; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia e Innovación (MICIN). España
The formation of a diazotrophic cyanobacterial filament represents a simple example of biological development. In Anabaena, a non-random pattern of one nitrogen-fixing heterocyst separated by about 10 photosynthetic vegetative cells results from lateral inhibition elicited by the cells differentiating into heterocysts. Key to this process is the patS gene, which has been shown to produce an inhibitor of heterocyst differentiation that involves the C-terminal RGSGR pentapeptide. Complementation of a ΔpatSAnabaena mutant with different versions of PatS, including point mutations or tag fusions, showed that patS is translated into a 17-amino acid polypeptide. Alterations in the N-terminal part of PatS produced inhibition of heterocyst differentiation, thus this part of the peptide appears necessary for proper processing and self-immunity in the producing cells. Alterations in the C-terminal part of PatS led to over-differentiation, thus supporting its role in inhibition of heterocyst differentiation. A polypeptide, produced in proheterocysts, consisting of a methionine followed by the eight, but not the five, terminal amino acids of PatS recreated the full activity of the native peptide. Immunofluorescence detection showed that an RGSGR-containing peptide accumulated in the cells adjacent to the producing proheterocysts, illustrating intercellular transfer of a morphogen in the cyanobacterial filaments.
Self-aggregation in Aqueous Solution of Amphiphilic Cationic Calix[4]arenes. Potential Use as Vectors and Nanocarriers
(Elsevier, 2020) Ostos Marcos, Francisco José; Lebrón Romero, José Antonio; López-Cornejo, María del Pilar; López López, Manuel; García Calderón, Margarita; García Calderón, Clara Beatriz; Valle Rosado, Iván; Kalchenko, Vitaly I.; Rodik, Roman V.; Moyá Morán, María Luisa; Universidad de Sevilla. Departamento de Química Física; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular; Junta de Andalucía; Universidad de Sevilla; Ministerio de Ciencia, Innovación y Universidades (MICINN). España; European Union (UE)
The self-aggregation of four amphiphilic cationic calix[4]arenes, CALIX, in aqueous solutions was investigated in this work. The nature of the polar group present at the upper rim as well as the length of the hydrophobic tails attached to the lower rim was varied. All the calixarenes present two critical aggregation concentrations, CAC1 and CAC2. For [CALIX] < CAC1 only micelles are present, within the range CAC1 < [CALIX] < CAC2 micelles as well as vesicles are observed, and for [CALIX] > CAC2 micelles and a wide distribution of vesicles were found. Cell viability experiments show that calixarene micelles and several of the calixarene vesicles investigated could be used as biocompatible nanocarriers. On this basis, the study of the interactions between the cationic calixarene aggregates (micelles and vesicles) and calf thymus DNA, ctDNA, were done and the results indicated that most of them strongly interact with the polynucleotide, inverting its charge. Micelles totally compact the ctDNA, while vesicles only partially cause conformational changes in the nucleic acid. Therefore, the CALIX micelles show potential as vectors in gene therapy. The encapsulation of the antineoplastic drug doxorubicin into the calixarene aggregates was also investigated. A high encapsulation efficiency was found for micelles and, specially, for vesicles. However, DOX-loaded calixarene vesicles present low stability at 37 °C, which is a serious restriction in their use as nanocarriers for this drug. The release of DOX from the calixarene micelles shows that they could lengthen the half-life of free doxorubicin in the body and, as a result, lower amounts of drug could be used in the cancer treatments diminishing the important side effects of DOX.
A complex and dynamic redox network regulates oxygen reduction at photosystem I in Arabidopsis
(Oxford Academic Press, 2024-09-26) Hani, Umama; Naranjo Río-Miranda, Belén; Shimakawa, Ginga; Espinasse, Christophe; Vanacker, Helene; Setif, Pierre; Rintamaki, Eevi; Issakidis-Bourguet, Emmanuelle; Krieger-Liszkay, Anja; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular; Ecole Universitaire de Recherche de Sciences des Plantes de Paris-Saclay (SPS-GSR); Agence Nationale de la Recherche. France; Ministerio de Ciencia e Innovación (MICIN). España; Agencia Estatal de Investigación. España
Thiol-dependent redox regulation of enzyme activities plays a central role in regulating photosynthesis. Besides the regulation of metabolic pathways, alternative electron transport is subjected to thiol-dependent regulation. We investigated the regulation of O2 reduction at photosystem I. The level of O2 reduction in leaves and isolated thylakoid membranes depends on the photoperiod in which plants are grown. We used a set of Arabidopsis (Arabidopsis thaliana) mutant plants affected in the stromal, membrane, and lumenal thiol network to study the redox protein partners involved in regulating O2 reduction. Light-dependent O2 reduction was determined in leaves and thylakoids of plants grown in short-day and long-day conditions using a spin-trapping electron paramagnetic resonance assay. In wild-type samples from short-day conditions, reactive oxygen species generation was double that of samples from long-day conditions, while this difference was abolished in several redoxin mutants. An in vitro reconstitution assay showed that thioredoxin m, NADPH-thioredoxin reductase C, and NADPH are required for high O2-reduction levels in thylakoids from plants grown in long-day conditions. Using isolated photosystem I, we also showed that reduction of a photosystem I protein is responsible for the increase in O2 reduction. Furthermore, differences in the membrane localization of m-type thioredoxins and 2-Cys peroxiredoxin were detected between thylakoids of short-day and long-day plants. Overall, we propose a model of redox regulation of O2 reduction according to the reduction power of the stroma and the ability of different thiol-containing proteins to form a network of redox interactions.
The nucleolus: Coordinating Stress Response and Genomic Stability
(Elsevier, 2024) González Arzola, Katiuska; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular
The perception that the nucleoli are merely the organelles where ribosome biogenesis occurs is challenged. Only around 30 % of nucleolar proteins are solely involved in producing ribosomes. Instead, the nucleolus plays a critical role in controlling protein trafficking during stress and, according to its dynamic nature, undergoes continuous protein exchange with nucleoplasm under various cellular stressors. Hence, the concept of nucleolar stress has evolved as cellular insults that disrupt the structure and function of the nucleolus. Considering the emerging role of this organelle in DNA repair and the fact that rDNAs are the most fragile genomic loci, therapies targeting the nucleoli are increasingly being developed. Besides, drugs that target ribosome synthesis and induce nucleolar stress can be used in cancer therapy. In contrast, agents that regulate nucleolar activity may be a potential treatment for neurodegeneration caused by abnormal protein accumulation in the nucleolus. Here, I explore the roles of nucleoli beyond their ribosomal functions, highlighting the factors triggering nucleolar stress and their impact on genomic stability.
Persulfidome of Sweet Pepper Fruits during Ripening: The Case Study of Leucine Aminopeptidase That Is Positively Modulated by H2S
(Multidisciplinary Digital Publishing Institute (MDPI), 2024-06-13) Muñoz-Vargas, María A.; González-Gordo, Salvador; Aroca Aguilar, Ángeles; Romero, Luis C.; Gotor, Cecilia; Palma, José M.; Corpas, Francisco J.; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia, Innovación y Universidades (MICINN). España; Agencia Estatal de Investigación (AEI)
Protein persulfidation is a thiol-based oxidative posttranslational modification (oxiPTM) that involves the modification of susceptible cysteine thiol groups present in peptides and proteins through hydrogen sulfide (H2S), thus affecting their function. Using sweet pepper (Capsicum annuum L.) fruits as a model material at different stages of ripening (immature green and ripe red), endogenous persulfidated proteins (persulfidome) were labeled using the dimedone switch method and identified using liquid chromatography and mass spectrometry analysis (LC-MS/MS). A total of 891 persulfidated proteins were found in pepper fruits, either immature green or ripe red. Among these, 370 proteins were exclusively present in green pepper, 237 proteins were exclusively present in red pepper, and 284 proteins were shared between both stages of ripening. A comparative analysis of the pepper persulfidome with that described in Arabidopsis leaves allowed the identification of 25% of common proteins. Among these proteins, glutathione reductase (GR) and leucine aminopeptidase (LAP) were selected to evaluate the effect of persulfidation using an in vitro approach. GR activity was unaffected, whereas LAP activity increased by 3-fold after persulfidation. Furthermore, this effect was reverted through treatment with dithiothreitol (DTT). To our knowledge, this is the first persulfidome described in fruits, which opens new avenues to study H2S metabolism. Additionally, the results obtained lead us to hypothesize that LAP could be involved in glutathione (GSH) recycling in pepper fruits.
Persulfidome of Sweet Pepper Fruits during Ripening: The Case Study of Leucine Aminopeptidase That Is Positively Modulated by H2S
(Multidisciplinary Digital Publishing Institute (MDPI), 2024) Muñoz Vargas, M. A.; González Gordo, S.; Aroca Aguilar, Ángeles; Romero, L. C.; Gotor, C.; Palma, J. M.; Corpas, F. J.; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia e Innovación (MICIN). España; Agencia Estatal de Investigación. España
Protein persulfidation is a thiol-based oxidative posttranslational modification (oxiPTM) that involves the modification of susceptible cysteine thiol groups present in peptides and proteins through hydrogen sulfide (H2S), thus affecting their function. Using sweet pepper (Capsicum annuum L.) fruits as a model material at different stages of ripening (immature green and ripe red), endogenous persulfidated proteins (persulfidome) were labeled using the dimedone switch method and identified using liquid chromatography and mass spectrometry analysis (LC-MS/MS). A total of 891 persulfidated proteins were found in pepper fruits, either immature green or ripe red. Among these, 370 proteins were exclusively present in green pepper, 237 proteins were exclusively present in red pepper, and 284 proteins were shared between both stages of ripening. A comparative analysis of the pepper persulfidome with that described in Arabidopsis leaves allowed the identification of 25% of common proteins. Among these proteins, glutathione reductase (GR) and leucine aminopeptidase (LAP) were selected to evaluate the effect of persulfidation using an in vitro approach. GR activity was unaffected, whereas LAP activity increased by 3-fold after persulfidation. Furthermore, this effect was reverted through treatment with dithiothreitol (DTT). To our knowledge, this is the first persulfidome described in fruits, which opens new avenues to study H2S metabolism. Additionally, the results obtained lead us to hypothesize that LAP could be involved in glutathione (GSH) recycling in pepper fruits.
Antisense RNA Regulates Glutamine Synthetase in a Heterocyst-forming Cyanobacterium
(American Society of Plant Biologists, 2024) Álvarez Escribano, Isidro; Suárez Murillo, Belén; Brenes Álvarez, Manuel; Vioque Peña, Agustín; Muro Pastor, Alicia M.; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia e Innovación (MICIN). España; Junta de Andalucía
Glutamine synthetase (GS) is a key enzyme involved in nitrogen assimilation and the maintenance of C/N balance, and it is strictly regulated in all bacteria. In cyanobacteria, GS expression is controlled by nitrogen control A (NtcA) transcription factor, which operates global nitrogen regulation in these photosynthetic organisms. Furthermore, posttranslational regulation of GS is operated by protein–protein interaction with GS inactivating factors (IFs). In this study, we describe an additional regulatory mechanism involving an antisense RNA. In Nostoc sp. PCC 7120, the gifA gene (encoding GS inactivating factor IF7) is transcribed downstream of the GS (glnA) gene, from the opposite strand, and the gifA mRNA extends into the glnA coding sequence in antisense orientation. Therefore, the dual RNA transcript that encodes gifA constitutes two functional regions: a 5′ protein-coding region, encoding IF7, and a 3′ untranslated region that acts as an antisense to glnA. By increasing the levels of such antisense RNA either in cis or in trans, we demonstrate that the amount of GS activity can be modulated by the presence of the antisense RNA. The tail-to-tail disposition of the glnA and gifA genes observed in many cyanobacterial strains from the Nostocales clade suggests the prevalence of such antisense RNA-mediated regulation of GS in this group of cyanobacteria.
CONSTANS alters the circadian clock in Arabidopsis thaliana
(Elsevier, 2024-06-17) Reyes, Pedro de los; Serrano Bueno, Gloria; Romero Campero, Francisco José; Gao, He; Romero Rodríguez, José María; Valverde, Federico; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular; Universidad de Sevilla. Departamento de Ciencias de la Computación e Inteligencia Artificial; Ministerio de Ciencia, Innovación y Universidades (MICINN). España
Plants are sessile organisms that have acquired highly plastic developmental strategies to adapt to the environment. Among these processes, the floral transition is essential to ensure reproductive success and is finely regulated by several internal and external genetic networks. The photoperiodic pathway, which controls plant response to day length, is one of the most important pathways controlling flowering. In Arabidopsis photoperiodic flowering, CONSTANS (CO) is the central gene activating the expression of the florigen FLOWERING LOCUS T (FT) in the leaves at the end of a long day. The circadian clock strongly regulates CO expression. However, to date, no evidence has been reported regarding a feedback loop from the photoperiod pathway back to the circadian clock. Using transcriptional networks, we have identified relevant network motifs regulating the interplay between the circadian clock and the photoperiod pathway. Gene expression, chromatin immunoprecipitation experiments, and phenotypic analysis allowed us to elucidate the role of CO over the circadian clock. Plants with altered CO expression showed a different internal clock period, measured by daily leaf rhythmic movements. We showed that CO upregulates the expression of key genes related to the circadian clock, such as CCA1, LHY, PRR5, and GI, at the end of a long day by binding to specific sites on their promoters. Moreover, a high number of PRR5-repressed target genes are upregulated by CO, and this could explain the phase transition promoted by CO. The CO-PRR5 complex interacts with the bZIP transcription factor HY5 and helps to localize the complex in the promoters of clock genes. Taken together, our results indicate that there may be a feedback loop in which CO communicates back to the circadian clock, providing seasonal information to the circadian system.
Properties of polyplexes formed between a cationic polymer derived from l-arabinitol and nucleic acids
(Royal Society of Chemistry, 2021-05-14) Pérez Alfonso, David; López López, Manuel; López-Cornejo, María del Pilar; Romero Azogil, Lucía; Benito Hernández, Elena María; García Martín, María de Gracia; García Calderón, Clara Beatriz; Valle Rosado, Iván; Romero Balestra, Fernando; Huertas Sánchez, Pablo; García Calderón, Margarita; Moyá Morán, María Luisa; Universidad de Sevilla. Departamento de Química Física; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular; Universidad de Sevilla. Departamento de Química Orgánica y Farmacéutica; Universidad de Sevilla. Departamento de Genética; Junta de Andalucía; Universidad de Sevilla; Ministerio de Ciencia, Innovación y Universidades (MICINN). España; European Union (UE)
In this work a sugar-based cationic polymer derived from L-arabinitol, PUArab, was prepared and its interactions with the linear calf thymus DNA and with the circular plasmid pEGFP-C1 were investigated at different N/P ratios. The polyplexes were characterized by using several techniques. For both nucleic acids, a charge inversion was observed, together with a conformational change from a coiled structure to a more compacted one. However, the N/P ratio required to observe the DNA condensation depended on the nucleic acid architecture. PUArab presents low toxicity in several cell lines. The transfection efficiency, TE, of the PUArab/pEGFP-C1 polyplexes was investigated at several N/P ratios in order to study their potential as vectors in gene transfection.
Phosphorylation of cytochrome c at tyrosine 48 finely regulates its binding to the histone chaperone SET/TAF-Iβ in the nucleus
(Wiley, 2024-11-16) Tamargo Azpilicueta, Joaquín; Casado Combreras, Miguel Ángel; Giner Arroyo, Rafael Luis; Velázquez Campoy, Adrián; Márquez Escudero, Inmaculada; Olloqui Sariego, José Luis; Rosa Acosta, Miguel Ángel de la; Díaz Moreno, Irene; Universidad de Sevilla. Departamento de Química Física; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia, Innovación y Universidades (MICINN). España; European Union (UE); Junta de Andalucía
Post-translational modifications (PTMs) of proteins are ubiquitous processes present in all life kingdoms, involved in the regulation of protein stability, subcellular location and activity. In this context, cytochrome c (Cc) is an excellent case study to analyze the structural and functional changes induced by PTMS as Cc is a small, moonlighting protein playing different roles in different cell compartments at different cell-cycle stages. Cc is actually a key component of the mitochondrial electron transport chain (ETC) under homeostatic conditions but is translocated to the cytoplasm and even the nucleus under apoptotic conditions and/or DNA damage. Phosphorylation does specifically alter the Cc redox activity in the mitochondria and the Cc non-redox interaction with apoptosis-related targets in the cytoplasm. However, little is known on how phosphorylation alters the interaction of Cc with histone chaperones in the nucleus. Here, we report the effect of Cc Tyr48 phosphorylation by examining the protein interaction with SET/TAF-Iβ in the nuclear compartment using a combination of molecular dynamics simulations, biophysical and structural approaches such as isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) and in cell proximity ligation assays. From these experiments, we infer that Tyr48 phosphorylation allows a fine-tuning of the Cc-mediated inhibition of SET/TAF-Iβ histone chaperone activity in vitro. Our findings likewise reveal that phosphorylation impacts the nuclear, stress-responsive functions of Cc, and provide an experimental framework to explore novel aspects of Cc post-translational regulation in the nucleus.
Analysis of sulfide signaling in rice highlights specific drought responses
(Oxford University Press, 2024-08-28) Zhang, Jing; Aroca Aguilar, Ángeles; Hervás Morón, Manuel; Navarro, José A.; Moreno, Inmaculada; Xie, Yanjie; Romero, Luis C.; Gotor Martínez, Cecilia; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia e Innovación (MICIN). España; Consejo Superior de Investigaciones Científicas (CSIC); Junta de Andalucía
Hydrogen sulfide regulates essential plant processes, including adaptation responses to stress situations, and the best characterized mechanism of action of sulfide consists of the post-translational modification of persulfidation. In this study, we reveal the first persulfidation proteome described in rice including 3443 different persulfidated proteins that participate in a broad range of biological processes and metabolic pathways. In addition, comparative proteomics revealed specific proteins involved in sulfide signaling during drought responses. Several proteins are involved in the maintenance of cellular redox homeostasis, the tricarboxylic acid cycle and energy-related pathways, and ion transmembrane transport and cellular water homeostasis, with the aquaporin family showing the highest differential levels of persulfidation. We revealed that water transport activity is regulated by sulfide which correlates with an increasing level of persulfidation of aquaporins. Our findings emphasize the impact of persulfidation on total ATP levels, fatty acid composition, levels of reactive oxygen species, antioxidant enzymatic activities, and relative water content. Interestingly, the role of persulfidation in aquaporin transport activity as an adaptation response in rice differs from current knowledge of Arabidopsis, which highlights the distinct role of sulfide in improving rice tolerance to drought.
Use of a Biostimulant Obtained from Slaughterhouse Sludge in a Greenhouse Tomato Crop
(MDPI, 2022) Ávila Pozo, Paloma; Parrado Rubio, Juan; Caballero Jiménez, Pablo; Tejada Moral, Manuel; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular; Universidad de Sevilla. Departamento de Cristalografía, Mineralogía y Química Agrícola; Junta de Andalucía; European Union (UE). H2020; Ministerio de Ciencia, Innovación y Universidades (MICINN). España
Currently, the use of biostimulants is widespread in sustainable agriculture because they represent an alternative to chemical fertilizers. In this manuscript, we investigate the response of a greenhouse tomato crop grown in pots to a biostimulant obtained from slaughterhouse sludge applied continuously to the substrate under which the tomato crop was grown or applied directly via the foliar route. Two doses of biostimulant (0.7 and 1.4 g L−1), applied four times throughout the crop growth period (120 days), were used. During this period, the height of the plants, number of flowers and number of fruits obtained were measured. After 90 days of the growth period, the nutrient contents in leaves as well as the chlorophyll a and b contents were analyzed. For fruits, nutrient and lycopene contents were determined, and the equatorial diameter and average fresh and dry weight were measured. The results indicate a higher content of nutrients and chlorophyll a and b in the leaves of plants treated with the highest dose of biostimulant and applied continuously to the substrate. This higher content of photosynthesis pigments in the plant is possibly responsible for a greater absorption of N by the plant and, consequently, for better growth.
Persulfidation of ATG18a regulates autophagy underER stress in Arabidopsis
(National Academy of Sciences, 2021-05) Aroca Aguilar, Ángeles; Yruela Guerrero, Inmaculada; Gotor Martínez, Cecilia; Bassham, Diane C.; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular; European Union (UE). H2020; European Commission (EC). Fondo Europeo de Desarrollo Regional (FEDER); Agencia Estatal de Investigación. España; National Institutes of Health. United States
Hydrogen sulfide (H2S) is an endogenously generated gaseous signaling molecule, which recently has been implicated in autophagy regulation in both plants and mammals through persulfidation of specific targets. Persulfidation has been suggested as the molecular mechanism through which sulfide regulates autophagy in plant cells. ATG18a is a core autophagy component that is required for bulk autophagy and also for reticulophagy during endoplasmic reticulum (ER) stress. In this research, we revealed the role of sulfide in plant ER stress responses as a negative regulator of autophagy. We demonstrate that sulfide regulates ATG18a phospholipid-binding activity by reversible persulfidation at Cys103, and that this modification activates ATG18a binding capacity to specific phospholipids in a reversible manner. Our findings strongly suggest that persulfidation of ATG18a at C103 regulates autophagy under ER stress, and that the impairment of persulfidation affects both the number and size of autophagosomes.

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