Investigación

URI permanente para esta comunidadhttps://hdl.handle.net/11441/10690

Esta comunidad enfocada en la investigación recoge artículos, capítulos de libros, libros, ponencias y datos fuentes de investigación.

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A conserved C-terminal peptide of sorghum phosphoenolpyruvate carboxylase promotes its proteolysis, which is prevented by Glc-6P or the phosphorylation state of the enzyme
(Springer Nature, 2021) Gandullo Tovar, Jacinto Manuel; Álvarez Morales, Rosario; Feria Bourrellier, Ana Belén; Monreal Hermoso, José Antonio; Díaz, Isabel; Vidal, Jean; Echevarría Ruiz de Vargas, Cristina; Universidad de Sevilla. Departamento de Biología Vegetal y Ecología
Main conclusion: A synthetic peptide from the C-terminal end of C4-phosphoenolpyruvate carboxylase is implicated in the proteolysis of the enzyme, and Glc-6P or phosphorylation of the enzyme modulate this effect. Abstract: Phosphoenolpyruvate carboxylase (PEPC) is a cytosolic, homotetrameric enzyme that performs a variety of functions in plants. Among them, it is primarily responsible for CO2 fixation in the C4 photosynthesis pathway (C4-PEPC). Here we show that proteolysis of C4-PEPC by cathepsin proteases present in a semi-purified PEPC fraction was enhanced by the presence of a synthetic peptide containing the last 19 amino acids from the C-terminal end of the PEPC subunit (pC19). Threonine (Thr)944 and Thr948 in the peptide are important requirements for the pC19 effect. C4-PEPC proteolysis in the presence of pC19 was prevented by the PEPC allosteric effector glucose 6-phosphate (Glc-6P) and by phosphorylation of the enzyme. The role of these elements in the regulation of PEPC proteolysis is discussed in relation to the physiological context.
Altered Cortical Palmitoylation Induces Widespread Molecular Disturbances in Parkinson’s Disease
(MDPI, 2022) Cervilla-Martínez, Juan F.; Rodríguez Gotor, Juan J.; Wypijewski, Krzysztof J.; Fontán Lozano, Ángela del Carmen; Wang, Tao; Santamaria, Enrique; Fuller, William; Mejías Estévez, Rebeca María; National Institutes of Health. United States
The relationship between Parkinson’s disease (PD), the second-most common neurodegenerative disease after Alzheimer’s disease, and palmitoylation, a post-translational lipid modification, is not well understood. In this study, to better understand the role of protein palmitoylation in PD and the pathways altered in this disease, we analyzed the differential palmitoyl proteome (palmitome) in the cerebral cortex of PD patients compared to controls (n = 4 per group). Data-mining of the cortical palmitome from PD patients and controls allowed us to: (i) detect a set of 150 proteins with altered palmitoylation in PD subjects in comparison with controls; (ii) describe the biological pathways and targets predicted to be altered by these palmitoylation changes; and (iii) depict the overlap between the differential palmitome identified in our study with protein interactomes of the PD-linked proteins α-synuclein, LRRK2, DJ-1, PINK1, GBA and UCHL1. In summary, we partially characterized the altered palmitome in the cortex of PD patients, which is predicted to impact cytoskeleton, mitochondrial and fibrinogen functions, as well as cell survival. Our study suggests that protein palmitoylation could have a role in the pathophysiology of PD, and that comprehensive palmitoyl-proteomics offers a powerful approach for elucidating novel cellular pathways modulated in this neurodegenerative disease.
Broad Transcriptomic Impact of Sorafenib and Its Relation to the Antitumoral Properties in Liver Cancer Cells
(Multidisciplinary Digital Publishing Institute (MDPI), 2022) Contreras Bernal, Laura; Rodríguez Gil, Alfonso; Muntané Relat, Jordi; Cruz Díaz, Jesús de la; Universidad de Sevilla. Departamento de Genética; Universidad de Sevilla. Departamento de Fisiología Médica y Biofísica; Instituto de Salud Carlos III PI19/01266; Centro de Investigación Biomédica en Red Cáncer (CIBERONC) CB16/12/00480
Hepatocellular carcinoma (HCC) is one of the most frequent and essentially incurable cancers in its advanced stages. The tyrosine kinase inhibitor Sorafenib (Sfb) remains the globally accepted treatment for advanced HCC. However, the extent of its therapeutic benefit is limited. Sfb exerts antitumor activity through its cytotoxic, anti-proliferative and pro-apoptotic roles in HCC cells. To better understand the molecular mechanisms underlying these effects, we used RNA sequencing to generate comprehensive transcriptome profiles of HepG2 and SNU423, hepatoblastoma-(HB) and HCC-derived cell lines, respectively, following a Sfb treatment at a pharmacological dose. This resulted in similar alterations of gene expression in both cell lines. Genes functionally related to membrane trafficking, stress-responsible and unfolded protein responses, circadian clock and activation of apoptosis were predominantly upregulated, while genes involved in cell growth and cycle, DNA replication and repair, ribosome biogenesis, translation initiation and proteostasis were downregulated. Our results suggest that Sfb causes primary effects on cellular stress that lead to upregulation of selective responses to compensate for its negative effect and restore homeostasis. No significant differences were found specifically affecting each cell line, indicating the robustness of the Sfb mechanism of action despite the heterogeneity of liver cancer. We discuss our results on terms of providing rationalization for possible strategies to improve Sfb clinical outcomes.
Cisplatin-induced Cell Death Increases the degradation of the MRE11-RAD50-NBS1 Complex Through the Autophagy/lysosomal Pathway
(Springer Nature, 2023) Belmonte Fernández, Alejandro; Herrero Ruiz, Joaquín; Galindo Moreno, María; Limón Mortés, María Cristina; Mora Santos, María del Mar; Sáez, Carmen; Japón, Miguel A.; Tortolero García, María Dolores; Romero Portillo, Francisco; Universidad de Sevilla. Departamento de Microbiología; Ministerio de Ciencia e Innovación (MICIN). España; Junta de Andalucía
Cisplatin and other platinum-based anticancer agents are among the most widely used chemotherapy drugs in the treatment of different types of cancer. However, it is common to find patients who respond well to treatment at first but later relapse due to the appearance of resistance to cisplatin. Among the mechanisms responsible for this phenomenon is the increase in DNA damage repair. Here, we elucidate the effect of cisplatin on the MRN (MRE11-RAD50-NBS1) DNA damage sensor complex. We found that the tumor suppressor FBXW7 is a key factor in controlling the turnover of the MRN complex by inducing its degradation through lysosomes. Inhibition of lysosomal enzymes allowed the detection of the association of FBXW7-dependent ubiquitylated MRN with LC3 and the autophagy adaptor p62/SQSTM1 and the localization of MRN in lysosomes. Furthermore, cisplatin-induced cell death increased MRN degradation, suggesting that this complex is one of the targets that favor cell death. These findings open the possibility of using the induction of the degradation of the MRN complex after genotoxic damage as a potential therapeutic strategy to eliminate tumor cells.
DDX47, MeCP2, and other functionally heterogeneous factors protect cells from harmful R loops
(Elsevier, 2023) Marchena Cruz, Esther; Camino, Lola P.; Bhandari, Jay; Pinela Da Silva, Sonia Cristina; Marqueta Gracia, José Javier; Amdeen, Shahad A.; Guillén Mendoza, Cristina; García Rubio, María Luisa; Calderón Montaño, José Manuel; Xue, Xiaoyu; Luna Varo, Rosa María; Aguilera López, Andrés; Universidad de Sevilla. Departamento de Genética; Universidad de Sevilla. Departamento de Farmacología; Ministerio de Ciencia e Innovación (MICIN). España; European Research Council (ERC); Ministerio de Economía y Competitividad (MINECO). España
Unscheduled R loops can be a source of genome instability, a hallmark of cancer cells. Although targeted proteomic approaches and cellular analysis of specific mutants have uncovered factors potentially involved in R-loop homeostasis, we report a more open screening of factors whose depletion causes R loops based on the ability of activation-induced cytidine deaminase (AID) to target R loops. Immunofluorescence analysis of gH2AX caused by small interfering RNAs (siRNAs) covering 3,205 protein-coding genes identifies 59 potential candidates, from which 13 are analyzed further and show a significant increase of R loops. Such candidates are enriched in factors involved in chromatin, transcription, and RNA biogenesis and other processes. A more focused study shows that the DDX47 helicase is an R-loop resolvase, whereas the MeCP2 methyl-CpG-binding protein uncovers a link between DNA methylation and R loops. Thus, our results suggest that a plethora of gene dysfunctions can alter cell physiology via affecting R-loop homeostasis by different mechanisms.
Deviations from temporal scaling support a stage-specific regulation for C. elegans postembryonic development
(BMC, 2022) Mata Cabana, Alejandro; Romero Expósito, Francisco Javier; Geibel, Mirjam; Piubeli, Francine; Merrow, Martha; Olmedo López, María; Universidad de Sevilla. Departamento de Genética; Universidad de Sevilla. Departamento de Microbiología y Parasitología; Ministerio de Economía y Competitividad (MINECO). España; FEDER Andalucía; Junta de Andalucía
Background After embryonic development, Caenorhabditis elegans progress through for larval stages, each of them finishing with molting. The repetitive nature of C. elegans postembryonic development is considered an oscillatory process, a concept that has gained traction from regulation by a circadian clock gene homologue. Nevertheless, each larval stage has a defined duration and entails specific events. Since the overall duration of development is controlled by numerous factors, we have asked whether different rate-limiting interventions impact all stages equally. Results We have measured the duration of each stage of development for over 2500 larvae, under varied environmental conditions known to alter overall developmental rate. We applied changes in temperature and in the quantity and quality of nutrition and analysed the effect of genetically reduced insulin signalling. Our results show that the distinct developmental stages respond differently to these perturbations. The changes in the duration of specific larval stages seem to depend on stage-specific events. Furthermore, our high-resolution measurement of the effect of temperature on the stage-specific duration of development has unveiled novel features of temperature dependence in C. elegans postembryonic development. Conclusions Altogether, our results show that multiple factors fine tune developmental timing, impacting larval stages independently. Further understanding of the regulation of this process will allow modelling the mechanisms that control developmental timing.
Isolation of Plant Growth Promoting Rhizobacteria from Spartina densiflora and Sarcocornia perennis in San Antonio polluted salt marsh, Patagonian Argentina
(Elsevier, 2021) Idaszkin, Yanina L.; Polifroni, Rosana; Mesa Marín, Jennifer; Universidad de Sevilla. Departamento de Biología Vegetal y Ecología; Universidad de Sevilla; European Commission (EC). Fondo Europeo de Desarrollo Regional (FEDER)
With the purpose of Plant Growth Promoting Rhizobacteria (PGPR) isolation, several authors have sampled different wetlands in the northern half of Argentina. However, to date, we do not know the existence of microbiological studies conducted in the Patagonian salt marshes, and concretely, concerning isolation of PGPR. The present work was conducted in the heavy metal polluted San Antonio salt marsh, in two areas with different grade of pollution. In those areas, we isolated cultivable bacteria from the rhizosphere of the halophytes Spartina densiflora and Sarcocornia perennis, and look for several plant growth promoting (PGP) properties among them. In total, 60 different cultivable bacteria were isolated. 50% of the rhizobacterial strains demonstrated at least one of the PGP properties assayed, 25% of them produced siderophores, 16% were able to solubilize phosphate, 11% were able to produce auxins and 7% chitinase. We could observe that PGP properties were more abundant among bacteria growing in polluted soils. Also, bacteria inhabiting S. densiflora rhizosphere showed more PGP properties related to heavy metal phytostabilization mechanisms, in line with the phytoremediation strategy of this halophyte. Overall, these findings support the idea that coastal hazardous scenarios may be a good opportunity to seek for PGPR. Indeed, some of the strains isolated in this work presented more than one PGP property, so they may be selected for further formulation of inoculants for different applications. For further research, it would be interesting to analyse other PGP properties in these strains, as well as to isolate rhizobacteria from other halophytes in diverse Patagonian salt marshes.
Knock-down of phosphoenolpyruvate carboxylase 3 negatively impacts growth, productivity, and responses to salt stress in sorghum (Sorghum bicolor L.)
(Wiley, 2022) Osa Fernández, Clara de la; Pérez López, Jesús; Feria Bourrellier, Ana Belén; Baena Vaca, Guillermo; Marino, Daniel; Coleto, Inmaculada; Pérez Montaño, Francisco de Asís; Gandullo Tovar, Jacinto Manuel; Echevarría Ruiz de Vargas, Cristina; García-Mauriño Ruiz-Berdejo, Sofía; Monreal Hermoso, José Antonio; Universidad de Sevilla. Departamento de Biología Vegetal y Ecología; Universidad de Sevilla. Departamento de Microbiología; Junta de Andalucía; Gobierno Vasco; Ministerio de Economia, Industria y Competitividad (MINECO). España
Phosphoenolpyruvate carboxylase (PEPC) is a carboxylating enzyme with important roles in plant metabo-lism. Most studies in C4plants have focused on photosynthetic PEPC, but less is known about non-photosynthetic PEPC isozymes, especially with respect to their physiological functions. In this work, weanalyzed the precise roles of the sorghum (Sorghum bicolor) PPC3 isozyme by the use of knock-down lineswith the SbPPC3gene silenced (Ppc3lines).Ppc3plants showed reduced stomatal conductance and plantsize, a delay in flowering time, and reduced seed production. In addition, silenced plants accumulated stressindicators such as Asn, citrate, malate, and sucrose in roots and showed higher citrate synthase activity,even in control conditions. Salinity further affected stomatal conductance and yield and had a deeperimpact on central metabolism in silenced plants compared to wild type, more notably in roots, withPpc3plants showing higher nitrate reductase and NADH-glutamate synthase activity in roots and the accumula-tion of molecules with a higher N/C ratio. Taken together, our results show that although SbPPC3 is pre-dominantly a root protein, its absence causes deep changes in plant physiology and metabolism in rootsand leaves, negatively affecting maximal stomatal opening, growth, productivity, and stress responses insorghum plants. The consequences of SbPPC3silencing suggest that this protein, and maybe orthologs inother plants, could be an important target to improve plant growth, productivity, and resistance to saltstress and other stresses where non-photosynthetic PEPCs may be implicated.
MIF versus SIF motoneurons, what are their respective contribution in the oculomotor medial rectus pool?
(Society for Neuroscience, 2021) Carrero Rojas, Génova; García Hernández, Rosendo Miguel; Blumer, Roland; Rodríguez de la Cruz, Rosa María; Pastor Loro, Ángel Manuel; Universidad de Sevilla. Departamento de Fisiología; Ministerio de Ciencia e Innovación (MICIN). España; Austrian Science Fund
Multiply-innervated muscle fibers (MIFs) are peculiar to the extraocular muscles as they are non-twitch but produce a slow build up in tension on repetitive stimulation. The motoneurons innervating MIFs establish en grappe terminals along the entire length of the fiber, instead of the typical en plaque terminals that singly-innervated muscle fibers (SIFs) motoneurons establish around the muscle belly. MIF motoneurons have been proposed to participate only in gaze holding and slow eye movements. We aimed to discern the function of MIF motoneurons by recording medial rectus motoneurons of the oculomotor nucleus. Single-unit recordings in awake cats demonstrated that electrophysiologically-identified medial rectus MIF motoneurons participated in different types of eye movements, including fixations, rapid eye movements or saccades, convergences, and the slow and fast phases of the vestibulo-ocular nystagmus, the same as SIF motoneurons did. However, MIF medial rectus motoneurons presented lower firing frequencies, were recruited earlier and showed lower eye position (EP) and eye velocity (EV) sensitivities than SIF motoneurons. MIF medial rectus motoneurons were also smaller, had longer antidromic latencies and a lower synaptic coverage than SIF motoneurons. Peristimulus time histograms (PSTHs) revealed that electrical stimulation to the myotendinous junction, where palisade endings are located, did not recurrently affect the firing probability of medial rectus motoneurons. Therefore, we conclude there is no division of labor between MIF and SIF motoneurons based on the type of eye movement they subserve.
Relation between CarS expression and activation of carotenogenesis by stress in Fusarium fujikuroi
(Frontiers Media S.A., 2022) Ruger Herreros, Macarena; Nordzieke, Steffen; Vega Álvarez, Carmen; Ávalos Cordero, Francisco Javier; Limón Mirón, María del Carmen; Universidad de Sevilla. Departamento de Genética; Ministerio de Economía y Competitividad (MINECO). España; Ministerio de Ciencia e Innovación (MICIN). España; Junta de Andalucía
Fusarium fujikuroi, a model organism for secondary metabolism in fungi, produces carotenoids, terpenoid pigments with antioxidant activity. Previous results indicate that carotenoid synthesis in F. fujikuroi is stimulated by light or by different stress conditions and downregulated by a RING finger protein encoded by carS gene. Here, we have analyzed the effects of three stressors, nitrogen scarcity, heat shock, and oxidative stress. We compared them with the effect of light in the wild type, a carS mutant that overproduces carotenoids, and its complemented strain. The assayed stressors increase the synthesis of carotenoids in the three strains, but mRNA levels of structural genes of carotenogenesis, carRA and carB, are only enhanced in the presence of a functional carS gene. In the wild-type strain, the four conditions affect in different manners the mRNA levels of carS: greater in the presence of light, without significant changes in nitrogen starvation, and with patent decreases after heat shock or oxidative stress, suggesting different activation mechanisms. The spores of the carS mutant are more resistant to H2O2 than those of the wild type; however, the mutant shows a greater H2O2 sensitivity at the growth level, which may be due to the participation of CarS in the regulation of genes with catalase domains, formerly described. A possible mechanism of regulation by heat stress has been found in the alternative splicing of the intron of the carS gene, located close to its 3′ end, giving rise to the formation of a shorter protein. This action could explain the inducing effect of the heat shock, but not of the other inducing conditions, which may involve other mechanisms of action on the CarS regulator, either transcriptionally or post-transcriptionally.
Salmonella Type III Secretion Effector SrfJ: A Glucosylceramidase Affecting the Lipidome and the Transcriptome of Mammalian Host Cells
(MDPI, 2023) Aguilera Herce, Julia; Panadero Medianero, Concepción; Sánchez Romero, María Antonia; Balbontín Soria, Roberto; Bernal Bayard, Joaquín; Ramos Morales, Francisco; Universidad de Sevilla. Departamento de Genética; Universidad de Sevilla. Departamento de Microbiología y Parasitología; European Commission (EC). Fondo Europeo de Desarrollo Regional (FEDER); Generalitat de Catalunya
Type III secretion systems are found in many Gram-negative pathogens and symbionts of animals and plants. Salmonella enterica has two type III secretion systems associated with virulence, one involved in the invasion of host cells and another involved in maintaining an appropriate intracellular niche. SrfJ is an effector of the second type III secretion system. In this study, we explored the biochemical function of SrfJ and the consequences for mammalian host cells of the expression of this S. enterica effector. Our experiments suggest that SrfJ is a glucosylceramidase that alters the lipidome and the transcriptome of host cells, both when expressed alone in epithelial cells and when translocated into macrophages in the context of Salmonella infection. We were able to identify seventeen lipids with higher levels and six lipids with lower levels in the presence of SrfJ. Analysis of the forty-five genes, the expression of which is significantly altered by SrfJ with a fold-change threshold of two, suggests that this effector may be involved in protecting Salmonella from host immune defenses.
The ceramide synthase subunit lac1 regulates cell growth and size in fission yeast
(Multidisciplinary Digital Publishing Institute (MDPI), 2022) Flor Parra, Ignacio; Sabido Bozo, Susana; Ikeda, Atsuko; Hanaoka, Kazuki; Aguilera Romero, María Auxiliadora; Funato, Kouichi; Muñiz Guinea, Manuel; Lucena Hernández, Rafael; Universidad de Sevilla. Departamento de Biología Celular
Cell division produces two viable cells of a defined size. Thus, all cells require mechanisms to measure growth and trigger cell division when sufficient growth has occurred. Previous data suggest a model in which growth rate and cell size are mechanistically linked by ceramide-dependent signals in budding yeast. However, the conservation of mechanisms that govern growth control is poorly understood. In fission yeast, ceramide synthase is encoded by two genes, Lac1 and Lag1. Here, we characterize them by using a combination of genetics, microscopy, and lipid analysis. We showed that Lac1 and Lag1 co-immunoprecipitate and co-localize at the endoplasmic reticulum. However, each protein generates different species of ceramides and complex sphingolipids. We further discovered that Lac1, but not Lag1, is specifically required for proper control of cell growth and size in Schizosaccharomyces pombe. We propose that specific ceramide and sphingolipid species produced by Lac1 are required for normal control of cell growth and size in fission yeast.
Transcriptional regulation by the velvet protein VE-1 during asexual development in the fungus neurospora crassa.
(American Society for Microbiology, 2022) Cea Sánchez, Sara; Corrochano Luque, María; Gutiérrez Pozo, Gabriel; Glass, N. Louise; Cánovas López, David; Corrochano Peláez, Luis María; Universidad de Sevilla. Departamento de Genética; Ministerio de Ciencia e Innovación (MICIN). España; Agencia Estatal de Investigación. España; European Commission (EC). Fondo Europeo de Desarrollo Regional (FEDER)
Asexual reproduction in fungi facilitates the dispersal and colonization of new substrates and, in pathogenic fungi, allows infection of plants and animals. The velvet complex is a fungus-specific protein complex that participates in the regulation of gene expression in response to environmental signals like light, as well as developmental processes, pathogenesis, and secondary metabolism. The velvet complex in the fungus Neurospora crassa is composed of three proteins, VE-1, VE-2, and LAE-1. Mutations in ve-1 or ve-2, but not in lae-1, led to shorter heights of aerial tissue, a mixture of aerial hyphae and developing macroconidia, and increased microconidiation when they were combined with mutations in the transcription factor gene fl. VE-2 and LAE-1 were detected during vegetative growth and conidiation, unlike VE-1, which was mostly observed in samples obtained from submerged vegetative hyphae. We propose that VE-1 is the limiting component of the velvet complex during conidiation and has a major role in the transcriptional regulation of conidiation. Characterization of the role of VE-1 during mycelial growth and asexual development (conidiation) by transcriptome sequencing (RNA-seq) experiments allowed the identification of a set of genes regulated by VE-1 that participate in the regulation of conidiation, most notably the transcription factor genes vib-1 and fl. We propose that VE-1 and VE-2 regulate the development of aerial tissue and the balance between macro- and microconidiation in coordination with FL and VIB-1.

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