Relationships between the ABC-exporter HetC and peptides that regulate the spatiotemporal pattern of heterocyst distribution in Anabaena

In the model cyanobacterium Anabaena sp. PCC 7120, cells called heterocysts that are specialized in the fixation of atmospheric nitrogen differentiate from vegetative cells of the filament in the absence of combined nitrogen. Heterocysts follow a specific distribution pattern along the filament, and a number of regulators have been identified that influence the heterocyst pattern. PatS and HetN, expressed in the differentiating cells, inhibit the differentiation of neighboring cells. At least PatS appears to be processed and transferred from cell to cell. HetC is similar to ABC exporters and is required for differentiation. We present an epistasis analysis of these regulatory genes and of genes, hetP and asr2819, successively downstream from hetC, and we have studied the localization of HetC and HetP by use of GFP fusions. Inactivation of patS, but not of hetN, allowed differentiation to proceed in a hetC background, whereas inactivation of hetC in patS or patS hetN backgrounds decreased the frequency of contiguous proheterocysts. A HetC-GFP protein is localized to the heterocysts and especially near their cell poles, and a putative HetC peptidase domain was required for heterocyst differentiation but not for HetC-GFP localization. hetP is also required for heterocyst differentiation. A HetP-GFP protein localized mostly near the heterocyst poles. ORF asr2819, which we denote patC, encodes an 84-residue peptide and is induced upon nitrogen step-down. Inactivation of patC led to a late spreading of the heterocyst pattern. Whereas HetC and HetP appear to have linked functions that allow heterocyst differentiation to progress, PatC may have a role in selecting sites of differentiation, suggesting that these closely positioned genes may be functionally relatedGobierno de España BFU2010-1798

The Role of SepF in Cell Division and Diazotrophic Growth in the Multicellular Cyanobacterium Anabaena sp. Strain PCC 7120

The cyanobacterium Anabaena forms filaments of cells that grow by intercalary cell division producing adjoined daughter cells connected by septal junction protein complexes that provide filament cohesion and intercellular communication, representing a genuine case of bacterial multicellularity. In spite of their diderm character, cyanobacterial genomes encode homologs of SepF, a protein normally found in Gram-positive bacteria. In Anabaena, SepF is an essential protein that localized to the cell division ring and the intercellular septa. Overexpression of sepF had detrimental effects on growth, provoking conspicuous alterations in cell morphology that resemble the phenotype of mutants impaired in cell division, and altered the localization of the division-ring. SepF interacted with FtsZ and with the essential FtsZ tether ZipN. Whereas SepF from unicellular bacteria generally induces the bundling of FtsZ filaments, Anabaena SepF inhibited FtsZ bundling, reducing the thickness of the toroidal aggregates formed by FtsZ alone and eventually preventing FtsZ polymerization. Thus, in Anabaena SepF appears to have an essential role in cell division by limiting the polymerization of FtsZ to allow the correct formation and localization of the Z-ring. Expression of sepF is downregulated during heterocyst differentiation, likely contributing to the inhibition of Z-ring formation in heterocysts. Finally, the localization of SepF in intercellular septa and its interaction with the septal-junction related proteins SepJ and SepI suggest a role of SepF in the formation or stability of the septal complexes that mediate cell-cell adhesion and communication, processes that are key for the multicellular behavior of Anabaena.Ministerio de Ciencia e Innovación PID2020-118595GB-10

ZipN is an essential FtsZ membrane tether and contributes to the septal localization of SepJ in the flamentous cyanobacterium Anabaena

The organismic unit of heterocyst-forming cyanobacteria is a filament of communicating cells connected by septal junctions, proteinaceous structures bridging the cytoplasms of contiguous cells. This distinct bacterial organization is preserved during cell division. In Anabaena, deletion of the zipN gene could not be segregated. We generated strain CSL109 that expresses zipN from a synthetic regulatable promoter. Under conditions of ZipN depletion, cells progressively enlarged, reflecting restricted cell division, and showed drastic morphological alterations including cell detachment from the filaments, to finish lysing. In contrast to the wild-type localization in midcell Z-rings, FtsZ was found in delocalized aggregates in strain CSL109. Consistently, the proportion of membrane-associated to soluble FtsZ in fractionated cell extracts was lower in CSL109. Bacterial two-hybrid analysis showed that ZipN interacts with FtsZ and other cell-division proteins including cytoplasmic Ftn6 and SepF, and polytopic FtsW, FtsX, FtsQ and FtsI. Additionally, ZipN interacted with the septal protein SepJ, and in CSL109 depletion of ZipN was concomitant with a progressive loss of septal specificity of SepJ. Thus, in Anabaena ZipN represents an essential FtsZ membrane tether and an organizer of the divisome, and it contributes to the conformation of septal structures for filament integrity and intercellular communication.Agencia Estatal de Investigación BFU2013-44686-P, BFU2016-77097-

Proteomics: translating genes into cellular functions to understand biology and disease

The sequencing of the human genome has provided a privileged envision of our own biology. However, much remains to be explored to fully define a complete map of cell functions, their regulatory mechanisms and the alterations participating in disease. As proteins are primary effectors of cellular pathways, the study of the proteome based on a systems biology strategy has opened new perspectives in biomedical research. Proteome complexity, the wide dynamic range of its components as well as their inherent physicochemical nature precludes the coverage of the entire protein set of a living organism on a single experiment. However, the astonishing progress in protein/peptide fractionation combined with mass spectrometry in the past decade allow unprecedented proteome coverage and measurement accuracy. Proteomics emerges, therefore, as an exciting tool in biomedical research. In this seminar basic concepts and state of the art methods in proteomics will be revised as well as their application to the analysis of complex cell and tissue proteomes. One carbon metabolism (1CM) was prioritized as a central pathway associated to the progression of liver disorders. Impairment of the methylation capacity of liver cells lead to the identification of 216 differential proteins that suggest deregulation of cellular pathways as those mediated by ERK or NFκB. R-methyl proteome analysis lead to the identification of 74 differentially methylated proteins, including 116 new methylation sites. Inhibition of RNA binding proteins methylation is especially relevant. Moreover, quantification of 1CM enzymes by SRM in the liver revealed a tissue specific expression profile as well as a significant remodelling in mice upon CCl4 induced liver injury and in liver tumours. It is then tempting to suggest that impairment of 1CM is a bad prognostic hallmark in cancer. The systematic monitorization of one carbon metabolism in the liver may probe its usefulness for the assessment of liver parenchymal cells homeostasis.Fil: Paradela, Alberto. Consejo Superior de Investigaciones Científicas; EspañaFil: Ambao, Veronica Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; ArgentinaFil: Granero, Ignacio. Consejo Superior de Investigaciones Científicas; EspañaFil: Guerrero, Laura. Consejo Superior de Investigaciones Científicas; EspañaFil: Corrales, Fernando J.. Consejo Superior de Investigaciones Científicas; España7th WorkshopMadridEspañaConsejo Superior de Investigaciones CientíficasCentre National de la Recherche Scientifiqu

FtsZ of filamentous, heterocyst-forming cyanobacteria has a conserved N-Terminal peptide required for normal FtsZ polymerization and cell division

Filamentous cyanobacteria grow by intercalary cell division, which should involve distinct steps compared to those producing separate daughter cells. The N-terminal region of FtsZ is highly conserved in the clade of filamentous cyanobacteria capable of cell differentiation. A derivative of the model strain Anabaena sp. PCC 7120 expressing only an FtsZ lacking the amino acids 2-51 of the N-terminal peptide (1N-FtsZ) could not be segregated. Strain CSL110 expresses both 1N-FtsZ, from the endogenous ftsZ gene promoter, and the native FtsZ from a synthetic regulated promoter. Under conditions of 1N-FtsZ predominance, cells of strain CSL110 progressively enlarge, reflecting reduced cell division, and show instances of asymmetric cell division and aberrant Z-structures notably differing from the Z-ring formed by FtsZ in the wild type. In bacterial 2-hybrid assays FtsZ interacted with 1N-FtsZ. However, 1N-FtsZ-GFP appeared impaired for incorporation into Z-rings when expressed together with FtsZ. FtsZ, but not 1N-FtsZ, interacted with the essential protein SepF. Both FtsZ and 1N-FtsZ polymerize in vitro exhibiting comparable GTPase activities. However, filaments of FtsZ show a distinct curling forming toroids, whereas 1N-FtsZ form thick bundles of straight filaments. Thus, the N-terminal FtsZ sequence appears to contribute to a distinct FtsZ polymerization mode that is essential for cell division and division plane location in Anabaena.Agencia Estatal de Investigación BFU2013-44686-P BFU2016-77097-

Monitoring one-carbon metabolism by mass spectrometry to assess liver function and disease

Precision medicine promises to overcome the constraints of the traditional “one-for-all” healthcare approach through a clear understanding of the molecular features of a disease, allowing for innovative and tailored treatments. State-of-the-art proteomics has the potential to accurately explore the human proteome to identify, quantify, and characterize proteins associated with disease progression. There is a pressing need for informative biomarkers to diagnose liver disease early in its course to prevent severe disease for which no efficient treatment is yet available. Here, we propose the concept of a cellular pathway as a functional biomarker, whose monitorization may inform normal and pathological status. We have developed a standardized targeted selected-reaction monitoring assay to detect and quantify 13 enzymes of one-carbon metabolism (1CM). The assay is compliant with Clinical Proteomics Tumor Analysis Consortium (CPTAC) guidelines and has been included in the protein quantification assays that can be accessed through the assay portal at the CPTAC web page. To test the feasibility of the assay, we conducted a retrospective, proof-of-concept study on a collection of liver samples from healthy controls and from patients with cirrhosis or hepatocellular carcinoma (HCC). Our results indicate a significant reconfiguration of 1CM upon HCC development resulting from a process that can already be identified in cirrhosis. Our findings indicate that the systematic and integrated quantification of 1CM enzymes is a promising cell function-based biomarker for patient stratification, although further experiments with larger cohorts are needed to confirm these findings.Fil: Guerrero, Laura. Consejo Superior de Investigaciones Científicas; EspañaFil: Sangro, Bruno. Universidad de Navarra; EspañaFil: Ambao, Veronica Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; ArgentinaFil: Granero, José Ignacio. Consejo Superior de Investigaciones Científicas; EspañaFil: Ramos Fernández, Antonio. No especifíca;Fil: Paradela, Alberto. Consejo Superior de Investigaciones Científicas; EspañaFil: Corrales, Fernando J.. Consejo Superior de Investigaciones Científicas; Españ

Spatial Fluctuations in Expression of the Heterocyst Differentiation Regulatory Gene hetR in Anabaena Filaments

Under nitrogen deprivation, filaments of the cyanobacterium Anabaena undergo a process of development, resulting in a one-dimensional pattern of nitrogen-fixing heterocysts separated by about ten photosynthetic vegetative cells. Many aspects of gene expression before nitrogen deprivation and during the developmental process remain to be elucidated. Furthermore, the coupling of gene expression fluctuations between cells along a multicellular filament is unknown. We studied the statistics of fluctuations of gene expression of HetR, a transcription factor essential for heterocyst differentiation, both under steady-state growth in nitrogen-rich conditions and at different times following nitrogen deprivation, using a chromosomally-encoded translational hetR-gfp fusion. Statistical analysis of fluorescence at the individual cell level in wild-type and mutant filaments demonstrates that expression fluctuations of hetR in nearby cells are coupled, with a characteristic spatial range of circa two to three cells, setting the scale for cellular interactions along a filament. Correlations between cells predominantly arise from intercellular molecular transfer and less from cell division. Fluctuations after nitrogen step-down can build up on those under nitrogen-replete conditions. We found that under nitrogen-rich conditions, basal, steady-state expression of the HetR inhibitor PatS, cell-cell communication influenced by the septal protein SepJ and positive HetR auto-regulation are essential determinants of fluctuations in hetR expression and its distribution along filaments. A comparison between the expression of hetR-gfp under nitrogen-rich and nitrogen-poor conditions highlights the differences between the two HetR inhibitors PatS and HetN, as well as the differences in specificity between the septal proteins SepJ and FraC/FraD. Activation, inhibition and cell-cell communication lie at the heart of developmental processes. Our results show that proteins involved in these basic ingredients combine together in the presence of inevitable stochasticity in gene expression, to control the coupled fluctuations of gene expression that give rise to a one-dimensional developmental pattern in this organism

The Histone Chaperones SET/TAF-1β and NPM1 Exhibit Conserved Functionality in Nucleosome Remodeling and Histone Eviction in a Cytochrome c-Dependent Manner

Chromatin homeostasis mediates essential processes in eukaryotes, where histone chaperones have emerged as major regulatory factors during DNA replication, repair, and transcription. The dynamic nature of these processes, however, has severely impeded their characterization at the molecular level. Here, fluorescence optical tweezers are applied to follow histone chaperone dynamics in real time. The molecular action of SET/template-activating factor-Iβ and nucleophosmin 1—representing the two most common histone chaperone folds—are examined using both nucleosomes and isolated histones. It is shown that these chaperones present binding specificity for fully dismantled nucleosomes and are able to recognize and disrupt non-native histone-DNA interactions. Furthermore, the histone eviction process and its modulation by cytochrome c are scrutinized. This approach shows that despite the different structures of these chaperones, they present conserved modes of action mediating nucleosome remodeling.Ministerio de Educación y Cultura FPU016/01513European Commission PAIDI-DOCTOR 2020 DOC_00796European Molecular Biology Organization 8541European ARBRE-Mobieu consortium CA15126Ministerio de Ciencia e Innovación PGC 2018-096049-BI00, PID2021-126663NB-100Junta de Andalucía BIO198, US-1254317, P18-FR-3487, P18HO-409

Autonomous cortisol secretion in patients with primary aldosteronism: prevalence and implications on cardiometabolic profile and on surgical outcomes

Purpose: The aim of this study was to evaluate the prevalence of autonomous cortisol secretion (ACS) in patients with primary aldosteronism (PA) and its implications on cardiometabolic and surgical outcomes. Methods: This is a retrospective multicenter study of PA patients who underwent 1 mg dexamethasone-suppression test (DST) during diagnostic workup in 21 Spanish tertiary hospitals. ACS was defined as a cortisol post-DST >1.8 μg/dL (confirmed ACS if >5 μg/dL and possible ACS if 1.8–5 μg/dL) in the absence of spe cific clinical features of hypercortisolism. The cardiometabolic profile was compared with a control group with ACS without PA (ACS group) matched for age and DST levels. Results: The prevalence of ACS in the global cohort of patients with PA (n = 176) was 29% (ACS–PA; n = 51). Ten patients had confirmed ACS and 41 possible ACS. The cardiometabolic profile of ACS–PA and PA-only patients was simil ar, except for older age and larger tumor size of the adrenal lesion in the ACS–PA group. When comparing the ACS–PA group (n = 51) and the ACS group (n = 78), the prevalence of hypertension (OR 7.7 (2.64–22.32)) and cardiovascular events (OR 5.0 (2.29–11.07)) was higher in ACS–PA patients than in ACS patients. The coexistence of ACS in patien ts with PA did not affect the surgical outcomes, the proportion of biochemical cure and clinical cure being similar between ACS–PA and PA-only groups. Conclusion: Co-secretion of cortisol and aldosterone affects almost one-thi rd of patients with PA. Its occurrence is more frequent in patients with larger tumors and advanced age. However, the cardiometabolic and surgical outcomes of patients with ACS–PA and PA-only are similar

Acción de reguladores de la diferenciación en el establecimiento del patrón de heterocistos en la cianobacteria anabaena SP. PCC 7120

Texto completo descargado desde TeseoAnabaena sp. PCC 7120 es una cianobacteria multicelular capaz de producir dos tipos celulares diferentes cuando el ambiente lo propicia. En condiciones de bajo nivel de nitrógeno combinado, algunas células se diferencian a heterocistos, que están especializados en la fijación del nitrógeno molecular. Esta diferenciación sigue un patrón definido por el cual sólo una de cada 10 células se diferencia, lo cual requiere una regulación compleja en la que están involucradas más de 30 proteínas reguladoras. En esta tesis se han estudiado 6 de ellas, intentando obtener una visión global de sus relaciones durante el proceso de diferenciación. La proteína HetR está considerada el principal regulador de la diferenciación, siendo un factor de transcripción que se pensaba funcionaba en los primeros momentos del proceso. Aquí hemos demostrado que la proteína HetR ejerce su regulación desde antes de que el ambiente cambie. También se han estudiado los reguladores negativos del proceso, PatS y HetN. Se ha demostrado la función de cada aminoácido del péptido PatS, así como su procesamiento y posterior exporte y la concreta localización de HetN en los polos de los heterocistos. Por otro lado, se ha estudiado la proteína HetC, que ejerce un papel positivo en el desarrollo de los heterocistos y su relación con sus vecinos genómicos, HetP y Asr2819, también relacionados positivamente con la diferenciación de los heterocistos