Examinando por Autor "Bernardo Seisdedos, Ganeko"

Mostrando 1 - 3 de 3
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    Ítem
    Enhanced standard operating procedures for 31P NMR-based metabolomics in tissue extracts
    (American Chemical Society, 2025-04-13) Martín Ramos, Sara; Bilbao García, Jon; Diercks, Tammo; Mato, José María; Bernardo Seisdedos, Ganeko; Millet, Óscar
    Phosphorylated metabolites, here referred to as phosphometabolites, are sufficiently abundant and widely distributed to provide a condensed representation of metabolism that can be readily accessed through NMR spectroscopy. This study addresses the challenge of precisely quantifying phosphometabolites via quantitative 31P NMR from tissue extracts. We optimized standard operating procedures for enhanced spectral resolution, signal intensity, and accuracy. By amply evaluating solvent and buffer conditions, reference compounds, and paramagnetic relaxation enhancers, we identified optimal conditions for metabolite analysis, including the use of trimethylphosphine oxide for accurate signal referencing due to its short T1 relaxation time and minimal offset and glycine buffer (in D2O) at pD 9.5, where virtually invariant 31P signal frequencies and sample osmolarities are observed, along with maximal NMR detection sensitivity and temperature stability of the pH. These methodological advancements significantly improve the reliability and reproducibility of phosphometabolite characterization, allowing the assignment of up to 60 independent signals in the one-dimensional (1D) 31P spectrum (of a total of 94 peaks), that resulted in the proper quantification of 44 phosphometabolites from different tissular samples.
  • Miniatura
    Ítem
    Pseudophosphorylation of single residues of the J-domain of DNAJA2 regulates the holding/folding balance of the Hsc70 system
    (John Wiley and Sons Inc, 2024-08) Velasco Carneros, Lorea; Bernardo Seisdedos, Ganeko; Maréchal, Jean-Didier; Millet, Óscar; Moro Pérez, Fernando; Muga Villate, Arturo
    The Hsp70 system is essential for maintaining protein homeostasis and comprises a central Hsp70 and two accessory proteins that belong to the J-domain protein (JDP) and nucleotide exchange factor families. Posttranslational modifications offer a means to tune the activity of the system. We explore how phosphorylation of specific residues of the J-domain of DNAJA2, a class A JDP, regulates Hsc70 activity using biochemical and structural approaches. Among these residues, we find that pseudophosphorylation of Y10 and S51 enhances the holding/folding balance of the Hsp70 system, reducing cochaperone collaboration with Hsc70 while maintaining the holding capacity. Truly phosphorylated J domains corroborate phosphomimetic variant effects. Notably, distinct mechanisms underlie functional impacts of these DNAJA2 variants. Pseudophosphorylation of Y10 induces partial disordering of the J domain, whereas the S51E substitution weakens essential DNAJA2-Hsc70 interactions without a large structural reorganization of the protein. S51 phosphorylation might be class-specific, as all cytosolic class A human JDPs harbor a phosphorylatable residue at this position.
  • Miniatura
    Ítem
    Quantitative analysis of the human semen phosphorometabolome by 31P-NMR
    (Multidisciplinary Digital Publishing Institute (MDPI), 2024-01-30) Serrano Pérez, Rebeca; Martín Hidalgo, Martín; Bilbao García, Jon; Bernardo Seisdedos, Ganeko; Millet, Óscar; García Marín, Luis Jesús; Bragado González, María Julia
    Phosphorus-containing metabolites occupy a prominent position in cell pathways. The phosphorometabolomic approach in human sperm samples will deliver valuable information as new male fertility biomarkers could emerge. This study analyzed, by 31P-NMR, seminal plasma and whole semen from asthenozoospermic and normozoospermic samples (71% vs. 27% and 45% vs. 17%, total and progressive sperm motility, respectively), and also ejaculates from healthy donors. At least 16 phosphorus-containing metabolites involved in central energy metabolism and phospholipid, nucleotide, and nicotinamide metabolic pathways were assigned and different abundances between the samples with distinct sperm quality was detected. Specifically, higher levels of phosphocholine, glucose-1-phosphate, and to a lesser degree, acetyl phosphate were found in the asthenozoospermic seminal plasma. Notably, the phosphorometabolites implicated in lipid metabolism were highlighted in the seminal plasma, while those associated with carbohydrate metabolism were more abundant in the spermatozoa. Higher levels of phosphocholine, glucose-1-phosphate, and acetyl phosphate in the seminal plasma with poor quality suggest their crucial role in supporting sperm motility through energy metabolic pathways. In the seminal plasma, phosphorometabolites related to lipid metabolism were prominent; however, spermatozoa metabolism is more dependent on carbohydrate-related energy pathways. Understanding the presence and function of sperm phosphorylated metabolites will enhance our knowledge of the metabolic profile of healthy human sperm, improving assessment and differential diagnosis.