Biology subjects

Castro-Fernandez, V.

Publications and source records attributed to Castro-Fernandez, V..

3 recordsLinked to original sources

bioRxiv2025

Clade-wide proteome analysis shows widespread non-canonical Dcr proteins in fungi.

Dicers (Dcrs) are central proteins involved in the biogenesis of small RNAs (sRNAs) in eukaryotes. Most of the knowledge on Dcr structure, function and evolution comes from studies conducted in animal and plant species. Comparatively, much less is known in fungi, which are a genetically and ecologically diverse group with important roles in ecosystems, agriculture, medicine, and biotechnology. While canonical Dcrs in plants and animals contain a well-defined domain architecture, most fungal Dcrs with experimentally validated functions lack one or more identifiable canonical domains, raising questions about how RNA-binding and precise sRNA processing is retained. Here, we conducted the most extensive survey of fungal Dcr proteins, analyzing 1,593 proteomes across eight phyla. We found a diversity of Dcr domain architectures, with some of them lacking an identifiable PAZ, Helicase, and/or double-stranded RNA binding domains. Phylogenetic analyses showed that different Dcr classes are distributed across distinct clades that often align with fungal taxonomic groups. Despite the lack of canonical domain architectures, we found that fungal Dcrs fold into a characteristic L-shaped structure and show PAZ-like folds, even in proteins without detectable PAZ sequences. Molecular docking and electrostatic analyses further indicate that these divergent Dcrs maintain key RNA-binding surfaces for proper sRNA processing. Our results indicate a remarkable evolutionary plasticity of Dcr in fungi, showing that essential sRNA processing functions can be retained through structural conservation, and highlighting fungi as models to study the modular evolution of the RNAi machinery in eukaryotes. Significance statementDicer (Dcr) proteins are central to RNA interference (RNAi), a gene regulatory mechanism conserved across eukaryotes. However, current models of Dcr structure, function, and evolution are largely based on studies in animals and plants. Here, we present the most comprehensive analysis to date of Dcr proteins in fungi, a diverse eukaryotic group including many societally important pathogens and symbiotes which are reliant on RNAi. Our findings reveal that despite widespread divergence from canonical Dcr architecture, fungal Dcrs conserve critical folds and RNA-binding features, further suggesting that core RNAi functions are maintained. This work establishes fungi as key models for studying the evolution and functional robustness of the RNAi machinery, offering broader insight into the diversity and plasticity of sRNA biogenesis pathways across eukaryotes.

genomics↗

bioRxiv2025

Tracing the evolution of prestin's area-motor activity through ancestral sequence reconstruction and structural modeling

Prestin, a member of the SLC26A family, is essential for the electromotility of mammalian outer hair cells, converting voltage changes into mechanical work. In contrast, nonmammalian orthologues function as anion transporters. To investigate the molecular and structural basis of this functional divergence, we performed ancestral sequence reconstruction (ASR) of prestin across vertebrates, followed by structural modeling using AlphaFold2-multimer and molecular dynamics simulations. We identified more than 200 amino acid substitutions along the lineage that lead to placental mammals, with early substitutions concentrated in the transmembrane domain (TMD) and late substitutions clustering in the STAS domain, particularly in the intervening sequence (IVS). Structural modeling and simulation revealed that early substitutions modulate protein-lipid interactions and interhelical contacts. In placental mammals, the IVS-loop adopts a distinct conformation that places a negatively charged patch near the chloride access pathway, potentially affecting the ion dynamics and voltage responsiveness. These structural transitions occurred without major rearrangements of the global fold of prestin, supporting a notion in which the novel function evolved through distributed substitutions within a conserved scaffold. Our findings illustrate how molecular exaptation, and incremental structural remodeling enabled the repurposing of an ancestral anion transporter into a voltage-sensitive area-motor, providing a framework for understanding the molecular evolution of complex biophysical traits central to auditory neuroscience.

evolutionary biology↗

bioRxiv2024

Glycogen phosphorylase from the methanogenic archaeon Methanococcus maripaludis: Unique regulatory properties of a pyridoxal 5'-phosphate independent phosphorylase

Glycogen phosphorylase (GP) is a critical enzyme in glycogen metabolism. Even though methanogens from the archaeal orders Methanosarcinales and Methanococcales are unable to grow on sugars, they store glycogen, which is metabolized through the glycogenolysis and glycolytic pathways when the carbon source for methanogenesis is depleted. Under these metabolic conditions, the activity of the GP enzyme is essential. To be active, all phosphorylases characterized to date require the cofactor pyridoxal 5-phosphate (PLP). This cofactor is covalently bound via Schiff base to a strictly conserved lysine residue at the active site. Extensive GP sequence analysis of organisms from different domains of life shows strict conservation of active site residues despite significant differences in sequence length. Interestingly, in GP sequences of organisms from the order Methanococcales of archaea, a threonine residue replaces the conserved lysine involved in PLP binding. The purification and characterization of recombinant GP from Methanococcus maripaludis show that the enzyme exhibits glycogen phosphorylase activity and high specificity for glycogen as a substrate. Analysis of the PLP content performed by several methods, such as absorbance, fluorescence, cyanohydrin adduct formation, and mass spectrometry, confirmed the absence of PLP. The results demonstrate that an archaeal GP from the order Methanococcales performs catalysis without the PLP cofactor, deviating from the well-established phosphorylase catalytic mechanism and revealing new scenarios for the glucosyltransferase reaction. Moreover, analysis of enzyme regulation shows that the activity is affected by various molecules, including nucleotides, intermediates of carbon metabolism, and phosphate species. Most of these molecules have not previously been identified as regulators of glycogen phosphorylases in prokaryotes. These results suggest that other GPs from Methanococcales can undergo complex regulation.

biochemistry↗