Biology subjects

Del Cano-Ochoa, F.

Publications and source records attributed to Del Cano-Ochoa, F..

5 recordsLinked to original sources

bioRxiv2026

A cold-adapted Cas9 nuclease from the deep ocean

The abundance of CRISPR systems in bacteria and archaea fuels the search for Cas nucleases that perform where established enzymes fail. Deep Ocean 1 Cas9 (DO1Cas9) is to our knowledge the first Cas9 nuclease characterized from the deep ocean, recovered from a metagenomic sample at 1,500 m depth in the Atlantic Ocean. DO1Cas9 shows cold-adapted structural flexibility, thermal susceptibility, salt-dependent stability, higher specificity than SpCas9 and cleaves double-stranded DNA efficiently under cold conditions where mesophilic SpCas9 retains only residual activity. DO1Cas9 edits genomes in diverse organisms such as Caenorhabditis elegans, zebrafish, the marine tunicate Oikopleura dioica, and the malaria parasite Plasmodium falciparum. In the marine bacterium Marinomonas mediterranea, DO1Cas9 programmed against an invading phage reduced plaquing efficiency about 10-fold more than SpCas9. These results expand the CRISPR-Cas9 toolbox with a nuclease for cold-chain editing and marine biotechnology.

genetics↗

bioRxiv2025

High conformational flexibility of phosphomannomutase 2: Implications for functioning mechanisms, stability and pharmacological chaperone design

Phosphomannomutase 2 (PMM2) is a critical enzyme in the N-glycosylation pathway, and its defect is the cause of the most common congenital disorder of glycosylation. Despite its biological relevance, the understanding of PMM2 is limited, as the catalytic mechanism and required conformational dynamics remain unknown. In this study, we investigated murine PMM2 (Pmm2) to elucidate its structural flexibility and functional insights. High-resolution crystal structures of Pmm2 in both apo and activator-bound forms provided a more detailed model of the protein, underscoring the role of three ionic cofactors that are essential for dimerization, catalysis, and stability. The Pmm2 structures also provided eight distinct conformations of the protein subunit, highlighting its dynamic nature. Structural comparisons among Pmm2, human PMM2 and other phosphomannomutases helped define the architecture of the enzyme as a dimer assembled by the rigid association of the cap domains, which provide a flat platform from which the core domains of each subunit protrude in a flexible manner. Molecular dynamics (MD) simulations of the human and murine PMM2s further emphasized the enzymes substantial conformational flexibility, revealing extensive core domain movements and suggesting potential inter-subunit communication within the dimer. This study refines the model of PMM2 function, demonstrating its dynamic role in substrate binding, intermediate reorientation, and product release. The observed flexibility provides new opportunities to target specific enzyme states, enabling the development of pharmacological chaperones.

biochemistry↗

bioRxiv2020

UMP inhibition and sequential firing in aspartate transcarbamoylase open ways to regulate plant growth

Pyrimidine nucleotides are essential to plant development. We proved that Arabidopsis growth can be inhibited or enhanced by down- or upregulating aspartate transcarbamoylase (ATC), the first committed enzyme for de novo biosynthesis of pyrimidines in plants. To understand the unique mechanism of feedback inhibition of this enzyme by uridine 5-monophosphate (UMP), we determined the crystal structure of the Arabidopsis ATC trimer free and bound to UMP, demonstrating that the nucleotide binds and blocks the active site. The regulatory mechanism relies on a loop exclusively conserved in plants, and a single-point mutation (F161A) turns ATC insensitive to UMP. Moreover, the structures in complex with a transition-state analog or with carbamoyl phosphate proved a mechanism in plant ATCs for sequential firing of the active sites. The disclosure of the unique regulatory and catalytic properties suggests new strategies to modulate ATC activity and to control de novo pyrimidine synthesis and plant growth.

plant biology↗

bioRxiv2020

Cell-based analysis of CAD variants identifies individuals likely to benefit from uridine therapy

PurposePathogenic autosomal recessive variants in CAD, encoding the multienzymatic protein initiating pyrimidine de novo biosynthesis, cause a severe inborn metabolic disorder treatable with a dietary supplement of uridine. This condition is difficult to diagnose given the large size of CAD with over 1000 missense variants and the non-specific clinical presentation. We aimed to develop a reliable and discerning assay to assess the pathogenicity of CAD variants and to select affected individuals that might benefit from uridine therapy. MethodsUsing CRISPR/Cas9, we generated a human CAD-knockout cell line that requires uridine supplements for survival. Transient transfection of the knockout cells with recombinant CAD restores growth in absence of uridine. This system determines missense variants that inactivate CAD and do not rescue the growth phenotype. ResultsWe identified 25 individuals with biallelic variants in CAD and a phenotype consistent with a CAD deficit. We used the CAD-knockout complementation assay to test a total of 34 variants, identifying 16 as deleterious for CAD activity. Combination of these pathogenic variants confirmed 11 subjects with a CAD deficit, for whom we describe the clinical phenotype. ConclusionsWe designed a cell-based assay to test the pathogenicity of CAD variants, identifying 11 CAD deficient individuals, who could benefit from uridine therapy.

genetics↗

bioRxiv2019

The GATA3 X308_Splice breast cancer mutation is a hormone context-dependent oncogenic driver

As the catalogue of oncogenic driver mutations is expanding, it is becoming clear that alterations in a given gene should not be lumped into one single class, since they might have different functions. The transcription factor GATA3 is a paradigm of this. Here, we address the functions of the most common GATA3 mutation (X308_Splice) which generates a neoprotein that we designate as neoGATA3, associated with good patient prognosis. Based on extensive analyses of molecular and clinical data from approximately 3000 breast cancer patients, supported by mechanistic studies in vitro, we show that neoGATA3 interferes with the transcriptional programs controlled by estrogen and progesterone receptors, without fully abrogating them. This has opposite outputs in the pre- or post-menopausal hormonal context, having pro- or anti-proliferative effects, respectively. NeoGATA3 is an example of a context- and stage-dependent driver mutation. Our data call for functional analyses of putative cancer drivers to guide clinical application.

cancer biology↗