Person: Navas Hernández, María Ángeles
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First Name
María Ángeles
Last Name
Navas Hernández
Affiliation
Universidad Complutense de Madrid
Faculty / Institute
Medicina
Department
Bioquímica y Biología Molecular
Area
Bioquímica y Biología Molecular
Identifiers
7 results
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Now showing 1 - 7 of 7
Item A novel reverse two-hybrid method for the identification of missense mutations that disrupt protein–protein binding(Scientific Reports, 2020) Vincent, Olivier; Gutiérrez Nogués, Ángel; Trejo Herrero, Adrián; Navas Hernández, María ÁngelesThe reverse two-hybrid system is a powerful method to select mutations that disrupt the interaction between two proteins and therefore to identify the residues involved in this interaction. However, the usefulness of this technique has been limited by its relative complexity when compared to the classical two-hybrid system, since an additional selection step is required to eliminate the high background of uninformative truncation mutants. We have developed a new method that combines the classical and reverse two-hybrid systems to select loss-of-binding missense mutations in a single step. The strategy used to select against truncation mutants is based on the two-hybrid interaction between a C-terminal fusion peptide and the Tsg101 protein. We have applied this method to identify mutations in human glucokinase (GK) that disrupt glucokinase regulatory protein (GKRP) binding. Our results indicate that this method is very efficient and eliminates all the truncation mutants and false positives. The mutated residues identified in GK are involved in the GKRP binding interface or in stabilizing the super-open conformation of GK that binds GKRP. This technique offers an improvement over existing methods in terms of speed, efficiency and simplicity and can be used to study any detectable protein interaction in the two-hybrid system.Item Effects of glucose and insulin on glucokinase activity in rat hypothalamus(Journal of Endocrinology, 2007) Sanz Miguel, María Del Carmen; Roncero Rincón, Isabel; Vázquez Pérez, Patricia; Navas Hernández, María Ángeles; Blázquez Fernández, EnriqueIn an attempt to study the role of glucokinase (GK) and the effects of glucose and peptides on GK gene expression and on the activity of this enzyme in the hypothalamus, we used two kinds of biological models: hypothalamic GT1-7 cells and rat hypothalamic slices. The expression of the GK gene in GT1-7 cells was reduced by insulin (INS) and was not modified by different glucose concentrations, while GK enzyme activities were significantly reduced by the different peptides. Interestingly, a distinctive pattern of GK activities between the ventromedial hypothalamus (VMH) and lateral hypothalamus (LH) were found, with higher enzyme activities in the VMH as the glucose concentrations rose, while LH enzyme activities decreased at 2.8 and 20 mM glucose, the latter effect being prevented by incubation with INS. These effects were produced only by d-glucose and the modifications found were due to GK, but not to other hexokinases. In addition, GK activities in the VMH and the LH were reduced by glucagon-like peptide 1, leptin, orexin B, INS, and neuropeptide Y (NPY), but this effect was only statistically significant for NPY in LH. Our results indicate that the effects of both glucose and peptides occur on GK enzyme activities rather than on GK gene transcription. Moreover, the effects of glucose and INS on GK activity suggest that in the brain GK behaves in a manner opposite to that in the liver, which might facilitate its role in glucose sensing. Finally, hypothalamic slices seem to offer a good physiological model to discriminate the effects between different areas.Item Coiled-coil-mediated dimerization of Atg16 is required for binding to the PROPPIN Atg21(Open Biology, 2023) Bueno Arribas, Miranda; Cruz Cuevas, Celia; Navas Hernández, María Ángeles; Escalante, Ricardo; Vincent, OlivierPROPPINs/WIPIs are β-propeller proteins that bind phosphoinositides and contribute to the recruitment of protein complexes involved in membrane remodelling processes such as autophagosome formation and endosomal trafficking. Yeast Atg21 and mammalian WIPI2 interact with Atg16/ATG16L1 to mediate recruitment of the lipidation machinery to the autophagosomal membrane. Here, we used the reverse double two-hybrid method (RD2H) to identify residues in Atg21 and Atg16 critical for protein–protein binding. Although our results are generally consistent with the crystal structure of the Atg21-Atg16 complex reported previously, they also reveal that dimerization of the Atg16 coiled-coil domain is required for Atg21 binding. Furthermore, most of the residues identified in Atg21 are conserved in WIPI2 and we showed that these residues also mediate ATG16L1 binding. Strikingly, these residues occupy the same position in the β-propeller structure as residues in PROPPINs/WIPIs Hsv2 and WIPI4 that mediate Atg2/ATG2A binding, supporting the idea that these proteins use different amino acids at the same position to interact with different autophagic proteins. Finally, our findings demonstrate the effectiveness of the RD2H system to identify critical residues for protein–protein interactions and the utility of this method to generate combinatory mutants with a complete loss of binding capacity.- Project number: PIMCD33/23-24
Item Orientación a los estudiantes de bioquímica en su transición desde la universidad a la carrera profesional(2024) Navas Hernández, María Ángeles; Martínez Quiles, Narcisa; Herrera González, Blanca María; Fernández Arquero, Miguel; Vidal López, Juan; Martin Quiroga, Ana Isabel; Madariaga López, Anne; García-Loygorri Arias, Virginia; Racionero Gonzalez, Xiana María Item A conserved ATG2 binding site in WIPI4 and yeast Hsv2 is disrupted by mutations causing β-propeller protein-associated neurodegeneration(Human Molecular Genetics, 2021) Bueno Arribas, Miranda; Blanca, Irene; Cruz Cuevas, Celia; Escalante, Ricardo; Navas Hernández, María Ángeles; Vincent, OlivierPROPPINs are phosphoinositide-binding β-propeller proteins that mediate membrane recruitment of other proteins and are involved in different membrane remodeling processes. The main role of PROPPINs is their function in autophagy, where they act at different steps in phagophore formation. The human PROPPIN WIPI4 (WDR45) forms a complex with ATG2 involved in phagophore elongation, and mutations in this gene cause β-propeller protein-associated neurodegeneration (BPAN). The yeast functional counterpart of WIPI4 is Atg18, although its closest sequence homolog is another member of the PROPPIN family, Hsv2, whose function remains largely undefined. Here, we provide evidence that Hsv2, like WIPI4 and Atg18, interacts with Atg2. We show that Hsv2 and a pool of Atg2 colocalize on endosomes under basal conditions and at the pre-autophagosomal structure (PAS) upon autophagy induction. We further show that Hsv2 drives the recruitment of Atg2 to endosomes while Atg2 mediates Hsv2 recruitment to the PAS. HSV2 overexpression results in mis-sorting and secretion of carboxypeptidase CPY, suggesting that the endosomal function of this protein is related to the endosome-to-Golgi recycling pathway. Furthermore, we show that the Atg2 binding site is conserved in Hsv2 and WIPI4 but not in Atg18. Notably, two WIPI4 residues involved in ATG2 binding are mutated in patients with BPAN, and there is a correlation between the inhibitory effect of these mutations on ATG2 binding and the severity of the diseaseItem Effect of rapamycin on lysosomal accumulation in a CRISPR/Cas9-based cellular model of VPS13A deficiency(Journal of Cellular and Molecular Medicine, 2023) Tornero Écija, Alba Rocío; Navas Hernández, María Ángeles; Muñoz Braceras, Sandra; Vincent, Olivier; Escalante Hernández, RicardoVPS13A is a lipid transfer protein localized at different membrane contact sites between organelles, and mutations in the corresponding gene produce a rare neurodegenerative disease called chorea-acanthocytosis (ChAc). Previous studies showed that VPS13A depletion in HeLa cells results in an accumulation of endosomal and lysosomal markers, suggesting a defect in lysosomal degradation capacity leading to partial autophagic dysfunction. Our goal was to determine whether compounds that modulate the endo-lysosomal pathway could be beneficial in the treatment of ChAc. To test this hypothesis, we first generated a KO model using CRISPR/Cas9 to study the consequences of the absence of VPS13A in HeLa cells. We found that inactivation of VPS13A impairs cell growth, which precludes the use of isolated clones due to the undesirable selection of edited clones with residual protein expression. Therefore, we optimized the use of pool cells obtained shortly after transfection with CRISPR/Cas9 components. These cells are a mixture of wild-type and edited cells that allow a comparative analysis of phenotypes and avoids the selection of clones with residual level of VPS13A expression after long-term growth. Consistent with previous observations by siRNA inactivation, VPS13A inactivation by CRISPR/Cas9 resulted in accumulation of the endo-lysosomal markers RAB7A and LAMP1. Notably, we observed that rapamycin partially suppressed the difference in lysosome accumulation between VPS13A KO and WT cells, suggesting that modulation of the autophagic and lysosomal pathway could be a therapeutic target in the treatment of ChAc.Item The WIPI Gene Family and Neurodegenerative Diseases: Insights From Yeast and Dictyostelium Models(Frontiers in Cell and Developmental Biology, 2021) Vincent, Olivier; Antón Esteban, Laura; Bueno Arribas, Miranda; Tornero Écija, Alba; Navas Hernández, María Ángeles; Escalante, RicardoWIPIs are a conserved family of proteins with a characteristic 7-bladed β-propeller structure. They play a prominent role in autophagy, but also in other membrane trafficking processes. Mutations in human WIPI4 cause several neurodegenerative diseases. One of them is BPAN, a rare disease characterized by developmental delay, motor disorders, and seizures. Autophagy dysfunction is thought to play an important role in this disease but the precise pathological consequences of the mutations are not well established. The use of simple models such as the yeast Saccharomyces cerevisiae and the social amoeba Dictyostelium discoideum provides valuable information on the molecular and cellular function of these proteins, but also sheds light on possible pathways that may be relevant in the search for potential therapies. Here, we review the function of WIPIs as well as disease-causing mutations with a special focus on the information provided by these simple models.