CryoEM structural exploration of catalytically active enzyme pyruvate carboxylase

Pyruvate carboxylase (PC) is a tetrameric enzyme that contains two active sites per subunit that catalyze two consecutive reactions. A mobile domain with an attached prosthetic biotin links both reactions, an initial biotin carboxylation and the subsequent carboxyl transfer to pyruvate substrate to produce oxaloacetate. Reaction sites are at long distance, and there are several co-factors that play as allosteric regulators. Here, using cryoEM we explore the structure of active PC tetramers focusing on active sites and on the conformational space of the oligomers. The results capture the mobile domain at both active sites and expose catalytic steps of both reactions at high resolution, allowing the identification of substrates and products. The analysis of catalytically active PC tetramers reveals the role of certain motions during enzyme functioning, and the structural changes in the presence of additional cofactors expose the mechanism for allosteric regulation.This study was supported by grants from the HFSP (RGP0062), and from the Spanish Ministerio de Ciencia e Innovacion (PGC2018-098996-B-100) to M.V., and grants from the NIGMS (R35GM118093) and the NIAID (R01AI116669) to L.T

Effect of ATG12-ATG5-ATG16L1 autophagy E3 complex on the ability of LC3/GABARAP proteins to induce

Trabajo presentado en el 44º Congreso Nacional de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM), celebrado en Málaga entre el 6 y el 9 de septiembre de 2022.In macroautophagy, the autophagosome (AP) engulfs portions of cytoplasm to allow their lysosomal degradation. AP formation in humans requires the concerted action of the ATG12 and LC3/GABARAP conjugation systems. The ATG12–ATG5-ATG16L1 (E3) complex acts as a ubiquitin-like E3 ligase enzyme, promoting LC3/GABARAP protein anchoring to the AP membrane. The role of the various proteins in the AP expansion process is still unclear, in part because there are no studies comparing LC3/GABARAP-family member roles under the same conditions, and also because the full human E3 complex was only recently available. In the present study, the lipidation of six members of the LC3/GABARAP family has been reconstituted in the presence and absence of E3, and the mechanisms by which E3 and LC3/GABARAP proteins participate in vesicle tethering and fusion have been investigated. In the absence of E3, GABARAP and GABARAPL1 showed the highest activities. Differences found within LC3/GABARAP proteins suggest the existence of a lipidation threshold, lower for the GABARAP subfamily, as a requisite for tethering and inter-vesicular lipid mixing. E3 increases and speeds up lipidation and LC3/GABARAP-promoted tethering. However E3 hampers LC3/GABARAP capacity to induce intervesicular lipid mixing or subsequent fusion, presumably through formation of a rigid scaffold on the vesicle surface. Our results suggest a model of AP expansion in which the growing regions would be areas where the LC3/GABARAP proteins involved should be susceptible to lipidation in the absence of E3, or else a regulation should exist to inhibit the formation of an E3 immobile scaffold

E3 autophagy complex and LC3/GABARAP proteins in autophagosome generation

Póster presentado al 8th International Iberian Biophysics Congress celebrado en Bilbao los días 20 y 21 de junio de 2022.In macroautophagy, autophagosome (AP) formation in humans requires the concerted action of the LC3/GABARAP and ATG12 ubiquitin-like conjugation systems. In the present study, the conjugation to the membrane of six members of the LC3/GABARAP family has been reconstituted in the presence and absence of the product of the ATG12 system (the E3 complex). In addition, the mechanisms by which the different LC3/GABARAP proteins and E3 could participate in vesicle tethering and fusion during AP expansion have been investigated. In the absence of E3, GABARAP and GABARAPL1 showed the highest activities. E3 increases and speeds up lipidation and LC3/GABARAP-promoted tethering. However, E3 hampers LC3/GABARAP capacity to induce inter-vesicular lipid mixing or subsequent fusion, presumably through formation of a rigid scaffold on the vesicle surface. Our results suggest a model of AP expansion in which the growing regions would be areas where the LC3/GABARAP proteins involved should be susceptible to lipidation in the absence of E3, or else a regulation should exist to inhibit the formationof an E3 immobile scaffold

Thiabendazole adsorption on montmorillonite, octadecyltrimethylammonium- and Acremonium sp.-loaded products and their copper complexes

This study is a preliminary approach to develop novel montmorillonite (Mt) based adsorbents for removing inorganic and organic contaminants in a step-like process. Biomass (fungi) and surfactant (octadecyltrimethylammonium bromide) modified montmorillonites (BMt and OMt, respectively) were obtained. In a prior step, Cu2+ was loaded into Mt, BMt and OMt. In a second step, the materials (Mt, Mt-Cu, BMt, BMt-Cu, OMt, OMt-Cu) were used as thiabendazole (TBZ) adsorbents. TBZ adsorption isotherms were performed, and Langmuir, Freundlich and Langmuir-Freundlich mathematical models were evaluated. TBZ removal efficiency of the materials was also tested using three adsorbent dosages. The adsorbents and TBZ-adsorbed products were characterized by X-ray diffraction and thermal analysis, and by electron paramagnetic resonance (EPR) and solid-state nuclear magnetic resonance measurements (ss-NMR). To determine the subsequent arrangement of the adsorption products, leaching experiments were also conducted. In the BMt sample, TBZ adsorption was enhanced compared to the raw Mt sample. An opposite behaviour was observed for OMt samples. All the Cu2+-loaded materials showed better TBZ removal efficiencies than the same materials without Cu2+. Furthermore, Cu2+ chelation through the imidazolic and thiazolic nitrogen atoms of TBZ in all Cu2+-loaded samples allowed TBZ and Cu2+ to be resistant to migration in environmental leaching conditions.Centro de Tecnología de Recursos Minerales y Cerámic

Effect of ATG12–ATG5-ATG16L1 autophagy E3-like complex on the ability of LC3/GABARAP proteins to induce vesicle tethering and fusion

In macroautophagy, the autophagosome (AP) engulfs portions of cytoplasm to allow their lysosomal degradation. AP formation in humans requires the concerted action of the ATG12 and LC3/GABARAP conjugation systems. The ATG12–ATG5-ATG16L1 or E3-like complex (E3 for short) acts as a ubiquitin-like E3 enzyme, promoting LC3/GABARAP proteins anchoring to the AP membrane. Their role in the AP expansion process is still unclear, in part because there are no studies comparing six LC3/GABARAP family member roles under the same conditions, and also because the full human E3 was only recently available. In the present study, the lipidation of six members of the LC3/GABARAP family has been reconstituted in the presence and absence of E3, and the mechanisms by which E3 and LC3/GABARAP proteins participate in vesicle tethering and fusion have been investigated. In the absence of E3, GABARAP and GABARAPL1 showed the highest activities. Differences found within LC3/GABARAP proteins suggest the existence of a lipidation threshold, lower for the GABARAP subfamily, as a requisite for tethering and inter-vesicular lipid mixing. E3 increases and speeds up lipidation and LC3/GABARAP-promoted tethering. However, E3 hampers LC3/GABARAP capacity to induce inter-vesicular lipid mixing or subsequent fusion, presumably through the formation of a rigid scaffold on the vesicle surface. Our results suggest a model of AP expansion in which the growing regions would be areas where the LC3/GABARAP proteins involved should be susceptible to lipidation in the absence of E3, or else a regulatory mechanism would allow vesicle incorporation and phagophore growth when E3 is present.This work was supported in part by the Spanish Ministerio de Ciencia e Innovación (MCI), Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) (grants No. PGC2018-099857-B-I00 and PID2021-124461NB-I00), by the Basque Government (grants No. IT1625-22 and IT1270-19), by Fundación Biofísica Bizkaia, by Fundación Ramón Areces (grant No. CIVP20A6619), and by the Basque Excellence Research Centre (BERC) program of the Basque Government. MI and YV were recipients of predoctoral FPU fellowships from the Spanish Ministry of Science, Innovation and Universities (FPU16/05873, FPU18/00799), UB thanks the University of the Basque Country for a predoctoral contract. This work was supported by Human Frontiers Science Program RGP0026/2017 (S.M.)

Kinetic and equilibrium adsorption of two post-harvest fungicides onto copper-exchanged montmorillonite: synergic and antagonistic effects of both fungicides’ presence

The simultaneous adsorption of both imazalil (IMZ) and thiabendazole (TBZ) fungicides in a Cu2+-exchanged Mt was studied in this work. Kinetic studies were used to determine the rate law which describes the adsorption of individual fungicides onto the adsorbent. Adsorption isotherm of individual and combined fungicides was done to evaluate synergic or antagonistic effects. The Mt-Cu material considerably improved TBZ and/or IMZ adsorption from aqueous suspensions with respect to raw Mt, leading to removal efficiencies higher than 99% after 10 min of contact time for TBZ and IMZ Ci = 15 and 40 mg/L, respectively, when a solid dosage = 1 g/L was used. The adsorption sites involved were determined by a combination of X-ray diffraction (XRD) determinations and electron paramagnetic resonance (EPR), indicating that fungicides were bonded to Cu2+ cations, while the rate limiting step was the formation of coordination bonds. The adsorption mechanism proposed is that of ligand exchange between water and fungicide molecules in the metal coordination sphere. The single-crystal structure for the IMZ-Cu2+ complex indicated that four molecules were bounded to the copper centers, while two molecules of TBZ are bounded to copper explaining the higher IMZ uptake capacity for the Mt-Cu material.Centro de Tecnología de Recursos Minerales y CerámicaConsejo Nacional de Investigaciones Científicas y Técnica

Effect of ATG12-ATG5-ATG16L1 autophagy E3-like complex on the ability of LC3/GABARAP proteins to induce vesicle tethering and fusion

In macroautophagy, the autophagosome (AP) engulfs portions of cytoplasm to allow their lysosomal degradation. AP formation in humans requires the concerted action of the ATG12 and LC3/GABARAP conjugation systems. The ATG12-ATG5-ATG16L1 or E3-like complex (E3 for short) acts as a ubiquitin-like E3 enzyme, promoting LC3/GABARAP proteins anchoring to the AP membrane. Their role in the AP expansion process is still unclear, in part because there are no studies comparing six LC3/GABARAP family member roles under the same conditions, and also because the full human E3 was only recently available. In the present study, the lipidation of six members of the LC3/GABARAP family has been reconstituted in the presence and absence of E3, and the mechanisms by which E3 and LC3/GABARAP proteins participate in vesicle tethering and fusion have been investigated. In the absence of E3, GABARAP and GABARAPL1 showed the highest activities. Differences found within LC3/GABARAP proteins suggest the existence of a lipidation threshold, lower for the GABARAP subfamily, as a requisite for tethering and inter-vesicular lipid mixing. E3 increases and speeds up lipidation and LC3/GABARAP-promoted tethering. However, E3 hampers LC3/GABARAP capacity to induce inter-vesicular lipid mixing or subsequent fusion, presumably through the formation of a rigid scaffold on the vesicle surface. Our results suggest a model of AP expansion in which the growing regions would be areas where the LC3/GABARAP proteins involved should be susceptible to lipidation in the absence of E3, or else a regulatory mechanism would allow vesicle incorporation and phagophore growth when E3 is present.Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work was supported in part by the Spanish Ministerio de Ciencia e Innovación (MCI), Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) (grants No. PGC2018-099857-B-I00 and PID2021-124461NB-I00), by the Basque Government (grants No. IT1625-22 and IT1270-19), by Fundación Biofísica Bizkaia, by Fundación Ramón Areces (grant No. CIVP20A6619), and by the Basque Excellence Research Centre (BERC) program of the Basque Government. MI and YV were recipients of predoctoral FPU fellowships from the Spanish Ministry of Science, Innovation and Universities (FPU16/05873, FPU18/00799), UB thanks the University of the Basque Country for a predoctoral contract. This work was supported by Human Frontiers Science Program RGP0026/2017 (S.M.).Peer reviewe

Supplementary Information for Effect of ATG12–ATG5-ATG16L1 autophagy E3-like complex on the ability of LC3/GABARAP proteins to induce vesicle tethering and fusion

15 pages. -- This file includes: Supp. Fig. 1-13. -- Supp. Table 1. List of protein constructs used in this work: vector, expression system, detailed protein encoded, and reference study.Peer reviewe

¿Qué queda de mí?

Este libro es una reclamación a quienes hemos sido, somos o seremos docentes. A quienes no hemos respetado a las personas que se han puesto junto a nosotros y nosotras, confiando su bien más preciado: la libertad. Estas páginas denuncian cada vez que convertimos una visión en la visión, una emoción en la emoción, un saber en el saber, un comportamiento en el comportamiento. Es un grito contra la imposición, la normalización, la neutralización y la universalización de una perspectiva particular. Una pugna contra cada proceso que no se ha conectado con las vidas de los aprendices. Un texto colaborativo realizado por alumnado de Educación y Cambio Social en el Grado en Educación Infantil de la Universidad de Málaga y coordinado por Ignacio Calderón Almendros