Activation of the DnaK-ClpB Complex is Regulated by the Properties of the Bound Substrate

The chaperone ClpB in bacteria is responsible for the reactivation of aggregated proteins in collaboration with the DnaK system. Association of these chaperones at the aggregate surface stimulates ATP hydrolysis, which mediates substrate remodeling. However, a question that remains unanswered is whether the bichaperone complex can be selectively activated by substrates that require remodeling. We find that large aggregates or bulky, native-like substrates activates the complex, whereas a smaller, permanently unfolded protein or extended, short peptides fail to stimulate it. Our data also indicate that ClpB interacts differently with DnaK in the presence of aggregates or small peptides, displaying a higher affinity for aggregate-bound DnaK, and that DnaK-ClpB collaboration requires the coupled ATPase-dependent remodeling activities of both chaperones. Complex stimulation is mediated by residues at the beta subdomain of DnaK substrate binding domain, which become accessible to the disaggregase when the lid is allosterically detached from the beta subdomain. Complex activation also requires an active NBD2 and the integrity of the M domain-ring of ClpB. Disruption of the M-domain ring allows the unproductive stimulation of the DnaK-ClpB complex in solution. The ability of the DnaK-ClpB complex to discriminate different substrate proteins might allow its activation when client proteins require remodeling.A.A. thanks the Basque Government for a Predoctoral Fellowship. The excellent technical assistance of N. Orozco is gratefully acknowledged. We also thank Mathias P. Mayer for the plasmid encoding the DnaK SBD. This work was supported by grants BFU2016-75983 (AEI/FEDER, UE) and IT709-13 (Basque Government)

Truncation-Driven Lateral Association of α-Synuclein Hinders Amyloid Clearance by the Hsp70-Based Disaggregase

The aggregation of α-synuclein is the hallmark of a collective of neurodegenerative disorders known as synucleinopathies. The tendency to aggregate of this protein, the toxicity of its aggregation intermediates and the ability of the cellular protein quality control system to clear these intermediates seems to be regulated, among other factors, by post-translational modifications (PTMs). Among these modifications, we consider herein proteolysis at both the N- and C-terminal regions of α-synuclein as a factor that could modulate disassembly of toxic amyloids by the human disaggregase, a combination of the chaperones Hsc70, DnaJB1 and Apg2. We find that, in contrast to aggregates of the protein lacking the N-terminus, which can be solubilized as efficiently as those of the WT protein, the deletion of the C-terminal domain, either in a recombinant context or as a consequence of calpain treatment, impaired Hsc70-mediated amyloid disassembly. Progressive removal of the negative charges at the C-terminal region induces lateral association of fibrils and type B* oligomers, precluding chaperone action. We propose that truncation-driven aggregate clumping impairs the mechanical action of chaperones, which includes fast protofilament unzipping coupled to depolymerization. Inhibition of the chaperone-mediated clearance of C-truncated species could explain their exacerbated toxicity and higher propensity to deposit found in vivo.This work was supported by grants PID2019-111068GB-I00 (to A.M.) (AEI/FEDER, UE) and PID2019-105872GB-I00 (to J.M.V.) (AEI/FEDER, UE) from the Ministry of Science and Innovation and by the Basque Government (grant IT1201-19 to AM). The Centro Nacional de Biotecnología (CNB) is a Severo Ochoa Center of Excellence (MINECO award SEV 2017-0712). N.O. holds a contract funded by Fundacion Biofisika Bizkaia. Acknowledgment

Extraction and Refolding Determinants of Chaperone-Driven Aggregated Protein Reactivation

Reactivation of protein aggregates plays a fundamental role in numerous situations, including essential cellular processes, hematological and neurological disorders, and biotechnological applications. The molecular details of the chaperone systems involved are known to a great extent but how the overall reactivation process is achieved has remained unclear. Here, we quantified reactivation over time through a predictive mechanistic model and identified the key parameters that control the overall dynamics. We performed new targeted experiments and analyzed classical data, covering multiple types of non-ordered aggregates, chaperone combinations, and experimental conditions. We found that, irrespective of the behavior observed, the balance of surface disaggregation and refolding in solution universally determines the reactivation dynamics, which is broadly described by two characteristic times. This characterization makes it possible to use activity measurements to accurately infer the underlying loss of aggregated protein and to quantify, for the first time, the refolding rates of the soluble intermediates.This work was supported by the Spanish Ministry of Economy and Innovation (MICINN/FEDER) under grants FIS2015-68722-R, PGC2018-101282-B-I00, and BFU2016-75983 and by the Basque Government under grant IT1201-19.Peer reviewe

Human LDL Structural Diversity Studied by IR Spectroscopy

Lipoproteins are responsible for cholesterol traffic in humans. Low density lipoprotein (LDL) delivers cholesterol from liver to peripheral tissues. A misleading delivery can lead to the formation of atherosclerotic plaques. LDL has a single protein, apoB-100, that binds to a specific receptor. It is known that the failure associated with a deficient protein-receptor binding leads to plaque formation. ApoB-100 is a large single lipid-associated polypeptide difficulting the study of its structure. IR spectroscopy is a technique suitable to follow the different conformational changes produced in apoB-100 because it is not affected by the size of the protein or the turbidity of the sample. We have analyzed LDL spectra of different individuals and shown that, even if there are not big structural changes, a different pattern in the intensity of the band located around 1617 cm 21 related with strands embedded in the lipid monolayer, can be associated with a different conformational rearrangement that could affect to a protein interacting region with the receptor.This work has been supported in part by MINECO grant (BFU 2006-14423) and Programa INNPACTO (grant NuIPT-2011-0817-010000). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional funding received for this study

The self-association equilibrium of DNAJA2 regulates its interaction with unfolded substrate proteins and with Hsc70

Abstract J-domain proteins tune the specificity of Hsp70s, engaging them in precise functions. Despite their essential role, the structure and function of many J-domain proteins remain largely unknown. We explore human DNAJA2, finding that it reversibly forms highly-ordered, tubular structures that can be dissociated by Hsc70, the constitutively expressed Hsp70 isoform. Cryoelectron microscopy and mutational studies reveal that different domains are involved in self-association. Oligomer dissociation into dimers potentiates its interaction with unfolded client proteins. The J-domains are accessible to Hsc70 within the tubular structure. They allow binding of closely spaced Hsc70 molecules that could be transferred to the unfolded substrate for its cooperative remodelling, explaining the efficient recovery of DNAJA2-bound clients. The disordered C-terminal domain, comprising the last 52 residues, regulates its holding activity and productive interaction with Hsc70. These in vitro findings suggest that the association equilibrium of DNAJA2 could regulate its interaction with client proteins and Hsc70

All-or-none amyloid disassembly via chaperone-triggered fibril unzipping favors clearance of α-synuclein toxic species

α-synuclein aggregation is present in Parkinson's disease and other neuropathologies. Among the assemblies that populate the amyloid formation process, oligomers and short fibrils are the most cytotoxic. The human Hsc70-based disaggregase system can resolve α-synuclein fibrils, but its ability to target other toxic assemblies has not been studied. Here, we show that this chaperone system preferentially disaggregates toxic oligomers and short fibrils, while its activity against large, less toxic amyloids is severely impaired. Biochemical and kinetic characterization of the disassembly process reveals that this behavior is the result of an all-or-none abrupt solubilization of individual aggregates. High-speed atomic force microscopy explicitly shows that disassembly starts with the destabilization of the tips and rapidly progresses to completion through protofilament unzipping and depolymerization without accumulation of harmful oligomeric intermediates. Our data provide molecular insights into the selective processing of toxic amyloids, which is critical to identify potential therapeutic targets against increasingly prevalent neurodegenerative disorders

Enseñar y aprender en época de cambios : XXVI Premios Francisco Giner de los Ríos a la Mejora de la Calidad Educativa

En esta edición de los Premios Giner de los Ríos se ha reconocido con el Premio Especial el trabajo llevado a cabo a lo largo de quince años por el profesorado del área de ciencias, junto a sus alumnos, en el estudio de la calidad de las aguas del río Guadalquivir a su paso por Sevilla. En Educación Infantil se ha premiado una webquest basada en la metodología constructivista que consigue el desarrollo de todas las competencias del alumnado e investiga cómo es la vida en la sabana, tundra-polo, selva y desierto. La primera experiencia premiada en Educación Primaria reconoce el esfuerzo de una comunidad educativa en la elaboración de un largometraje sobre Astronomía. La segunda es la creación y puesta en práctica del programa ELIGe©, que ayuda a los alumnos con TEA a la elección de actividades cotidianas, y a la comprensión y expresión de emociones básicas. En Ciencia y Tecnología, se ha premiado el Proyecto bambú, bosquete con variedades de esta planta para trabajar. En Humanidades y Ciencias Sociales, se ha reconocido el valor de una experiencia que transmite al alumnado la idea de que la lengua es la herramienta que permite proyectar una imagen de lo que somos, queremos y anhelamos. En Otras Materias y Áreas Curriculares se ha galardonado un trabajo cuyo objetivo es la enseñanza al alumnado del trabajo autónomo y el desarrollo de la competencia comunicativa. En la modalidad de Trabajos de Aplicación de Conocimientos en Distintos Ámbitos Personales o Sociales, se ha galardonado un proyecto de Formación Profesional que aborda tres objetivos: la integración curricular del desarrollo de proyectos de empresa y simulaciones de entornos reales de trabajo; el cambio en la dinámica del aula con el uso intensivo de la web 2.0; y el cambio en el rol del alumno, que pasa de receptor a creador de conocimiento.MECDES

Promoción turística sostenible de la reserva de la biosfera Tajo-Tejo Internacional

Convocatoria proyectos de innovación de Extremadura 2020/2021Se describe un proyecto llevado acabo por varios centros educativos ubicados en la zona de la Reserva de la Biosfera Tajo-Tejo Internacional (RBTTI) que pretendía contribuir a la transformación sostenible del entorno mediante su conocimiento y promoción, implementando las competencias digital, social y ciudadana y la cultura emprendedora mediante metodologías activas como el aprendizaje servicio. Entre los objetivos principales del proyecto destacan: dar a conocer las implicaciones de la RBTTI; diseñar una campaña de promoción de la RBTTI mediante trípticos y vídeos promocionales; conocer la Reserva a través de las principales vías pecuarias y caminos que comunican los pueblos; descubrir los principales elementos socioculturales, históricos y tradicionales de la Reserva; valorar la importancia del territorio para conservar la biodiversidad: paisajes, ecosistemas, fauna y flora representativa; relacionar la trashumancia y las vías pecuarias como rasgos identificativos de la Reserva, vinculándolo con la historia y rasgos culturales de los pueblos y valorar el emprendimiento y la iniciativa personal, el asosiacionismo y creación de redes de cooperación en y entre pueblos como motor de desarrolloExtremaduraES