Regulation of the Hsc70 system by the cochaperone DnaJA2

270 p.El sistema Hsc70 humano controla funciones esenciales para la viabilidad celular, como la neutralización de conformaciones proteicas mal plegadas y la reactivación de agregados formados cuando la agregación es inevitable. La Hsc70 es el componente principal del sistema, pero requiere de dos proteínas accesorias: un miembro de la familia Hsp40 y un intercambiador de nucleótidos de la familia Hsp110. La familia Hsp40 es la más numerosa y diversa desde el punto de vista funcional y estructural, y sus miembros regulan la especificidad funcional de Hsc70. A pesar de su importancia, no existen estructuras de Hsp40s completas a alta resolución y se desconoce la función de muchas de ellas, sobre todo de las que pueden formar oligómeros. En esta Tesis se ha caracterizado la estructura y función de DnaJA2, una Hsp40 humana de clase A implicada en la homeostasis proteica, y cómo la fosforilación de residuos específicos regula su actividad y la colaboración con Hsc70. Demostramos que DnaJA2 se autoasocia formando estructuras ordenadas y tubulares que pueden interactuar productivamente con Hsc70. La alta eficiencia del sistema Hsc70 para replegar proteínas desplegadas unidas a DnaJA2 indica que el ensamblaje oligomérico podría servir como un andamio que facilita la unión simultánea de varias Hsc70 al sustrato para su remodelado eficiente. Por otro lado, demostramos que la fosforilación de residuos específicos localizados en diferentes dominios de DnaJA2 podría regular su estado oligomérico, actividad holdasa y colaboración con Hsc70. Esta regulación mediada por fosforilación podría servir como un interruptor para mejorar el equilibrio entre la protección de proteínas desplegadas y su replegamiento por el sistema Hsc70 en ciertas circunstancias, sin alterar las funciones esenciales en las que Hsc70 está implicada

Dieta bidez zilar eta kuprez kutsaturiko Crassostrea gigas ostraren azterketa histologikoa eta histokimikoa

Crassostrea gigas ostra dieta bidez kupre eta zilarrarekin kutsatu ondoren burututako azterketa histologiko eta histokimikoa

Unzipping the Secrets of Amyloid Disassembly by the Human Disaggregase

Neurodegenerative diseases (NDs) are increasingly positioned as leading causes of global deaths. The accelerated aging of the population and its strong relationship with neurodegeneration forecast these pathologies as a huge global health problem in the upcoming years. In this scenario, there is an urgent need for understanding the basic molecular mechanisms associated with such diseases. A major molecular hallmark of most NDs is the accumulation of insoluble and toxic protein aggregates, known as amyloids, in extracellular or intracellular deposits. Here, we review the current knowledge on how molecular chaperones, and more specifically a ternary protein complex referred to as the human disaggregase, deals with amyloids. This machinery, composed of the constitutive Hsp70 (Hsc70), the class B J-protein DnaJB1 and the nucleotide exchange factor Apg2 (Hsp110), disassembles amyloids of α-synuclein implicated in Parkinson’s disease as well as of other disease-associated proteins such as tau and huntingtin. We highlight recent studies that have led to the dissection of the mechanism used by this chaperone system to perform its disaggregase activity. We also discuss whether this chaperone-mediated disassembly mechanism could be used to solubilize other amyloidogenic substrates. Finally, we evaluate the implications of the chaperone system in amyloid clearance and associated toxicity, which could be critical for the development of new therapies.This research was funded by MCI/AEI/FEDER, UE (grant PID2019-111068GB-I00) and by the Basque Government (grant IT1201-19). L.V.-C. is the recipient of a predoctoral fellowship from the UPV/EHU and N.O. holds a contract funded by Fundacion Biofisika Bizkaia

Dieta bidez zilar eta kuprez kutsaturiko Crassostrea gigas ostraren azterketa histologikoa eta histokimikoa

Crassostrea gigas ostra dieta bidez kupre eta zilarrarekin kutsatu ondoren burututako azterketa histologiko eta histokimikoa

The Complex Phosphorylation Patterns That Regulate the Activity of Hsp70 and Its Cochaperones

Proteins must fold into their native structure and maintain it during their lifespan to display the desired activity. To ensure proper folding and stability, and avoid generation of misfolded conformations that can be potentially cytotoxic, cells synthesize a wide variety of molecular chaperones that assist folding of other proteins and avoid their aggregation, which unfortunately is unavoidable under acute stress conditions. A protein machinery in metazoa, composed of representatives of the Hsp70, Hsp40, and Hsp110 chaperone families, can reactivate protein aggregates. We revised herein the phosphorylation sites found so far in members of these chaperone families and the functional consequences associated with some of them. We also discuss how phosphorylation might regulate the chaperone activity and the interaction of human Hsp70 with its accessory and client proteins. Finally, we present the information that would be necessary to decrypt the effect that post-translational modifications, and especially phosphorylation, could have on the biological activity of the Hsp70 system, known as the chaperone code.The Agencia Espanola de Investigacion/Fondos de Desarrollo Regional (AEI/FEDER, UE), [BFU2016-75983] and the Basque Government [IT1201-19] provided financial support for this work. L.V. and L.D. are supported by predoctoral grants from the University of the Basque Country and the Spanish Ministry of Economy, Industry and Competitiveness respectively

OMMProtocol: A Command Line Application to Launch Molecular Dynamics Simulations with OpenMM

OpenMM is a free and GPU-accelerated Molecular Dynamics (MD) engine written as a layered and reusable library. This approach allows maximum flexibility to configure MD simulations and develop new molecular mechanics (MM) methods. However, this powerful versatility comes at a cost: the user is expected to write Python scripts to run a simulation. OMMProtocol aims to fill this gap by stitching OpenMM and additional third-party modules together, providing an easy way to create an input file to configure a full multi-stage simulation protocol, from minimization to equilibration and production. OMMProtocol is LGPL-licensed and freely available at https://github.com/insilichem/ommprotocol. </p

BioMetAll: Identifying Metal-Binding Sites in Proteins from Backbone Preorganization

With a large amount of research dedicated to decoding how metallic species bind to protein, in silico methods are interesting allies for experimental procedures. To date, computational predictors mostly work by identifying the best possible sequence or structural match of the target protein with metal binding templates. These approaches are fundamentally focused on the first coordination sphere of the metal. Here, we present the BioMetAll predictor that is based on a different postulate: the formation of a potential metal-binding site is related to the geometric organization of the protein backbone. We first report the set of convenient geometric descriptors of the backbone needed for the algorithm and their parametrization from a statistical analysis. Then, the successful benchmark of BioMetAll on a set of more than 50 metal-binding X-Ray structures is presented. Because BioMetAll allows structural predictions regardless of the exact geometry of the side chains, it appears extremely valuable for systems which structures (either experimental or theoretical) are not optimal for metal binding sites. We report here its application on three different challenging cases i) the modulation of metal-binding sites during conformational transition in human serum albumin, ii) the identification of possible routes of metal migration in hemocyanins, and iii) the prediction of mutations to generate convenient metal-binding sites for de novo biocatalysts. This study shows that BioMetAll offers a versatile platform for numerous fields of research at the interface between inorganic chemistry and biology, and allows to highlight the role of the preorganization of the protein backbone as a marker for metal binding.</div

Inhibition of the Human Hsc70 System by Small Ligands as a Potential Anticancer Approach

Heat shock protein (Hsp) synthesis is upregulated in a wide range of cancers to provide the appropriate environment for tumor progression. The Hsp110 and Hsp70 families have been associated to cancer cell survival and resistance to chemotherapy. In this study, we explore the strategy of drug repurposing to find new Hsp70 and Hsp110 inhibitors that display toxicity against melanoma cancer cells. We found that the hits discovered using Apg2, a human representative of the Hsp110 family, as the initial target bind also to structural regions present in members of the Hsp70 family, and therefore inhibit the remodeling activity of the Hsp70 system. One of these compounds, the spasmolytic agent pinaverium bromide used for functional gastrointestinal disorders, inhibits the intracellular chaperone activity of the Hsp70 system and elicits its cytotoxic activity specifically in two melanoma cell lines by activating apoptosis. Docking and molecular dynamics simulations indicate that this compound interacts with regions located in the nucleotide-binding domain and the linker of the chaperones, modulating their ATPase activity. Thus, repurposing of pinaverium bromide for cancer treatment appears as a promising novel therapeutic approach

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