Biophysical characterization of a chaperone complex involved in macroautophagy
EntityUAM. Departamento de Biología Molecular; CSIC. Centro Nacional de Biotecnología (CNB)
SubjectsCélulas - Degeneración - Tesis doctorales; Homeostasis - Alteración - Enfermedades degenerativas - Tesis doctorales; Biología y Biomedicina / Biología
NoteTesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura:31-01-2020
Esta tesis tiene embargado el acceso al texto completo hasta el 31-07-2021
Esta obra está bajo una licencia de Creative Commons Reconocimiento-NoComercial-SinObraDerivada 4.0 Internacional.
Eukaryotic cells have developed regulated pathways to maintain a fine balance between protein synthesis, folding, trafficking and degradation (proteostasis). Disruption of this homeostasis network may lead to the onset of pathologies such as cancer or neurodegenerative disorders. Therefore, cells count on several pathways to remove and prevent accumulation and aggregation of potentially toxic proteins, namely the ubiquitin-proteasome system (UPS) and autophagy. Three types of autophagy have been described so far: chaperone-mediated autophagy (CMA), microautophagy and macroautophagy. The chaperone Hsp70 has a central role in all of them. In macroautophagy, Hsp70, in collaboration with other partners (CHIP, Bag3, HspB8), forms a complex that recognizes, ubiquitinates and delivers aggregate-prone proteins towards special locations in cells (aggresomes), for further degradation. This project focuses on the biophysical characterization of the HspB8:Bag3:Hsp70 complex and its components. HspB8, Bag3 and Hsp70 form a stable ternary complex in vitro, which we have studied by different biophysical techniques and also by standard electron microscopy (negative staining) and cryoelectron microscopy (cryoEM). Using cryoelectron tomography, we have analyzed the distribution of the ternary complex in the specimen grids, and have found a tendency of the complex not to distribute evenly within the ice layer, but in a single stratum probably the air-water interface. Using single particle analysis, a low-resolution map has been obtained by negative staining, which combined with crosslinking-mass spectrometry data, has allowed us to build a pseudo-atomic model of the ternary complex. Additionally, we have obtained by cryoEM a low-resolution 3D map where only the atomic models of Hsp70SBD and BAG:Hsp70NBD could be accommodated. The model supports the previously described bimodal character of the Bag3:Hsp70 interaction.
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Texto de la Tesis Doctoral
Google Scholar:Gragera Cabezudo, Marcos
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