Biophysical characterization of a chaperone complex involved in macroautophagy

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura:31-01-2020Esta tesis tiene embargado el acceso al texto completo hasta el 31-07-2021Eukaryotic 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

The development of microfluidic paper-based analytical devices for point-of-care diagnosis of sheep scab

The recent growing interest and development of microfluidic paper-based analytical devices (μPADs) for point-of-care (POC) testing in human health in low-resource settings has great potential for the exploitation of these technologies in animal disease diagnosis. Sheep scab is a highly infectious, widespread and notifiable disease of sheep, which poses major economic and welfare concerns for the UK farming industry. The possibility of diagnosing sheep scab at the POC is, consequently, very important to controlling this disease. The overall aim of this project was, therefore, to develop μPADs based on a novel method of fabrication, in order to translate the existing lab-based sheep scab ELISA (Pso o 2) and a biomarker test for haptoglobin (Hp) into paper-based ELISA (P-ELISA), to enable POC diagnosis of this animal disease. In Chapter 3, the novel fabrication method is described, in Chapters 4 and 5, the translation of the lab-based ELISAs (Hp and Pso o 2 respectively) are explained and in Chapter 6 the development of a μPAD for incorporation of the POC tests into a multiplexed, rapid assay is covered. Experiments showed that both ELISAs were successfully transferred onto paper and that the devices developed were suitable for POC testing. This study has resulted in a novel fabrication method for μPADs, in successfully translated existing ELISAs to P-ELISA and in novel solutions for the POC diagnosis of an important veterinary disease

The development of microfluidic paper-based analytical devices for point-of-care diagnosis of sheep scab

The recent growing interest and development of microfluidic paper-based analytical devices (μPADs) for point-of-care (POC) testing in human health in low-resource settings has great potential for the exploitation of these technologies in animal disease diagnosis. Sheep scab is a highly infectious, widespread and notifiable disease of sheep, which poses major economic and welfare concerns for the UK farming industry. The possibility of diagnosing sheep scab at the POC is, consequently, very important to controlling this disease. The overall aim of this project was, therefore, to develop μPADs based on a novel method of fabrication, in order to translate the existing lab-based sheep scab ELISA (Pso o 2) and a biomarker test for haptoglobin (Hp) into paper-based ELISA (P-ELISA), to enable POC diagnosis of this animal disease. In Chapter 3, the novel fabrication method is described, in Chapters 4 and 5, the translation of the lab-based ELISAs (Hp and Pso o 2 respectively) are explained and in Chapter 6 the development of a μPAD for incorporation of the POC tests into a multiplexed, rapid assay is covered. Experiments showed that both ELISAs were successfully transferred onto paper and that the devices developed were suitable for POC testing. This study has resulted in a novel fabrication method for μPADs, in successfully translated existing ELISAs to P-ELISA and in novel solutions for the POC diagnosis of an important veterinary disease

The Inducible Intein-Mediated Self-Cleaving Tag (IIST) System: A Novel Purification and Amidation System for Peptides and Proteins

An efficient self-cleavable purification tag could be a powerful tool for purifying recombinant proteins and peptides without additional proteolytic processes using specific proteases. Thus, the intein-mediated self-cleavage tag was developed and has been commercially available as the IMPACT™ system. However, uncontrolled cleavages of the purification tag by the inteins in the IMPACT™ system have been reported, thereby reducing final yields. Therefore, controlling the protein-splicing activity of inteins has become critical. Here we utilized conditional protein splicing by salt conditions. We developed the inducible intein-mediated self-cleaving tag (IIST) system based on salt-inducible protein splicing of the MCM2 intein from the extremely halophilic archaeon, Halorhabdus utahensis and applied it to small peptides. Moreover, we described a method for the amidation using the same IIST system and demonstrated 15N-labeling of the C-terminal amide group of a single domain antibody (VHH)

Structural characterisation of proteins at the cohesion/replication interface

Cohesion establishment and DNA synthesis are tightly regulated processes occurring at the replication fork. DNA synthesis is under the control of the replisome; a complex machinery of numerous proteins which mediate DNA unwinding and DNA synthesis. Factors interacting with the replisome facilitate replication-related events ahead and behind the fork. Arising impediments ahead of the replisome are resolved by specialised helicases and DNA repair complexes. Conversely, events occurring behind the fork focus not only on DNA synthesis but also on the joining of the newly synthesised sister chromatids. The protein complex cohesin is responsible for ensuring that the sisters are joined immediately after replication and remain held together until anaphase. The precise spatiotemporal relations of proteins at the replication fork have not been fully elucidated. This thesis addresses two important questions of cohesion establishment using structural biology tools. The first focuses on a long outstanding question on the mechanisms of cohesin loading onto DNA. It exposes insights into the folding mechanism of cohesin upon loading, governed by its accessory complex, the cohesin loader. The thesis further describes the variety of modifications which can be applied to study cohesin, and characterises the overall architectures of the loader complexes. The second question studied in this thesis describes the link between cohesion and DNA replication via the small helicase Chl1. There is currently no structure of Chl1 available and therefore the presented Chl1 envelope is the first structural characterisation of this helicase, pointing to a conserved architecture amongst the XPD subfamily of helicases. Additional work focuses on studying a potential auto-inhibition mechanism contributing to the function of this helicase in response to replication stress

The Inducible Intein-Mediated Self-Cleaving Tag (IIST) System: A Novel Purification and Amidation System for Peptides and Proteins

An efficient self-cleavable purification tag could be a powerful tool for purifying recombinant proteins and peptides without additional proteolytic processes using specific proteases. Thus, the intein-mediated self-cleavage tag was developed and has been commercially available as the IMPACT™ system. However, uncontrolled cleavages of the purification tag by the inteins in the IMPACT™ system have been reported, thereby reducing final yields. Therefore, controlling the protein-splicing activity of inteins has become critical. Here we utilized conditional protein splicing by salt conditions. We developed the inducible intein-mediated self-cleaving tag (IIST) system based on salt-inducible protein splicing of the MCM2 intein from the extremely halophilic archaeon, Halorhabdus utahensis and applied it to small peptides. Moreover, we described a method for the amidation using the same IIST system and demonstrated 15N-labeling of the C-terminal amide group of a single domain antibody (VHH)

Quadratic Binary Programming Models in Computational Biology

In this paper we formulate four problems in computational molecular biology as 0-1 quadratic programs. These problems are all NP-hard and the current solution methods used in practice consist of heuristics or approximation algorithms tailored to each problem. Using test problems from scientific databases, we address the question, “Can a general-purpose solver obtain good answers in reasonable time?” In addition, we use the latest heuristics as incumbent solutions to address the question, “Can a general-purpose solver confirm optimality or find an improved solution in reasonable time?” Our computational experiments compare four different reformulation methods: three forms of linearization and one form of quadratic convexification