Caracterización bioquímica del complejo humano entre Rpn1 y Bag-1

Alonso Rodríguez, Melisa (2019). Caracterización bioquímica del complejo humano entre Rpn1 y Bag-1. Proyecto Fin de Carrera / Trabajo Fin de Grado, E.T.S. de Ingeniería Agronómica, Alimentaria y de Biosistemas (UPM), Madrid.

Description

Title: Caracterización bioquímica del complejo humano entre Rpn1 y Bag-1
Author/s:
  • Alonso Rodríguez, Melisa
Contributor/s:
  • Valpuesta, José María
  • Fernández Pacios, Luis
Item Type: Final Project
Degree: Grado en Biotecnología
Date: July 2019
Subjects:
Faculty: E.T.S. de Ingeniería Agronómica, Alimentaria y de Biosistemas (UPM)
Department: Biotecnología - Biología Vegetal
Creative Commons Licenses: Recognition - No derivative works - Non commercial

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Abstract

The maintenance of proteostasis is essential for cell viability. The native state of proteins can be affected by mutations, stress, etc., which are factors that can make them unstable. This triggers cell death, aging processes or pathological states in cell organisms and this is why there are mechanisms involved in the control of proteostasis, such as protein folding or even protein degradation, in case they affect the correct functioning of the cell. Molecular chaperones contribute to proteostasis not only by helping other proteins in the folding and correct assembly process but also by participating in degradation pathways. This is the case of the chaperone Hsp70, which uses its conformational changes, driven by ATP hydrolysis, to play an important role in protein degradation. Hsp70 was identified as the main chaperone link to the proteasome-ubiquitin pathway. The Bag-1 cochaperone family binds Hsp70 to the proteasome, delivering the ubiquitinated protein to the proteolytic machinery. It is known that the NBD domain of Hsp70 interacts with the BAG domain of Bag-1. The research group where this study has been carried out recently discovered that this cochaperone interacts with the subunit 1 of the regulatory particle of the proteasome (Rpn1). The goal of this study was to delimit the interaction regions between the Bag-1 cochaperone and Rpn1, the subunit 1 of the proteasome. In order to do that, we obtained a mutant of Rpn1 that contained a deletion in the N-terminal domain of 259 aminoacids, and two isolated Bag-1 domains: the UBL domain and the BAG domain. The full proteins and their truncated versions were purified. The production and purification of the truncated versions were purified. Once the purified proteins were obtained, we characterised the interaction between the different versions of Bag-1 and Rpn1 through gel filtration and pulldown assays. The results obtained in this experiment show that Bag-1 interacts with Rpn1 through its PC repetitions and the UBL domain of Bag-1. This double interaction of Bag-1 with Hsp70 and Rpn1 forms a very important complex that links two essential components of the proteostasis control: the protein folding and degradation pathways. This link can be relevant in diseases caused by aggregation of proteins or problems in their folding. Moreover, obtaining the truncated proteins purified and information about their interaction can be used for a future structural characterisation.

More information

Item ID: 57455
DC Identifier: http://oa.upm.es/57455/
OAI Identifier: oai:oa.upm.es:57455
Deposited by: Biblioteca ETSI Agrónomos
Deposited on: 10 Dec 2019 11:24
Last Modified: 10 Dec 2019 11:24
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