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Structural and functional characterization of chaperones involved in the solubilization of protein aggregates : the role of CHIP from "Sorghum bicolor" and HspB1 in the human disaggregation system

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Author(s):
Gonçalves, Conrado de Campos
Total Authors: 1
Document type: Doctoral Thesis
Institution: Universidade Estadual de Campinas, Instituto de Biologia
Defense date:
Advisor: Carlos Henrique Inácio Ramos
Abstract

The maintenance of cellular homeostasis by the protein quality control (PQC) system is very complex and relies on multiple processes governed by molecular chaperones and ubiquitin-proteasome system. During normal metabolic conditions, but especially during stress conditions, it is extremely important that all these processes occur efficiently in the cell, avoiding the deposition of non-functional polypeptides which may cause damage. Therefore, the investigation of structural and functional aspects of proteins that belong to PQC system is quite relevant to gain information to terapheutic and biotechnology innovation. Thus, in this work, we aim to understand two very important proteins, whose functions are directly related to protein triage decisions in the cell. First, we investigate details about the co-chaperone CHIP from Sorghum bicolor (SBCHIP) and report on the sequence of the gene of interest identified through homology with the ortholog present in Arabidopsis. Additionally, the gene was cloned, used for superexpression in E. coli and the protein was purified and characterized as a dimer, rich in alpha-helix secondary structure and stable under thermal stress conditions. We also characterized the assembly of SbCHIP and HsHsc70 in complexes and evaluated SbCHIP activity of auto-ubiquitination and ubiquitination of other protein substrates. In addition, our research focus was on the role of the human HspB1 in the protein disaggregation activity of Hsp70 system. Taken together, our results support a model of disaggregation in which unfolded polypeptide co-aggregates with oligomers of activated HspB1. Hsp70 and co-chaperones (Hsp40 and Hsp110) extract the substrate from the aggregates, but also help to disassemble HspB1 into dimers, and this is required for efficient disaggregation. Activation of HspB1 by phosphorylation is important to destabilize oligomers, but not for the immediate formation of dimers. We propose that after disassembly aided by chaperones, HspB1 dimers reassemble spontaneously into oligomers, which allows the cycle to restart for a new round of disaggregation, except for the GPG mutant. In conclusion, the data presented here provides a better understanding of the relationship between structure and function of CHIP from a plant and also contributes to the understanding of the mechanism of solubilization of protein aggegates in human cells (AU)

FAPESP's process: 14/00076-0 - Study of important chaperone-interaction from the celular protein quality control system in higher eukaryotes.
Grantee:Conrado de Campos Gonçalves
Support type: Scholarships in Brazil - Doctorate