Expression, purification, conformational and functional characterization of HSPH1, a human HSP110 co-chaperone, with emphasis on its role in protein disaggregation systems

Author(s)

Gabriela de Mello Gandelini

• Document type: Master's Dissertation
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Química
• Defense date: 2018-04-12

Examining board members

Carlos Henrique Inacio Ramos; Taicia Pacheco Fill; Julio Cesar Borges

• Advisor: Carlos Henrique Inacio Ramos

Abstract

The incorrect protein folding can cause many diseases and even aging. It can lead to loss of function or to the formation of protein amyloid aggregates, which are involved in several diseases. In order to protect from these aggregates, many organisms express the Hsp100 chaperone (ClpB in bacteria, Hsp104 in yeast and Hsp101 in plants), which is able to disaggregate other proteins leading to their reactivation. Surprisingly, metazoa does not have a gene for the Hsp100. Nevertheless, recent studies have suggested that Hsp110 co-chaperone have disaggregation function. Thus, the aim of this dissertation is to characterize HSPH1, a human Hsp110 co-chaperone, by expressing, purifying and performing structure and functional characterization. The hsph1 gene in pPROEX-Htb vector expressed a soluble protein into Escherichia coli (E. coli) BL21 (DE3) and two chromatography steps were used to purify de protein (affinity chromatography followed by size exclusion chromatography). Circular dichroism (CD) and fluorescence emission spectroscopy assays showed that HSPH1 was expressed and purified as a folded and stable protein, with ? helical content of about 34% and it kept its secondary structure content up to about 48 °C and 3 mol/L of urea. Analysis of the emission fluorescence spectroscopy indicated that at least one of the tryptophan residues was partially buried in the native protein. Hydrodynamics properties (molecular mass, diffusion coefficient and Stokes radius) obtained from analytical gel filtration (AGF), size exclusion chromatography combined with multi-angle light (SEC-MALS) and dynamic light scattering (DLS) techniques results suggested that HSPH1 was an elongated monomer. Functional experiments showed that the protein has selective chaperone activity with different protection effects depend on the client-protein tested. The results have the potential to increase significantly the knowledge about protein homeostasis in the cell, which can generate inputs that have medical and biotechnological application (AU)

• FAPESP's process: 16/04246-2 - Cloning, expression, purification, biophysical and functional characterization of members of the human Hsp110 family co-chaperone with emphasis on its role in protein disaggregation systems
• Grantee: Gabriela de Mello Gandelini
• Support Opportunities: Scholarships in Brazil - Master