Functional and structural characterization of truncated mutants of human Hsp70-escort protein 1

• Grant number: 21/04799-0
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: September 01, 2021
• End date: November 25, 2023
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Julio Cesar Borges
• Grantee: Maria Júlia Mattos Gomes
• Host Institution: Instituto de Química de São Carlos (IQSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil
• Associated research grant: 17/26131-5 - The chaperome: study of the relationship of the structure of its components and the maintenance of proteostasis, AP.TEM

Abstract

The 70 kDa Heat shock protein (Hsp70 or HSPA) is part of a family of molecular chaperones that, in addition to being highly conserved, is expressed in several cell compartments. HSPAs are associated with several pathologies, such as neurodegenerative diseases and cancer. In humans, mitochondrial HSPA (HSPA9, GRP75, or mortalin) acts on the import of proteins synthesized in the cytosol into the mitochondria and requires a co-chaperone to remain functional and soluble. This co-chaperone is called human Hsp70-escort protein 1 (hHep1) and has an L-shaped structure with a "zinc finger" domain, which is essential for interaction with its ligands. Group studies have shown that hHep1 is capable of maintaining the solubility of HSPA9 and HSPA1A (human Hsp701A), remodeling their supramolecular complexes into smaller particles and has a capacity to stimulate the ATPase activity of these HSPAs. In addition to being found in the mitochondria, hHep1 is present in the cell nucleus and can therefore act on other human HSPAs such as HSPA1A which is present in the cytoplasm and nucleus. Given the importance that hHep1 has for HSPA homeostasis, we propose to carry out the structural and functional characterization of hHep1 mutants, whose N- and C- terminal regions will be truncated in order to identify the main regions of this protein responsible for their function. Another hHep1 mutant, hHep1_core64-160, which contains the zinc finger domain essential to the interaction with HSPA9, is being characterized in our group and the data of this proposal will contribute to elucidating the structural and functional roles of the N- and C-terminal regions of the hHep1. For this purpose, analyzes of its secondary and tertiary structure, chemical, thermal and temporal stability will be carried out. In addition, functional characterization will be carried out through ATPásica activity, as well as its role in the remodeling of the supramolecular complexes of HSPA9 and HSPA1A.(AU)