Obtaining and Biophysical characterization of R81A, H107A and D111A mutants of Human Hsp70 Escort Protein 1 (hHep1)

• Grant number: 24/18160-9
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: December 01, 2024
• End date: November 30, 2025
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Julio Cesar Borges
• Grantee: Bruna Espiño dos Santos
• 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

hHep1 (human Hsp70-escort protein 1) is a small molecular chaperone often found in the mitochondria, aiding in the import of nuclear-encoded proteins. Under heat stress conditions, it is also found in the nucleus and cytoplasm, playing a crucial role in maintaining cellular proteostasis. hHep1 directly interacts with Hsp70s, increasing their solubility, preventing its aggregation and stimulating their ATPase activity: essential processes for protein homeostasis. Alterations in Hsp70 expression or activity are associated with various diseases, including neurodegenerative disorders. Studies in yeast have shown that Hep1 depletion results in mitochondrial biogenesis defects due to issues in mitochondrial Hsp70 folding. The structure of hHep1 includes an N-terminal mitochondrial targeting signal and a DNL-type Zinc finger domain, which is essential for its interaction with HSPA9 (human mitochondrial Hsp70). This project aims to investigate the structural and functional properties of hHep1 through point mutations in three amino acids of its Zinc finger domain. The goal is to characterize the impact of these mutations on Hsp70 interaction and ATPase activity, aiming to identify mutants that disrupt hHep1's effects on Hsp70. Seven hHep1 variants (three single mutants, three double mutants and one triple mutant) will be functionally evaluated for their ability to interact with Hsp70s, stimulate their ATPase activity and prevent the aggregation of HSPA9, HSPA1A and model client proteins. The measurement of these activities is standardized within the research group. The results obtained will provide a deeper understanding of hHep1's regulatory mechanisms and the importance of its structure in maintaining cellular proteostasis. Mutants that interfere with Hsp70 interaction will be candidates for in cellulo experiments to elucidate whether hHep1's action in the nucleus is dependent or independent of its partnership with human Hsp70s.