Biochemical evaluation of the redox state in the formation and stability of HSPA5 supramolecular complexes (BiP)

• Grant number: 24/04401-4
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Effective date (Start): July 01, 2024
• Effective date (End): June 30, 2025
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Julio Cesar Borges
• Grantee: Mariana Oliveira Tavares
• 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

HSP70s represent a crucial class of Molecular Chaperones playing vital roles in cellular protein homeostasis. Within this family, HSPA5 stands out, also known as BiP (Binding immunoglobulin Protein), the most abundantly expressed form of Hsp70 and the only one found in the lumen of the endoplasmic reticulum (ER) in eukaryotes. In addition to the ER, HSPA5 can be found in mitochondria, cytoplasm, nucleoplasm, cell surface, and exosomes. Structurally, this chaperone consists of two domains connected by a hydrophobic linker: the nucleotide-binding domain (NBD) and the substrate-binding domain (SBD). Although predominantly monomeric, HSPA5 can also exist as oligomers and supramolecular complexes (CSM), a process associated with intermolecular disulfide bridge interactions involving cysteines in the NBD (Cys41) and SBD (Cys420). Preliminary results from the Protein Biochemistry and Biophysics group (IQSC/USP) indicate that, in the presence of a reducing environment, monomeric HSPA5 catalyzes the disassembly of formed CSMs. In contrast, in an oxidative environment, the monomeric form apparently leads to the degradation of these complexes. Redox variations in the ER are known, and therefore, these changes may modulate the in vivo function of HSPA5. The purpose of this project is to understand how the redox environment modulates HSPA5 CSMs via intermolecular disulfide bridges and how this may activate or enhance its autoproteolytic action. To achieve this goal, recombinant human HSPA5 will be expressed, purified, and subjected to heating to form CSMs. These will be repurified to obtain the oligomerized fraction, free from the monomeric fraction. The CSMs will be analyzed for their secondary, tertiary, and quaternary structure using spectroscopic techniques (circular dichroism and fluorescence) and hydrodynamics (size-exclusion chromatography, dynamic light scattering, and electrophoretic methods). Functionality will be assessed through ATPase activity assays. The action of the monomeric fraction on the complexes will be chronologically analyzed by analytical size-exclusion chromatography under variable redox conditions. This in-depth study aims to elucidate the self-modulation of HSPA5 through redox variations, potentially revealing the catalytic mechanism underlying self-purification.