Mapping and mechanistic characterization of the interaction of the human HspA5 (BiP/Grp78/erHsp70) protein with the human Hep1 co-chaperone and with the Spike protein of the SARS-CoV-2 Coronavirus

Abstract

The 70 kDa Heat shock proteins (Hsp70) are molecular chaperones that act in the control of cell protein quality. They are monomeric, ubiquitous, highly conserved proteins and are composed of a Nucleotide Binding Domain (NBD) and a Peptide-substrate Binding Domain (PBD). To direct their functional cycle, Hsp70 rely on the help of adenosine nucleotides and auxiliary proteins: co-chaperones. HspA5 (Grp78/BiP/erHsp70) is human Hsp70 isoform that resides mainly in the Endoplasmic Reticulum (ER) and plays several essential cellular roles, such as protein folding and refolding, regulation of cell signaling, response to unraveling proteins, regulation of calcium homeostasis, etc. Due to these roles, HspA5 has been widely studied as a therapeutic target in different types of diseases, including COVID-19. It is known that under stressful conditions, HspA5 can move to the plasma membrane and act as a cell receptor, interacting with viral proteins, such as the Spike protein of the new Coronavirus. Different studies have also shown that HspA5 is present in mitochondria, however, its network of interactions in mitochondria is poorly established. Experimental data from Protein Biochemistry and Biophysics group (IQSC/USP) demonstrated, in an unprecedented way, the interaction between NBD_HspA5 and human Hsp70-escort protein 1 (hHep1). Although hHep1 has been reported as an exclusive co-chaperone of the human mitochondrial Hsp70 (HspA9, mortalin), data from the research group demonstrate that hHep1 is also present in the nucleoplasm, and that it modulates the ATPase activity of HspA1A (main inducible human Hsp70). Therefore, hHep1 can modulate other Hsp70 present in the nucleoplasm and mitochondria. In this context, this project proposes: i) to map the in vitro interaction of HspA5 with the SARS-Cov2 Spike protein Receptor Binding Domain (RBD) and ii) to characterize the interaction mechanism of human proteins HspA5 and hHep1, as well as obtain the interactome of these proteins. For this, the recombinant human HspA5 proteins and the viral RBD_Spike will be expressed and purified, inorder to study the formation of the protein complex through biophysical techniques. In this context, the recombinant HspA1A and HspA8 proteins will be also used for comparative purposes. In addition, express and purify the heterologous NBD and PBD domains of human HspA5 and hHep1, in order to carry out the comparative mechanistic characterization of the interaction of hHep1 with HspA5 and its domains. The effects of hHep1 on the ATPase activity of HspA5 will also be evaluated, in addition to comparative assays of the action of hHep1 on the prevention of proteolysis and aggregation/oligomerization of the DLN of the HspA1A and HspA5 proteins. Additionally, cell assays will be carried out in order to evaluate the colocalization between HspA5 and hHep1, and to map the potential interaction between them and their cellular partners, through the BioID method combined with MS. Subsequently, it also aims to characterize the interaction between NBD_HspA5 and hHep1 through protein crystallography. Data from the research group indicate that evaluation of the interaction of hHep1 with HspA1A or HspA9 has technical limitations (unstable interactions or limited amounts of Hsp70). Therefore, the stable interaction with HspA5 can allow a comparative analysis of different human Hsp70 and allow considerable advances in the understanding of the molecular mechanisms of hHep1 over human Hsp70. Thus, an in-depth study of HspA5 will be carried out in order to elucidate processes of differential protein interactions and, consequently, obtain an integrated analysis of the cell processes in which hHep1 is involved. Therefore, the knowledge resulting from this project could serve as a basis for the development of new approaches for the treatment of human diseases, including COVID-19. (AU)