Interaction mapping of the three cytosolic heat-induced human HSP70 proteins HSPA1A, HSPA1L and HSPA6: identification of co-chaperone partnership and client selectivity

Abstract

Heat shock proteins (HSPs) are important components of cell protein quality control, acting on protein folding, intracellular traffic, protein degradation, etc. Central in protein quality control is the HSP70 family that consists of highly conserved, ubiquitous and ATP-dependent molecular chaperones. In humans, there are several HSP70 isoforms, most of these are found in the cytosol while others are organelle specific. Previous studies have shown that, although they have high sequence identity and overlapping functions, HSP70 isoforms seem not always interchangeable and display specificity towards clients and co-chaperones (HAGEMAN et al 2011; SERLIDAKI et al., 2020). Our first hypothesis is that in different cell and environmental conditions, each human nucleus/cytosolic HSP70 protein has at least a set of non-overlapping client and partner proteins. In addition, we propose that cytosolic HSP70s can act together on the same clients, maybe at different state of (un)folding and that there is the possibility of transferring client proteins between them (KNIGHTON et al., 2019). Using a well-established BioID assay implemented in the Kampinga's laboratory for other chaperones, we propose to map the interaction network of the following HSP70: HSPA1A, HSPA1L, and HSPA6 in HeLa cells. These 3 human proteins are the more or exclusively stress-inducible members of the family and were shown to have differential regulation and functionalities. We will detect their interactome upon their overexpression with the Bio-ID tag in normal growing cells and upon heat shock and recovery. It's common knowledge that HSP70 chaperones are not working in isolation but are (co)regulated by co-chaperones; for a minimal machinery it is assumed that they need at least interaction with one J-Domain Protein (JDP), albeit this has been disputed for HSPA6 (HAGEMAN et al 2011), and one of the so-called NEFs, for which e.g. differential affinities were found between e.g. HSPA1A and HSPA1L (SERLIDAKI et al., 2020). In this sense, to confine the experimental set-up, we will test the impact of overexpression or depletion of one from each group. DNAJB1 and HSP110, on the interactome of the 3 HSP70s. In addition, we will also test the impact of the human HSP70-escort protein 1 (hHep1) on the interactome. hHep1 was identified in our group as potential co-chaperone of HSPA1A, able to activate and remodel its supramolecular assemblies and increase its ATPase activity (DORES-SILVA et al., 2021b; KIRALY et al., 2020). The hHep1 role for such HSP70 assemblies in e.g. substrate interaction is still unknown, nor is it know whether it also impedes the formation and similarly acts on HSPA1L or HSPA6. To our knowledge, this will be the first approach to assess 1) differential substrate affinity of cytosolic isoforms; 2) importance of co-chaperones expression on HSP70 interactomes and how it may differ between 3 highly homologous, all heat-inducible isoforms; 3) the role of HSP70 supramolecular assemblies and its regulation for substrate interactions; 4) the further discovery of co-chaperones-dependent cytosolic HSP70 functions. The results will reveal the putative functional relevant of why cells have evolved 3 highly homologous proteins, all heat-inducible to combat stress-induced damages. (AU)