Caracterização estrutural, funcional e estabilidade térmica das proteínas humanas HspA5 e HspA8: uma abordagem comparativa

Abstract

Heat shock proteins (Hsps) are important components of cell protein quality control (PQC), acting on protein synthesis, folding, traffic and degradation. The central component in the PQC is the Hsp70 family, which consists of highly conserved, ubiquitous and ATP-dependent molecular chaperones. These chaperones can assume different conformations that are regulated by their dynamic association with the adenosine nucleotides (ATP and ADP), as well as by their interaction with cochaperones and client proteins. In humans there are several Hsp70 proteins, most of which are found in the cytoplasm, while others are specific for organelles. The objective of this work was to structurally and functionally characterize the cytosolic (HspA8) and endoplasmic reticulum (HspA5) human Hsp70 proteins, using similar methodologies.For this, the recombinant hHsp70 proteins were expressed, purified and then characterized for structure, stability and function by means of biophysical tests. The results show that the HspA5 and HspA8 proteins were produced in their folded form, with secondary structure content rich in α-helices and as slightly elongated monomers in solution. Such Hsp70 showed similar chemical and thermal stability, however two thermal transitions (Tm) were observed for HspA5 and three for HspA8, indicating structural differences that result in distinct thermal stability. Changes in secondary and tertiary structure were observed in the proteins under study in the presence of nucleotides, which also caused the thermal stabilization of hHsp70. The presence of Ca 2+ and Mg 2+ ions favored the binding of hHsp70 to ATP and ADP nucleotides in different ways, and unlike HspA5, the HspA8 protein interacted with Ca 2+ even in the absence of nucleotides. A process thermodynamically governed by enthalpy and entropy was observed for the interaction of the adenosine nucleotides with the HspA5 and HspA8 proteins. hHsp70 showed similar and relatively low ATP activity, however the affinity of HspA5 for ATP was higher than that of HspA8. Additionally, the HspA5 and HspA8 proteins were effective in preventing the aggregation of model client proteins, with different specificities. In summary, the data obtained demonstrate that, although they share 66% of identity, the human proteins HspA5 and HspA8 present structural and functional peculiarities, thus enriching the knowledge about such macromolecules.