During the period of this grant we made significant progress in understanding the interactions between the Hsp40 chaperones and their unfolded, misfolded, and aggregated client proteins
We discovered that Hsp40 members from class A (DNAJA1 and DNAJA2) and class B (DNAJB1 and DNAJB4) present markedly different substrate specificities, despite the high homology between the two groups. For example, while class A Hsp40s can bind to monomeric tau protein and prevent its incorporation into the amyloid fibers, while class B chaperones specifically bind to tau seeds / nuclei and, by doing so, prevent the nucleation and growth of tau fibers. We further uncovered that, while the C-terminal domain II of both DNAJA2 and DNAJB1 recognizes tau nuclei and mature fibers, the diverging specificities between the chaperones stem from fundamental differences in the functionality of their C-terminal domain I binding sites - with only CTDI of DNAJA2 being capable of selectively binding to the exposed hydrophobic regions in the aggregation-prone repeats of tau monomers. Using a combination of kinetic assays and negative stain EM, we then found that these distinct binding profiles allow the two chaperone classes to interrupt the tau aggregation pathways in different ways - DNAJB1 efficiently prevents the formation of tau nuclei (seeds), while DNAJA2 mainly prevents the incorporation of tau monomers into both nuclei and the growing tau fibers. These findings suggest that the two classes of the Hsp40 chaperones play distinct roles in the prevention of a number of neurodegenerative disorders (such as Alzheimer’s disease) associated with tau aggregation.
In addition, using advanced NMR methods we found that Hsp40s utilize their C-terminal domain-I binding sites to recognize exposed hydrophobic residues present in unfolded and misfolded residues. Once bound to the chaperone, the clients adopt a largely unfolded extended conformation that enhances the breakage of any transient secondary structures, and by doing so prepares the client protein for recognition and refolding by the Hsp70 chaperone system.
Lastly, we found that Class A Hsp40 chaperones, in addition to recognizing largely exposed hydrophobic regions through their CTDI client binding domain, can also function through a novel chaperoning mechanism. These chaperones use unique β-hairpin binding sites, located in their zinc finger-like region (ZFLR), to sense the very initial stages of misfolding in β-sheet rich proteins. The chaperones recognize the increase in protein dynamics and transient breakage of hydrogen bonds that happen due to destabilization of the client protein and, through the β-hairpin sites, bind and stabilize these proteins, protecting them from further misfolding and aggregation.
