HSP-1-specific nanobodies alter chaperone function in vitro and in vivo

Targeted regulation of 70 kDa heat shock protein (HSP70) chaperones, particularly the essential cognate heat shock protein (HSC70) and its Caenorhabditis elegans ortholog (HSP-1), may hold the key to improving cellular proteostasis and ameliorating aging-associated conditions linked to protein misfolding and aggregation. However, tools to selectively alter HSP70 chaperone activity remain elusive. In this study, we pioneer the development of two novel nanobodies, B12 and H5, which specifically bind to both recombinant and endogenous HSP-1. We show that these nanobodies, differing by only two amino acids in their complementarity-determining regions, bind specifically to HSP-1 and effectively reduce both HSP-1 ATPase activity and protein folding capacity in a dose-dependent manner in vitro. We further demonstrate in vivo expression of B12, but not H5, in transgenic C. elegans strains reduces heat-stress survival and proteotoxic-stress resistance, mirroring the effects of hsp-1 knockdown via RNA interference. Our findings suggest that these nanobodies can serve as effective and specific tools for inhibiting HSP-1 chaperone activity in vivo. These discoveries provide a foundation for future research exploring the therapeutic potential of HSP70-targeting nanobodies in aging and protein misfolding diseases.