Neuromyopathy-associated frameshift mutations in the heat shock protein b8 cause its aggregation and protostasis impairment

The small heat shock protein B8 (HSPB8) is a chaperone that assures proteostasis of striatal muscles and neurons by promoting the clearance of misfolded and damaged proteins through chaperone-assisted selective autophagy (CASA). This is achieved by the interaction of HSPB8 with the cochaperone BAG3, the heat shock protein family A (HSPA) and the E3-ubiquitin ligase STUB1, forming the CASA complex. Client proteins recognized by HSPB8 and HSPAs undergo refolding, and, if this process fails, STUB1 promotes their ubiquitination for subsequent autophagic degradation. Frameshift mutations in the last exon of the HSPB8 gene have been identified in patients affected by myopathies, with or without neurogenic involvement. These HSPB8 frameshift mutations determine the elongation of the HSPB8 protein at the carboxy terminus. Here, we show that the HSPB8 frameshift mutants display high aggregation propensity. Nevertheless, these HSPB8 mutants retain the ability to interact with CASA complex members, causing the sequestration of the HSPB8 wild-type and the other components of the CASA complex. Consequently, misfolded and ubiquitinated substrates are entrapped in HSPB8 mutant aggregates together with autophagy receptors. Notably, we show that HSPB8 mutant aggregation is driven neither by the CASA members nor autophagy receptors. Instead, the mutated C-terminal sequence of the HSPB8 mutants possesses intrinsic properties to aggregation, negatively affecting muscle cell differentiation. In conclusion, we report a gain of toxic function mechanism through which HSPB8 frameshift mutations are detrimental to muscle and neuronal cells.