Shared mechanisms of pathogenicity of frameshift mutations in the heat shock protein B8 associated with neuromyopathies

Introduction: The heat shock protein B8 (HSPB8) is a chaperone highly expressed in muscle cells, where it favors the degradation of damaged structural proteins through chaperone-assisted selective autophagy (CASA). Additionally, in neurons, HSPB8 enhances the clearance of misfolded proteins associated with motoneuron diseases. This is achieved by the interaction of HSPB8 with the cochaperone BAG3, forming the CASA complex together with the heat shock protein family A (HSPA) and the E3-ubiquitin ligase STUB1. The chaperones of the CASA complex recognize the substrate, which undergoes refolding or STUB1-mediated ubiquitination. Once ubiquitinated, the client protein is routed to autophagic degradation. Different HSPB8 frameshift mutations (p.P173Sfs*43, p.Q170Gfs*45, and p.T194Sfs*23) have been identified in myopathies, with or without neuropathy. These HSPB8 frameshift mutations cause the same elongation of the HSPB8 protein at the carboxy-terminus and a variable modification of the carboxy-terminal region. Results: Our results indicate that HSPB8 frameshift mutants are characterized by insolubility and cytoplasmic aggregation when expressed in cells. Despite the elongated carboxy-terminus, the HSPB8 frameshift mutants retain the ability to interact with the HSPB8 wild-type and the partner BAG3, causing the sequestration of all the CASA complex members. As a result, misfolded and ubiquitinated client proteins are entrapped in HSPB8-mutants aggregates together with autophagy receptors, indicating a general failure of proteostasis. Notably, we show that HSPB8 mutant aggregation is driven neither by the CASA members nor by autophagy receptors. Instead, the mutated carboxy-terminal sequence of the HSPB8 mutants possesses intrinsic properties promoting aggregation. Conclusion: In summary, here we describe a common pathomechanism through which different HSPB8 frameshift mutations negatively affect muscle and neuronal cell biology, causing neuromyopathies.