Pro209 mutations on the Bcl-2-Associated Athanogene 3 BAG3 : common molecular behaviors of mutants linked to distinct diseases

Proteostasis is essential for cells and is the result of a dynamic equilibrium between protein synthesis and disposal. Protein disposal can occur through the autophagic system, which consists of different pathways converging to the digestion of substrates into lysosomes. Among these pathways, the Chaperone Assisted Selective Autophagy (CASA) plays a fundamental role in the disposal of damaged/misfolded proteins in neurons and muscle cells. CASA is mediated by a heteromeric complex in which the Bcl-2-associated athanogene 3 (BAG3) acts as a scaffold protein by binding HSPB8 and damaged/misfolded proteins for their ubiquitination and routing to autophagosomes. Mutations in BAG3 are linked to diseases that affect muscle and neuronal cells. Noteworthy, P209 substitutions (P209L/Q/S) have been linked to three different diseases that range from myopathies to neuropathies with different onsets and severities. These mutations locate in one of the two IPV domains involved in HSPB8 binding. Here, we investigate the behaviour of BAG3 P209 mutations in respect to the wild-type (wt) and the E455K mutation located in the HSP70 interacting region and associated to dilated cardiomyopathy. To this aim, we transfected BAG3 mutants in HSPB8 stable transfected HEK293T cells. Our results indicates that P209 mutants are characterized by a decreased solubility. In fact, Western Blot (WB) showed low soluble levels of P209 mutants, while these mutant proteins were enriched in insoluble fractions compared to wt BAG3 and the E455K mutant. Similarly, Filter Retardation Assay (FRA) indicated high insoluble levels of P209 mutants. Interestingly, its functional partner HSPB8 shows a similar trend in protein levels, both in WB and FRA. Immunofluorescence assay confirmed the presence of big and multiple aggregates of P209 mutants. Immunoprecipitation assays revealed that P209 mutations do not cause a loss of interaction with HSPB8. Evaluation of the proautophagic activity of P209 mutants against the Superoxide Dismutase 1 G93A mutant, which is an Amyotrophic Lateral Sclerosis-linked misfolded protein and substrate of the CASA complex, revealed a loss of function of P209 mutants in misfolded protein clearance. In conclusion, despite the different phenotypes associated to P209 mutants, they share common aberrant behaviour and dysfunction, suggesting the presence of other BAG3 functions, cell specific features, genetic/environmental factors that may impact on the development of the diseases.