Aberrant biochemical and functional behaviour of P209 mutants of the Bcl-2-Associated Athanogene 3

The chaperone-assisted selective autophagy (CASA) acts in the maintenance of a normal proteome in neurons, particularly motoneurons, and muscle cells. CASA has been firstly described in muscle cells, where it assures the disposal of damaged structural proteins. In neurons, CASA acts in the removal of misfolded proteins which, if not eliminated, accumulate as oligomeric toxic species. CASA relies on the formation of CASA complex, which consists of the Bcl-2-Associated Athanogene 3 (BAG3), the Heat Shock Proteins HSPB8 and HSP70 and the ubiquitin E3-ligase CHIP. BAG3 is the scaffold protein that permits the assembly of CASA complex and the dynein-mediated targeting of substrates to the microtubule-organizing center, where aggresomes form. Mutations in CASA complex members are associated to diseases that affect neurons and muscle cells. Regarding BAG3, three mutations at position P209 in one of the two IPV domains, which enable the interaction with HSPB8, cause myopathies (P209Q/L) or neuropathy (P209S). Instead, a mutation in the BAG domain (E455K) is associated to cardiomyopathy. Here, by analysing the activity of BAG3 in the clearance of SOD1-G93A, which causes Amyotrophic Lateral Sclerosis and is a well-known misfolded substrate of the CASA complex, we showed that P209 mutations are associated with a detrimental function of BAG3 in the removal of misfolded prone proteins. In addition, we demonstrated that BAG3 P209 mutants are themselves prone to aggregate. BAG3 P209 mutants form high molecular weight species insoluble to mild detergents and detectable by complementary techniques. Our results also showed that P209 mutant aggregates localize in the cytoplasm and concentrate in the perinuclear region, site of aggresome formation. Instead, the BAG3 E455K mutant is characterized by high solubility and no aggregate formation. In conclusion, our data show that Proline at position 209 in the IPV domain is critical for BAG3 solubility and activity against misfolded proteins.