P209 point mutations in the Bcl-2 Associated Athanogene 3 impact in the chaperone assisted selective autophagy

Misfolded and aggregated proteins are the major hallmark of neurodegenerative diseases (NDs), suggesting that proteostasis failure occurs in affected neurons. To cope with proteotoxic stress, cells, including neurons, rely on the Protein Quality Control (PQC) system, a network of proteins that acts in the refolding or the disposal of damaged and misfolded proteins. The Chaperone Assisted Selective Autophagy (CASA) pathway protects neurons and muscle cells from proteotoxicity, by promoting the disposal of misfolded and aggregate prone substrates. Essential players of CASA are the Bcl-2-Associated Athanogene 3 (BAG3) and the small Heat Shock Protein B8 (HSPB8), which interact together through two IPV domains of BAG3. By HSPB8 recognition and dynein-mediated retrograde transport, misfolded substrates are concentrated to the perinuclear aggresome. Mutations of BAG3 and HSPB8 are related to myopathies and neuropathies, while the induction or the overexpression of the WT forms of these CASA members enhance the clearance of proteins aggregates both in cell and animal models of Amyotrophic Lateral Sclerosis (ALS) and other NDs. Here, we investigate the biochemical behaviour of three BAG3 mutations in the second IPV domain (P209L/S/Q) responsible of myopathies or neuropathies. By using complementary techniques, we show that P209 mutants accumulate forming high molecular weight species, which are insoluble in mild detergents. By analysing protein localization, we show that P209 mutants form aggregates, partly dispersed in the cytoplasm and often associated to the nuclear envelope which resembles aggresomes. Furthermore, we show that the presence of these mutants is associated with an increase of insoluble species of the ALS-related misfolded SOD1G93A protein, which is a substrate of CASA complex. In conclusion, mutations at position P209 are characterized by a CASA dysfunction, which could dysregulate proteostasis maintenance.