Bcl2-Associated Athanogene 3 mutants : solubility and aggregation prone behaviour assessment by different techniques

Mutations in the Bcl2-Associated Athanogene 3 (BAG3) have been reported in neuropathies and myopathies. BAG3 is a co-chaperone of the BAG family. Its multidomain structure lets BAG3 to interact with different partners, making BAG3 involved in several pathways, such as apoptosis, migration and protein quality control (PQC) system. BAG3 role in PQC relies in its ability to act as a scaffold protein in the Chaperone Assisted Selective Autophagy (CASA) complex, which removes misfolded proteins. In particular, BAG3 directly interacts with the Heat Shock Proteins HSPB8 and HSP70, through two IPV domains and a BAG domain, respectively, and indirectly, with the ubiquitin E3 ligase CHIP and the dynein motor complex. The pathway is based on misfolded substrate recognition by HSPB8 and ubiquitination by CHIP. Once the substrate is ubiquitinated, it is routed to the aggresome by dynein-mediated retrograde transport, for autophagic disposal. CASA is fundamental in misfolded/damaged proteins clearance both in motoneurons and muscle cells. The IPVs are relevant for BAG3 since their mutations are related to early onset severe (P209L) to mild, adult onset myopathy (P209Q) or late-onset Charcot Marie Tooth CMT2 (P209S). Mutation in the BAG domain (E455K) is linked to cardiomyopathy. Here, we assessed the aggregation prone behaviour of BAG3 mutants in respect to the wild type (WT) BAG3, using different and complementary assays. First, we performed an NonidetP40 soluble/insoluble protein extraction on HEK293T expressing a V5-tag HSPB8 and transiently transfected with GFP-tag BAG3 WT or mutants, followed by Western Blot (WB) and Filter Retardation Assay (FRA) that allows the immunodetection of insoluble oligomeric/aggregated species. We observed higher levels of P209 mutants in the NP-40 insoluble fraction, which correlated with lower protein levels in the NP-40 soluble fraction. Instead, E455K was present only in the soluble fraction. The formation of aggregates was confirmed in immunofluorescence (IF) assay: P209 mutants formed multiple small aggregates or big aggregates in the perinuclear region. Because of the low sensitivity of IF/FRA assays in the detection of small differences among P209 mutants, we performed Flow cytometric analyses of inclusion and trafficking (FLoIT) assay, which allows to quantify cytoplasmic aggregates by analysing cell lysates with intact nuclei. By FLoIT assay, we confirmed the presence of cytoplasmic aggregates of P209 mutants, but the number of P209L mutant inclusions were statistically higher in respect to P209S/Q, highlighting a mutation-phenotype relationship. Finally, we tested the capability of BAG mutants to remove misfolded proteins: using the misfolded Amyotrophic Lateral Sclerosis-associated G93A-SOD1 paradigm. We observed a loss of function of P209 BAG3 mutants in the clearance of G93A-SOD1 insoluble species. In conclusion, we demonstrated a different solubility and aggregation prone behaviour of P209 BAG3 mutants by different and complementary techniques and their loss of function in the removal of misfolded prone proteins.