BAG3 Is a Modular, Scaffolding Protein that physically Links Heat Shock Protein 70 (Hsp70) to the Small Heat Shock Proteins

Abstract

Small heat shock proteins (sHsps) are a family of ATP-independent molecular chaperones that are important for binding and stabilizing unfolded proteins. In this task, the sHsps have been proposed to coordinate with ATP-dependent chaperones, including heat shock protein 70 (Hsp70). However, it isn’t yet clear how these two important components of the chaperone network are linked. We report that the Hsp70 co-chaperone, BAG3, is a modular, scaffolding factor to bring together sHsps and Hsp70s. Using domain deletions and point mutations, we confirmed that BAG3 uses both of its IPV motifs to interact with sHsps, including Hsp27 (HspB1), αB-crystallin (HspB5), Hsp22 (HspB8) and Hsp20 (HspB6). BAG3 does not appear to be a passive scaffolding factor; rather, its binding promoted de-oligomerization of Hsp27, likely by competing for the self-interactions that normally stabilize large oligomers. BAG3 bound to Hsp70 at the same time as either Hsp22, Hsp27 or αB-crystallin, suggesting that it might physically bring the chaperone families together into a complex. Indeed, addition of BAG3 coordinated the ability of Hsp22 and Hsp70 to refold denatured luciferase in vitro. Together, these results suggest that BAG3 physically and functionally links Hsp70 and sHsps.

Loss-of-function mutations in co-chaperone BAG3 destabilize small HSPs and cause cardiomyopathy

“…This stringent cutoff yielded 123 potential binding partners. Interestingly, several ATPase-independent sHSP family members (HSPB5, -6, and -8), and ATPase-dependent HSP70 family members (HSP70 and HSC70), previously identified as part of the BAG3 complex (35), were among the 123 potential binding partners identified in this manner. Notably, of the 123 potential binding partners, 39 were overlapping with proteins identified as being increased within the insoluble protein fraction from BAG3-CKO hearts ( Figure 4, B and C, Figure 3, B and E, and Supplemental Table 1).…”

“…Notably, the BAG3 E455K mutation did not affect BAG3 binding to sHSPs ( Figure 5C) (35). Taken together, we concluded that the ability of BAG3 to interact with HSP70 was essential to the stability of sHSPs.…”

“…As a co-chaperone protein, BAG3 interacts with both ATP-dependent high-molecular-weight HSPs and ATP-independent small HSPs (sHSPs) in large, functionally distinct multi-chaperone complexes (18,(32)(33)(34). BAG3 interacts with sHSPs through its 2 IPV motifs (32,33,35). To determine the effects of Bag3 deletion on components of the chaperone complex, we evaluated protein samples from isolated adult cardiomyocytes of 2-month-old CKO mice and control mice.…”

“…Compared with the 3XFlag-HA- GFP control affinity purification, multiple bands were present in the immune-precipitated BAG3-containing complex ( Figure 4A). Using gel digestion MS, we identified the most intense band at 70 kDa as HSP70 and HSC70, which are known interactors of BAG3 (35). To comprehensively profile the BAG3 interactome, we performed on-bead digestion following TAP and subjected the immunoprecipitated products to label-free proteomics (51).…”

A BAG3 chaperone complex maintains cardiomyocyte function during proteotoxic stress

“…As a co-chaperone, the BAG3 protein modulates ATP turnover and the protein folding activity of HSC70 (constitutive) and HSP70 (induced) (18,19). It also physically and functionally interacts with a separate family of chaperones, the small heat shock proteins (HSPB genes), and acts as a scaffold that links the functions of HSP70 and HSPB families (20)(21)(22). In human cells, the BAG3-HSP70-HSPB complex regulates degradation of ubiquitinated proteins via the proteasome and autophagy pathways (23)(24)(25)(26).…”