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Even under conditions that threaten protein integrity, cell and organism survival depend on the ability to maintain the proteome. In stressed cells, BCL2-associated athanogene 3 (BAG3) is essential for protein homeostasis (proteostasis). Due to its multi-domain structure, its involved in a variety of processes that are critical to proteome maintenance. BAG3 promotes molecular chaperone activity, sequesters and concentrates of misfolded proteins, initiates autophagy processing, and balances transcription, translation and degradation.
BAG3 promotes chaperone activity
BAG3 is tightly integrated into the cell chaperone network, where the competition and cooperation between the complexes are essential for the formation of functional different chaperone complexes. BAG3 belongs to the family of co-chaperones having the BAG domain, which bind to the N-terminal ATPase domain of the 70 kDa heat shock protein (HSP70s). HSP70s are highly versatile chaperone proteins that recognize non-native proteins by transiently hydrophobic regions regulated by ATP. The BAG domain accessory partner acts as a nucleotide exchange factor (NEF) for HSP70 to facilitate client release and accelerate the chaperone cycle along with the J-domain co-chaperones, such as DNAJB6. Importantly, BAG3 exerts its HSP70 modulating activity in multicomponent chaperone complexes. Two IPV motifs mediate the binding of BAG3 to small heat shock proteins (sHSPs), such as HSPB6 and HSPB8. sHSP forms an oligomer assembly that isolates misfolded proteins in a state that allows for refolding or further processing. The interaction of sHSPs with BAG3 provides a means to couple their retention function to the folding activity of the multifunctional HSP70.
The most usual defense strategy against the toxic consequences of protein aggregation in cells is concentrate and isolate of misfolded proteins. The sHSPs play an important role in this sequestration, BAG3 regulate this process by interating with sHSPs, and BAG3 also promoting intracellular trafficking of misfolded proteins. Two RSQS motifs and a proline-rich PxxP domain contribute to the binding of the co-chaperone to the microtubule-motor kinesin; this involves 14-3-3 adaptor proteins and appears to be regulated by co-chaperone phosphorylation. Associated with kinesin movement allows BAG3 to induce HSP70 customers to retrograde along the microtubules to the perinuclear sites, so-called aggregates. The deposition of misfolded conformers in aggregates minimizes toxic interference to essential cellular processes and promotes efficient aggregate clearance by macroautophagy.
Fig. 1. BAG3 plays a critical role in the formation of multi-chaperone complexes and the stability of sHSPs. (Fang, et al.The Journal of Clinical Investigation. 2017).
BAG3 initiates an autophagy pathway
Autophagy is a pathway by which lysosomal hydrolases degrade excess or damaged cellular components. The cellular contents are engulfed by the double membrane, resulting in the formation of autophagosomes that eventually fuse with the lysosomal vesicles to achieve degradation of the contents. The first time autophagy was found in starved cells was a rather non-selective process. However, in recent years, an increasing number of selective autophagy pathways have been described which depend on the specific choice of cargo for autophagy delivery to lysosomes. Selection typically involves the initial labeling of the cargo with a degradation marker ubiquitin and subsequent recognition by an autophagy ubiquitin adaptor that provides linkage to an autophagosome precursor membrane. BAG3 and HSP70-associated ubiquitin ligase CHIP cooperate to induce this selectivity, ubiquitin-dependent autophagy pathway to treat chaperone clients. CHIP interacts with the C-terminus of HSP70, which covers the peptide binding site of the chaperone, and cooperates with ubiquitin conjugating enzymes (UBCs) in the ubiquitylation of HSP70-bound clients. In this case, the chaperone protein HSP70 becomes a degradation factor. In fact, CHIP-mediated ubiquitination has been shown to direct a wide range of HSP70 clients to different degradation pathways.
Cytosol clients are typically target to the proteasome, a protease complex specifically designed for the conversion of ubiquitinated proteins, and the plasma of the membrane is identical to its CHIP-mediated ubiquitination. However, when CHIP ubiquitylates clients that are presented by HSP70-BAG3-HSPB8 complexes, their autophagic degradation is favoured. The reason for this is not completely understood, but efficient recruitment of the autophagic ubiquitin adaptor SQSTM1 appears to be a decisive step, which promotes client loading onto phagophore membranes. Of note, other chaperone-associated ubiquitin ligases, such as the quality control ligase parkin (PRKN), may substitute for CHIP during the initiation of autophagy. Indeed, a recent study reveals a cooperation of BAG3 with parkin in the autophagic clearance of damaged mitochondria. It is also important to note that the pathway induced by BAG3 is distinct from chaperone-mediated autophagy (CMA). CMA does not depend on autophagosome formation, but instead involves a direct transfer of HSP70 clients across the lysosomal membrane. To emphasize this distinction, researchers coined the term chaperone-assisted selective autophagy (CASA) for the BAG3-induced pathway.
Fig. 2. BAG3 and Parkin translocate from cytosol to mitochondria upon depolarization. (Tahrir, et al. Journal of cellular physiology. 2017).
BAG3-mediated cytoskeleton maintenance is not restricted to striated muscle
In smooth muscle cells and non-muscle cells, filamin is associated with actin stress fibers, which form actin stress fibers when force is applied from cells or forces are generated within the cells during adhesion and migration. Stretching of smooth muscle cells and adhesion, spread and migration of lymphoblasts lead to the induction of BAG3, accompanied by an increase in autophagy renewal of filament proteins. In addition, BAG3 is critical for the movement and adhesion of a variety of cancer cells. Thus, CASA machine-mediated protein quality control appears to represent a broad and common mechanism for maintaining the cytoskeleton in muscle and non-muscle cells. Notably, HSPB8 and BAG3-dependent autophagy is also essential in dividing cells at stages when profound changes in cell tension occur, for instance during proper spindle orientation and the disassembly of the actin-based contractile ring during cytokinesis.