The role of BAG3 in dilated cardiomyopathy and its association with Charcot-Marie-Tooth disease type 2.

Figures

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  Figure 1.. BAG3 consists of many domains, allowing the multifunctional protein to attach to various proteins and participate in several cellular functions. The different regions of BAG3 include the WW domain, two conserved IPV motifs, two 14-3-3 binding motifs, PxxP region, and the conserved BAG domain, which are encoded by four exons. The WW domain is located within the N-terminal region, while the conserved BAG domain is located within the C-terminal region. The phosphorylation sites of BAG3 have been marked in six locations at serine and tyrosine amino acids. The PxxP region contains a caspase cleavage site (KEVD) along with the C-terminal region (LEAD) (reproduced from the open source, from Stürner, Behl, 2017, mod.) 3.

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  Figure 2.. sHSP-substrate complexes are formed in which the outer surface consists of dynamic sHSPs in equilibrium shifted toward sHSPs being bound. These assemblies contain a stable sHSP-substrate center, which initially remains intact when Hsp70 and cochaperones displace the dynamic sHSPS in the outer surface. After successfully outcompeting the superficial sHSPs in binding to substrates, Hsp70 and Hsp100 identify the misfolded proteins and begin refolding, causing the sHSP–substrate assemblies to disassociate (reproduced from the open source, from Żwirowski et al., 2017, mod.) 17.

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  Figure 3.. ATPase cycle of Hsp70. When ADP binds to Hsp70, SBD is configured to its closed conformation and has higher affinity for substrates. The lid domain closes onto the substrate that is bound to the SBD. NEFs catalyze the release of ADP and the binding of ATP to the NBD, which induces an open configuration of the lid domain. Hsp70 now has lower affinity for the substrate, leading to substrate release from the SBD. J-domain proteins are important for facilitating ATP hydrolysis to produce ADP. The ADP-bound-state once again configures for the lid domain to close upon a new substrate within the cleft (reproduced from the open source, from Kabani, Martineau, 2008, mod.) 24.

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  Figure 4.. Under proteotoxic stress conditions, native proteins can become unfolded into a misfolded protein state. Molecular chaperones, such as the HSP family, bind to the misfolded proteins inhibiting aggregation and initiating refolding. If successfully remodeled, the misfolded protein will become a native protein. If refolding fails, non-native proteins may also be degraded by UPS, involving the binding of BAG1 to the Hsp70-substrate complex. Upon aggregation of misfolded proteins, protein aggregates are directed to the autophagy system with the recruitment of BAG3 (reproduced from the open source, from Stürner, Behl, 2017, mod.) 3.

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  Figure 5.. p62 overexpression protects the proteasome stability of a cell by increasing the number of ubiquitinated protein aggregates that are sent to the autophagy lysosomal pathway. A deficiency in UPS decreases the number of ubiquitinated proteins degraded by UPS, leading to increased p62 expression and phosphorylation on S405 and S409. Upon UPS inhibition, the levels of Nrf2 increases, inducing p62 overexpression. The greater concentration of p62 better enables it to compete with Nrf2 for Kea1, forming p62-Keap1 complexes, which assist with the formation of ubiquitinated protein aggregates for degradation by autophagy. HDAC6 is critical in the autophagy lysosomal pathway, but its synthesis is directly inhibited by UPS inhibition and p62 overexpression. However, p62 overexpression can be inhibited by an increase in autophagy. Therefore, the ratio of p62 to HDAC6 is essential for cellular stability (reproduced from the open source, from Liu et al., 2016, mod.) 23.

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  Figure 6.. Ultrastructural analysis of cardiac tissue from control 1 (A-C) and BAG3 P209L-mutant patient (D-I). (D) BAG3-mutant cardiac tissue has altered Z-disk structure and extreme myofibrillar disintegration with cells of varying diameters and vacuoles. (E) Recurring Z-disk alterations and substantial electron dense material originating from Z-disks. (F) Central accumulation of mitochondria and visible lipofuscin granules. (G,H) Bundles of electron-dense material are abundant and partly surrounded by electron dense vacuoles. (I) In spaces between myofibrillar bundles, empty vacuoles and glycogen or electron dense vacuoles are present. Scale: 2.5 μm (A,D,G) and 0.5 μm (B,C,E,F,H,I) (reproduced from the open source, from Schänzer et al., 2018, mod.) 33.

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  Figure 7.. Protein quality control in neurons and other mechanically strained tissue cells. Upon the misfolding or unfolding of a protein, molecular chaperones bind to these non-native proteins and facilitate the process of refolding. However, if refolding is unsuccessful, the HSC70 chaperone is responsible for routing the misfolded protein to one of two pathways for degradation. The binding of BAG1, a nucleotide exchange factor from the Bcl2-associated athanogene family to the HSC70 CHIP complex allows for the inhibition of HSC70’s chaperone activity and the polyubiquitination of the client by CHIP. This interaction directs the misfolding protein to UPS, in which a proteasome breaks down the client. Conversely, the binding of BAG3 and HSPB8 to the complex reroutes the substrate to CASA. In this pathway, 14-3-3 protein and dynein are responsible for facilitating the transportation of the CASA complex along microtubules to MTOC, site of autophagosome formation. Here, autophagy receptor p62/SQSTM1 attaches to the polyubiquitin chain of the misfolded protein and directs the client to the autophagosome through its interaction with the LC3 receptor. The misfolded protein is degraded once a…

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  Figure 8.. In the presence of BAG3 wild type, the CASA complex assembles, attaches to a protein aggregate, to an autophagosome in MTOC, where the aggregate becomes degraded. The P209L mutation increases the aggregation propensity of BAG3 and may impair the ATPase cycle of Hsp70, causing Hsp70 to be unable to release the protein aggregate. As a result, the protein aggregate may remain bound to the CASA complex and not be delivered to an autophagosome, leading to the accumulation of CASA complexes in MTOC. These complexes become relocated to aggresomes, decreasing the accessibility of essential and functional proteins, including Hsp70, HSPB8, and p62 (reproduced from the open source, from Adriaenssens et al., 2020, mod.) 13.

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  Figure 9.. Model for sHSP chaperone activity. (i) sHSP dimers and monomers continuously alternate between being bound in an oligomer or released into the environment. (ii) Misfolded and unfolded proteins expose hydrophobic regions to which sHSPs bind using a hydrophobic groove in the ACD, forming large substrate-sHSP complexes (iii). (iv) The denatured substrate can be refolded by Hsp70, ATP, and a co-chaperone which displace the sHSP-substrate complex and attach to the substrate. (v) Lack of adequate sHSPs can result in the formation of protein aggregates, which are harder to eliminate. (vi) Cellular proteases can also degrade the substrate from an sHSP-substrate complex, but this pathway is less well-known. This model does not elucidate all possible sHSP proteins actions (reproduced with the permission of the publisher, from Basha et al., 2012, mod.) 21.

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  Figure 10.. Structure and grooves of alpha-crystallin domain (ACD) within sHSPs. (A) ACD dimer with β-strand and L5/6 loops connecting β5 strands to β6+7 strands. The dashed line represents the dimer interface. The β-strand is divided into a “bottom” sheet with 6β strands (in gray) consisting of β4, β5, and β6+7 from both sHSP monomers 6. The “top” sheet is composed of 3β strands (in magenta), including β3, β8, and β9 6. (B) Surface front view of the dimer interface groove formed by the β6+7 strands. The L5/6 loops on either side of the groove are in “loop-up” position 6. The 6-stranded β sheet is indicated by the dashed yellow line and the 3-stranded β sheets are indicated by the solid yellow line. (C) Color coded portions of the dimer interface groove from the same front view. Cyan residues represent the floor of the groove and buff residues represent the sides of the groove. (D) Top view of ACD structure shows hydrophobic β4/β8 groove (edge groove) with the two holes and wall identified (reproduced from the open source, from Klevit, 2020, mod.) 6.

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  Figure 11.. Model for subunit exchange within sHSPs. Unbound ACD dimer is white, and within the oligomer, NTR is blue, CTR is red, and ACD is green. (1) Free CTR within the oligomer recruits an unbound ACD dimer to the oligomer. (2) A subunit within the oligomer and the recruited subunit dissociates. (3) A heterodimer is formed with one new subunit and one original subunit (reproduced from the open source, from Delbecq et al., 2015, mod.) 51.

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  Figure 12.. Representation of the NTR-ACD interactions. (A,B) intrachain, (C) intradimer, and (D) interdimer interactions between sHSPs. (E,F) Structure of NTR with distal region in red, aromatic region in green, conserved region in orange, Trp-rich region in yellow, insertion region in purple, and boundary region in blue. ACD is in gray and CTR is in white. Interactions indicated by solid lines are supported by evidence from the study and the dotted lines are interactions that the researchers “believe are likely to occur” 46. This figure demonstrates the degree of flexibility in NTR’s interactions within an sHSP or with other sHSPs (reproduced from the open source, from Clouser et al., 2019, mod.) 46.