BAG3 directly interacts with mutated alphaB-crystallin to suppress its aggregation and toxicity.
ABSTRACT: A homozygous disruption or genetic mutation of the bag3 gene causes progressive myofibrillar myopathy in mouse and human skeletal and cardiac muscle disorder while mutations in the small heat shock protein ?B-crystallin gene (CRYAB) are reported to be responsible for myofibrillar myopathy. Here, we demonstrate that BAG3 directly binds to wild-type ?B-crystallin and the ?B-crystallin mutant R120G, via the intermediate domain of BAG3. Peptides that inhibit this interaction in an in vitro binding assay indicate that two conserved Ile-Pro-Val regions of BAG3 are involved in the interaction with ?B-crystallin, which is similar to results showing BAG3 binding to HspB8 and HspB6. BAG3 overexpression increased ?B-crystallin R120G solubility and inhibited its intracellular aggregation in HEK293 cells. BAG3 suppressed cell death induced by ?B-crystallin R120G overexpression in differentiating C2C12 mouse myoblast cells. Our findings indicate a novel function for BAG3 in inhibiting protein aggregation caused by the genetic mutation of CRYAB responsible for human myofibrillar myopathy.
Project description:The R120G mutation in the small heat shock-like protein alphaB-crystallin (CryAB(R120G)) causes desmin-related myopathy (DRM), which is characterized by the formation of desmin- and CryAB-containing aggregates within muscle fibers. Mice with cardiac-specific overexpression of CryAB(R120G) develop cardiomyopathy at 3 months and die at 6-7 months from heart failure (HF). Previous studies showed that overexpression of CryAB(R120G) results in accumulation of preamyloid oligomer (PAO). PAO is considered to be the cytotoxic entity in many of the protein misfolding-based neurodegenerative diseases. On the basis of data from mouse models of neurodegenerative diseases showing that exercise or environmental enrichment reduces the amyloid oligomer level and improves cognitive ability, we hypothesized that CryAB(R120G)-induced DRM would also respond favorably to prolonged voluntary exercise, reducing HF symptoms and rescuing the mice from premature death. Six months of voluntary exercise in CryAB(R120G) animals resulted in 100% survival at a time when all unexercised mice had died. After 22 weeks of exercise, PAO levels were decreased by 47% compared with the unexercised CryAB(R120G) control mice (P = 0.00001). Although CryAB(R120G) expression led to decreased levels of the metallomembrane endopeptidase neprilysin, normal levels were maintained in the exercised CryAB(R120G) mice, and in vitro loss-of-function and gain-of-function experiments using adenovirus-infected cardiomyocytes confirmed the importance of neprilysin in ameliorating PAO accumulation. The data demonstrate that voluntary exercise slows the progression to HF in the CryAB(R120G) DRM model and that PAO accumulation is mediated, at least in part, by decreased neprilysin activity.
Project description:HSPB5 (also called ?B-crystallin) is a ubiquitously expressed small heat shock protein. Mutations in HSPB5 have been found to cause cataract, but are also associated with a subgroup of myofibrillar myopathies. Cells expressing each of these HSPB5 mutants are characterized by the appearance of protein aggregates of primarily the mutant HSPB5. Like several members of the HSPB family, HSPB5 can form both homo-oligomeric and hetero-oligomeric complexes. Previous studies showed that co-expression of HSPB1 and HSPB8 can prevent the aggregation associated with the HSPB5 (R120G) mutant in cardiomyocytes and in transgenic mice. In this study, we systematically compared the effect of co-expression of each of the members of the human HSPB family (HSPB1-10) on the aggregation of three different HSPB5 mutants (R120G, 450 ? A, 464 ? CT). Of all members, co-expression of HSPB1, HSPB4 and HSPB5 itself, most effectively prevent the aggregation of these 3 HSPB5 mutants. HSPB6 and HSPB8 were also active but less, whilst the other 5 HSPB members were ineffective. Co-expression of Hsp70 did not reduce the aggregation of the HSPB5 mutants, suggesting that aggregate formation is most likely not related to a toxic gain of function of the mutants per se, but rather related to a loss of chaperone function of the oligomeric complexes containing the HSPB5 mutants (dominant negative effects). Our data suggest that the rescue of aggregation associated with the HSPB5 mutants is due to competitive incorporation of its partners into hetero-oligomers hereby negating the dominant negative effects of the mutant on the functioning of the hetero-oligomer.
Project description:Transgenic mice with cardiac specific overexpression of mutated alphaB-crystallin (CryAB(R120G)) display Desmin-related myopathy (DRM) with dilated cardiomyopathy and heart failure. Our previous studies showed the presence of progressive mitochondrial abnormalities and activation of apoptotic cell death in CryAB(R120G) transgenic hearts. However, the role of mitochondrial dysfunction and apoptosis in the overall course of the disease was unclear.We tested the hypothesis that prevention of apoptosis would ameliorate CryAB(R120G) pathology and decrease morbidity.We crossed CryAB(R120G) mice to transgenic mice with cardiac specific overexpression of Bcl-2. Sustained Bcl-2 overexpression in CryAB(R120G) hearts prolonged CryAB(R120G) transgenic mice survival by 20%. This was associated with decreased mitochondrial abnormalities, restoration of cardiac function, prevention of cardiac hypertrophy, and attenuation of apoptosis. CryAB(R120G) misfolded protein aggregation was significantly reduced in the double transgenic. However, inhibition of apoptotic signaling resulted in the upregulation of autophagy and alternative death pathways, the net result being increased necrosis.Although Bcl-2 overexpression prolonged life in this DRM model, in the absence of apoptosis, another death pathway was activated.
Project description:During cell life, proteins often misfold, depending on particular mutations or environmental changes, which may lead to protein aggregates that are toxic for the cell. Such protein aggregates are the root cause of numerous diseases called "protein conformational diseases," such as myofibrillar myopathy and familial amyotrophic lateral sclerosis. To fight against aggregates, cells are equipped with protein quality control mechanisms. Here we report that NF?B transcription factor is activated by misincorporation of amino acid analogues into proteins, inhibition of proteasomal activity, expression of the R120G mutated form of HspB5 (associated with myofibrillar myopathy), or expression of the G985R and G93A mutated forms of superoxide dismutase 1 (linked to familial amyotrophic lateral sclerosis). This noncanonical stimulation of NF?B triggers the up-regulation of BAG3 and HspB8 expression, two activators of selective autophagy, which relocalize to protein aggregates. Then NF?B-dependent autophagy allows the clearance of protein aggregates. Thus NF?B appears as a central and major regulator of protein aggregate clearance by modulating autophagic activity. In this context, the pharmacological stimulation of this quality control pathway might represent a valuable strategy for therapies against protein conformational diseases.
Project description:Myofibrillar myopathy (MFM) is a group of inherited muscular disorders characterized by myofibrils dissolution and abnormal accumulation of degradation products. So far causative mutations have been identified in nine genes encoding Z-disk proteins, including ?B-crystallin (CRYAB), a small heat shock protein (also called HSPB5). Here, we report a case study of a 63-year-old Polish female with a progressive lower limb weakness and muscle biopsy suggesting a myofibrillar myopathy, and extra-muscular multisystemic involvement, including cataract and cardiomiopathy. Five members of the proband's family presented similar symptoms. Whole exome sequencing followed by bioinformatic analysis revealed a novel D109A mutation in CRYAB associated with the disease. Molecular modeling in accordance with muscle biopsy microscopic analyses predicted that D109A mutation influence both structure and function of CRYAB due to decreased stability of oligomers leading to aggregate formation. In consequence disrupted sarcomere cytoskeleton organization might lead to muscle pathology. We also suggest that mutated RQDE sequence of CRYAB could impair CRYAB chaperone-like activity and promote aggregation of lens crystallins.
Project description:Skeletal muscle and the nervous system depend on efficient protein quality control, and they express chaperones and cochaperones at high levels to maintain protein homeostasis. Mutations in many of these proteins cause neuromuscular diseases, myopathies, and hereditary motor and sensorimotor neuropathies. In this review, we cover mutations in DNAJB6, DNAJB2, ?B-crystallin (CRYAB, HSPB5), HSPB1, HSPB3, HSPB8, and BAG3, and discuss the molecular mechanisms by which they cause neuromuscular disease. In addition, previously unpublished results are presented, showing downstream effects of BAG3 p.P209L on DNAJB6 turnover and localization.
Project description:To report novel disease and pathology due to HSPB8 mutations in 2 families with autosomal dominant distal neuromuscular disease showing both myofibrillar and rimmed vacuolar myopathy together with neurogenic changes.We performed whole-exome sequencing (WES) in tandem with linkage analysis and candidate gene approach as well as targeted next-generation sequencing (tNGS) to identify causative mutations in 2 families with dominant rimmed vacuolar myopathy and a motor neuropathy. Pathogenic variants and familial segregation were confirmed using Sanger sequencing.WES and tNGS identified a heterozygous change in HSPB8 in both families: c.421A > G p.K141E in family 1 and c.151insC p.P173SfsX43 in family 2. Affected patients had a distal myopathy that showed myofibrillar aggregates and rimmed vacuoles combined with a clear neurogenic component both on biopsy and neurophysiologic studies. MRI of lower limb muscles demonstrated diffuse tissue changes early in the disease stage progressing later to fatty replacement typical of a myopathy.We expand the understanding of disease mechanisms, tissue involvement, and phenotypic outcome of HSPB8 mutations. HSPB8 is part of the chaperone-assisted selective autophagy (CASA) complex previously only associated with Charcot-Marie-Tooth type 2L (OMIM 60673) and distal hereditary motor neuronopathy type IIa. However, we now demonstrate that patients can develop a myopathy with histologic features of myofibrillar myopathy with aggregates and rimmed vacuoles, similar to the pathology in myopathies due to gene defects in other compounds of the CASA complex such as BAG3 and DNAJB6 after developing the early neurogenic effects.
Project description: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 is not 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 found that BAG3 uses both of its IPV motifs to interact with sHsps, including Hsp27 (HspB1), ?B-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 Hsp22, Hsp27, or ?B-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.
Project description:Disease-causing mutations of genes encoding small MW heat shock proteins (sHSPs) constitute a growing family of inherited myofibrillar disorders. In the present work, we found that three structurally-distinct CryAB mutants R120G, 450delA and 464delCT are mostly present in the detergent-insoluble fractions when overexpressed in H9c2 rat heart cells. We found that either over-expression or knockdown of HSPB1, a related sHSP, affects the solubility, stability, and degradation of aggregation-prone CryAB mutants. HSPB1 overexpression has negligible effects on the solubility and protein aggregates of either R120G and/or 450delA but increased the solubility and prevented formation of 464delCT aggregates. HSPB1 knockdown decreased solubility and increased protein aggregates of all CryAB mutants, indicating a key role for HSPB1 in clearance of CryAB mutants under basal conditions. We provide four lines of evidence that such selective clearance of R120G, 450delA and 464delCT mutants by HSPB1 is mediated by the ubiquitin-proteasome system (UPS). First, we found that treatment with the proteasome inhibitors increased the levels of all CryAB mutants. Second, R120G and 450delA overexpression corresponded to the accumulation of their specific ubiquitin conjugates in H9c2 cells. Third, HSPB1 knockdown directly increased the levels of all polyubiquitin conjugates. And fourth, the selective attenuation of 464delCT expression by HSPB1 over-expression was abrogated by the proteasome inhibition. We conclude that such selective interactions between CryAB mutants and HSPB1 overexpression might have important implications for the clinical manifestations and potential treatment.
Project description:Human alpha B-crystallin and small heat shock proteins HspB6 and HspB8 were mutated so that all endogenous Cys residues were replaced by Ser and the single Cys residue was inserted in a position homologous to that of Cys137 of human HspB1, i.e. in a position presumably located in the central part of beta 7 strand of the alpha-crystallin domain. The secondary, tertiary, and quaternary structures of thus obtained Cys-mutants as well as their chaperone-like activity were similar to those of their wild-type counterparts. Mild oxidation of Cys-mutants leads to formation of disulfide bond crosslinking neighboring monomers thus indicating participation of the beta 7 strand in intersubunit interaction. Oxidation weakly affects the secondary and tertiary structure, does not affect the quaternary structure of alpha B-crystallin and HspB6, and shifts equilibrium between monomer and dimer of HspB8 towards dimer formation. It is concluded that the beta 7 strand participates in the intersubunit interaction of four human small heat shock proteins (alpha B-crystallin, HspB1, HspB6, HspB8) having different structure of beta2 strand of alpha-crystallin domain and different length and composition of variable N- and C-terminal tails.