Bcl-2-associated Athanogene 3 Protein Research Articles

The Role of BAG3 Protein Interactions in Cardiomyopathies.

Bcl-2-associated athanogene 3 (BAG3) plays an important function in cellular protein quality control (PQC) maintaining proteome stability. Mutations in the BAG3 gene result in cardiomyopathies. Due to its roles in cardiomyopathies and the complexity of BAG3-protein interactions, it is important to understand these protein interactions given the importance of the multifunctional cochaperone BAG3 in cardiomyocytes, using an in vitro cardiomyocyte model. The experimental assay was conducted using high pressure liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in the human AC16 cardiomyocyte cell line with BioID technology. Proteins with BAG3-interaction were identified in all the 28 hallmark gene sets enriched in idiopathic cardiomyopathies and/or ischemic disease. Among the 24 hallmark gene sets enriched in both idiopathic cardiomyopathies and ischemic disease, 15 gene sets had at least 3 proteins with BAG3-interaction. This study highlights BAG3 protein interactions, unveiling the key gene sets affected in cardiomyopathies, which help to explain the molecular mechanisms of the cardioprotective effects of BAG3. In addition, this study also highlighted the complexity of proteins with BAG3 interactions, implying unwanted effects of BAG3.

Tuning the B-CLL microenvironment: evidence for BAG3 protein- mediated regulation of stromal fibroblasts activity

The Bcl2-associated athanogene-3 (BAG3) protein, a critical regulator of cellular survival, has been identified as a potential therapeutic target in various malignancies. This study investigates the role of BAG3 within stromal fibroblasts and its interaction with B-cell chronic lymphocytic leukemia (B-CLL) cells. Previous research demonstrated that BAG3 maintains the active state of pancreatic stellate cells (PSCs) and aids pancreatic ductal adenocarcinoma (PDAC) spread via cytokine release. To explore BAG3’s role in bone marrow-derived stromal fibroblasts, BAG3 was silenced in HS-5 cells using siRNA. In co-culture experiments with PBMCs from B-CLL patients, BAG3 silencing in HS-5 cells increased apoptosis and decreased phosphorylation of BTK, AKT, and ERK in B-CLL cells, thus disrupting their pro-survival key signaling pathways. The observation of fibroblast-activated protein (FAP) positive cells in infiltrated bone marrow specimens co-expressing BAG3 further support the involvement of the protein in fibroblast-mediated tumor survival. Additionally, BAG3 appears to support B-CLL survival by modulating cytokine networks, including IL-10 and CXCL12, which are essential for leukemic cell survival and proliferation. A robust correlation between BAG3 expression and the levels of CXCL12 and IL-10 was observed in both co-cultures and patient specimens. These findings point out the need for a more in-depth comprehension of the intricate network of interactions within the tumor microenvironment and provide valuable insights for the selection of new potential therapeutic targets in the medical treatment of CLL.

Acetylation-dependent deubiquitinase USP26 stabilizes BAG3 to promote breast cancer progression

Deubiquitylases (DUBs) have emerged as promising targets for cancer therapy due to their role in stabilizing substrate proteins within the ubiquitin machinery. Here, we identified ubiquitin-specific protease 26 (USP26) as an oncogene via screening prognostic DUBs in breast cancer. Through in vitro and in vivo experiments, we found that depletion of USP26 inhibited breast cancer cell proliferation and invasion, and suppressed tumor growth and metastasis in nude mice. Further investigation identified co-chaperone Bcl-2-associated athanogene 3 (BAG3) as the direct substrate of USP26, and ectopic expression of BAG3 partially reversed antitumor effect induced by USP26 knockdown. Mechanistically, the lysine acetyltransferase Tip60 targeted USP26 at K134 for acetylation, which enhanced USP26 binding affinity to BAG3, leading to BAG3 deubiquitination and increased protein stability. Importantly, we employed a structure-based virtual screening and discovered a drug-like molecule called 5813669 that targets USP26, destabilizing BAG3 and effectively mitigating tumor growth and metastasis in vivo. Clinically, high expression levels of USP26 were correlated with elevated BAG3 levels and poor prognosis in breast cancer patients. Overall, our findings highlight the critical role of USP26 in BAG3 protein stabilization and provide a promising therapeutic target for breast cancer.

Design, synthesis and biological evaluation of novel 2,4-thiazolidinedione derivatives able to target the human BAG3 protein

The Bcl-2-associated athanogene 3 (BAG3) protein plays multiple roles in controlling cellular homeostasis, and it has been reported to be deregulated in many cancers, leading tumor cell apoptosis escape. BAG3 protein is then an emerging target for its oncogenic activities in both leukemia and solid cancers, such as medulloblastoma. In this work a series of forty-four compounds were designed and successfully synthesized by the modification and optimization of a previously reported 2,4-thiazolidinedione derivative 28. Using an efficient cloning and transfection in human embryonic kidney HEK-293T cells, BAG3 was collected and purified by chromatographic techniques such as IMAC and SEC, respectively. Subsequently, through Surface Plasmon Resonance (SPR) all the compounds were evaluated for their binding ability to BAG3, highlighting the compound FB49 as the one having the greatest affinity for the protein (Kd = 45 ± 6 μM) also against the reference compound 28. Further analysis carried out by Saturation Transfer Difference (STD) Nuclear Magnetic Resonance (NMR) spectroscopy further confirmed the highest affinity of FB49 for the protein. In vitro biological investigation showed that compound FB49 is endowed with an antiproliferative activity in the micromolar range in three human tumoral cell lines and more importantly is devoid of toxicity in human peripheral mononuclear cell deriving from healthy donors. Moreover, FB49 was able to block cell cycle in G1 phase and to induce apoptosis as well as autophagy in medulloblastoma HD-MB03 treated cells. In addition, FB49 demonstrated a synergistic effect when combined with a chemotherapy cocktail of Vincristine, Etoposide, Cisplatin, Cyclophosphamide (VECC). In conclusion we have demonstrated that FB49 is a new derivative able to bind human BAG3 with high affinity and could be used as BAG3 modulator in cancers correlated with overexpression of this protein.

Regulation of BAG3 localization and sarcomeric protein turnover in the cardiac myocyte.

Bcl2-associated athanogene-3 (BAG3) is a pro-autophagy co-chaperone that we have previously shown localizes to the cardiac sarcomere and is critical for proteostasis and maintenance of normal sarcomeric function. Indeed, BAG3 loss in heart failure (HF) results in accumulation of ubiquitinated sarcomeric proteins, and depressed maximum force generating capacity (Fmax). However, how BAG3 is regulated in the cell and how it coordinates sarcomeric protein turnover is unknown. During our analysis of human heart tissue, BAG3 appears as a “doublet”, with one band at 74 kDa (BAG3-L) and a second at a higher 84 kDa (BAG3-H). Previous studies hypothesized the BAG3-H band was due to phosphorylation. To test this, we treated human myocardial tissue with 10 units of alkaline phosphatase for 1 hour, which decreased troponin I phosphorylation by ∼60%, but had no impact on the intensity of BAG3-H or BAG3-L bands, suggesting these two bands are not due to phosphorylation. When we ran purified human BAG3 protein, it ran near the same molecular weight as the BAG3-H band, suggesting the lighter BAG3-L band is the modified version. These results suggest the BAG3-L band has been cleaved at either the N- or C-termini, which would be around 90 amino acids to cause the observed mass shift. Mass spectrometry analysis is being performed to determine the cleavage site and the domains being interrupted by this cleavage. Importantly, the BAG3-H/BAG3-L is higher in the myofilament compared to the cytoplasm of cardiomyocytes, suggesting the heavier form of BAG3 binds with sarcomere with increased affinity. We are also using high resolution live-cell imaging reveal the kinetics of the sarcomeric protein quality control system orchestrated by BAG3. These findings reveal novel regulation of the critical BAG3 protein and provides potential for BAG3-based HF therapies.

Therapeutic induction of Bcl2-associated athanogene 3-mediated autophagy in idiopathic pulmonary fibrosis.

BackgroundExaggerated fibroblast proliferation is a well‐known feature in idiopathic pulmonary fibrosis (IPF) which may be – in part – due to insufficient autophagy, a lysosome dependent cellular surveillance pathway. Bcl2‐associated athanogene 3 (BAG3) is a pivotal co‐chaperone of the autophagy pathway. Here, we studied whether therapeutic modulation of BAG3‐mediated autophagy can rescue insufficient autophagy and impact IPF fibroblast proliferation.MethodsPrimary interstitial fibroblasts or precision cut lung slices (PCLS) of IPF lungs were treated with (1) the antifibrotic drug pirfenidone (Pirf), (2) the demethylating agent 5‐azacytidine (Aza), (3) the BAG3 modulator cantharidin (Ctd). Autophagy flux was measured following pretreatment with the autophagy inhibitors or by GFP‐RFP‐LC3B transfection followed by drug treatments. Proliferation was measured by 5‐bromo‐2′‐deoxyuridine assay. BAG3, filamin C (FLNC), proliferating‐cell‐nuclear‐antigen (PCNA), collagen1A1 (COL1A1) and autophagy proteins were assessed by immunoblotting or immunofluorescence. Loss of function experiments were performed by siRNA mediated knockdown of BAG3.ResultsIn comparison with healthy donors, increased BAG3 protein was observed in IPF lung homogenates and IPF fibroblasts. In addition, the substrate of BAG3‐mediated autophagy, FLNC, was increased in IPF fibroblasts, implying insufficient activation of BAG3‐dependent autophagy. Therapeutic modulation of this pathway using Aza and Ctd alone or in combination with the IPF therapy drug Pirf rescued the insufficient BAG3‐mediated autophagy and decreased fibroblast proliferation. Such effects were observed upon therapeutic modulation of BAG3 but not upon knock down of BAG3 per se in IPF fibroblasts. Similarly, PCLS of IPF patients showed a significant decrease in collagen deposition in response to these drugs, either alone or in a more potent form in combination with Pirf.ConclusionsOur study reveals that repurposing drugs that modulate autophagy regulating proteins render therapeutic benefits in IPF. Fine tuning of this pathway may hence signify a promising therapeutic strategy to ameliorate antifibrotic properties and augment the efficacy of current IPF therapy.

Different roles of BAG3 in cardiac physiological hypertrophy and pathological remodeling

Heart failure is one of the leading causes of death worldwide. A stimulated heart undergoes either adaptive physiological hypertrophy, which can maintain a normal heart function, or maladaptive pathological remodeling, which can deteriorate heart function. These 2 kinds of remodeling often co-occur at the early stages of many heart diseases and have important effects on cardiac function. The Bcl2-associated athanogene 3 (BAG3) protein is highly expressed in the heart and has many functions. However, it is unknown how BAG3 is regulated and what its function is during physiological hypertrophy and pathological remodeling. We generated tamoxifen-induced, heart-specific heterozygous and homozygous BAG3 knockout mouse models (BAG3 protein level decreased by approximately 40% and 80% in the hearts after tamoxifen administration). BAG3 knockout models were subjected to swimming training or phenylephrine (PE) infusion to induce cardiac physiological hypertrophy and pathological remodeling. Neonatal rat ventricular cardiomyocytes (NRVCs) were used to study BAG3 functions and mechanisms in vitro. We found that BAG3 was upregulated in physiological hypertrophy and in pathological remodeling both in vivo and in vitro. Heterozygous or homozygous knockout BAG3 in mouse hearts and knockdown of BAG3 in the NRVCs blunted physiological hypertrophy and aggravated pathological remodeling, while overexpression of BAG3 promoted physiological hypertrophy and inhibited pathological remodeling in NRVCs. Mechanistically, BAG3 overexpression in NRVCs promoted physiological hypertrophy by activating the protein kinase B (AKT)/mammalian (or mechanistic) target of rapamycin (mTOR) pathway. BAG3 knockdown in NRVCs aggravated pathological remodeling through activation of the calcineurin/nuclear factor of activated T cells 2 (NFATc2) pathway. Because BAG3 has a dual role in cardiac remodeling, heart-specific regulation of BAG3 may be an effective therapeutic strategy to protect against deterioration of heart function and heart failure caused by many heart diseases.

SAT-632 Involvement of NF-κB-p65 in BAG3 Regulation After Stress Stimuli

We previously identified the limb salvage QTL1 (LSQ-1) on mouse chromosome 7 as a locus that offered protection against ischemic injury following induction of hind limb ischemia (HLI) a model of experimental peripheral arterial disease (PAD) in mice. To better understand the role of the LSQ-1 locus in post ischemic adaptation we characterized several genes within this locus and identified a number of genes that were important in tissue adaptation to ischemia, including BCL2-associated athanogene 3 (BAG3). BAG3 is an anti-apoptotic protein that plays an important role in cell survival through the regulation of autophagy. BAG3 expression is induced in the gastrocnemius muscles of mice after hind limb ischemia but how ischemia regulates BAG3 expression is poorly understood. Additionally, the activation of NF-κB transcription factor is essential for cell survival after stress stimuli. We hypothesized that BAG3 upregulation following stress stimuli is regulated by NF-κB. We determined whether NF-κB is involved in BAG3-mediated survival of primary human skeletal muscle cells (HSMC) during ischemia and diabetic conditions. Within 6 hours of treatment, ischemia induced BAG3 mRNA (no ischemia vs ischemia: 1.0 ±0.09 vs 1.41±0.02; p<0.01) and protein expression (BAG3/total protein, no ischemia vs ischemia 1.0±0.01 vs 1.38±0.06; p<0.01). Knockdown of BAG3 expression by shRNA induced early cell damage in HMSC under ischemic conditions as measured by HMGB1 expression (HMGB1/total protein; control plasmid vs shRNA, 1.0±0.09 vs 1.71±0.04; p<0.01). Knockdown of p65 subunit of the NF-κB by shRNA significantly diminished BAG3 mRNA expression after ischemia (control plasmid vs shRNA: 2.11±0.18 vs 1.48±0.05; p<0.05). Moreover, treatment of HSMC with 750 uM palmitic acid (PA) and 100nM insulin for 3 days to mimic diabetic conditions significantly increased the expression of BAG3 mRNA (control vs PAL+Ins, 1.0±0.14 vs 2.27±0.08; p<0.01) and protein (BAG3/total protein, control vs PAL+Ins, 1.0±0.03 vs 1.39±0.11, p<0.01). Knocking down p65 attenuated these increase in BAG3 mRNA (PAL+Ins vs shRNA+PAL+Ins, 2.27±0.08 vs 1.56±0.02 p<0.01) and protein (BAG3/total protein; PAL+Ins vs shRNA+PAL+Ins, 1.39±0.11 vs 0.99±0.1; p<0.05) Thus, 1) BAG3 expression is important in cell survival under ischemic conditions, and 2) NF-kB plays a key role in upregulating the expression of BAG3 under diabetic and ischemic conditions.

BAG3P215L/KO Mice as a Model of BAG3P209L Myofibrillar Myopathy

BCL-2-associated athanogene 3 (BAG3) is a co-chaperone to heat shock proteins important in degrading misfolded proteins through chaperone-assisted selective autophagy. The recurrent dominant BAG3-P209L mutation results in a severe childhood-onset myofibrillar myopathy (MFM) associated with progressive muscle weakness, cardiomyopathy, and respiratory failure. Because a homozygous knock-in (KI) strain for the mP215L mutation homologous to the human P209L mutation did not have a gross phenotype, compound heterozygote knockout (KO) and KI mP215L mice were generated to establish whether further reduction in BAG3 expression would lead to a phenotype. The KI/KO mice have a significant decrease in voluntary movement compared with wild-type and KI/KI mice in the open field starting at 7 months. The KI/KI and KI/KO mice both have significantly smaller muscle fiber cross-sectional area. However, only the KI/KO mice have clear skeletal muscle histologic changes in MFM. As in patient muscle, there are increased levels of BAG3-interacting proteins, such as p62, heat shock protein B8, and αB-crystallin. The KI/KO mP215L strain is the first murine model of BAG3 myopathy that resembles the human skeletal muscle pathologic features. The results support the hypothesis that the pathologic development of MFM requires a significant decrease in BAG3 protein level and not only a gain of function caused by the dominant missense mutation.

The PXXP domain is critical for the protective effect of BAG3 in cardiomyocytes.

Bcl-2-associated athanogene3(BAG3) protects the heart and cardiomyocytes from ischaemia/reperfusion (I/R) injury. Although the anti-apoptosis effect of BAG3 has been demonstrated in multiple cell types, the structural domain of BAG3, which is responsible for its anti-apoptosis effect, is not well understood. BAG3 protein consists of various characteristic amino acid motifs/regions that permit the interaction of BAG3 with numerous proteins involved in many cellular key pathways. The purpose of this study is to determine whether the proline-rich (PXXP) domain of BAG3 is necessary for its cellular protection against hypoxia-reoxygenation (H/R) stress by binding to its chaperone, heat shock cognate 71 kDa protein (HSC70). Cell apoptosis induced by H/R was evaluated using propidium iodide (PI) staining, caspase 3/7 activation and TUNEL staining in cultured H9C2 cells. The expression levels of BAG3 and HSC70 were manipulated, where BAG3 or its mutant, which lacked the PXXP domain, was overexpressed using a plasmid and adenovirus vector, and HSC70 expression was silenced using siRNA. Co-immunoprecipitation (co-IP) followed by western blot was employed to define the complex of BAG3 binding to its chaperones. The PXXP domain of BAG3 was determined to be critical for BAG3-mediated attenuation of H9C2 cell apoptosis induced by H/R through the binding of PXXP with HSC70. The abolished cellular protection of BAG3 induced by the knockdown of HSC70 is associated with reduced binding to HSC70. Given that the structural domain PXXP of BAG3 is necessary for the cellular protection of BAG3 from I/R injury, the mechanism revealed in this study indicates that BAG3 may be a therapeutic target in patients undergoing reperfusion after myocardial infarction.

BAG3 contributes to HGF-mediated cell proliferation, migration, and invasion via the Egr1 pathway in gastric cancer.

Bcl2-associated athanogene 3 (BAG3) is elevated in several types of cancers. However, the role of BAG3 in progression of gastric cancer is unknown. Therefore, the present study aims to find out the role of BAG3 in hepatocyte growth factor (HGF)-mediated tumor progression and the molecular mechanisms by which HGF regulates BAG3 expression. BAG3 mRNA and protein were measured using reverse transcription polymerase chain reaction and Western blot in the 2 human gastric cancer cell lines, NUGC3 and MKN28, treated with or without HGF. The effects of BAG3 knockdown on cell proliferation, cell invasion, and apoptosis were analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, the in vitro 2-chamber invasion assay, and flow cytometry in BAG3 short hairpin RNA (shRNA)-transfected cells and control cells. The signaling pathways involved in BAG3 that are regulated by HGF were analyzed. The chromatin immunoprecipitation assay was used to determine binding of Egr1 to the BAG3 promoter. BAG3 mRNA and protein levels were increased following treatment with HGF. HGF-mediated BAG3 upregulation increased cell proliferation and cell invasion; however, it decreased apoptosis. HGF-mediated BAG3 upregulation is regulated by an ERK and Egr1-dependent pathway. BAG3 may have an important role in HGF-mediated cell proliferation and metastasis in gastric cancer through an ERK and Egr1-dependent pathway. This pathway may provide novel therapeutic targets and provide information for further identification of other targets of therapeutic significance in gastric cancer.

Regulation of angiotensin II-induced B-cell lymphoma-2-associated athanogene 3 expression in vascular smooth muscle cells.

Previous studies have demonstrated that angiotensin II (Ang II) is involved in the process of atherosclerosis and vascular restenosis through its proinflammatory effect. Bcl‑2‑associated athanogene 3 (BAG3) had been suggested to be associated with proliferation, migration and invasion in many types of tumor. However, the role of BAG3 among the proliferative process of vascular smooth muscle cells (VSMCs) induced by Ang II, to the best of our knowledge, remains to be investigated. The present study demonstrated that in growth‑arrested VSMCs, Ang II‑induced VSMC proliferation, accompanied by increased BAG3 mRNA and protein expression levels in a dose‑ and time‑dependent manner. BAG3 expression levels were measured in VSMCs treated in the presence or absence of Ang II. The proliferation of VSMCs was assessed using manual cell counting and Cell Counting kit‑8 assays. mRNA and protein expression levels of BAG3, Toll‑like receptor 4 (TLR4), proliferating cell nuclear antigen, nuclear factor (NF)‑κB p65, smooth muscle protein22α and phosphorylated NF‑κB p65 were assessed by reverse transcription‑quantitative polymerase chain reaction and western blotting, respectively. In non‑transfected or scramble short hairpin RNA (shRNA)‑transfected VSMCs cells, Ang II significantly induced VSMC proliferation. However, this Ang II‑induce proliferation was attenuated when BAG3 was silenced, suggesting that inhibition of BAG3 may somehow reduce proliferation in Ang II‑induced VSMCs. Furthermore, the TLR4/NF‑κB p65 signaling pathway was involved in BAG3 gene upregulation. In conclusion, to the best of our knowledge, the present study demonstrated for the first time that inhibition of BAG3 attenuates cell proliferation. Furthermore, Ang II induced VSMCs proliferation through regulation of BAG3 expression via the TLR4/NF‑κB p65 signaling pathway.

Bcl-2 associated athanogene 3 affects the epithelial-mesenchymal transition in human cervical cancer

Objective: To investigate the expression of Bcl-2 associated athanogene 3 (BAG3) in cervical cancer tissues and cells and its role in epithelial mesenchymal transition (EMT) of cervical cancer. Methods: (1) Cervical cancer samples were collected from September 2015 to March 2017 in the Qilu Hospital of Shandong University and Shangdong Provincial Hospital. While, 50 normal tissues were collected from August 2015 to March 2017 in the Dezhou Municiple Hospital, which were obtained from patients with uterine myoma underwent hysterectomy and patients with cervical biopsy. Reverse transcription (RT)-PCR and western blot were used to detect the expression of BAG3 mRNA and protein, and their clinical significances were analyzed. (2) The expression of BAG3 mRNA and protein was detected using RT-PCR and western blot method in HeLa and SiHa cell lines and normal cervical epithelial cells. The experiment was divided into two groups, BAG3 small interfering RNA transfected group (si-BAG3) and the control group transfected with small interfering RNA (siRNA). Cell counting kit 8 (CCK-8) analysis was used to detect cell proliferation of two groups. Wound-healing and transwell assay were used to detect the migration and invasion ability of HeLa and SiHa cells. The xenograft model of cervical cancer in nude mice was used to observe the effect of BAG3 on tumor xenografts and the tumor-related biomarkers were tested by western blot. Results: (1) The expression levels of BAG3 mRNA and protein in cervical carcinoma tissues were 1.20±0.15 and 1.10±0.16, which were significantly higher than that in normal cervical tissue, 0.23±0.04 and 0.29±0.03 (both P<0.01). The results showed that the expression levels of BAG3 mRNA and protein were significantly correlated with cervical carcinoma staging and lymph node metastasis (P<0.05).However, its expression was not correlated with the patient's age, pathological grade, and diameter of tumor (all P>0.05). (2) Compared with normal cervical epithelial cells, the expression of BAG3 mRNA and protein levels in HeLa and SiHa cells were significantly increased (P<0.01), the expression levels of BAG3 mRNA and protein in HeLa and SiHa cells transfected with si-BAG3 were significantly lower than that in control group (all P<0.01). After post-transfected 72 hours, A value of HeLa and SiHa with transfection were significantly lower than those in control group [(0.88±0.08) vs (1.22±0.13), (0.92±0.09) vs (1.35±0.12); both P<0.01]. After post-transfected 24 hours, the migration level of HeLa and SiHa cells with transfection were significantly lower than those in the control group [(20.1±2.1)% vs (58.6±5.6)%, and (21.1±2.1)% vs (61.7±5.4)%; both P<0.01]. The transmembrane cell number in HeLa and SiHa cells with transfection were 76±11 and 71±8, which were significantly less than those in control group (131±12 and 129±14; both P<0.01). After the inoculation into nude mice, tumor formation time of HeLa and SiHa cells with transfection were (9.5±0.5) and (10.5±1.3) days, respectively, which were significantly longer than those in control group [(4.5±0.5) and (5.2±1.1) days; both P<0.05]. Compared with those in the control group, the expression level of Slug, N-cadherin and matrix metalloproteinase-2 (MMP-2) protein in HeLa and SiHa cells with transfected in tumor tissues were significantly decreased (all P<0.01), while the expression level of E-cadherin protein was significantly increased (P<0.01). Conclusion: BAG3 could be involved in the proliferation, migration and invasion of cervical cancer cells by affecting cervical cancer EMT, and BAG3 may be an effective target for the treatment of cervical cancer.

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

Defective protein quality control (PQC) systems are implicated in multiple diseases. Molecular chaperones and co-chaperones play a central role in functioning PQC. Constant mechanical and metabolic stress in cardiomyocytes places great demand on the PQC system. Mutation and downregulation of the co-chaperone protein BCL-2-associated athanogene 3 (BAG3) are associated with cardiac myopathy and heart failure, and a BAG3 E455K mutation leads to dilated cardiomyopathy (DCM). However, the role of BAG3 in the heart and the mechanisms by which the E455K mutation leads to DCM remain obscure. Here, we found that cardiac-specific Bag3-KO and E455K-knockin mice developed DCM. Comparable phenotypes in the 2 mutants demonstrated that the E455K mutation resulted in loss of function. Further experiments revealed that the E455K mutation disrupted the interaction between BAG3 and HSP70. In both mutants, decreased levels of small heat shock proteins (sHSPs) were observed, and a subset of proteins required for cardiomyocyte function was enriched in the insoluble fraction. Together, these observations suggest that interaction between BAG3 and HSP70 is essential for BAG3 to stabilize sHSPs and maintain cardiomyocyte protein homeostasis. Our results provide insight into heart failure caused by defects in BAG3 pathways and suggest that increasing BAG3 protein levels may be of therapeutic benefit in heart failure.

The Role of the Multifunctional BAG3 Protein in Cellular Protein Quality Control and in Disease.

In neurons, but also in all other cells the complex proteostasis network is monitored and tightly regulated by the cellular protein quality control (PQC) system. Beyond folding of newly synthesized polypeptides and their refolding upon misfolding the PQC also manages the disposal of aberrant proteins either by the ubiquitin-proteasome machinery or by the autophagic-lysosomal system. Aggregated proteins are primarily degraded by a process termed selective macroautophagy (or aggrephagy). One such recently discovered selective macroautophagy pathway is mediated by the multifunctional HSP70 co-chaperone BAG3 (BCL-2-associated athanogene 3). Under acute stress and during cellular aging, BAG3 in concert with the molecular chaperones HSP70 and HSPB8 as well as the ubiquitin receptor p62/SQSTM1 specifically targets aggregation-prone proteins to autophagic degradation. Thereby, BAG3-mediated selective macroautophagy represents a pivotal adaptive safeguarding and emergency system of the PQC which is activated under pathophysiological conditions to ensure cellular proteostasis. Interestingly, BAG3-mediated selective macroautophagy is also involved in the clearance of aggregated proteins associated with age-related neurodegenerative disorders, like Alzheimer’s disease (tau-protein), Huntington’s disease (mutated huntingtin/polyQ proteins), and amyotrophic lateral sclerosis (mutated SOD1). In addition, based on its initial description BAG3 is an anti-apoptotic protein that plays a decisive role in other widespread diseases, including cancer and myopathies. Therefore, in the search for novel therapeutic intervention avenues in neurodegeneration, myopathies and cancer BAG3 is a promising candidate.

Overexpression of the anti-apoptotic protein BAG3 in human choroidal melanoma: A case report.

Bcl-2-associated athanogene 3 (BAG3), a co-chaperone of heat shock protein 70 (HSP70), exerts anti-apoptotic effects in various malignant tumors. However, relationships between choroidal melanoma and BAG3 are poorly studied. This study investigated the expression of BAG3 in a case of human choroidal melanoma. Funduscopy, computed tomography, and single-photon emission computed tomography with the intravenous injection of N-isopropyl-p-[123I] iodoamphetamine strongly indicated choroidal melanoma in a 68-year-old woman. Accordingly, we carried out an enucleation and pathological diagnosis. Proteins and total RNA were extracted from normal retinochoroidal and tumor tissues. Proteins were also extracted from ocular nevus tissues of other patients. We examined the expression of BAG3 protein and mRNA using Western blotting and the real-time quantitative polymerase chain reaction, respectively. Immunohistochemical stains were positive for melan-A, HMB-45, and S-100. Histopathology confirmed a choroidal melanoma. The expression of BAG3 protein and mRNA in the choroidal melanoma tissue was upregulated with respect to both normal retinochoroidal tissue and ocular nevus tissues from other patients. Because BAG3 may inhibit apoptosis of choroidal melanoma and facilitate its survival, overexpression of this gene product may be a prognostic marker and therapeutic target.

BAG3 regulates contractility and Ca2 + homeostasis in adult mouse ventricular myocytes

Bcl2-associated athanogene 3 (BAG3) is a 575 amino acid anti-apoptotic protein that is constitutively expressed in the heart. BAG3 mutations, including mutations leading to loss of protein, are associated with familial cardiomyopathy. Furthermore, BAG3 levels have been found to be reduced in end-stage non-familial failing myocardium. In contrast to neonatal myocytes in which BAG3 is found in the cytoplasm and involved in protein quality control and apoptosis, in adult mouse left ventricular (LV) myocytes BAG3 co-localized with Na+–K+–ATPase and L-type Ca2+ channels in the sarcolemma and t-tubules. BAG3 co-immunoprecipitated with β1-adrenergic receptor, L-type Ca2+ channels and phospholemman. To simulate decreased BAG3 protein levels observed in human heart failure, we targeted BAG3 by shRNA (shBAG3) in adult LV myocytes. Reducing BAG3 by 55% resulted in reduced contraction and [Ca2+]i transient amplitudes in LV myocytes stimulated with isoproterenol. L-type Ca2+ current (ICa) and sarcoplasmic reticulum (SR) Ca2+ content but not Na+/Ca2+ exchange current (INaCa) or SR Ca2+ uptake were reduced in isoproterenol-treated shBAG3 myocytes. Forskolin or dibutyryl cAMP restored ICa amplitude in shBAG3 myocytes to that observed in WT myocytes, consistent with BAG3 having effects upstream and at the level of the receptor. Resting membrane potential and action potential amplitude were unaffected but APD50 and APD90 were prolonged in shBAG3 myocytes. Protein levels of Ca2+ entry molecules and other important excitation–contraction proteins were unchanged in myocytes with lower BAG3. Our findings that BAG3 is localized at the sarcolemma and t-tubules while modulating myocyte contraction and action potential duration through specific interaction with the β1-adrenergic receptor and L-type Ca2+ channel provide novel insight into the role of BAG3 in cardiomyopathies and increased arrhythmia risks in heart failure.

HSPB8 and the Cochaperone BAG3 Are Highly Expressed During the Synthetic Phase of Rat Myometrium Programming During Pregnancy1

The small heat shock protein (HSP) B family of proteins are a group of molecular chaperones that enable tissues to adapt to changes in their local environments during differentiation, stress, or disease conditions. The objective of this research was to characterize the expression of HSPB8 and its cochaperone Bcl2-associated athanogene 3 (BAG3) in nonpregnant (NP) and pregnant rat myometrium during myometrial programming. Rat myometrium was collected from NP and pregnant rats as well as 1 day postpartum (PP) and samples prepared for immunoblot and immunofluorescence analysis. Immunoblot analysis determined that HSPB8 protein expression was significantly elevated at Day (D) 15, D17, and D19 compared to expression at NP and D6, while BAG3 expression was significantly elevated at D15 compared to NP, and D17 compared to NP, D6, D23, and PP time points (P < 0.05). In situ, HSPB8 and BAG3 were predominantly localized to myometrial cells throughout pregnancy, with intense cytoplasmic HSPB8 and BAG3 detection on D15 and D17 in both longitudinal and circular muscle layers. Immunoblot analysis of HSPB8 and BAG3 protein expression in myometrium from unilateral pregnancies also revealed that expression of both proteins was significantly increased at D15 in gravid compared to nongravid horns. Thus, HSPB8 and BAG3 are highly expressed during the synthetic phase of myometrial differentiation marked by initiation of uterine distension and myometrial hypertrophy. HSPB8 and BAG3 could be regulators of the protein quality control required for this process.

NIK is required for NF-κB-mediated induction of BAG3 upon inhibition of constitutive protein degradation pathways.

Recently, we reported that induction of the co-chaperone Bcl-2-associated athanogene 3 (BAG3) is critical for recovery of rhabdomyosarcoma (RMS) cells after proteotoxic stress upon inhibition of the two constitutive protein degradation pathways, that is, the ubiquitin-proteasome system by Bortezomib and the aggresome-autophagy system by histone deacetylase 6 (HDAC6) inhibitor ST80. In the present study, we investigated the molecular mechanisms mediating BAG3 induction under these conditions. Here, we identify nuclear factor-kappa B (NF-κB)-inducing kinase (NIK) as a key mediator of ST80/Bortezomib-stimulated NF-κB activation and transcriptional upregulation of BAG3. ST80/Bortezomib cotreatment upregulates mRNA and protein expression of NIK, which is accompanied by an initial increase in histone H3 acetylation. Importantly, NIK silencing by siRNA abolishes NF-κB activation and BAG3 induction by ST80/Bortezomib. Furthermore, ST80/Bortezomib cotreatment stimulates NF-κB transcriptional activity and upregulates NF-κB target genes. Genetic inhibition of NF-κB by overexpression of dominant-negative IκBα superrepressor (IκBα-SR) or by knockdown of p65 blocks the ST80/Bortezomib-stimulated upregulation of BAG3 mRNA and protein expression. Interestingly, inhibition of lysosomal activity by Bafilomycin A1 inhibits ST80/Bortezomib-stimulated IκBα degradation, NF-κB activation and BAG3 upregulation, indicating that IκBα is degraded via the lysosome in the presence of Bortezomib. Thus, by demonstrating a critical role of NIK in mediating NF-κB activation and BAG3 induction upon ST80/Bortezomib cotreatment, our study provides novel insights into mechanisms of resistance to proteotoxic stress in RMS.

Decreased Levels of BAG3 in a Family With a Rare Variant and in Idiopathic Dilated Cardiomyopathy

The most common cause of dilated cardiomyopathy and heart failure (HF) is ischemic heart disease; however, in a third of all patients the cause remains undefined and patients are diagnosed as having idiopathic dilated cardiomyopathy (IDC). Recent studies suggest that many patients with IDC have a family history of HF and rare genetic variants in over 35 genes have been shown to be causative of disease. We employed whole-exome sequencing to identify the causative variant in a large family with autosomal dominant transmission of dilated cardiomyopathy. Sequencing and subsequent informatics revealed a novel 10-nucleotide deletion in the BCL2-associated athanogene 3 (BAG3) gene (Ch10:del 121436332_12143641: del. 1266_1275 [NM 004281]) that segregated with all affected individuals. The deletion predicted a shift in the reading frame with the resultant deletion of 135 amino acids from the C-terminal end of the protein. Consistent with genetic variants in genes encoding other sarcomeric proteins there was a considerable amount of genetic heterogeneity in the affected family members. Interestingly, we also found that the levels of BAG3 protein were significantly reduced in the hearts from unrelated patients with end-stage HF undergoing cardiac transplantation when compared with non-failing controls. Diminished levels of BAG3 protein may be associated with both familial and non-familial forms of dilated cardiomyopathy.