BCL2-associated Athanogene Research Articles

BAG3 mutation in a patient with atypical phenotypes of myofibrillar myopathy and Charcot-Marie-Tooth disease.

Bcl2-associated athanogene 3 (BAG3) mutations have been reported to cause the myofibrillar myopathy (MFM) which shows progressive limb muscle weakness, respiratory failure, and cardiomyopathy. Myopathy patients with BAG3 mutation are very rare. We described a patient showing atypical phenotypes. We aimed to find the genetic cause of Korean patients with sensory motor polyneuropathy, myopathy and rigid spine. We performed whole exome sequencing (WES) with 423 patients with sensory motor polyneuropathy. We found BAG3 mutation in one patient with neuropathy, myopathy and rigid spine syndrome, and performed electrophysiological study, whole body MRI and muscle biopsy on the patient. A de novo heterozygous p.Pro209Leu (c.626C>T) mutation in BAG3 was identified in a female myopathy. She first noticed a gait disturbance and spinal rigidity at the age of 11, and serum creatine kinase levels were elevated ninefolds than normal. She showed an axonal sensory-motor polyneuropathy like Charcot-Marie-Tooth disease (CMT), myopathy, rigid spine and respiratory dysfunction; however, she did not show any cardiomyopathy, which is a common symptom in BAG3 mutation. Lower limb MRI and whole spine MRI showed bilateral symmetric fatty atrophy of muscles at the lower limb and paraspinal muscles. When we track traceable MRI 1year later, the muscle damage progressed slowly. As far as our knowledge, this is the first Korean patient with BAG3 mutation. We described a BAG3 mutation patient with atypical phenotype of CMT and myopathy, and those are expected to broaden the clinical spectrum of the disease and help to diagnose it.

Identification of genes and genetic networks associated with BAG3‑dependent cell proliferation and cell survival in human cervical cancer HeLa cells.

Bcl‑2‑associated athanogene (BAG) 3, is a member of the BAG protein family and a known co‑chaperone of heat shock protein (HSP) 70. BAG3 serves a role in regulating a variety of cellular functions, including cell growth, proliferation and cell death including apoptosis. BAG3 is a stress‑inducible protein, however the constitutive expression level of BAG3 is increased in cancer cells compared with healthy cells. Recent proteomics technology combined with bioinformatics has revealed that BAG3 participates in an interactome with a number of proteins other than its typical partner HSP70. The functional types represented in the interactome included nucleic acid binding proteins and transcription factors, as well as chaperones, which indicated that overexpression of BAG3 may contribute to proliferation and cell survival through the alteration of gene transcription. While an increasing number of studies have addressed the function of BAG3 as a co‑chaperone protein, BAG3‑dependent alteration of gene transcription has not been studied extensively. The present study established two BAG3 knockout human cervical cancer HeLa cell clones and addressed the role of BAG3 in cell proliferation and survival through gene transcription, using DNA microarray‑based transcriptome analysis and bioinformatics. The present study also identified two genetic networks associated with 'cellular growth and proliferation' and 'cell death and survival', which are dysregulated in the absence of BAG3, and may therefore be linked to BAG3 overexpression in cancer. These findings provide a molecular basis for understanding of BAG3‑dependent cell proliferation and survival from the aspect of alteration of gene expression.

Bcl-2-associated athanogene 3(BAG3) is associated with tumor cell proliferation, migration, invasion and chemoresistance in colorectal cancer

BackgroundCRC is one of the most common malignancies worldwide, and its molecular mechanisms remain unclear. Elevated levels of BAG3 have been reported in various tumors. The present study aimed to explore the expression and function of BAG3 in CRC.MethodsBAG3 protein expression was evaluated in 90 CRC specimens using immunohistochemistry in tissue microarrays, and the correlation between BAG3 expression and the clinicopathological features were assessed. In HCT116 cells BAG3 overexpression cell models were constructed, and CRISPR/Cas9 was used for BAG3 knockout. Western blotting and quantitative real-time PCR were used to determine BAG3 expression in HCT-116 Cells. Cell proliferation, migration and invasion were analyzed by cell counting, colony formation assay, EdU cell proliferation assay, RTCA growth curve assays, wound-healing migration assay and transwell invasion assay. The influence of BAG3 expression level on chemoresistance in HCT-116 cells was examined. Gene expression microarray and IPA analyses were employed to explore signaling pathways associated with the control of BAG3.ResultsUsing immunohistochemistry, this study found that BAG3 was markedly upregulated in colorectal cancer tissues and that BAG3 levels were significantly associated with tumor size and gender. BAG3 overexpression promoted HCT-116 cell growth, migration and invasion in vitro. In contrast, BAG3 knockout inhibited HCT-116 cell growth, migration and invasion. HCT-116 cells with high expression of BAG3 had higher cell viability and lower apoptosis rate than control cells after treatment with 5-FU, while the BAG3 knockout group demonstrated the opposite effects. So BAG3 expression level was associated with chemoresistance to 5-FU in HCT-116 cells. Gene expression microarrays and bioinformatics analyses of HCT-116 cells with BAG3 knockout demonstrated the involvement of BAG3 in signaling pathways associated with the control of cell proliferation, migration, invasion and chemoresistance in CRC.ConclusionsIn conclusion, this study provided evidence that BAG3 has a relevant role in CRC biology, and defined potential molecular pathways and networks. So BAG3 may be considered as a potential therapeutic target for anti-tumor therapy in colorectal cancer.

Silencing of the Hsp70-specific nucleotide-exchange factor BAG3 corrects the F508del-CFTR variant by restoring autophagy

The protein chaperones heat shock protein 70 (Hsp70) and Hsp90 are required for de novo folding of proteins and protect against misfolding-related cellular stresses by directing misfolded or slowly folding proteins to the ubiquitin/proteasome system (UPS) or autophagy/lysosomal degradation pathways. Here, we examined the role of the Bcl2-associated athanogene (BAG) family of Hsp70-specific nucleotide-exchange factors in the biogenesis and functional correction of genetic variants of the cystic fibrosis transmembrane conductance regulator (CFTR) whose mutations cause cystic fibrosis (CF). We show that siRNA-mediated silencing of BAG1 and -3, two BAG members linked to the clearance of misfolded proteins via the UPS and autophagy pathways, respectively, leads to functional correction of F508del-CFTR and other disease-associated CFTR variants. BAG3 silencing was the most effective, leading to improved F508del-CFTR stability, trafficking, and restoration of cell-surface function, both alone and in combination with the FDA-approved CFTR corrector, VX-809. We also found that the BAG3 silencing-mediated correction of F508del-CFTR restores the autophagy pathway, which is defective in F508del-CFTR-expressing cells, likely because of the maladaptive stress response in CF pathophysiology. These results highlight the potential therapeutic benefits of targeting the cellular chaperone system to improve the functional folding of CFTR variants contributing to CF and possibly other protein-misfolding-associated diseases.

Host Protein BAG3 is a Negative Regulator of Lassa VLP Egress.

Lassa fever virus (LFV) belongs to the Arenaviridae family and can cause acute hemorrhagic fever in humans. The LFV Z protein plays a central role in virion assembly and egress, such that independent expression of LFV Z leads to the production of virus-like particles (VLPs) that mimic egress of infectious virus. LFV Z contains both PTAP and PPPY L-domain motifs that are known to recruit host proteins that are important for mediating efficient virus egress and spread. The viral PPPY motif is known to interact with specific host WW-domain bearing proteins. Here we identified host WW-domain bearing protein BCL2 Associated Athanogene 3 (BAG3) as a LFV Z PPPY interactor using our proline-rich reading array of WW-domain containing mammalian proteins. BAG3 is a stress-induced molecular co-chaperone that functions to regulate cellular protein homeostasis and cell survival via Chaperone-Assisted Selective Autophagy (CASA). Similar to our previously published findings for the VP40 proteins of Ebola and Marburg viruses, our results using VLP budding assays, BAG3 knockout cells, and confocal microscopy indicate that BAG3 is a WW-domain interactor that negatively regulates egress of LFV Z VLPs, rather than promoting VLP release. Our results suggest that CASA and specifically BAG3 may represent a novel host defense mechanism, whereby BAG3 may dampen egress of several hemorrhagic fever viruses by interacting and interfering with the budding function of viral PPxY-containing matrix proteins.

MiR-206 Inhibits Cell Proliferation, Migration, and Invasion by Targeting BAG3 in Human Cervical Cancer.

miR-206 and Bcl-2-associated athanogene 3 (BAG3) have been suggested as important regulators in various cancer types. However, the biological role of miR-206 and BAG3 in cervical cancer (CC) remains unclear. We investigated the expressions and mechanisms of miR-206 and BAG3 in CC using in vitro and in vivo assays. In the present study, miR-206 expression was expressed at a lower level in CC tissues and cells than adjacent normal tissues and NEECs. By contrast, BAG3 mRNA and protein were expressed at higher levels in CC tissues and cells. Furthermore, miR-206 overexpression repressed cell proliferation, migration, and invasion in vitro, and the 3′-untranslated region (3′-UTR) of BAG3 was a direct target of miR-206. miR-206 overexpression also inhibited EGFR, Bcl-2, and MMP2/9 protein expression, but promoted Bax protein expression. Besides, BAG3 overexpression partially abrogated miR-206-inhibited cell proliferation and invasion, while BAG3 silencing enhanced miR-206-mediated inhibition. In vivo assay revealed that miR-206 repressed tumor growth in nude mice xenograft model. In conclusion, miR-206 inhibits cell proliferation, migration, and invasion by targeting BAG3 in human CC. Thus, miR-206-BAG3 can be used as a useful target for CC.

HIV-1 Tat increases BAG3 via NF-κB signaling to induce autophagy during HIV-associated neurocognitive disorder

ABSTRACT The human immunodeficiency virus-1 (HIV-1) regulatory protein Tat plays an important role during HIV-1-associated neurocognitive disorders (HAND) by inducing neuronal autophagy. In this study, we used immunohistochemistry, immunofluorescence, western blot, qRT-PCR, and RNA interference to elucidate the involvement of Bcl-2-associated athanogene 3 (BAG3) in the pathogenesis of HIV-1 Tat-induced autophagy during HAND. We found that BAG3 expression is elevated in astrocytes in frontal cortex of macaques infected with simian immunodeficiency virus-human immunodeficiency chimeric virus (SHIV). In addition, in human primary glioblastoma cells (U87), HIV-1 Tat upregulated BAG3 in an NF-κB-dependent manner to induce autophagy. Importantly, suppression of BAG3 or inhibition of NF-κB activity reversed the HIV-1 Tat-induced autophagy. These results indicate that HIV-1 Tat induces autophagy by upregulating BAG3 via NF-κB signaling, which suggests BAG3 and NF-κB could potentially serve as novel targets for HAND therapies.

The VCP-UBXN1 Complex Mediates Triage of Ubiquitylated Cytosolic Proteins Bound to the BAG6 Complex.

A balance between protein synthesis and degradation is necessary to maintain cellular homeostasis. Failure to triage aberrant proteins may result in their accumulation and aggregation in the cytosol. The valosin-containing protein (VCP)-BCL2-associated athanogene 6 (BAG6) complex facilitates a wide variety of ubiquitin-mediated quality control events at the endoplasmic reticulum (ER), both prior to ER translocation and during ER-associated degradation (ERAD). However, how ubiquitylated clients associated with BAG6 are recognized by VCP for proteasomal degradation is presently unknown. We have identified UBXN1 as the VCP adaptor in BAG6-dependent processes occurring prior to ER insertion but not during ERAD. The loss of VCP-UBXN1 results in the inappropriate stabilization of ubiquitylated BAG6 clients and their accumulation in insoluble aggregates and sensitizes cells to proteotoxic stress. Our results identify how VCP is specifically targeted to ubiquitylated substrates in the BAG6 triage pathway and suggest that the degradation of ubiquitylated clients by the proteasome is reliant on the association of UBXN1 with ubiquitylated substrates and the catalytic activity of VCP.

P209L mutation in Bag3 does not cause cardiomyopathy in mice.

Bcl-2-associated athanogene 3 (BAG3) is a cochaperone protein and a central player of the cellular protein quality control system. BAG3 is prominently expressed in the heart and plays an essential role in cardiac protein homeostasis by interacting with chaperone heat shock proteins (HSPs) in large, functionally distinct multichaperone complexes. The BAG3 mutation of proline 209 to leucine (P209L), which resides in a critical region that mediates the direct interaction between BAG3 and small HSPs (sHSPs), is associated with cardiomyopathy in humans. However, the mechanism by which the BAG3 P209L missense mutation leads to cardiomyopathy remains unknown. To determine the molecular basis underlying the cardiomyopathy caused by the BAG3 P209L mutation, we generated a knockin (KI) mouse model in which the endogenous Bag3 gene was replaced with mutant Bag3 containing the P215L mutation, which is equivalent to the human P209L mutation. We performed physiological, histological, and biochemical analyses of Bag3 P209L KI mice to determine the functional, morphological, and molecular consequences of the P209L mutation. We found that Bag3 P209L KI mice exhibited normal cardiac function and morphology up to 16 mo of age. Western blot analysis further revealed that levels of sHSPs, stress-inducible HSPs, ubiquitinated proteins, and autophagy were unaffected in P209L mutant mouse hearts. In conclusion, the P209L mutation in Bag3 does not cause cardiomyopathy in mice up to 16 mo of age under baseline conditions. NEW & NOTEWORTHY Bcl-2-associated athanogene 3 (BAG3) P209L mutation is associated with human cardiomyopathy. A recent study reported that transgenic mice overexpressing human BAG3 P209L in cardiomyocytes have cardiac dysfunction. In contrast, our P209L mice that express mutant BAG3 at the same level as that of wild-type mice displayed no overt phenotype. Our results suggest that human cardiomyopathy may result from species-specific requirements for the conserved motif that is disrupted by P209L mutation or from genetic background-dependent effects.

A Bcl-2 Associated Athanogene (bagA) Modulates Sexual Development and Secondary Metabolism in the Filamentous Fungus Aspergillus nidulans.

The Bcl-2 associated athanogene (Bag) family is a multifunctional group of proteins distinguished by a conserved region known as the Bag domain (BD). Herein, we discuss the discovery and characterization of a Bag protein in the model genetic fungus Aspergillus nidulans, we designated BagA. BagA shares striking similarities in 3D structure, domain organization, amino acid properties, and Hsp70 binding surfaces to animal and plant Bags. While Hsp70 binding is a common feature of Bag proteins, our experimental evidence shows that BagA does not cooperate with A. nidulans Hsp70s, suggesting this association may not be a universal feature of Bag proteins. Gene expression of bagA was strongly induced during sexual development suggesting a role in developmental processes. Accordingly, the deletion of bagA (ΔbagA) negatively impacted sexual development, while its overexpression resulted in constitutive induction of sexual fruiting bodies and spores. Asexual and sexual development was linked to secondary metabolism in A. nidulans. Our data show that the deletion of bagA also provoked an altered secondary metabolite (SM) profile in both sexual and vegetative growth phases. Indeed, LC-MS analysis showed a significant enrichment of SMs in ΔbagA, including novel metabolites not produced by wild type strain. Enrichment of SMs in ΔbagA strain is particularly intriguing and suggest that altering cellular homeostasis can be used as a provocative strategy to activate cryptic metabolites and uncover novel bioactive compounds. Overall, our results indicate that Bag proteins in filamentous fungi share developmental regulatory roles with their animal and plant counterparts. We also show a potentially unique role for BagA in modulating secondary metabolism in A. nidulans. To our knowledge, this study provides a first insight into Bag function in filamentous fungi.

Regulation of the expression of tumor necrosis factor‑related genes by abnormal histone H3K27 acetylation: Implications for neural tube defects.

The association between apoptosis and neural tube defects (NTDs) is recognized as important, however, the precise link remains to be elucidated. Epigenetic modifications in human NTDs have been detected previously. In the present study, the occurrence of epigenetic modifications in apoptosis-related genes was investigated in a retinoic acid (RA)-induced mouse NTD model. Among 84 key genes involved in programmed cell death, 13 genes, including tumor necrosis factor (Tnf), annexin A5, apoptosis inhibitor 5, Bcl2-associated athanogene 3, baculoviral IAP repeat-containing 3, caspase (Casp)12, Casp4, Casp8, lymphotoxin β receptor, NLR family, apoptosis inhibitory protein 2, TNF receptor superfamily (Tnfrsf)1a, TNF superfamily (Tnfs)f10 and Tnfsf12, were downregulated, whereas nucleolar protein 3 was upregulated in the RA-induced NTD mice. Chromatin immunoprecipitation assays revealed that the regulatory regions of these differentially expressed TNF-related genes showed reduced histone H3K27 acetylation in NTDs, compared with control mice without NTDs. Reverse transcription-quantitative polymerase chain reaction revealed that H3K27ac-binding to the differentially regulated genes was markedly decreased in the NTD mice, whereas binding to the unchanged genes Casp3 and Nfkb1 was unaffected. In conclusion, certain TNF-related genes appeared to be downregulated in NTDs, possibly as a result of abnormal histone H3K27 acetylation. These results shed new light on the epigenetic dysregulation of apoptosis-related genes in NTDs.

Near-Infrared Light-Activated Thermosensitive Liposomes as Efficient Agents for Photothermal and Antibiotic Synergistic Therapy of Bacterial Biofilm.

Biofilm is closely related to chronic infections and is difficult to eradicate. Development of effective therapy strategies to control biofilm infection is still challenging. Aiming at biofilm architecture, we designed and prepared near-infrared-activated thermosensitive liposomes with photothermal and antibiotic synergistic therapy capacity to eliminate Pseudomonas aeruginosa biofilm. The liposomes with positive charge and small size aided to enter the biofilm microchannels and locally released antibiotics in infection site. The liposomes could remain stable at 37 °C and release about 80% antibiotics over 45 °C. The biofilm dispersion rate was up to 80%, which was a 7- to 8-fold rise compared to excess antibiotic alone, indicating that the localized antibiotic release and photothermal co-therapy improved the antimicrobial efficiency. In vivo drug-loaded liposomes in treating P. aeruginosa-induced abscess exhibited an outstanding therapeutic effect. Furthermore, photothermal treatment could stimulate the expression of bcl2-associated athanogene 3 to prevent normal tissue from thermal damage. The near-infrared-activated nanoparticle carriers had the tremendous therapeutic potential to dramatically enhance the efficacy of antibiotics through thermos-triggered drug release and photothermal therapy.

Transcriptomic profiling of mitochondrial dysfunction induced apoptosis in accelerated cardiovascular disease

IntroductionVascular calcification is a significant contributor to accelerated cardiovascular disease and mortality in patients with chronic kidney disease (CKD). We previously reported that heat shock protein 70 (HSP70) induction can inhibit human aortic smooth muscle cell (HA‐SMC) calcification in CKD. In addition, our data indicated mitochondrial complexes II, IV, NADH cytochrome c reductase (NCCR), succinate cytochrome c reductase (SCCR), and cytochrome c oxidase (CCO) activities were preserved by HSP70 induction leading to reduced apoptosis. Apoptosis occurs with either receptor mediated extrinsic pathway or the mitochondrial‐dependent intrinsic pathway. The molecular mechanisms of HSP70 vasculo‐protective effects involving mitochondrial dysfunction induced apoptosis are currently unknown.HypothesisThe goal of the present study was to investigate the transcriptomic profile of genes involved in mitochondrial function and vasculo‐protective mechanisms of HSPs in apoptosis induced vascular calcification.MethodsHuman arteries were collected from healthy and CKD patients. Human aortic endothelial cells (HAECs) was treated in calcification medium (CM: 5mM β‐glycerolphosphate and 5mM CaCl2) in time‐course experiments (0–48 hours), in vitro. Total RNA from human arteries and HAECs was extracted using the Nucleospin RNA isolation kit. cDNA was synthesized and fragmented using SMART‐seq2 and Nextera XT (Illumina), respectively to generate libraries for sequencing on Hi‐seq 2000 (Illumina). Five million single‐ended raw reads from each sample were mapped to the human reference genome consortium GRCh38 by Burrows‐Wheeler Aligner (BWA). Gene analysis were performed by the combinations of fold‐changes on log 2 ratio, and p value < 0.05.ResultsIn healthy human arteries, we found that HSPA5 (fold change, FC, 3.85), HSPA1 (FC=3.64), and HSPA9 (FC=3.85) expression was preserved and Bcl2 associated Athanogene 1 (BAG1) (FC=7.44) was upregulated. However, mitochondrial fusion regulation genes MFN1 (FC=1.83) and OPA1 (FC=2.33) were down‐regulated in CKD arteries together with downregulation of downstream anti‐apoptotic gene Bcl2 (FC=4.52) and up‐regulation of pro‐apoptotic gene BAX (FC=2.77) and P53 (FC=5.35). Lastly, caspase 3 (FC=2.6) and cytochrome c (FC=2.29) expression were significantly increased in CKD arteries compared to control. In the primary HAECs, our data indicates that BAG1 was significantly increased at 6 hours prior to upregulation of Bcl2 at 12 hours in HAECs after CM treatment. BAG1 is a co‐chaperone for heat‐shock protein 70 (HSP70) that coordinates signals for cell growth in response to stress. Stress respondent genes, DNAJB6 (HSP40) and HSPA5 (HSP70 member5) was significantly increased at 24 hours and 12 hours respectively in CM‐treated HAECs.ConclusionComplex differential gene regulation involving preservation of mitochondrial function and cell apoptosis may be involved in the pathogenesis of accelerated vascular calcification. Decreased HSPs in CKD arteries may play the key role in mitochondrial dysfunction induced endothelial cells' apoptosis, which further leading to vascular calcification. Our findings may serve to inform the rational development of therapeutic strategies for vascular calcification involving therapeutic targeting of HSPs.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Haplo-insufficiency of Bcl2-associated athanogene 3 in mice results in progressive left ventricular dysfunction, β-adrenergic insensitivity, and increased apoptosis.

Bcl2-associated athanogene 3 (BAG3) is a 575 amino acid protein that is found predominantly in the heart, skeletal muscle, and many cancers. Deletions and truncations in BAG3 that result in haplo-insufficiency have been associated with the development of dilated cardiomyopathy. To study the cellular and molecular events attributable to BAG3 haplo-insufficiency we generated a mouse in which one allele of BAG3 was flanked by loxP recombination sites (BAG3fl/+ ). Mice were crossed with α-MHC-Cre mice in order to generate mice with cardiac-specific haplo-insufficiency (cBAG3+/-) and underwent bi-weekly echocardiography to assess their cardiac phenotype. By 10 weeks of age, cBAG3+/- mice demonstrated increased heart size and diminished left ventricular ejection fraction when compared with non-transgenic littermates (Cre-/- BAG3fl/+ ). Contractility in adult myocytes isolated from cBAG3+/- mice were similar to those isolated from control mice at baseline, but showed a significantly decreased response to adrenergic stimulation. Intracellular calcium ([Ca2+ ]i ) transient amplitudes in myocytes isolated from cBAG3+/- mice were also similar to myocytes isolated from control mice at baseline but were significantly lower than myocytes from control mice in their response to isoproterenol. BAG3 haplo-insufficiency was also associated with decreased autophagy flux and increased apoptosis. Taken together, these results suggest that mice in which BAG3 has been deleted from a single allele provide a model that mirrors the biology seen in patients with heart failure and BAG3 haplo-insufficiency.

Genes involved in stress response influence lithium efficacy in bipolar patients.

In mood disorders, chronic stimulation with stress results in aberrant regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Lithium was shown to influence HPA axis function. The underlying genetic background as well as environmental context may influence the stress response, and therefore lithium efficacy. The aim of the present study was to analyze if genetic variants located in genes involved in HPA axis regulation affect the response to long-term lithium treatment in bipolar patients. We included 93 patients with bipolar disorder (32 males and 61 females), aged 31-80years. The patients had been treated with lithium carbonate for at least 5years. The magnitude of the lithium response was assessed using the Alda scale. Genotyping was performed for 28 polymorphisms in the genes encoding the following proteins involved in HPA axis regulation: corticotropin-releasing hormone receptor 1 (CRHR1), arginine vasopressin receptor 1B (AVPR1b), FK506 binding protein (FKBP) 5, FKBP4, BCL2-associated athanogene 1 (BAG1), stress induced phosphoprotein 1 (STIP1), glucocorticoid-induced transcript 1 (GLCC1), dual specificity phosphatase 1 (DUSP1) serine and arginine rich splicing factor (SRSF) 3, SRSF9, SRSF5, and acid phosphatase 1 (ACP1). Linkage disequilibrium and haplotype analysis were then performed, followed by statistical analysis (Statistica v.12; Stasoft, Krakow, Poland). We found a correlation between stressful life events at first episode and worse response to lithium (P=.019). In single marker analysis, we observed a significant association between three FKBP5 polymorphisms (rs1360780, rs7748266 and rs9296158), one ACP1 variant (rs300774) and one glucocorticoid-induced transcript 1 gene (GLCC1) variant (rs37972) and the degree of lithium response. Five out of seven FKBP5 polymorphisms showed strong linkage with one haplotype demonstrating an association with lithium efficacy (P=.008). No relationship was found between the other analyzed polymorphisms and lithium response. The response to lithium may depend on the variants of genes regulating the HPA axis and stressful life events in bipolar patients.

MiR‑494‑BAG‑1 axis is involved in cinobufacini‑induced cell proliferation and apoptosis in gastric cancer.

Cinobufacini is widely used in the treatment of advanced cancers. It has been previously reported that microRNA (miR)‑494 was upregulated in cinobufacini‑treated gastric cancer cells; however, the detailed role of miR‑494 in the anti‑tumor activity of cinobufacini is unclear. The present study aimed to clarify the function of miR‑494 in cinobufacini‑induced cell behavior changes. Cell viability and proliferation ability were investigated using a Cell Counting Kit‑8 assay. Flow cytometry was performed to investigate the apoptosis rate of gastric cancer (GC) cells. The mRNA expression levels of microRNA (miR)‑494 and BCL2 associated athanogene1 (BAG‑1) were investigated using reverse transcription‑quantitative polymerase chain reaction, and the protein expression level of BAG‑1 was investigated using western blot assays. The results demonstrated that treatment with cinobufacini suppressed proliferation and promoted apoptosis of gastric cancer cells. miR‑494 acts as a tumor suppressor gene in gastric cancer. In cinobufacini‑treated cells, miR‑494 and BAG‑1 exhibited opposing expression trends. Furthermore, knockdown of miR‑494 in cinobufacini‑treated cells upregulated the protein expression level of BAG‑1, promoted cell proliferation and inhibited cell apoptosis. In addition, inhibition of BAG‑1 using small interfering RNA in cinobufacini‑treated cells partially abrogated the effects of miR‑494 inhibitor on cell proliferation and apoptosis. Thus, these results suggest that cinobufacini suppresses GC cells proliferation and promotes apoptosis partially through the regulation of miR‑494‑BAG‑1 axis, which may provide a novel insight into the functional mechanism of cinobufacini.

BAG3 promotes tumour cell proliferation by regulating EGFR signal transduction pathways in triple negative breast cancer

Triple-negative breast cancer (TNBC), is a heterogeneous disease characterised by absence of expression of the estrogen receptor (ER), progesterone receptor (PR) and lack of amplification of human epidermal growth factor receptor 2 (HER2). TNBC patients can exhibit poor prognosis and high recurrence stages despite early response to chemotherapy treatment. In this study, we identified a pro-survival signalling protein BCL2- associated athanogene 3 (BAG3) to be highly expressed in a subset of TNBC cell lines and tumour tissues. High mRNA expression of BAG3 in TNBC patient cohorts significantly associated with a lower recurrence free survival. The epidermal growth factor receptor (EGFR) is amplified in TNBC and EGFR signalling dynamics impinge on cancer cell survival and disease recurrence. We found a correlation between BAG3 and EGFR expression in TNBC cell lines and determined that BAG3 can regulate tumour cell proliferation, migration and invasion in EGFR expressing TNBC cells lines. We identified an interaction between BAG3 and components of the EGFR signalling networks using mass spectrometry. Furthermore, BAG3 contributed to regulation of proliferation in TNBC cell lines by reducing the activation of components of the PI3K/AKT and FAK/Src signalling subnetworks. Finally, we found that combined targeting of BAG3 and EGFR was more effective than inhibition of EGFR with Cetuximab alone in TNBC cell lines. This study demonstrates a role for BAG3 in regulation of distinct EGFR modules and highlights the potential of BAG3 as a therapeutic target in TNBC.

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.

HSPB8 and BAG3 cooperate to promote spatial sequestration of ubiquitinated proteins and coordinate the cellular adaptive response to proteasome insufficiency.

BCL2-associated athanogene (BAG)-3 is viewed as a platform that would physically and functionally link distinct classes of molecular chaperones of the heat shock protein (HSP) family for the stabilization and clearance of damaged proteins. In this study, we show that HSPB8, a member of the small heat shock protein subfamily, cooperates with BAG3 to coordinate the sequestration of harmful proteins and the cellular adaptive response upon proteasome inhibition. Silencing of HSPB8, like depletion of BAG3, inhibited targeting of ubiquitinated proteins to the juxtanuclear aggresome, a mammalian system of spatial quality control. However, aggresome targeting was restored in BAG3-depleted cells by a mutant BAG3 defective in HSPB8 binding, uncoupling HSPB8 function from its binding to BAG3. Depletion of HSPB8 impaired formation of ubiquitinated microaggregates in an early phase and interfered with accurate modifications of the stress sensor p62/sequestosome (SQSTM)-1. This impairment correlated with decreased coupling of BAG3 to p62/SQSTM1 in response to stress, hindering Kelch-like ECH-associated protein (KEAP)-1 sequestration and stabilization of nuclear factor E2-related factor (Nrf)-2, an important arm of the antioxidant defense. Notably, the myopathy-associated mutation of BAG3 (P209L), which lies within the HSPB8-binding motif, deregulated the association between BAG3 and p62/SQSTM1 and the KEAP1-Nrf2 signaling axis. Together, our findings support a so-far-unrecognized role for the HSPB8-BAG3 connection in mounting of an efficient stress response, which may be involved in BAG3-related human diseases.-Guilbert, S. M., Lambert, H., Rodrigue, M.-A., Fuchs, M., Landry, J., Lavoie, J. N. HSPB8 and BAG3 cooperate to promote spatial sequestration of ubiquitinated proteins and coordinate the cellular adaptive response to proteasome insufficiency.

Bcl-2-associated athanogene 3 (BAG3) is an enhancer of small heat shock protein turnover via activation of autophagy in the heart

Bcl-2-associated athanogene 3 (BAG3) is strongly expressed in both cardiac and skeletal muscle. A recent study showed that BAG3 may play a protective role in muscles. Little is known, however, regarding the detailed role of BAG3 in cardiac muscle. To better understand the functional role of cardiac BAG3 in the heart, we generated transgenic (TG) mice that overexpress BAG3. A decrease in fractional shortening, and the induction of cardiac atrial natriuretic peptide, were observed in BAG3 TG mice. Moreover, a marked reduction in the protein level of small HSPs was detected in BAG3 TG mouse hearts. We analyzed the cardiac small HSP levels when either the ubiquitin-proteasome system (UPS) or the autophagy system (AS) was inhibited in BAG3 TG mice. The protein turnovers of small HSPs by the AS were activated in BAG3 TG mouse hearts. Thus, BAG3 is critical for the protein turnover of small HSPs via activation of autophagy in the heart.