Construction of an autophagy-related genes risk model as predicting prognosis: BAG1 suppresses growth of clear cell renal cell carcinoma.

BACKGROUND: The incidence of clear cell renal cell carcinoma (ccRCC) is increasing annually. While the cure rate and prognosis of early ccRCC are promising, the 5-year survival rate of patients with metastatic ccRCC is below 12%. Autophagy disfunction is closely related to infection, cancer, neurodegeneration and aging. Nevertheless, there has been limited exploration of the association between autophagy and ccRCC through bioinformatics analysis. METHODS: A novel risk model of autophagy-related genes (ARGs) was constructed to predict the prognosis of patients with ccRCC and guide the individualized treatment to some extent. Relevant data samples were obtained from the TCGA database, and ccRCC-related ARGs were identified by Pearson correlation analysis, leading to the establishment of a risk model covering 10 ccRCC-related ARGs. Many indicators were used to assess the accuracy of the risk model. RESULTS: Receiver operating characteristic (ROC) curve analysis showed that the risk model had high accuracy, indicating that the risk model could predict the prognosis of ccRCC patients. Moreover, the findings revealed significant differences about immune and metabolic features in low- and high-risk groups. The study also found that BAG1 within the risk model was closely related to the prognosis of ccRCC and an independent risk factor. In vitro and in vivo experiments validated for the first time that BAG1 could suppress the proliferation, migration, and invasion of ccRCC. CONCLUSION: The construction of ARGs risk model, can well predict the prognosis of ccRCC patients, and provide guidance for individual therapy to patients. It was also found that BAG1 has significant prognostic value for ccRCC patients and acts as a tumor suppressor gene in ccRCC. These findings have crucial implications for the prognosis and treatment of ccRCC patients.

Toxoplasma gondii bradyzoite-specific BAG1 is nonessential for cyst formation due to compensation by other heat-shock proteins.

BACKGROUND: Toxoplasma gondii is an opportunistic pathogenic protozoan that infects all warm-blooded animals, including humans, and causes zoonotic toxoplasmosis. The bradyzoite antigen 1 (BAG1), known as heat-shock protein (HSP)30, is a specific antigen expressed during the early stage of T. gondii tachyzoite-bradyzoite conversion. METHODS: A bag1 gene knockout strain based on the T. gondii type II ME49 was constructed and designated as ME49Δbag1. The invasion, proliferation, and cyst formation efficiency in the cell model and survival in the mouse model were compared between the ME49 and ME49Δbag1 strains after infection. Quantitative polymerase chain reaction (qPCR) was used to detect the transcriptional level of important genes, and western-blot was used to detect protein levels. RESULTS: ME49Δbag1 displayed significantly inhibited cyst formation, although it was not completely blocked. During early differentiation induced by alkaline and starvation conditions in vitro, the proliferation of ME49Δbag1 was significantly accelerated relative to the ME49 strain. Meanwhile, the transcription of the HSP family and bradyzoite formation deficient 1 (bfd1) were significantly enhanced. The observed upregulation suggests a compensatory mechanism to counterbalance the impaired stress responses of T. gondii following bag1 knockout. On the other hand, the elevated transcription levels of several HSP family members, including HSP20, HSP21, HSP40, HSP60, HSP70, and HSP90, along with BFD1, implied the involvement of alternative regulatory factors in bradyzoite differentiation aside from BAG1. CONCLUSIONS: The data suggested that when bag1 was absent, the stress response of T. gondii was partially compensated by increased levels of other HSPs, resulting in the formation of fewer cysts. This highlighted a complex regulatory network beyond BAG1 influencing the parasite's transformation into bradyzoites, emphasizing the vital compensatory function of HSPs in the T. gondii life cycle adaptation.

Bag-1-mediated HSF1 phosphorylation regulates expression of heat shock proteins in breast cancer cells.

According to the World Health Organization in 2022, 2.3 million women were diagnosed with breast cancer. Investigating the interaction networks between Bcl-2-associated athanogene (Bag)-1 and other chaperone proteins may further the current understanding of the regulation of protein homeostasis in breast cancer cells and contribute to the development of treatment options. The present study aimed to determine the interactions between Bag-1 and heat shock proteins (HSPs); namely, HSP90, HSP70 and HSP27, to elucidate their role in promoting heat shock factor-1 (HSF1)-dependent survival of breast cancer cells. HER2-negative (MCF-7) and HER2-positive (BT-474) cell lines were used to examine the impact of Bag-1 expression on HSF1 and HSPs. We demonstrated that Bag-1 overexpression promoted HER2 expression in breast cancer cells, thereby resulting in the concurrent constitutive activation of the HSF1-HSP axis. The activation of HSP results in the stabilization of several tumor-promoting HSP clients such as AKT, mTOR and HSF1 itself, which substantially accelerates tumor development. Our results suggest that Bag-1 can modulate the chaperone activity of HSPs, such as HSP27, by directly or indirectly regulating the phosphorylation of HSF1. This modulation of chaperone activity can influence the activation of genes involved in cellular homeostasis, thereby protecting cells against stress.

Bag1 protein loss sensitizes mouse embryonic fibroblasts to glutathione depletion.

Bcl2-associated athanogene-1 protein (Bag1) acts as a co-chaperone of heat shock protein 70 and heat shock cognate 70 and regulates multiple cellular processes, including cell proliferation, apoptosis, environmental stress response, and drug resistance. Since Bag1 knockout mice exhibited fetal lethality, the in vivo function of Bag1 remains unclear. In this study, we established a mouse line expressing Bag1 gene missing exon 5, which corresponds to an encoding region for the interface of heat shock protein 70/heat shock cognate 70. Despite mice carrying homoalleles of the Bag1 mutant (Bag1Δex5) expressing undetectable levels of Bag1, Bag1Δex5 homozygous mice developed without abnormalities. Bag1Δex5 protein was found to be highly unstable in cells and in vitro. We found that the growth of mouse embryonic fibroblasts derived from Bag1Δex5-homo mice was attenuated by doxorubicin and a glutathione (GSH) synthesis inhibitor, buthionine sulfoximine. In response to buthionine sulfoximine, Bag1Δex5-mouse embryonic fibroblasts exhibited a higher dropping rate of GSH relative to the oxidized glutathione level. In addition, Bag1 might mitigate cellular hydrogen peroxide levels. Taken together, our results demonstrate that the loss of Bag1 did not affect mouse development and that Bag1 is involved in intracellular GSH homeostasis, namely redox homeostasis.

Supramolecular nanodrug targeting CDK4/6 overcomes BAG1 mediated cisplatin resistance in oral squamous cell carcinoma.

Chemoresistance to cisplatin remains a significant challenge affecting the prognosis of advanced oral squamous cell carcinoma (OSCC). However, the specific biomarkers and underlying mechanisms responsible for cisplatin resistance remain elusive. Through comprehensive bioinformatic analyses, we identified a potential biomarker, BCL2 associated athanogene-1 (BAG1), showing elevated expression in head and neck squamous cell carcinoma (HNSCC). Since OSCC represents the primary pathological type of HNSCC, we investigated BAG1 expression in human tumor tissues and cisplatin resistant OSCC cell lines, revealing that silencing BAG1 induced apoptosis in cisplatin-resistant cells both in vitro and in vivo. This effect led to impaired cell viability of cisplatin resistant OSCC cells and indicated a positive correlation between BAG1 expression and the G1/S transition during cell proliferation. Based on these insights, the administration of a CDK4/6 inhibitor in combination with cisplatin effectively overcame cisplatin resistance in OSCC through the CDK4/6-BAG1 axis. Additionally, to enable simultaneous drug delivery and enhance synergistic antitumor efficacy, we developed a novel supramolecular nanodrug LEE011-FFERGD/CDDP, which was validated in an OSCC orthotopic mouse model. In summary, our study highlights the potential of a combined administration of CDK4/6 inhibitor and cisplatin as a promising therapeutic regimen for treating advanced or cisplatin resistant OSCC.

Comparison of the immune response and protection against the experimental Toxoplasma gondii infection elicited by immunization with the recombinant proteins BAG1, ROP8, and BAG1-ROP8.

Toxoplasmosis is one of the most dangerous zoonotic diseases, causing serious economic losses worldwide due to abortion and reproductive problems. Vaccination is the best way to prevent disease; thus, it is imperative to develop a candidate vaccine for toxoplasmosis. BAG1 and ROP8 have the potential to become vaccine candidates. In this study, rTgBAG1, rTgROP8, and rTgBAG1-rTgROP8 were used to evaluate the immune effect of vaccines in each group by detecting the humoral and cellular immune response levels of BABL/c mice after immunization and the ability to resist acute and chronic infection with Toxoplasma gondii (T. gondii). We divided the mice into vaccine groups with different proteins, and the mice were immunized on days 0, 14, and 28. The protective effects of different proteins against T. gondii were analysed by measuring the cytokines, serum antibodies, splenocyte proliferation assay results, survival time, and number and diameter of brain cysts of mice after infection. The vaccine groups exhibited substantially higher IgG, IgG1, and IgG2a levels and effectively stimulated lymphocyte proliferation. The levels of IFN-γ and IL-2 in the vaccine group were significantly increased. The survival time of the mice in each vaccine group was prolonged and the diameter of the cysts in the vaccine group was smaller; rTgBAG1-rTgROP8 had a better protection. Our study showed that the rTgBAG1, rTgROP8, and rTgBAG1-rTgROP8 recombinant protein vaccines are partial but effective approaches against acute or chronic T. gondii infection. They are potential candidates for a toxoplasmosis vaccine.

Development and Validation of a New BAG-1L-Specific Antibody to Quantify BAG-1L Protein Expression in Advanced Prostate Cancer.

BCL-2-associated athanogene-1L (BAG-1L) is a critical co-regulator that binds to and enhances the transactivation function of the androgen receptor, leading to prostate cancer development and progression. Studies investigating the clinical importance of BAG-1L protein expression in advanced prostate cancer have been limited by the paucity of antibodies that specifically recognize the long isoform. In this study, we developed and validated a new BAG-1L-specific antibody using multiple orthogonal methods across several cell lines with and without genomic manipulation of BAG-1L and all BAG-1 isoforms. Following this, we performed exploratory immunohistochemistry to determine BAG-1L protein expression in normal human, matched castration-sensitive prostate cancer (CSPC) and castration-resistant prostate cancer (CRPC), unmatched primary and metastatic CRPC, and early breast cancer tissues. We demonstrated higher BAG-1L protein expression in CRPC metastases than in unmatched, untreated, castration-sensitive prostatectomies from men who remained recurrence-free for 5 years. In contrast, BAG-1L protein expression did not change between matched, same patient, CSPC and CRPC biopsies, suggesting that BAG-1L protein expression may be associated with more aggressive biology and the development of castration resistance. Finally, in a cohort of patients who universally developed CRPC, there was no association between BAG-1L protein expression at diagnosis and time to CRPC or overall survival, and no association between BAG-1L protein expression at CRPC biopsy and clinical outcome from androgen receptor targeting therapies or docetaxel chemotherapy. The limitations of this study include the requirement to validate the reproducibility of the assay developed, the potential influence of pre-analytical factors, timing of CRPC biopsies, relatively small patient numbers, and heterogenous therapies on BAG-1L protein expression, and the clinical outcome analyses performed. We describe a new BAG-1L-specific antibody that the research community can further develop to elucidate the biological and clinical significance of BAG-1L protein expression in malignant and nonmalignant diseases.

Bag-1 Protects Nucleus Pulposus Cells from Oxidative Stress by Interacting with HSP70.

Bcl-2-associated athanogene 1 (Bag-1) is a multifunctional prosurvival protein that binds to several intracellular targets and promotes cell survival. HSP70 and Raf-1 are important targets of Bag-1; however, the protective function of Bag-1 in nucleus pulposus (NP) cells remains unclear. In this study, we determined the effects of Bag-1 on NP cells under oxidative stress induced by treatment with hydrogen peroxide (H2O2). We found that Bag-1 was bound to HSP70, but Bag-1-Raf1 binding did not occur in NP cells. Bag-1 overexpression in NP cells enhanced cell viability and mitochondrial function and significantly suppressed p38/MAPKs phosphorylation during oxidative stress, although NP cells treated with a Bag-1 C-terminal inhibitor, which is the binding site of HSP70 and Raf-1, decreased cell viability and mitochondrial function during oxidative stress. Furthermore, the phosphorylation of the ERK/MAPKs was significantly increased in Bag-1 C-terminal inhibitor-treated NP cells without H2O2 treatment but did not change with H2O2 exposure. The phosphorylation of Raf-1 was not influenced by Bag-1 overexpression or Bag-1 C-terminal binding site inhibition. Overall, the results suggest that Bag-1 preferentially interacts with HSP70, rather than Raf-1, to protect NP cells against oxidative stress.

Downregulation of STAT3 in Epstein-Barr Virus-Positive Hodgkin Lymphoma.

STAT3 is a transcription factor which is activated via various signaling transduction pathways or Epstein-Barr virus (EBV) infection and plays an oncogenic role in lymphoid malignancies including Hodgkin lymphoma (HL). The tumor cells of HL are derived from germinal center B-cells and transformed by chromosomal rearrangements, aberrant signal transduction, deregulation of developmental transcription factors, and EBV activity. HL cell lines represent useful models to investigate molecular principles and deduced treatment options of this malignancy. Using cell line L-540, we have recently shown that constitutively activated STAT3 drives aberrant expression of hematopoietic NKL homeobox gene HLX. Here, we analyzed HL cell line AM-HLH which is EBV-positive but, nevertheless, HLX-negative. Consistently, AM-HLH expressed decreased levels of STAT3 proteins which were additionally inactivated and located in the cytoplasm. Combined genomic and expression profiling data revealed several amplified and overexpressed gene candidates involved in opposed regulation of STAT3 and EBV. Corresponding knockdown studies demonstrated that IRF4 and NFATC2 inhibited STAT3 expression. MIR155 (activated by STAT3) and SPIB (repressed by HLX) showed reduced and elevated expression levels in AM-HLH, respectively. However, treatment with IL6 or IL27 activated STAT3, elevated expression of HLX and MIR155, and inhibited IRF4. Taken together, this cell line deals with two conflicting oncogenic drivers, namely, JAK2-STAT3 signaling and EBV infection, but is sensitive to switch after cytokine stimulation. Thus, AM-HLH represents a unique cell line model to study the pathogenic roles of STAT3 and EBV and their therapeutic implications in HL.

The comparison of differentially expressed microRNAs in Bag-1 deficient and wild type MCF-7 breast cancer cells by small RNA sequencing.

The multifunctional BAG-1 (Bcl-2 athanogene-1) protein promotes breast cancer survival through direct or indirect interaction partners. The number of the interacting partners determines its cellular role in different conditions. As well as interaction partner variability, the amount of BAG-1 protein in the cells could cause dramatic alterations. According to previous studies, while the transient silencing of Bag-1 enhanced drug-induced apoptosis, deletion of BAG-1 could induce stemness properties and Akt-mediated actin remodeling in MCF-7 breast cancer cells. Considering the heterogeneity of breast cancer and the variability of BAG-1 -mediated cell response, it has become essential to determine microRNA (miRNA) functions in breast cancer depending on Bag-1 expression level. This study aims to compare microRNA expression levels in wt and Bag-1 knockout (KO) MCF-7 breast cancer cells. hsa-miR-429 was selected as a potential miRNA in BAG-1KO MCF-7 cells because of the downregulation both in bioinformatics and validation qRT-PCR assay. According to predicted mRNA targets and functional enrichment analysis the ten hub proteins that are phosphatidylinositol-4,5-biphosphate 3-kinase catalytic subunit alpha (PIK3CA), kinase insert domain receptor (KDR), GRB2 associated binding protein 1 (GAB1), Rac family small GTPase1 (RAC1), vascular endothelial growth factor A (VEGFA), Cbl proto-oncogene (CBL), syndecan 2 (SDC2), phospholipase C gamma 1 (PLCG1), E1A binding protein p300 (EP300), and CRK like proto-oncogene, adaptor protein (CRKL) were identified as targets of hsa-miR-429. The functional enrichment analysis showed that the most significant proteins were enriched in PI3K/Akt, focal adhesion, cytoskeleton regulation, proteoglycans in cancer, and Ras signaling pathways. It was determined that hsa-miR-429 targeted these pathways in Bag-1 deficient conditions and could be used as a potential therapeutic target in future studies.

Application of Toxoplasma gondii-specific SAG1, GRA7 and BAG1 proteins in serodiagnosis of animal toxoplasmosis.

Toxoplasmosis is a zoonotic disease caused by the obligate intracellular protozoan parasite T. gondii which is widely prevalent in humans and animals worldwide. The diagnosis of toxoplasmosis and distinguishing acute or chronic T. gondii infections have utmost importance for humans and animals. The TgSAG1, TgGRA7, and TgBAG1 proteins were used in the present study to develop the serological rSAG1-ELISA, rGRA7-ELISA and rBAG1-ELISA methods for the testing of T. gondii specific IgG and IgM antibodies and differentiating acute or chronic toxoplasmosis in 3733 animals, including Tibetan sheep, yaks, pigs, cows, cattle, horses, chickens, camels and donkeys from the Qinghai-Tibetan Plateau. The ELISA tests showed that the overall positivity of IgG antibody was 21.1% (786/3733), 15.3% (570/3733) and 18.2% (680/3733) for rSAG1-, rGRA7- and rBAG1-ELISA, respectively, and the positivity of IgM antibody was 11.8% (439/3733), 13.0% (486/3733) and 11.8% (442/3733) for rSAG1-, rGRA7- and rBAG1-ELISA, respectively. A total of 241 animals (6.5%) positive for all rSAG1-, rGRA7- and rBAG1-IgG were found in this study, and the 141 animals (3.8%) tested were anti-T. gondii IgM positive in all three ELISAs. Moreover, the 338, 284 and 377 animals were IgG positive in rSAG1 + rGRA7-, rBAG1 + rGRA7- and rSAG1 + rBAG1- ELISAs respectively, and the 346, 178 and 166 animals in rSAG1 + rGRA7-, rBAG1 + rGRA7- and rSAG1 + rBAG1-ELISAs were IgM positive respectively. The results confirmed that the application of SAG1, GRA7, and BAG1 recombinant antigens could successfully be used in the detection of specific IgG and IgM antibodies for distinguishing between acute or chronic T. gondii infections. It is inferred that the forms in which current animal species in the plateau area were infected with T. gondii, and the period of infection or the clinical manifestations of the current infections may be different. The present study provides substantial clinical evidence for the differential diagnosis of toxoplasmosis, and the classification of acute and chronic T. gondii infections.

BAG1 down-regulation increases chemo-sensitivity of acute lymphoblastic leukaemia cells.

BCL2-associated athanogene-1 (BAG1) is a multi-functional protein that is found deregulated in several solid cancers and in paediatric acute myeloid leukaemia. The investigation of BAG1 isoforms expression and intracellular localization in B-cell acute lymphoblastic leukaemia (B-ALL) patient-derived specimens revealed that BAG1 levels decrease during disease remission, compared to diagnosis, but drastically increase at relapse. In particular, at diagnosis both BAG1-L and BAG1-M isoforms are mainly nuclear, while during remission the localization pattern changes, having BAG1-M almost exclusively in the cytosol indicating its potential cytoprotective role in B-ALL. In addition, knockdown of BAG1/BAG3 induces cell apoptosis and G1-phase cell cycle arrest and, more intriguingly, shapes cell response to chemotherapy. BAG1-depleted cells show an increased sensitivity to the common chemotherapeutic agents, dexamethasone or daunorubicin, and to the BCL2 inhibitor ABT-737. Moreover, the BAG1 inhibitor Thio-2 induces a cytotoxic effect on RS4;11 cells both in vitro and in a zebrafish xenograft model and strongly synergizes with pan-BCL inhibitors. Collectively, these data sustain BAG1 deregulation as a critical event in assuring survival advantage to B-ALL cells.

The delayed death-causing nature of Rosmarinus officinalis leaf extracts and their mixture within experimental chronic toxoplasmosis: Therapeutic and prophylactic implications.

BACKGROUND: The restricted effect, significant toxicity, and emerging resistance of anti-toxoplasmosis synthetic agents impose the search for alternatives. The current research aimed to evaluate the prophylactic and therapeutic efficacy of Rosmarinus officinalis extracts and their mixtures against chronic murine toxoplasmosis and to clarify the phenomenon of delayed death. METHODS: This research included two experimental designs, the first to test the preventive and curative efficacy of the extracts and the second to assess delayed death in mice infected with the ME49 strain of Toxoplasma gondii. The essential oils of the plant were analyzed by gas chromatography/mass spectrometry. RESULTS: Treatment with a mixture of rosemary extracts displayed reduction rates of 81% for T. gondii cyst burden and 23% for cyst viability. The reinfected group with the pretreated cysts reported 93.4% reduction in cyst burden and 95.4% in cyst viability. Moreover, 90% reduction of the infectivity rate was obtained. The therapeutic efficacy of this mixture was superior to its valuable prophylactic effect. Histopathological examination of liver and brain tissue exhibited marked improvement. Both extracts possess free radical scavenging and antioxidant activities evidenced by high expression of iNOS stain. Our results were signified by low BAG-1 gene expression and massive mutilation of T. gondii cyst in the targeted group using scanning electron microscopy. Analysis of R. officinalis revealed the presence of isobornylformate as a novel ingredient. CONCLUSIONS: R. officinalis displays a therapeutic rather than prophylactic potential, indicating the emergence of an effective safe alternative therapy.

Expression of BAG1 is associated with prognosis in kidney renal clear cell carcinoma based on bioinformatics.

BACKGROUND: BCL2 associated Athano-Gene 1 (BAG1) has been described to be involved in the development and progression of cancer. But the role of BAG1 in kidney renal clear cell carcinoma (KIRC) has remained largely unknown. METHODS: We performed bioinformatic analysis of data from TCGA and GEO dataset. The role of BAG1 in KIRC was explored by Logistic and Cox regression model. The molecular mechanisms of BAG1 was revealed by GSEA. RESULTS: The current study found that the KIRC tumor samples have a low level of BAG1 mRNA expression compared to the matched normal tissues based on TCGA data and GEO databases. Low expression of BAG1 in KIRC was significantly associated with Sex, clinical pathological stage, tumor-node-metastasis (TNM) stage, hemoglobin levels, cancer status and history of neoadjuvant treatment. Kaplan-Meier survival analysis indicated that KIRC patients with BAG1 high expression have a longer survival time than those with BAG1 low expression (p < 0.000). Cox regression analysis showed that BAG1 remained independently associated with overall survival, with a hazard ratio (HR) of 1.75(CI:1.05-2.90; p = 0.029). GSEA indicated that the signaling pathways including fatty acid metabolism and oxidative phosphorylation were differentially enriched in high BAG1 expression phenotype. CONCLUSIONS: These findings suggested that BAG1 expression may act as a potential favorable prognostic marker and challenging therapeutic target.

Co-Chaperone Bag-1 Plays a Role in the Autophagy-Dependent Cell Survival through Beclin 1 Interaction.

Expression levels of the major mammalian autophagy regulator Beclin 1 and its interaction with Bcl-2 regulate the switch between autophagic cell survival and apoptotic cell death pathways. However, some of the regulators and the precise mechanisms of these processes still remain elusive. Bag-1 (Bcl-2 associated athanogene-1), a member of BAG family proteins, is a multifunctional pro-survival molecule that possesses critical functions in vital cellular pathways. Herein, we report the role of Bag-1 on Bcl-2/Beclin 1 crosstalk through indirectly interacting with Beclin 1. Pull-down experiments suggested a molecular interaction between Bag-1 and Beclin 1 in breast cancer cell lines. On the other hand, in vitro binding assays showed that Bag-1/Beclin 1 interaction does not occur directly but occurs through a mediator molecule. Bag-1 interaction with p-Beclin 1 (T119), indicator of early autophagy, is increased during nutrient starvation suggesting involvement of Bag-1 in the autophagic regulation. Furthermore, CRISPR/Cas9-mediated Bag-1 knock-out in MCF-7 cells hampered cell survival and proliferation and resulted in decreased levels of total LC3 under starvation. Collectively, we suggest that Bag-1 modulates cell survival/death decision through maintaining macroautophagy as a component of Beclin 1-associated complexes.

Bag-1 mediates glucocorticoid receptor trafficking to mitochondria after corticosterone stimulation: Potential role in regulating affective resilience.

Molecular abnormalities within the Glucocorticoid Receptor (GR) stress signaling pathway involved in dysfunction of mitochondria and confer vulnerability to stress-related psychiatric disorders. Bcl-2 associated athanogene (Bag-1) is a target for the actions of mood stabilizers. Bag-1 interacts with GR, thereby regulating glucocorticoid function. In this study, we investigate the potential role of Bag-1 in regulating GR translocation into mitochondria. Corticosterone (CORT) treatment significantly enhanced Bag-1/GR complex formation and GR mitochondrial translocation in cultured rat cortical neurons after treatment for 30 min and 24 hr. By contrast, after stimulation with CORT for 3 days, localization of the Bag-1/GR complex and mitochondrial GR were reduced. Similar results were obtained in mice, in which administrated CORT in drinking water for 21 days significantly impaired the GR levels in the mitochondria, while Bag-1 over-expression rescued this reduction. Furthermore, chronic CORT exposure led to anhedonia-like and depression-like behaviors in the sucrose-consumption test and forced swimming test, and these behaviors were rescued by Bag-1 over-expression. These results suggest that Bag-1 mediates GR trafficking to mitochondria and regulates affective resilience in response to a CORT increase and provide potential insight into the mechanisms by which Bag-1 and GR could contribute to the physiology and pathogenesis of psychiatric disorders in response to the change of stress hormone.

Mutation of the BAG-1 domain decreases its protective effect against hypoxia/reoxygenation by regulating HSP70 and the PI3K/AKT signalling pathway in SY-SH5Y cells.

BCL-2-associated athanogene-1 (BAG-1) is a multifunctional protein that was first identified as a binding partner of BCL-2. Our previous results indicated that BAG-1 large (BAG-1L) overexpression significantly increases cell viability and decreases apoptosis by upregulating HSP70 and p-AKT in response to hypoxia/reoxygenation in SY-SH5Y cells. However, the functional domain of BAG-1L that exerts these protective effects against hypoxic damage has not been identified. In this study, we examined changes in HSP70 and p-AKT protein levels in SH-SY5Y cells with or without BAG-1L domain mutation after six hours of hypoxia/reoxygenation treatment. The BAG-1 domain mutant (BAG-1MUT) attenuated neuronal viability and proliferation while enhancing apoptosis after hypoxia/reoxygenation, which was achieved in part by inhibiting the HSP70 and p-AKT signalling pathways. This evidence illustrates that the BAG-1 domain plays a key role in protecting cells from hypoxia/reoxygenation injury.

A Crucial Role for the Protein Quality Control System in Motor Neuron Diseases.

Motor neuron diseases (MNDs) are fatal diseases characterized by loss of motor neurons in the brain cortex, in the bulbar region, and/or in the anterior horns of the spinal cord. While generally sporadic, inherited forms linked to mutant genes encoding altered RNA/protein products have also been described. Several different mechanisms have been found altered or dysfunctional in MNDs, like the protein quality control (PQC) system. In this review, we will discuss how the PQC system is affected in two MNDs-spinal and bulbar muscular atrophy (SBMA) and amyotrophic lateral sclerosis (ALS)-and how this affects the clearance of aberrantly folded proteins, which accumulate in motor neurons, inducing dysfunctions and their death. In addition, we will discuss how the PQC system can be targeted to restore proper cell function, enhancing the survival of affected cells in MNDs.

Bag-1 stimulates Bad phosphorylation through activation of Akt and Raf kinases to mediate cell survival in breast cancer.

BACKGROUND: Bag-1 (Bcl-2-associated athanogene) is a multifunctional anti-apoptotic protein frequently overexpressed in cancer. Bag-1 interacts with a variety of cellular targets including Hsp70/Hsc70 chaperones, Bcl-2, nuclear hormone receptors, Akt and Raf kinases. In this study, we investigated in detail the effects of Bag-1 on major cell survival pathways associated with breast cancer. METHODS: Using immunoblot analysis, we examined Bag-1 expression profiles in tumor and normal tissues of breast cancer patients with different receptor status. We investigated the effects of Bag-1 on cell proliferation, apoptosis, Akt and Raf kinase pathways, and Bad phosphorylation by implementing ectopic expression or knockdown of Bag-1 in MCF-7, BT-474, MDA-MB-231 and MCF-10A breast cell lines. We also tested these in tumor and normal tissues from breast cancer patients. We investigated the interactions between Bag-1, Akt and Raf kinases in cell lines and tumor tissues by co-immunoprecipitation, and their subcellular localization by immunocytochemistry and immunohistochemistry. RESULTS: We observed that Bag-1 is overexpressed in breast tumors in all molecular subtypes, i.e., regardless of their ER, PR and Her2 expression profile. Ectopic expression of Bag-1 in breast cancer cell lines results in the activation of B-Raf, C-Raf and Akt kinases, which are also upregulated in breast tumors. Bag-1 forms complexes with B-Raf, C-Raf and Akt in breast cancer cells, enhancing their phosphorylation and activation, and ultimately leading to phosphorylation of the pro-apoptotic Bad protein at Ser112 and Ser136. This causes Bad's re-localization to the nucleus, and inhibits apoptosis in favor of cell survival. CONCLUSIONS: Overall, Bad inhibition by Bag-1 through activation of Raf and Akt kinases is an effective survival and growth strategy exploited by breast cancer cells. Therefore, targeting the molecular interactions between Bag-1 and these kinases might prove an effective anticancer therapy.

Bag-1 Silence Sensitizes Non-Small Cell Lung Cancer Cells To Cisplatin Through Multiple Gene Pathways.

PURPOSE: B-cell lymphoma-2 (Bcl-2) associated athanogene 1 (Bag-1) is a multifunctional protein, and Bag -1 overexpression is associated with progression, metastasis, and drug resistance in lung cancer. This study assessed the effects of Bag-1 siRNA on sensitization of cisplatin on non-small cell lung cancer (NSCLC) cells. MATERIAL AND METHODS: NSCLC A549 cell line was transfected with Bag-1 or negative control siRNA and then treated with cisplatin for cell viability, CCK-8, LDH, and flow cytometry assays. The Ca2+ levels were analyzed using Fluo-3/AM fluorescence staining, and the protein levels were assessed using Western blot analysis. RESULTS: Bag-1 siRNA significantly knocked down Bag-1 expression and inhibited cell invasion versus the negative control siRNA, while Bag-1 silence sensitized cisplatin to induce A549 cells to apoptosis by induction of cell cycle G1 arrest. At protein level, Bag-1 silence reduced the expression ratio of Bcl-2 to Bcl-2 associated X protein (Bax), downregulated activity of the PI3K/AKT and mitogen-activated protein kinase (MAPK) pathways, and potently upregulated the calcium signaling-mediated pathway. CONCLUSION: This study demonstrated that Bag-1 silencing sensitized A549 to cisplatin to enhance A549 cell apoptosis by modified multiple gene pathways. Further study will evaluate the usefulness of Bag-1 siRNA as a potential targeting therapy for NSCLC.