BCL2-associated Athanogene 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.

Neuronal BAG3 attenuates tau hyperphosphorylation, synaptic dysfunction, and cognitive deficits induced by traumatic brain injury via the regulation of autophagy-lysosome pathway

Growing evidence supports that early- or middle-life traumatic brain injury (TBI) is a risk factor for developing Alzheimer’s disease (AD) and AD-related dementia (ADRD). Nevertheless, the molecular mechanisms underlying TBI-induced AD-like pathology and cognitive deficits remain unclear. In this study, we found that a single TBI (induced by controlled cortical impact) reduced the expression of BCL2-associated athanogene 3 (BAG3) in neurons and oligodendrocytes, which is associated with decreased proteins related to the autophagy-lysosome pathway (ALP) and increased hyperphosphorylated tau (ptau) accumulation in excitatory neurons and oligodendrocytes, gliosis, synaptic dysfunction, and cognitive deficits in wild-type (WT) and human tau knock-in (hTKI) mice. These pathological changes were also found in human cases with a TBI history and exaggerated in human AD cases with TBI. The knockdown of BAG3 significantly inhibited autophagic flux, while overexpression of BAG3 significantly increased it in vitro. Specific overexpression of neuronal BAG3 in the hippocampus attenuated AD-like pathology and cognitive deficits induced by TBI in hTKI mice, which is associated with increased ALP-related proteins. Our data suggest that targeting neuronal BAG3 may be a therapeutic strategy for preventing or reducing AD-like pathology and cognitive deficits induced by TBI.

Bag2 protects against doxorubicin-induced cardiotoxicity by maintaining Pink1-mediated mitophagy

The clinical application of Doxorubicin (DOX) is limited due to its cardiotoxicity. Mitophagy dysfunction is the primary cause of DOX-induced cardiotoxicity (DIC). However, the precise mechanism by which DOX regulates mitophagy remains elusive. Bag2 (BCL2-associated athanogene 2) is a cochaperone implicated in multiple pathological states. The aim of this study was to investigate the potential cardio-protective effects of Bag2 in DIC. C57BL/6 mice and AC16 cells were used to establish DIC model. The expression of Bag2 were measured by western blotting and immunohistochemical. The effects of Bag2 on DIC were assessed through functional gain and loss experiments. Through in vitro and in vivo experiments, we found that Bag2 expression was significantly reduced after DOX treatment. Both Bag2 knockdown and DOX administration resulted in apoptosis, mitochondrial dysfunction, and impaired mitophagy. Conversely, Bag2 overexpression exerted protective effects against these phenotypes induced by DOX stimulation. Mechanistically, Bag2 maintained mitophagy activation by binding to Pink1 and protecting it from proteasome-dependent degradation, thereby preserving mitochondrial function and protecting against myocardial lesions. Our findings suggest that Bag2 may serve as a promising therapeutic target for the treatment of DIC.

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.

Overexpression of BAG3 (Bcl2-associated athanogene 3) in serum and skin of patients with systemic sclerosis.

BAG3 (Bcl2-associated athanogene3) is able to induce the transformation of cancer-associated fibroblasts to alpha smooth muscle actin (a-SMA) positive (+) myofibroblasts. In systemic sclerosis (SSc), a-SMA+ myofibroblasts also play an important role in the progression of fibrosis in the skin and involved internal organs. The aim of the study was to investigate whether BAG3 is overexpressed in SSc and may be a biomarker of fibrogenesis. BAG3 serum levels were measured in 106 patients with SSc, 47 with the limited (lc) and 59 the diffuse (dc) SSc, and in age- and sex-matched healthy controls (HC). BAG3 levels were then compared according to their clinical subset, nailfold video-capillaroscopic (NVC) patterns, interstitial lung disease (ILD, and correlated with modified Rodnan skin score (mRSS) and global disease activity. BAG3 expression was also investigated in skin biopsies of 8 dcSSc patients. BAG3 serum levels were significantly higher in dcSSc (143.3 pg/mL, 95%CI 78-208.5) than in HC (0.68 pg/mL, 95%CI 0.13-1.23), and were significantly higher in patients with late NVC pattern and ILD but did not correlate with disease activity and mRSS. Of note, BAG3 was strongly expressed in the skin biopsies of dcSSc patients. BAG3 is overexpressed in dcSSc patients and may contribute to skin and organ fibrosis by prompting the transition of fibroblasts into myofibroblasts and increasing their survival. Thus, BAG3 may play an important role in SSc fibrotic pathogenesis and be a potential biomarker of fibrosis. Further research on its role as a therapeutic target is warranted.

Exploring the Redox and pH Dimension of Carbonic Anhydrases in Cancer: A Focus on Carbonic Anhydrase 3.

Significance: Both redox and pH are important regulatory processes that underpin cell physiological functions, in addition to influencing cancer cell development and tumor progression. The thioredoxin (Trx) and glutathione redox systems and the carbonic anhydrase (CA) proteins are considered key regulators of cellular redox and pH, respectively, with components of the Trx system and CAs regarded as cancer therapeutic targets. However, the redox and pH axis in cancer cells is an underexplored topic of research. Recent Advances: Structural studies of a CA family member, CA3, localized two of its five cysteine residues to the protein surface. Redox-regulated modifications to CA3 have been identified, including glutathionylation. CA3 has been shown to bind to other proteins, including B cell lymphoma-2-associated athanogene 3, and squalene epoxidase, which can modulate autophagy and proinflammatory signaling, respectively, in cancer cells. Critical Issues: CA3 has also been associated with epithelial-mesenchymal transition processes, which promote cancer cell metastasis, whereas CA3 overexpression activates the phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway, which upregulates cell growth and inhibits autophagy. It is not yet known if CA3 modulates cancer progression through its reported antioxidant functions. Future Directions: CA3 is one of the least studied CA isozymes. Further studies are required to assess the cellular antioxidant role of CA3 and its impact on cancer progression. Identification of other binding partners is also required, including whether CA3 binds to Trx in human cells. The development of specific CA3 inhibitors will facilitate these functional studies and allow CA3 to be investigated as a cancer therapeutic target.

USP32 facilitates non-small cell lung cancer progression via deubiquitinating BAG3 and activating RAF-MEK-ERK signaling pathway

The regulatory significance of ubiquitin-specific peptidase 32 (USP32) in tumor is significant, nevertheless, the biological roles and regulatory mechanisms of USP32 in non-small cell lung cancer (NSCLC) remain unclear. According to our research, USP32 was strongly expressed in NSCLC cell lines and tissues and was linked to a bad prognosis for NSCLC patients. Interference with USP32 resulted in a significant inhibition of NSCLC cell proliferation, migration potential, and EMT development; on the other hand, USP32 overexpression had the opposite effect. To further elucidate the mechanism of action of USP32 in NSCLC, we screened H1299 cells for interacting proteins and found that USP32 interacts with BAG3 (Bcl2-associated athanogene 3) and deubiquitinates and stabilizes BAG3 in a deubiquitinating activity-dependent manner. Functionally, restoration of BAG3 expression abrogated the antitumor effects of USP32 silencing. Furthermore, USP32 increased the phosphorylation level of the RAF/MEK/ERK signaling pathway in NSCLC cells by stabilizing BAG3. In summary, these findings imply that USP32 is critical to the development of NSCLC and could offer a theoretical framework for the clinical diagnosis and management of NSCLC patients in the future.

Precision medicine in peripartum cardiomyopathy: advancing diagnosis and management through genomic and phenotypic integration.

Peripartum cardiomyopathy (PPCM) is a rare and life-threatening cardiac condition characterized by heart failure due to left ventricular systolic dysfunction, often developing in late pregnancy or the early postpartum period. Despite being a leading cause of maternal morbidity and mortality, clinical presentation of PPCM frequently overlaps with normal pregnancy-related physiological changes, causing diagnostic delays and increased complications. Current management strategies, primarily derived from general heart failure protocols, are evolving to address the unique aspects of PPCM. This includes the development of personalized medicine approaches that integrate genetic profiling, biomarker evaluation, and clinical phenotyping. Notable genes such as titin (TTN), Bcl2-associated athanogene 3 (BAG3), and lamin A/C (LMNA) are implicated in PPCM, revealing a complex genetic landscape similar to other cardiomyopathies. Biomarkers like N-terminal pro-brain-type natriuretic peptide (NT-proBNP) and cardiac troponin T (cTnT) are under investigation for their diagnostic and prognostic value, indicating that personalized treatments hold the promise of enhancing diagnostic precision and therapeutic outcomes by tailoring interventions to individual patient profiles. This review article aims to highlight how integrating genetic and phenotypic data can establish a novel framework for managing PPCM, potentially transforming treatment paradigms and improving long-term outcomes.

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.

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.

Sialyltransferase ST3GAL6 silencing reduces α2,3-sialylated glycans to regulate autophagy by decreasing HSPB8-BAG3 in the brain with hepatic encephalopathy.

End-stage liver diseases, such as cirrhosis and liver cancer caused by hepatitis B, are often combined with hepatic encephalopathy (HE); ammonia poisoning is posited as one of its main pathogenesis mechanisms. Ammonia is closely related to autophagy, but the molecular mechanism of ammonia's regulatory effect on autophagy in HE remains unclear. Sialylation is an essential form of glycosylation. In the nervous system, abnormal sialylation affects various physiological processes, such as neural development and synapse formation. ST3 β‍-galactoside α2,‍3-sialyltransferase 6 (ST3GAL6) is one of the significant glycosyltransferases responsible for adding α2,3-linked sialic acid to substrates and generating glycan structures. We found that the expression of ST3GAL6 was upregulated in the brains of mice with HE and in astrocytes after ammonia induction, and the expression levels of α2,3-sialylated glycans and autophagy-related proteins microtubule-associated protein light chain 3 (LC3) and Beclin-1 were upregulated in ammonia-induced astrocytes. These findings suggest that ST3GAL6 is related to autophagy in HE. Therefore, we aimed to determine the regulatory relationship between ST3GAL6 and autophagy. We found that silencing ST3GAL6 and blocking or degrading α2,3-sialylated glycans by way of Maackia amurensis lectin-II (MAL-II) and neuraminidase can inhibit autophagy. In addition, silencing the expression of ST3GAL6 can downregulate the expression of heat shock protein β8 (HSPB8) and Bcl2-associated athanogene 3 (BAG3). Notably, the overexpression of HSPB8 partially restored the reduced autophagy levels caused by silencing ST3GAL6 expression. Our results indicate that ST3GAL6 regulates autophagy through the HSPB8-BAG3 complex.

Abstract 3026: Genetic Deletion Of Skeletal Muscle Bag3 Exacerbates Ischemic Myopathy

Background: Bcl2-associated athanogene 3 (BAG3) is a multifunctional adaptor protein that regulates diverse cellular functions. Mutations in BAG3 are causally linked to myofibrillar myopathy and dilated cardiomyopathy in humans and now appear to be one of the most common to cause human disease. We previously identified a murine coding variant in Bag3 that contributes to hindlimb ischemia (HLI) pathology, however, no cell specific data on Bag3’s role in PAD pathology exists. Hypothesis: We hypothesized that muscle specific BAG3 is required for both the restoration of blood flow and muscle recovery following HLI. Methods: We generated a genetic model of lifelong muscle cell (mature fiber and progenitor) Bag3 deletion (Pax7-Cre; Bag3 -/- ) on a BL6 parental background. Male and Female mice (12-20 weeks; WT: n=45; KO: n=32) were evaluated at baseline and HLI d7 and d28 for blood flow via LDPI, isometric muscle force production, vascular density and muscle morphology. Bag3 deletion was validated by recombination, qRT-PCR and Western Blotting in whole tissue, isolated myofibers, and FACS muscle progenitor cells. Results: KO reduced muscle force production (n=18; 145.8mN Force ± 28.6 vs 56.9 ± 16.8 SEM; p=0.0208) and capillary perfusion (n=7; 0.17 CD31+/Lectin+ vessels ± 0.02 vs 0.09 ± 0.01 SEM p=0.0491) indicative of the multi-compartmental effect of BAG3 loss in skeletal muscle at HLI d7. Extended HLI (d28) revealed a KO phenotype characterized by reduced blood flow recovery (LDPI; n= 7; .68 ischemic/non limb ± 0.5 vs .38 ± 0.6 SEM p=0.0011) and sustained myopathy including reduced myofiber size ( n =7; 1585um 2 ± 161 vs 1093 ± 169 SEM; p =0.05) and increased tissue lesion size ( n =7; 2.5% muscle area ± 2.0 vs 48.3% ± 21.6; p =0.03). Conclusion: Loss of skeletal muscle Bag3 exacerbates myopathy and reduces capillary perfusion and blood flow recovery following HLI and demonstrate a requirement for BAG3 in recovery from limb ischemia.

BAG3 promotes proliferation and migration of arterial smooth muscle cells by regulating STAT3 phosphorylation in diabetic vascular remodeling

BackgroundDiabetic vascular remodeling is the most important pathological basis of diabetic cardiovascular complications. The accumulation of advanced glycation end products (AGEs) caused by elevated blood glucose promotes the proliferation and migration of vascular smooth muscle cells (VSMCs), leading to arterial wall thickening and ultimately vascular remodeling. Therefore, the excessive proliferation and migration of VSMCs is considered as an important therapeutic target for vascular remodeling in diabetes mellitus. However, due to the lack of breakthrough in experiments, there is currently no effective treatment for the excessive proliferation and migration of VSMCs in diabetic patients. Bcl-2-associated athanogene 3 (BAG3) protein is a multifunctional protein highly expressed in skeletal muscle and myocardium. Previous research has confirmed that BAG3 can not only regulate cell survival and apoptosis, but also affect cell proliferation and migration. Since the excessive proliferation and migration of VSMCs is an important pathogenesis of vascular remodeling in diabetes, the role of BAG3 in the excessive proliferation and migration of VSMCs and its molecular mechanism deserve further investigation.MethodsIn this study, BAG3 gene was manipulated in smooth muscle to acquire SM22αCre; BAG3FL/FL mice and streptozotocin (STZ) was used to simulate diabetes. Expression of proteins and aortic thickness of mice were detected by immunofluorescence, ultrasound and hematoxylin-eosin (HE) staining. Using human aorta smooth muscle cell line (HASMC), cell viability was measured by CCK-8 and proliferation was measured by colony formation experiment. Migration was detected by transwell, scratch experiments and Phalloidin staining. Western Blot was used to detect protein expression and Co-Immunoprecipitation (Co-IP) was used to detect protein interaction.ResultsIn diabetic vascular remodeling, AGEs could promote the interaction between BAG3 and signal transducer and activator of transcription 3 (STAT3), leading to the enhanced interaction between STAT3 and Janus kinase 2 (JAK2) and reduced interaction between STAT3 and extracellular signal-regulated kinase 1/2 (ERK1/2), resulting in accumulated p-STAT3(705) and reduced p-STAT3(727). Subsequently, the expression of matrix metallopeptidase 2 (MMP2) is upregulated, thus promoting the migration of VSMCs.ConclusionsBAG3 upregulates the expression of MMP2 by increasing p-STAT3(705) and decreasing p-STAT3(727) levels, thereby promoting vascular remodeling in diabetes. This provides a new orientation for the prevention and treatment of diabetic vascular remodeling.

Physiological processes underpinning the ubiquitous benefits and interactions of melatonin, butyrate and green tea in neurodegenerative conditions

There is a growing dissatisfaction at the lack of progress in treating neurodegenerative conditions, such as Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis. No current pharmaceuticals have any significant impact on the pathophysiological changes occurring in such neurodegenerative conditions. More promising has been the utilization of nutraceuticals, a number of which show preventative and treatment benefits. This article reviews the beneficial effects of melatonin, sodium butyrate and epigallocatechin gallate (EGCG) in the management of the pathophysiological changes underpinning neurodegenerative conditions. It is proposed that all three nutraceuticals upregulate the tryptophan-melatonin pathway, which may be particularly important in astrocytes given astrocyte regulation of neuronal energy supply and antioxidants, including released melatonin. Alterations in the tryptophan-melatonin pathway are intimately intertwined with changes in the kynurenine pathway and its neuroregulatory products, including kynurenic acid and quinolinic acid. This article places these changes in the tryptophan-melatonin pathways within a novel circadian-systemic interaction, involving the regulation of the night-time rise in cortisol culminating in the morning cortisol awakening response that mediates effects via glucocorticoid receptor (GR) activation. The night-time and morning GR activation is suppressed by melatonin, gut microbiome derived butyrate and bcl2-associated athanogene (BAG)-1. As melatonin, butyrate and BAG-1 decrease over age, there is a heightened level of GR nuclear translocation with age at night and early morning. This is exemplified by the 10-fold decrease in pineal melatonin in people in their 9th, versus 2nd, decade of life. The ‘battle’ of melatonin/butyrate/EGCG versus cortisol/GR for influence on cellular function, microenvironment homeostasis and systemic system (immune) regulation at night and early morning shapes how the body and brain are prepared for the coming day and drives the emergence of aging associated neurodegenerative conditions. It is upon such processes that melatonin, butyrate and EGCG have their impacts.

A new look at the fluorescent protein-based approach for identifying optimal coding sequence for recombinant protein expression in E. coli.

Due to the degeneracy of the genetic code, most amino acids are encoded by several codons. The choice among synonymous codons at the N-terminus of genes has a profound effect on protein expression in Escherichia coli. This is often explained by the different contributions of synonymous codons to mRNA secondary structure formation. Strong secondary structures at the 5'-end of mRNA interfere with ribosome binding and affect the process of translation initiation. In silico optimization of the gene 5'-end can significantly increase the level of protein expression; however, this method is not always effective due to the uncertainty of the exact mechanism by which synonymous substitutions affect expression; thus, it may produce nonoptimal variants as well as miss some of the best producers. In this paper, an alternative approach is proposed based on screening a partially randomized library of expression constructs comprising hundreds of selected synonymous variants. The effect of such substitutions was evaluated using the gene of interest fused to the reporter gene of the fluorescent protein with subsequent screening for the most promising candidates according to the reporter's signal intensity. The power of the approach is demonstrated by a significant increase in the prokaryotic expression of three proteins: canine cystatin C, human BCL2-associated athanogene 3 and human cardiac troponin I. This simple approach was suggested which may provide an efficient, easy, and inexpensive optimization method for poorly expressed proteins in bacteria.

BAG6 inhibits influenza A virus replication by inducing viral polymerase subunit PB2 degradation and perturbing RdRp complex assembly.

The interaction between influenza A virus (IAV) and host proteins is an important process that greatly influences viral replication and pathogenicity. PB2 protein is a subunit of viral ribonucleoprotein (vRNP) complex playing distinct roles in viral transcription and replication. BAG6 (BCL2-associated athanogene 6) as a multifunctional host protein participates in physiological and pathological processes. Here, we identify BAG6 as a new restriction factor for IAV replication through targeting PB2. For both avian and human influenza viruses, overexpression of BAG6 reduced viral protein expression and virus titers, whereas deletion of BAG6 significantly enhanced virus replication. Moreover, BAG6-knockdown mice developed more severe clinical symptoms and higher viral loads upon IAV infection. Mechanistically, BAG6 restricted IAV transcription and replication by inhibiting the activity of viral RNA-dependent RNA polymerase (RdRp). The co-immunoprecipitation assays showed BAG6 specifically interacted with the N-terminus of PB2 and competed with PB1 for RdRp complex assembly. The ubiquitination assay indicated that BAG6 promoted PB2 ubiquitination at K189 residue and targeted PB2 for K48-linked ubiquitination degradation. The antiviral effect of BAG6 necessitated its N-terminal region containing a ubiquitin-like (UBL) domain (17-92aa) and a PB2-binding domain (124-186aa), which are synergistically responsible for viral polymerase subunit PB2 degradation and perturbing RdRp complex assembly. These findings unravel a novel antiviral mechanism via the interaction of viral PB2 and host protein BAG6 during avian or human influenza virus infection and highlight a potential application of BAG6 for antiviral drug development.

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.

Thio-2 Inhibits Key Signaling Pathways Required for the Development and Progression of Castration-resistant Prostate Cancer.

Therapies that abrogate persistent androgen receptor (AR) signaling in castration-resistant prostate cancer (CRPC) remain an unmet clinical need. The N-terminal domain of the AR that drives transcriptional activity in CRPC remains a challenging therapeutic target. Herein we demonstrate that BCL-2-associated athanogene-1 (BAG-1) mRNA is highly expressed and associates with signaling pathways, including AR signaling, that are implicated in the development and progression of CRPC. In addition, interrogation of geometric and physiochemical properties of the BAG domain of BAG-1 isoforms identifies it to be a tractable but challenging drug target. Furthermore, through BAG-1 isoform mouse knockout studies, we confirm that BAG-1 isoforms regulate hormone physiology and that therapies targeting the BAG domain will be associated with limited "on-target" toxicity. Importantly, the postulated inhibitor of BAG-1 isoforms, Thio-2, suppressed AR signaling and other important pathways implicated in the development and progression of CRPC to reduce the growth of treatment-resistant prostate cancer cell lines and patient-derived models. However, the mechanism by which Thio-2 elicits the observed phenotype needs further elucidation as the genomic abrogation of BAG-1 isoforms was unable to recapitulate the Thio-2-mediated phenotype. Overall, these data support the interrogation of related compounds with improved drug-like properties as a novel therapeutic approach in CRPC, and further highlight the clinical potential of treatments that block persistent AR signaling which are currently undergoing clinical evaluation in CRPC.

A Mitochondrial Localized Chaperone Regulator OsBAG6 Functions in Saline-Alkaline Stress Tolerance in Rice

B-cell lymphoma 2 (Bcl-2)-associated athanogene (BAG) family genes play prominent roles in regulating plant growth, development, and stress response. Although the molecular mechanism underlying BAG’s response to abiotic stress has been studied in Arabidopsis, the function of OsBAG underlying saline-alkaline stress tolerance in rice remains unclear. In this study, OsBAG6, a chaperone regulator localized to mitochondria, was identified as a novel negative regulator of saline-alkaline stress tolerance in rice. The expression level of OsBAG6 was induced by high concentration of salt, high pH, heat and abscisic acid treatments. Overexpression of OsBAG6 in rice resulted in significantly reduced plant heights, grain size, grain weight, as well as higher sensitivity to saline-alkaline stress. By contrast, the osbag6 loss-of-function mutants exhibited decreased sensitivity to saline-alkaline stress. The transcriptomic analysis uncovered differentially expressed genes related to the function of “response to oxidative stress”, “defense response”, and “secondary metabolite biosynthetic process” in the shoots and roots of OsBAG6-overexpressing transgenic lines. Furthermore, cytoplasmic levels of Ca2+ increase rapidly in plants exposed to saline-alkaline stress. OsBAG6 bound to calcium sensor OsCaM1-1 under normal conditions, which was identified by comparative interactomics, but not in the presence of elevated Ca2+. Released OsCaM1-1 saturated with Ca2+ is then able to regulate downstream stress-responsive genes as part of the response to saline-alkaline stress. OsBAG6 also interacted with energy biosynthesis and metabolic pathway proteins that are involved in plant growth and saline-alkaline stress response mechanisms. This study reveals a novel function for mitochondrial localized OsBAG6 proteins in the saline-alkaline stress response alongside OsCaM1-1.

Involvement of HSP70 in BAG9-mediated thermotolerance in Solanum lycopersicum

Because of a high sensitivity to high temperature, both the yield and quality of tomato (Solanum lycopersicum L.) are severely restricted by heat stress. The Bcl-2-associated athanogene (BAG) proteins, a family of multi-functional co-chaperones, are involved in plant growth, development, and stress tolerance. We have previously demonstrated that BAG9 positively regulates thermotolerance in tomato. However, the BAG9-mediated mechanism of thermotolerance in tomato has remained elusive. In the present study, we report that BAG9 interacts with heat shock protein 70 (HSP70) in vitro and in vivo. Silencing HSP70 decreased thermotolerance of tomato plants, as reflected by the phenotype, relative electrolyte leakage and malondialdehyde. Furthermore, the photosystem activities, activities of antioxidant enzymes and expression of key genes encoding antioxidant enzymes were reduced in HSP70-silenced plants under heat stress. Additionally, silencing HSP70 decreased thermotolerance of overexpressing BAG9 plants, which was related to decreased photosynthetic rate, increased damage to photosystem I and photosystem II, decreased activity of antioxidant enzymes, and decreased expression of key genes encoding antioxidant enzymes. Taken together, the present study identified that HSP70 is involved in BAG9-mediated thermotolerance by protecting the photosystem stability and improving the efficiency of the antioxidant system in tomato. This knowledge can be helpful to breed improved crop cultivars that are better equipped with thermotolerance.