BCL2-associated Athanogene Research Articles

LncRNA TUG1 Promoted Stabilization of BAG5 by Binding DDX3X to Exacerbate Ketamine-Induced Neurotoxicity.

As a clinically widely used anesthetic, ketamine (KET) has been reported to cause neurotoxicity in patients. Our work aimed to probe the function of long-chain non-coding RNA taurine-upregulated gene 1 (lncRNA TUG1) in KET-induced neurotoxicity. HT22 cells were subjected to KET to build the cell model. 3-(4, 5-Dimethylthiazolyl2)-2, 5-diphenyltetrazolium bromide (MTT) assay was employed to determine cell viability. Additionally, cell apoptosis was evaluated by flow cytometry. The binding relationships among TUG1, DEAD-box RNA helicase 3X (DDX3X), and Bcl-2-associated athanogene 5 (BAG5) were verified by RIP and RNA pull-down assays. Cell viability was impaired and cell apoptosis was increased in KET-treated HT22 cells accompanied by increased TUG1, DDX3X, and BAG5 expressions. TUG1 knockdown dramatically enhanced cell viability and repressed the of KET-induced apoptosis in HT22 cells, while TUG1 overexpression presented the opposite effects. In addition, we found that TUG1 promoted DDX3X expression via directly binding with DDX3X. As expected, DDX3X overexpression abolished the palliative effect of TUG1 knockdown on KET-induced neurotoxicity. Further research proved that TUG1 increased the stability of BAG5 through interacting with DDX3X. Finally, as expected, the moderating effect of TUG1 knockdown on KET-induced neuron injury was abolished by BAG5 overexpression. Taken together, TUG1 promoted BAG5 expression by binding DDX3X to exacerbate KET-induced neurotoxicity.

BAG6 negatively regulates the RLR signaling pathway by targeting VISA/MAVS.

The virus-induced signaling adaptor protein VISA (also known as MAVS, ISP-1, Cardif) is a critical adaptor protein in the innate immune response to RNA virus infection. Upon viral infection, VISA self-aggregates to form a sizeable prion-like complex and recruits downstream signal components for signal transduction. Here, we discover that BAG6 (BCL2-associated athanogene 6, formerly BAT3 or Scythe) is an essential negative regulator in the RIG-I-like receptor signaling pathway. BAG6 inhibits the aggregation of VISA by promoting the K48-linked ubiquitination and specifically attenuates the recruitment of TRAF2 by VISA to inhibit RLR signaling. The aggregation of VISA and the interaction of VISA and TRAF2 are enhanced in BAG6-deficient cell lines after viral infection, resulting in the enhanced transcription level of downstream antiviral genes. Our research shows that BAG6 is a critical regulating factor in RIG-I/VISA-mediated innate immune response by targeting VISA.

Abstract P1055: Co-chaperone Bag5 Protects Cardiomyocytes From Oxidative Stress

Introduction: Cardiovascular disease is one of the leading causes of death globally. Cardiac ischemic can alter cellular homeostasis and potentially lead to heart failure. Ischemic cells modulate the expression of molecular chaperones, which assist with the folding/refolding of cellular proteins. Specifically, the heat-shock protein 70 (Hsp70) chaperone, and its co-chaperones in the Bcl-2-associated athanogene (BAG) protein family, help maintain protein homeostasis by protein folding and degradation. This project serves to explore the role of BAG5 in cardiac ischemia. Methods: Expression of BAG5 was determined in left ventricular tissue isolated from cardiomyopathy patients and in ischemic cardiomyocytes. Co-immunoprecipitation (IP) and mass spectrometry analysis were utilized to determine potential interacting partners of BAG5. For the generation of ischemic conditions, neonatal rat cardiomyocytes were incubated in a hypoxia chamber for 12 hours in DMEM media with no glucose, 0% serum, and 0.1% O2. The role of BAG5 in cellular viability, reactive oxygen species (ROS) production, and proteostasis during ischemia was determined through gain-in and knock-down approaches using adenoviruses. In addition, mitochondrial membrane potential and ROS production were determined by the TMRE and MitoSOX red staining, respectively. Mitochondrial function was determined by a Seahorse XF assay. Results: Co-immunoprecipitation and mass spectrometry analysis revealed that BAG5 interacts with Myosin and Hsp70. Expression of BAG5 in cardiomyocytes was significantly decreased during ischemia. Further, overexpression of BAG5 protects cardiomyocytes by inhibiting excessive autophagy and reducing ROS production. Moreover, BAG5 expression improves mitochondrial function during ischemia. Conclusion: Understanding the role of BAG5 within the cell may help establish the protein as a potential therapeutic molecule for the protection of the ischemic heart.

Identification of BAG5 as a Potential Biomarker for Parkinson's Disease Patients With R492X PINK1 Mutation.

Parkinson’s disease (PD) is a degenerative, progressive nervous system disorder with an unknown cause. PINK1 [phosphatase and tensin homolog deleted on chromosome 10 (PTEN)-induced putative kinase 1] causative mutations R492X may cause autosomal recessive early-onset parkinsonism. In this study, we utilized patient samples and cell line system to investigate roles of Bcl2-associated athanogene 5 (BAG5) in PD patients with R492X PINK1 mutation. We show that the expression levels of BAG5 in the skin tissues from PD patients with R492X PINK1 mutation are markedly lower than those from the healthy control subjects in a small cohort of patients, which has not been recognized before. In addition, we demonstrate that BAG5 physically binds to R492X mutated PINK1 protein. Furthermore, we reveal that BAG5 promotes the degradation of R492X mutated PINK1 protein via ubiquitin/proteasome-dependent pathway, suggesting that decreased level of BAG5 may lead to R492X mutated PINK1 protein accumulation. These findings suggest that BAG5 may serve as an early detection biomarker for PD patients with R492X PINK1 mutation and provide important new insights on how BAG5 affects R492X mutated PINK1 protein, highlighting therapeutic targets for this disease.

BAG9 Confers Thermotolerance by Regulating Cellular Redox Homeostasis and the Stability of Heat Shock Proteins in Solanum lycopersicum

The Bcl-2-associated athanogene (BAG) family, a group of co-chaperones that share conservative domains in flora and fauna, is involved in plant growth, development, and stress tolerance. However, the function of tomato BAG genes on thermotolerance remains largely unknown. Herein, we found that the expression of BAG9 was induced during heat stress in tomato plants. Knockout of the BAG9 gene by CRISPR/Cas9 reduced, while its overexpression increased thermotolerance in tomato plants as reflected by the phenotype, photosynthesis rate, and membrane peroxidation. Heat-induced reactive oxygen species and oxidative/oxidized proteins were further increased in bag9 mutants and were normalized in BAG9 overexpressing plants. Furthermore, the activities of antioxidant enzymes, ascorbic acid (AsA)/dehydroascorbic acid (DHA), and reduced glutathione (GSH)/oxidized glutathione (GSSG) were reduced in bag9 mutants and were increased in BAG9 overexpressing plants under heat stress. Additionally, BAG9 interacted with Hsp20 proteins in vitro and in vivo. Accumulation of Hsp proteins induced by heat showed a reduction in bag9 mutants; meanwhile, it was increased in BAG9 overexpressing plants. Thus, BAG9 played a crucial role in response to heat stress by regulating cellular redox homeostasis and the stability of heat shock proteins.

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

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

The role of BAG3 in dilated cardiomyopathy and its association with Charcot-Marie-Tooth disease type 2.

Bcl2-associated athanogene 3 (BAG3) is a multifunctional cochaperone responsible for protein quality control within cells. BAG3 interacts with chaperones HSPB8 and Hsp70 to transport misfolded proteins to the Microtubule Organizing Center (MTOC) and degrade them in autophagosomes in a process known as Chaperone Assisted Selective Autophagy (CASA). Mutations in the second conserved IPV motif of BAG3 are known to cause Dilated Cardiomyopathy (DCM) by inhibiting adequate removal of non-native proteins. The proline 209 to leucine (P209L) BAG3 mutant in particular causes the aggregation of BAG3 and misfolded proteins as well as the sequestration of essential chaperones. The exact mechanisms of protein aggregation in DCM are unknown. However, the similar presence of insoluble protein aggregates in Charcot-Marie-Tooth disease type 2 (CMT2) induced by the proline 182 to leucine (P182L) HSPB1 mutant points to a possible avenue for future research: IPV motif. In this review, we summarize the molecular mechanisms of CASA and the currently known pathological effects of mutated BAG3 in DCM. Additionally, we will provide insight on the importance of the IPV motif in protein aggregation by analyzing a potential association between DCM and CMT2.

Dissecting the progression of cardiac dysfunction in tumor-bearing mice

Abstract Funding Acknowledgements Type of funding sources: None. Background Cancer patients undergoing heart-related complications result in high incidences of mortality. Nevertheless, it is still not fully understood whether localized tumors affect heart function prior to the onset of cachexia, hence, making the heart more vulnerable for functional abnormalities in later stages of the disease. In addition to analyse heart function, we focus on the expression BCL-2–associated athanogene 3 (BAG3), a co-chaperone protein and Hsp70, which are highly expressed in tumor but decrease in cardiomyocytes (CM) in heart failure (HF). Methods Colon-26 adenocarcinoma cells (C26; n=22) with/without shIL-6 (C26 shIL-6; n=22) were injected subcutaneously into the right flank of 10-11 weeks old BALB/c male mice. Control mice were injected with vehicle (PBS; n=8). Cardiac function was assessed by echocardiography and invasive hemodynamic measurements 10 (early) and 20 (late) days after the injection, respectively. In addition, the expression of BAG3 and Hsp70 were determined by Western blot as well as the extend of cardiac fibrosis was determined by Masson-Goldner's trichrome staining. Results The tumor size was comparable between the two injected groups. However, only C26 group showed a significant loss of subcutaneous fat and skeletal muscle (p<0.05, respectively), suggesting cachexia. Heart weight normalized to tibia length was not changed in the injected groups as compared to controls (day 20). However, left ventricular ejection fraction (LVEF) showed a tendency to decline in the early phase (p~0.08) in both injected group and it reached significance at late stage (p<0.05). Invasive hemodynamic assessment also confirmed the contractile dysfunction, resulting in a decrease in LV systolic pressure and increase of LV end-diastolic pressure (p<0.05, respectively). Importantly, these functional changes in the heart in tumor-bearing mice were associated with a marked reduction in both BAG3 and Hsp70 in the myocardium. Furthermore, there was no sign of cardiac fibrosis in the injected groups. Discussion Our study shows for the first time that tumor rather than cancer cachexia plays a significant maladaptive role in the progression of cardiac dysfunction in a mouse model of C26 injection-induced cachexia. The progression of cardiac contractile dysfunction was associated with a decline in BAG3 and Hsp70 in tumor-bearing mice, suggesting changes of BAG3/Hsp 70 signalling may be a critical component as well as target.

BCL2-associated athanogene 6 exon24 contributes to testosterone synthesis and male fertility in mammals.

ObjectivesBCL2‐associated athanogene 6 (BAG6) plays critical roles in spermatogenesis by maintaining testicular cell survival. Our previous data showed porcine BAG6 exon24‐skipped transcript is highly expressed in immature testes compared with mature testes. The objective of this study is to reveal the functional significance of BAG6 exon24 in mammalian spermatogenesis.Materials and MethodsCRISPR/Cas9 system was used to generate Bag6 exon24 knockout mice. Testes and cauda epididymal sperm were collected from mice. TMT proteomics analysis was used to discover the protein differences induced by Bag6 exon24 deletion. Testosterone enanthate was injected into mice to generate a high‐testosterone mice model. H&E staining, qRT‐PCR, western blotting, vector/siRNA transfection, immunofluorescence, immunoprecipitation, transmission electron microscopy, TUNEL and ELISA were performed to investigate the phenotypes and molecular basis.Results Bag6 exon24 knockout mice show sub‐fertility along with partially impaired blood‐testis barrier, increased apoptotic testicular cell rate and abnormal sperm morphology. Endoplasmic reticulum stress occurs in Bag6 exon24‐deficient testes and sterol regulatory element‐binding transcription factor 2 is activated; as a result, cytochrome P450 family 51 subfamily A member 1 expression is up‐regulated, which causes a high serum testosterone level. Additionally, serine/arginine‐rich splicing factor 1 down‐regulates BAG6 exon24‐skipped transcripts in porcine Sertoli cells by binding to 35–51 nt on BAG6 exon24 via its N‐terminal RNA‐recognition domain.ConclusionsOur findings reveal the critical roles of BAG6 exon24 in testosterone biosynthesis and male fertility, which provides new insights into the regulation of spermatogenesis and pathogenesis of subfertility in mammals.

Stress routes clients to the proteasome via a BAG2 ubiquitin-independent degradation condensate

The formation of membraneless organelles can be a proteotoxic stress control mechanism that locally condenses a set of components capable of mediating protein degradation decisions. The breadth of mechanisms by which cells respond to stressors and form specific functional types of membraneless organelles, is incompletely understood. We found that Bcl2-associated athanogene 2 (BAG2) marks a distinct phase-separated membraneless organelle, triggered by several forms of stress, particularly hyper-osmotic stress. Distinct from well-known condensates such as stress granules and processing bodies, BAG2-containing granules lack RNA, lack ubiquitin and promote client degradation in a ubiquitin-independent manner via the 20S proteasome. These organelles protect the viability of cells from stress and can traffic to the client protein, in the case of Tau protein, on the microtubule. Components of these ubiquitin-independent degradation organelles include the chaperone HSP-70 and the 20S proteasome activated by members of the PA28 (PMSE) family. BAG2 condensates did not co-localize with LAMP-1 or p62/SQSTM1. When the proteasome is inhibited, BAG2 condensates and the autophagy markers traffic to an aggresome-like structure.

Determination of protoplast growth properties using quantitative single-cell tracking analysis

BackgroundAlthough quantitative single-cell analysis is frequently applied in animal systems, e.g. to identify novel drugs, similar applications on plant single cells are largely missing. We have exploited the applicability of high-throughput microscopic image analysis on plant single cells using tobacco leaf protoplasts, cell-wall free single cells isolated by lytic digestion. Protoplasts regenerate their cell wall within several days after isolation and have the potential to expand and proliferate, generating microcalli and finally whole plants after the application of suitable regeneration conditions.ResultsHigh-throughput automated microscopy coupled with the development of image processing pipelines allowed to quantify various developmental properties of thousands of protoplasts during the initial days following cultivation by immobilization in multi-well-plates. The focus on early protoplast responses allowed to study cell expansion prior to the initiation of proliferation and without the effects of shape-compromising cell walls. We compared growth parameters of wild-type tobacco cells with cells expressing the antiapoptotic protein Bcl2-associated athanogene 4 from Arabidopsis (AtBAG4).ConclusionsAtBAG4-expressing protoplasts showed a higher proportion of cells responding with positive area increases than the wild type and showed increased growth rates as well as increased proliferation rates upon continued cultivation. These features are associated with reported observations on a BAG4-mediated increased resilience to various stress responses and improved cellular survival rates following transformation approaches. Moreover, our single-cell expansion results suggest a BAG4-mediated, cell-independent increase of potassium channel abundance which was hitherto reported for guard cells only. The possibility to explain plant phenotypes with single-cell properties, extracted with the single-cell processing and analysis pipeline developed, allows to envision novel biotechnological screening strategies able to determine improved plant properties via single-cell analysis.

BAG6 prevents the aggregation of neurodegeneration-associated fragments of TDP43

SummaryNeurodegeneration is associated with the aggregation of proteins bearing solvent-exposed hydrophobicity as a result of their misfolding and/or proteolytic cleavage. An understanding of the cellular protein quality control mechanisms which prevent protein aggregation is fundamental to understanding the etiology of neurodegeneration. By examining the metabolism of disease-linked C-terminal fragments of the TAR DNA-binding protein 43 (TDP43), we found that the Bcl-2 associated athanogene 6 (BAG6) functions as a sensor of proteolytic fragments bearing exposed hydrophobicity and prevents their intracellular aggregation. In addition, BAG6 facilitates the ubiquitylation of TDP43 fragments by recruiting the Ub-ligase, Ring finger protein 126 (RNF126). Authenticating its role in preventing aggregation, we found that TDP43 fragments form intracellular aggregates in the absence of BAG6. Finally, we found that BAG6 could interact with and solubilize additional neurodegeneration-associated proteolytic fragments. Therefore, BAG6 plays a general role in preventing intracellular aggregation associated with neurodegeneration.

C-MYC-USP49-BAG2 axis promotes proliferation and chemoresistance of colorectal cancer cells in vitro

Deubiquitinases (DUBs) play critical roles in tumorigenesis and are emerging as potential therapeutic targets. However, it remains less clear which DUBs may play important roles and represent a realistic vulnerability for a particular type of tumor. Here we revealed that Ubiquitin Specific Peptidase 49 (USP49) is transcriptionally activated by c-MYC in colorectal cancer (CRC), and CRC patients with elevated USP49 levels exhibited significantly shorter survival. Knockdown of USP49 markedly inhibited CRC cell proliferation, colony formation, and chemotherapy resistance in vitro. Investigation of mechanisms unravels that USP49 deubiquitinates and stabilizes Bcl-2-Associated Athanogene 2 (BAG2), a well-known protein that antagonizes apoptosis and enables adaptive response of CRC cells. This study identified a novel mechanism by which USP49 promotes CRC cell survival by stabilizing BAG2 through the c-MYC-USP49-BAG2 axis, indicating that USP49 may become a potential therapeutic target for CRC.

Genome-Wide Identification of the Bcl-2 Associated Athanogene (BAG) Gene Family in Solanum lycopersicum and the Functional Role of SlBAG9 in Response to Osmotic Stress.

The Bcl-2-associated athanogene (BAG) proteins are a family of multi-functional group of co-chaperones regulators, modulating diverse processes from plant growth and development to stress response. Here, 10 members of SlBAG gene family were identified based on the available tomato (Solanum lycopersicum) genomic information and named as SlBAG1-10 according to their chromosomal location. All SlBAG proteins harbor a characteristic BAG domain, categorized into two groups, and SlBAG4, SlBAG7, and SlBAG9 of group I contain a plant-specific isoleucine glutamine (IQ) calmodulin-binding motif located in the N terminus. The quantitative real-time PCR expression analysis revealed that these SlBAG genes had organ-specific expression patterns and most SlBAG genes were differentially expressed in multiple abiotic stresses including drought, salt, high temperature, cold, and cadmium stress as well as abscisic acid and H2O2. In addition, heterologous overexpression of SlBAG9 increased the sensitivity of Arabidopsis to drought, salt, and ABA during seed germination and seedling growth. The decreased tolerance may be due to the downregulation of stress-related genes expression and severe oxidative stress. The expression levels of some stress and ABA-related genes, such as ABI3, RD29A, DREB2A, and P5CS1, were significantly inhibited by SlBAG9 overexpression under osmotic stress. Meanwhile, the overexpression of SlBAG9 inhibited the expression of FSD1 and CAT1 under stress conditions and the decreased levels of superoxide dismutase and catalase enzyme activities were detected accompanying the trends in the expression of both genes, which resulted in H2O2 accumulation and lipid peroxidation. Taken together, these findings lay a foundation for the future study of the biological function of SlBAG genes in tomato.

BAG family proteins contributes to autophagy-mediated multidrug resistance of tumor.

Multidrug resistance (MDR) is a significant cause of tumor treatment failure. Accumulating evidence suggests that autophagy plays a significant role in the development of MDR. Autophagy is a conserved mechanism that maintains tumor homeostasis by removing damaged mitochondria. However, the specific regulatory mechanism is unclear. Here, we summarize recent studies on the role of autophagy in the development of MDR and the initiation of mitophagy by Bcl-2-associated athanogene (BAG) family proteins. Additionally, this mini-review emphasizes the regulatory role of BAG family proteins, which maintain mitochondrial homeostasis by regulating the PINK1/Parkin pathway. Elucidation of the regulatory mechanisms of mitophagy may foster the development of clinical therapeutic strategies for MDR tumors.

BAG3 protects chondrocytes against lumbar facet joint osteoarthritis by regulating autophagy and apoptosis.

Bcl2-associated athanogene3 (BAG3) protein, mainly induced by stressful stimuli, has been confirmed to participate in apoptosis and autophagy. In recent studies, BAG3 has gradually become a key molecule in tumors. However, the role of BAG3 in the progression of lumbar facet joint osteoarthritis (FJOA) and whether it can regulate chondrocyte apoptosis and autophagy are still unknown. In both human and FJOA rat models, we observed an upregulation of BAG3 and apoptosis and autophagy-related proteins compared with healthy tissues. Then, we established the chondrocytes injury model in vitro by using IL-1β to stimulate human SW1353 cells. Western blot analysis data showed significant expression of BAG3, apoptosis, and autophagy-related proteins in SW1353 cells. Finally, by knocking down and overexpressing BAG3, we discovered possible anti-apoptotic and autophagy-promoted effects of BAG3 in FJOA through various experimental methods. This study demonstrated that BAG3 actively participates in regulating chondrocyte apoptosis and autophagy in FJOA and may be a highly interesting targetfor pharmacological interventions.

Functional characterization of BAG3 in orange-spotted grouper (Epinephelus coioides) during viral infection

Bcl-2-associated athanogene 3 (BAG3) is a cochaperone protein that interacts with Bcl-2 and mediate cell death. However, little is known about the roles of fish BAG3 during viral infection. In this study, we characterized a BAG3 homolog from orange-spotted grouper (Epinephelus coioides) (EcBAG3) and investigated its roles during viral infection. The EcBAG3 protein encoded 579 amino acids with typical WW, PXXP and BAG domains, which shared high identities with reported fish BAG3. Quantitative real-time PCR (qRT-PCR) analysis revealed that EcBAG3 was highly expressed in brain and heart. And the expression of EcBAG3 was significantly up-regulated after red-spotted grouper nervous necrosis virus (RGNNV) stimulation in vitro. EcBAG3 overexpression could promoted the expression of viral genes (coat protein (CP) and RNA-dependent RNA polymerase (RdRp)), which was enhanced by co-transfection with Hsp70 and Hsp22. Also, EcBAG3 overexpression up-regulated the expression of LC3-Ⅱ and down-regulated the expression of Bax and BNIP3, the IFN- (IRF1, IRF3, IRF7, IFP35, Mx1) or inflammation-related (IL-1β and TNFα) factors, as well as decreased the activities of NF-κB, ISRE and IFN-3. While knockdown of EcBAG3 decreased the transcripts of RGNNV CP gene and RdRp gene. Further studies showed that EcBAG3 knockdown impaired the expression level of autophagy factor LC3-Ⅱ, and promoted the expression level of Bax and BNIP3, inflammatory factors and interferon factors. These data indicate that EcBAG3 can affect viral infection through modulating virus-induced cell death, regulating the expression of IFN- and inflammation-related factors, which will be helpful to further explore the immune response of fish during viral infection.

Cytosolic Nipah Virus Inclusion Bodies Recruit Proteins Without Using Canonical Aggresome Pathways

Nipah virus (NiV) is a BSL-4 classified zoonotic paramyxovirus that causes respiratory or encephalitic diseases. A hallmark of NiV infections, as with all cell infections caused by non-segmented negative-strand RNA viruses, is the formation of cytoplasmic inclusion bodies (IBs). We previously showed that cytosolic NiV IBs, which are formed in infected cells or in cells minimally expressing the NiV nucleocapsid proteins, are associated with the microtubule-organizing center (MTOC) marker γ-tubulin. They also recruit overexpressed cytosolic proteins that are not functionally required for viral replication in IBs and that otherwise might form toxic protein aggregates. Therefore, NiV IBs are thought to share some functional properties with cellular aggresomes. The fact that aggresomes were not found in NiV-infected cells supports the idea that NiV IBs are successfully reducing the proteotoxic stress in infected cells. Only if the proteasome-ubiquitin system is artificially blocked by inhibitors, cellular aggresomes are formed in addition to IBs, but without colocalizing. Although both structures were positive for the classical aggresome markers histone deacetylase 6 (HDAC6) and Bcl-2-associated athanogene 3 (BAG3), they clearly differed in their cellular protein compositions and recruited overexpressed proteins to different extents. The further finding that inhibition of aggresome pathways by HDAC6 or microtubule (MT) inhibitors did neither interfere with IB formation nor with protein sequestration, strengthens the idea that cytosolic NiV IBs can assume some aggresome-like functions without involving active transport processes and canonical cellular aggresome pathways.

BAG Family Members as Mitophagy Regulators in Mammals.

The BCL-2-associated athanogene (BAG) family is a multifunctional group of co-chaperones that are evolutionarily conserved from yeast to mammals. In addition to their common BAG domain, these proteins contain, in their sequences, many specific domains/motifs required for their various functions in cellular quality control, such as autophagy, apoptosis, and proteasomal degradation of misfolded proteins. The BAG family includes six members (BAG1 to BAG6). Recent studies reported their roles in autophagy and/or mitophagy through interaction with the autophagic machinery (LC3, Beclin 1, P62) or with the PINK1/Parkin signaling pathway. This review describes the mechanisms underlying BAG family member functions in autophagy and mitophagy and the consequences in physiopathology.

Abstract P4-01-11: UBQLN4 regulates cisplatin-resistance in triple-negative breast cancer by targeting BAT3 for proteasomal degradation

Abstract Background: Triple-negative breast cancer (TNBC) is a heterogeneous breast cancer (BC) subtype. Cisplatin is one of the broad FDA-approved drugs to treat TNBC patients with recurrent and unresectable disease. However the treatment response is limited, and patients frequently develop resistance and recur locally and distantly. The aim of this study is to find molecular mechanisms driving cisplatin-resistance in TNBC. The ubiquitin proteasomal system (UPS) is a universal and conserved mechanism that helps to maintain cellular homeostasis. The aberrant expression of certain components of the UPS have been associated with cancer onset, progression, and metastasis as we and other groups have shown. UBQLN4 is part of the UPS and play a major role in controlling ubiquitinated proteins in response to genotoxic stress. BCL2-associated athanogene-6 (BAG6), alternately known as BAT3, is a chaperone that complex with other chaperones and ubiquitin ligases to regulate protein stability and insertion of tail-anchored membrane proteins into the endoplasmic reticulum. This is a critical step that reduce mislocalized proteins and proteotoxic stress. We hypothesized that UBQLN4 upregulation determine cisplatin-resistance in BC patients by targeting BAT3 for proteasomal degradation to reduce proteotoxic stress. Methods: TCGA BRCA database was utilized to determine clinical associations and outcomes in BC patients. A BC tissue microarray (TMA) was stained by IHC and protein levels quantified to confirm associations between UBQLN4 and TNBC patients. TNBC cell lines were CRISPR-engineered to generate UBQLN4 knockout (KO), and lentivirus transduction was used to recover the KO phenotype. Functional assays and confocal imaging were performed in BRCA mutant and wildtype human TNBC cell lines. Results: Increased UBQLN4 mRNA expression was observed in the TCGA BRCA database in primary BC tissues compared to normal adjacent tissues (p<0.0001). Using PAM50 classification, the BC patients were stratified and compared for UBQLN4 expression. BC patients with basal-like tumors showed significantly higher UBQLN4 expression than normal-like subtype (p< 0.0001). In the assessment of 5-years relapse-free survival (RFS), patients with high levels of UBQLN4 (n=221) showed significantly lower RFS rates compared to those with low UBQLN4 (n=221) (HR=1.67(1.21-2.3), log-rank test p<0.0016). These results were further evaluated by IHC using a BC TMA. Patients with TNBC showed a significantly increased UBQLN4 H-scores values compared to normal adjacent breast tissues. KO of UBQLN4 significantly increased cisplatin sensitivity in TNBC cell lines (p<0.0001). UBQLN4 overexpression restored the cisplatin-resistance in UBQLN4-KO cell line. In TGCA BRCA database, BAT3 was significantly upregulated in BC patients with basal-like tumors compared to normal-like subtype. Knockdown of BAT3 significantly increased cisplatin sensitivity in TNBC cell lines (p<0.0001). In TCGA BRCA database, BAT3 and UBQLN4 mRNA levels positively correlated (r= 0.26, p<0.0001). By using co-immunoprecipitation, endogenous BAT3 and UBQLN4 proteins interact in parental TNBC cell line, but only during cisplatin-treatment. These results were further validated using reciprocal co-immunoprecipitation and confocal imaging in TNBC cell lines with UBQLN4 overexpression. Conclusions: UBQLN4 expression controls cisplatin-resistance in TNBC cell lines. UBLQN4 interacts with BAT3 under cisplatin treatment. During cisplatin treatment, UBQLN4 targets BAT3 for proteasomal degradation to reduce proteotoxic stress induced by genotoxic cisplatin treatment. The UBQLN4 protein levels may represent a prognostic biomarker to predict RFS in TNBC patients. Citation Format: Yoshiaki Shoji, Takamichi Yokoe, Peter J Bostick, Yosef Shiloh, Dave SB Hoon, Matias A Bustos. UBQLN4 regulates cisplatin-resistance in triple-negative breast cancer by targeting BAT3 for proteasomal degradation [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-01-11.