Recent studies reveal a critical role of tumor cell-released extracellular vesicles (EVs) in pancreatic cancer (PC) progression. However, driver genes that direct EV function, the EV-recipient cells, and their cellular response to EV uptake remain to be identified. Therefore, we studied the role of Bcl-2-associated-anthanogene 6 (BAG6), a regulator of EV biogenesis for cancer progression. We used a Cre recombinase/LoxP-based reporter system in combination with single-cell RNA sequencing to monitor in vivo EV uptake and tumor microenvironment (TME) changes in mouse models for pancreatic ductal adenocarcinoma (PDAC) in a Bag6 pro- or deficient background. In vivo data were validated using mouse and human organoids and patient samples. Our data demonstrated that Bag6-deficient subcutaneous and orthotopic PDAC tumors accelerated tumor growth dependent on EV release. Mechanistically, this was attributed to mast cell (MC) activation via EV-associated IL33. Activated MCs promoted tumor cell proliferation and altered the composition of the TME affecting fibroblast polarization and immune cell infiltration. Tumor cell proliferation and fibroblast polarization were mediated via the MC secretome containing high levels of PDGF and CD73. Patients with high BAG6 gene expression and high protein plasma level have a longer overall survival indicating clinical relevance. The current study revealed a so far unknown tumor-suppressing activity of BAG6 in PDAC. Bag6-deficiency allowed the release of EV-associated IL33 which modulate the TME via MC activation promoting aggressive tumor growth. MC depletion using imatinib diminished tumor growth providing a scientific rationale to consider imatinib for patients stratified with low BAG6 expression and high MC infiltration. EVs derived from BAG6-deficient pancreatic cancer cells induce MC activation via IL33/Il1rl1. The secretome of activated MCs induces tumor proliferation and changes in the TME, particularly shifting fibroblasts into an inflammatory cancer-associated fibroblast (iCAF) phenotype. Blocking EVs or depleting MCs restricts tumor growth.

Chaperone-mediated autophagy (CMA) and endosomal microautophagy (eMI) are pathways for selective degradation of cytosolic proteins in lysosomes and late endosomes, respectively. These autophagic processes share as a first step the recognition of the same five-amino-acid motif in substrate proteins by the Hsc70 chaperone, raising the possibility of coordinated activity of both pathways. In this work, we show the existence of a compensatory relationship between CMA and eMI and identify a role for the chaperone protein Bag6 in triage and internalization of eMI substrates into late endosomes. Association and dynamics of Bag6 at the late endosome membrane change during starvation, a stressor that, contrary to other autophagic pathways, causes a decline in eMI activity. Collectively, these results show a coordinated function of eMI with CMA, identify the interchangeable subproteome degraded by these pathways, and start to elucidate the molecular mechanisms that facilitate the switch between them.

DERL2 (derlin 2) is a critical component of the endoplasmic reticulum quality control pathway system whose mutations play an important role in carcinogenesis, including cholangiocarcinoma (CHOL). However, its role and its underlying mechanism have yet to be elucidated. Herein, we revealed that DERL2 was highly expressed in CHOL and considered as an independent prognostic indicator for inferior survival in CHOL. DERL2 ectopically expressed in CHOL cells promoted cell proliferation and colony formation rates, and depleting DERL2 in CHOL cells curbed tumor growth in vitro and in vivo. More interestingly, the knockout of DERL2 augmented the growth-inhibitory effect of gemcitabine chemotherapy on CHOL cells by inducing cell apoptosis. Mechanistically, we discovered that DERL2 interacted with BAG6 (BAG cochaperone 6), thereby extending its half-life and reinforcing the oncogenic role of BAG6 in CHOL progression.

Inclusion body formation is associated with cytotoxicity in a number of neurodegenerative diseases. However, the molecular basis of the toxicity caused by the accumulation of aggregation-prone proteins remains controversial. In this study, we found that disease-associated inclusions induced by elongated polyglutamine chains disrupt the complex formation of BAG6 with UBL4A, a mammalian homologue of yeast Get5. UBL4A also dissociated from BAG6 in response to proteotoxic stresses such as proteasomal inhibition and mitochondrial depolarization. These findings imply that the cytotoxicity of pathological protein aggregates might be attributed in part to disruption of the BAG6-UBL4A complex that is required for the biogenesis of tail-anchored proteins.

Triple-negative breast cancer (TNBC), a heterogeneous subtype of breast cancer (BC), had poor prognosis. Endoplasmic reticulum (ER) stress was responsible for cellular processes and played a crucial role in the cell function. ER stress is a complex and dynamic process that can induce abnormal apoptosis and death. However, the underlying mechanism of ER stress involved in TNBC is not well defined.
We identified ubiquitin-specific protease 19 (USP19) as a TNBC negative regulator for further investigation. The effects of USP19 on BC proliferation were assessed in vitro using proliferation test and cell-cycle assays, while the effects in vivo were examined using a mouse tumorigenicity model. Through in vitro flow cytometric analyses and in vivo TUNEL assays, cell apoptosis was assessed. Proteomics was used to examine the proteins that interact with USP19.
Multiple in vitro and in vivo tests showed that USP19 decreases TNBC cell growth while increasing apoptosis. Then, we demonstrated that USP19 interacts with deubiquitinates and subsequently stabilises family molecular chaperone regulator 6 (BAG6). BAG6 can boost B-cell lymphoma 2 (BCL2) ubiquitination and degradation, thereby raising ER calcium (Ca2+ ) levels and causing ER stress. We also found that the N6 -methyladenosine (m6 A) \"writer\" methyltransferase-like 14 (METTL14) increased global m6 A modification.
Our study reveals that USP19 elevates the intracellular Ca2+ concentration to alter ER stress via regulation of BAG6 and BCL2 stability and may be a viable therapeutic target for TNBC therapy.

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.

Retinitis pigmentosa (RP) is the most common inherited retinal degenerative disease which is the major cause of vision loss. X-linked RP patients account for 5%-15% of all inherited RP cases and mutations in RP2 (Retinitis pigmentosa 2) were responsible for about 20% X-linked RP families. A majority of RP2 pathogenic mutations displayed a vulnerable protein stability and degraded rapidly through ubiquitin-proteasome system (UPS). Though the RP2 protein could be readily recovered by proteasome inhibitors, e.g., MG132, their applications for RP2-related RP therapy were limited by their nonspecific characterization. In the present study, we aimed to identify UPS-related factors, such as E3 ligases, which are specifically involved in degradation of RP2 pathogenic mutants. We identified several E3 ligases, such as HUWE1, and the co-chaperon BAG6 specifically interacting with RP2 pathogenic mutants. Knockdown of HUWE1 and BAG6 could partially rescue the reduced protein levels of RP2 mutants. BAG6 is required for recruitment of HUWE1 to ubiquitinate RP2 mutants at the K268 site. The HUWE1 inhibitor BI8622 could restore the levels of RP2 mutant and then the binding to its partner ARL3 in retina cell lines. This study revealed the details of UPS-related degradation of RP2 mutants and possibly provided a potential treatment for RP2-related RP.

The BAG proteins are a family of multi-functional co-chaperones. In plants, BAG proteins were found to play roles both in abiotic and biotic stress tolerance. However, the function of Arabidopsis BAG2 remains largely unknown, whereas BAG6 is required for plants\' defense to pathogens, although it remains unknown whether BAG6 is involved in plants\' tolerance to abiotic stresses. Here, we show that both BAG2 and BAG6 are expressed in various tissues and are upregulated by salt, mannitol, and heat treatments and by stress-related hormones including ABA, ethylene, and SA. Germination of bag2, bag6 and bag2 bag6 seeds is less sensitive to ABA compared to the wild type (WT), whereas BAG2 and BAG6 overexpression lines are hypersensitive to ABA. bag2, bag6, and bag2 bag6 plants show higher survival rates than WT in drought treatment but display lower survival rates in heat-stress treatment. Consistently, these mutants showed differential expression of several stress- and ABA-related genes such as RD29A, RD29B, NCED3 and ABI4 compared to the WT. Furthermore, these mutants exhibit lower levels of ROS after drought and ABA treatment but higher ROS accumulation after heat treatment than the WT. These results suggest that BAG2 and BAG6 are negatively involved in drought stress but play a positive role in heat stress in Arabidopsis.

NKp30 (Natural Cytotoxicity Receptor 1, NCR1) is a powerful cytotoxicity receptor expressed on natural killer (NK) cells which is involved in tumor cell killing and the regulation of antitumor immune responses. Ligands for NKp30, including BAG6 and B7-H6, are upregulated in virus-infected and tumor cells but rarely detectable on healthy cells. These ligands are released by tumor cells as part of the cellular secretome and interfere with NK cell activity. BAG6 is secreted via the exosomal pathway, and BAG6-positive extracellular vesicles (EV-BAG6) trigger NK cell cytotoxicity and cytokine release, whereas the soluble protein diminishes NK cell activity. However, the extracellular format and activity of B7-H6 remain elusive. Here, we used HEK293 as a model cell line to produce recombinant ligands and to study their impact on NK cell activity. Using this system, we demonstrate that soluble B7-H6 (sB7-H6), like soluble BAG6 (sBAG6), inhibits NK cell-mediated target cell killing. This was associated with a diminished cell surface expression of NKG2D and NCRs (NKp30, NKp40, and NKp46). Strikingly, a reduced NKp30 mRNA expression was observed exclusively in response to sBAG6. Of note, B7-H6 was marginally released in association with EVs, and EVs collected from B7-H6 expressing cells did not stimulate NK cell-mediated killing. The molecular analysis of EVs on a single EV level using nano flow cytometry (NanoFCM) revealed a similar distribution of vesicle-associated tetraspanins within EVs purified from wildtype, BAG6, or B7-H6 overexpressing cells. NKp30 is a promising therapeutic target to overcome NK cell immune evasion in cancer patients, and it is important to unravel how extracellular NKp30 ligands inhibit NK cell functions.

Bcl-2-associated athanogen-6 (BAG6) is a nucleocytoplasmic shuttling protein involved in protein quality control. We previously demonstrated that BAG6 is essential for autophagy by regulating the intracellular localization of the acetyltransferase EP300, and thus, modifying accessibility to its substrates (TP53 in the nucleus and autophagy-related proteins in the cytoplasm). Here, we investigated BAG6 localization and function in the cytoplasm. First, we demonstrated that BAG6 is localized in the mitochondria. Specifically, BAG6 is expressed in the mitochondrial matrix under basal conditions, and translocates to the outer mitochondrial membrane after mitochondrial depolarization with carbonyl cyanide m-chlorophenyl hydrazine, a mitochondrial uncoupler that induces mitophagy. In SW480 cells, the deletion of BAG6 expression abrogates its ability to induce mitophagy and PINK1 accumulation. On the reverse, its ectopic expression in LoVo colon cancer cells, which do not express endogenous BAG6, reduces the size of the mitochondria, induces mitophagy, leads to the activation of the PINK1/PARKIN pathway and to the phospho-ubiquitination of mitochondrial proteins. Finally, BAG6 contains two LIR (LC3-interacting Region) domains specifically found in receptors for selective autophagy and responsible for the interaction with LC3 and for autophagosome selectivity. Site-directed mutagenesis showed that BAG6 requires wild-type LIRs domains for its ability to stimulate mitophagy. In conclusion, we propose that BAG6 is a novel mitophagy receptor or adaptor that induces PINK1/PARKIN signaling and mitophagy in a LIR-dependent manner.