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BCL2-associated athanogene 6 (BAG-6) was discovered as a gene product of the major histocompatibility complex class III locus. The Xenopus ortholog Scythe was first identified to act as an anti-apoptotic protein. Subsequent studies unraveled that the large BAG-6 protein contributes to a number of cellular processes, including apoptosis, gene regulation, protein synthesis, protein quality control, and protein degradation.
BAG-6 in DNA damage response and gene regulation
BAG-6 has a nuclear localization signal (NLS) indicating nuclear localization. In fact, BAG-6 forms a complex with p300 of nucleoprotein in response to DNA damage, facilitating subsequent p53 acetylation. Activation of p53-induced p21 expression, preventing DNA cell division by DNA repair of complex CDK2, and puma, which promote p53-dependent DNA damage-induced apoptosis. In consequence, thymocytes of BAG-6-defcient ICR-mice exhibit reduced expression of puma and p21, and show impaired apoptosis after γ-irradiation. Interestingly, primary neuronal cells from 129SvJ × C57BL/6-derived BAG-6 knockout mice were resistant to apoptosis as well. The cellular localization of BAG-6 is diverse. Early studies showed that BAG-6 remains in the nucleus during staurosporine-induced apoptosis, whereas Others have shown that apoptosis requires repositioning of BAG-6 into the cytosol after different stimuli.
Recently, it has been reported that induction of apoptosis requires ATM/ATR BAG-6 nuclear localization and BAG-6 phosphorylation following treatment with ionizing radiation or DNA-damaging agents. In addition, after DNA damage, the BAG-6 / BRCA1 complex translocates to the site of injury, mediating DNA damage response signaling and homologous recombination-mediated repair. The formation of these BRCA1 lesions is strongly dependent on BAG-6. BAG-6 regulates gene expression through interaction with two histone methyltransferases SeT1A and DOT1L, which dimethylate histone H3K4 and H3K79, respectively. Interestingly, BAG-6 is able to induce expression of the DNA repair-promoting protein 53BP1 and of its interaction partner BRCA1 providing a feedback loop to boost DNA repair. Notably, the cellular localization of BAG-6 can be regulated by cell type-specific alternative RNA splicing. Furthermore, masking NLS by interacting proteins such as TRC35 may result in preferential solute localization of BAG-6. Recent reports indicate that BAG-6 can target the plasma membrane and exosomes, which are released in response to cellular stress. Although these pathways play an important role in immune surveillance of tumor cells and shaping of immune responses, the molecular details of BAG-6 re-routing are poorly understood.
BAG-6 in protein targeting and quality control
Recently, BAG-6 has been shown to play a key role in the regulation of many cells, such as facilitating protein targeting, protein quality control, and protein degradation. The N-terminal UBL domain of BAG-6 suggests an involvement in protein degradation and the C-terminal BAG domain suggests the ability to interact with HSP70. In fact, BAG-6 is required for HSP70 accumulation during heat shock, and once accumulated, HSP70 leads to the degradation of BAG-6 through the ubiquitin-proteasome system. These reciprocal inﬂuences suggest that BAG-6 is a central regulator of the cellular content of HSP70. Furthermore, it has been shown that BAG-6 regulates the stability of HSP2A in the case of spermatogenesis.
In addition to its function as a co-chaper, BAG-6 also regulates the biogenesis of the tail anchor (TA) protein. In contrast to signal-recognition particle-mediated co-translational membrane insertion of the majority of membrane proteins, post-translationally TA protein insert into ER membrane through a single c-terminal transmembrane domain (TMD). During delivery through the cytosol, the hydrophobic TMDs are shielded by chaperones order to prevent protein aggregation. The TA protein insertion pathway has been studied extensively in yeast. In a first step, the pre-targeting factor Sgt2, which assembles Get3, Get4, and Get5 to form the TMD recognition complex, and TA proteins are transferred to the homodimeric ATPase Get3. After transfer to the ER membrane, the TA protein is released into the ER membrane in an ATP-dependent manner by GET1 and Get2. Recently, BAG-6 was identifed as a central TMD-specific chaperone acting in a complex with TRC40, TRC35, and Ubl4a, the mammalian homologues of Get3, Get4, and Get5, respectively. The BAG-6 complex is recruited into the ribosome of the synthetic TA protein, and the released hydrophobic TMD is shielded from the aqueous cytosol and transferred to the TRC40 for ER membrane targeting. Another component of the BAG-6 complex is SGTA, the homologue of yeast sgt2, which is recruited by Ubl4a. Notably, molecular chaperone systems that protect newborn TA proteins from aggregation, inappropriate interactions, or other molecular chaperone segregation for ER membrane integration are highly conserved.
Fig. 1. The functional BAG-6 interactome. (Janina Binici et al. Cellular and Molecular Life Sciences. 2014).
BAG-6 is a multifunctional protein involved in a variety of non-related cellular pathways in health and disease. Consequently, BAG-6 has a diverse cellular localization. In the nucleus, BAG-6 associates with the nucleoprotein p300 in response to DNA damage, facilitating subsequent acetylation of p53 and DNA repair. Moreover, BAG-6 facilitates targeting of BRCA1 to sites of DNA damage for repair. In complex with the histone methyltransferases SeT1A or DOT1L, BAG-6 is involved in regulation of gene expression. BAG-6 chaperones the cytosolic class II trans-activator CIITA to the nucleus for regulation of gene expression of proteins of the HLA class II processing pathway. Phosphorylation of BAG-6 by ATM/ATR is a prerequisite for DNA damage induced apoptosis. BAG-6 is retained in the cytosol by shielding of the nuclear localization signal by a complex which contains TRC35. In the cytosol, BAG-6 is cleaved by caspase-3 after induction of intrinsic or extrinsic apoptosis generating a C-terminal fragment of BAG-6, which triggers apoptosis. BAG-6 is part of a complex together with TRC35, TRC40, and Ubl4a, which shields the C-terminal transmembrane domain of tail anchored proteins until post translational insertion into the ER membrane. BAG-6 associates with protein substrates dedicated to degradation and docks them to the 26S-proteasome subunit Rpn10c. Consequently, BAG-6 drives MHC class I antigen presentation and regulates the supply of antigenic peptides. Tumor cells and dendritic cells release BAG-6 on exosomes and/or as a soluble protein. exosomal BAG-6 activates NK cells, whereas soluble BAG-6 inhibits NK cell cytotoxicity upon ligation to the activating NK cell receptor NKp30. Furthermore, BAG-6 is found on the plasma membrane of malignantly transformed cells and dendritic cells and triggers killing of the BAG-6 presenting cell. The way how BAG-6 is attached to the exosomal or plasma membrane remains obscure.