Role of BAG3 protein in leukemia cell survival and response to therapy

The ability of BAG3, a member of the BAG family of heat shock protein (Hsp) 70 - cochaperones, to sustain the survival of human primary B-CLL and ALL cells was recognized about nine years ago. Since then, the anti-apoptotic activity of BAG3 has been confirmed in other tumor types, where it has been shown to regulate the intracellular concentration and localization of apoptosis-regulating factors, including NF-κB-activating (IKKγ) and Bcl2-family (Bax) proteins. Furthermore, growing evidences support its role in lymphoid and myeloid leukemia response to therapy. Moreover in the last years, the contribution of BAG3 to autophagy, a process known to be involved in the pathogenesis and response to therapy of leukemia cells, has been disclosed, opening a new avenue for the interpretation of the role of this protein in leukemias' biology.     

BAG1: the guardian of anti-apoptotic proteins in acute myeloid leukemia

BCL2 associated Athano-Gene 1 (BAG1) is a multifunctional protein that has been described to be involved in different cell processes linked to cell survival. It has been reported as deregulated in diverse cancer types. Here, BAG1 protein was found highly expressed in children with acute myeloid leukemia at diagnosis, and in a cohort of leukemic cell lines. A silencing approach was used for determining BAG1's role in AML, finding that its down-regulation decreased expression of BCL2, BCL-XL, MCL1, and phospho-ERK1/2, all proteins able to sustain leukemia, without affecting the pro-apoptotic protein BAX. BAG1 down-regulation was also found to increase expression of BAG3, whose similar activity was able to compensate the loss of function of BAG1. BAG1/BAG3 co-silencing caused an enhanced cell predisposition to death in cell lines and also in primary AML cultures, affecting the same proteins. Cell death was CASPASE-3 dependent, was accompanied by PARP cleavage and documented by an increased release of pro-apoptotic molecules Smac/DIABLO and Cytochrome c. BAG1 was found to directly maintain BCL2 and to protect MCL1 from proteasomal degradation by controlling USP9X expression, which appeared to be its novel target. Finally, BAG1 was found able to affect leukemia cell fate by influencing the expression of anti-apoptotic proteins crucial for AML maintenance.     

Ubiquitinylation of α-synuclein by carboxyl terminus Hsp70-interacting protein (CHIP) is regulated by Bcl-2-associated athanogene 5 (BAG5)

Parkinson's disease (PD) is a common neurodegenerative condition in which abnormalities in protein homeostasis, or proteostasis, may lead to accumulation of the protein α-synuclein (α-syn). Mutations within or multiplications of the gene encoding α-syn are known to cause genetic forms of PD and polymorphisms in the gene are recently established risk factors for idiopathic PD. α-syn is a major component of Lewy bodies, the intracellular proteinaceous inclusions which are pathological hallmarks of most forms of PD. Recent evidence demonstrates that α-syn can self associate into soluble oligomeric species and implicates these α-syn oligomers in cell death. We have previously shown that carboxyl terminus of Hsp70-interacting protein (CHIP), a co-chaperone molecule with E3 ubiquitin ligase activity, may reduce the levels of toxic α-syn oligomers. Here we demonstrate that α-syn is ubiquitinylated by CHIP both in vitro and in cells. We find that the products from ubiquitinylation by CHIP include both monoubiquitinylated and polyubiquitinylated forms of α-syn. We also demonstrate that CHIP and α-syn exist within a protein complex with the co-chaperone bcl-2-associated athanogene 5 (BAG5) in brain. The interaction of CHIP with BAG5 is mediated by Hsp70 which binds to the tetratricopeptide repeat domain of CHIP and the BAG domains of BAG5. The Hsp70-mediated association of BAG5 with CHIP results in inhibition of CHIP E3 ubiquitin ligase activity and subsequently reduces α-syn ubiquitinylation. Furthermore, we use a luciferase-based protein-fragment complementation assay of α-syn oligomerization to investigate regulation of α-syn oligomers by CHIP in living cells. We demonstrate that BAG5 mitigates the ability of CHIP to reduce α-syn oligomerization and that non-ubiquitinylated α-syn has an increased propensity for oligomerization. Thus, our results identify CHIP as an E3 ubiquitin ligase of α-syn and suggest a novel function for BAG5 as a modulator of CHIP E3 ubiquitin ligase activity with implications for CHIP-mediated regulation of α-syn oligomerization.     

BAG3 directly associates with guanine nucleotide exchange factor of Rap1, PDZGEF2, and regulates cell adhesion

BAG3, a member of the Hsc70 binding co-chaperone BAG-family proteins, has critical roles in regulating actin organization, cell adhesion, cell motility and tumor metastasis. The PDZ domain containing guanine nucleotide exchange factor 2 (PDZGEF2) was cloned as a BAG3-interacting protein. PDZGEF2 induces activation of Rap1 and increases integrin-mediated cell adhesion. The PPDY motif at the C-terminus of PDZGEF2 binds to the WW domain of BAG3 in vitro and in vivo. BAG3 deletion mutant lacking the WW domain lose its cell adhesion and motility activity. Gene knockdown of PDZGEF2 leads to the loss of cell adhesion on fibronectin-coated plates while BAG3 overexpression increases cell adhesion in Cos7 cells, but not in PDZGEF2 gene knockdown cells indicating that PDZGEF2 is a critical partner for BAG3 in regulating cell adhesion.     

BAG3 directly interacts with mutated alphaB-crystallin to suppress its aggregation and toxicity

A homozygous disruption or genetic mutation of the bag3 gene causes progressive myofibrillar myopathy in mouse and human skeletal and cardiac muscle disorder while mutations in the small heat shock protein αB-crystallin gene (CRYAB) are reported to be responsible for myofibrillar myopathy. Here, we demonstrate that BAG3 directly binds to wild-type αB-crystallin and the αB-crystallin mutant R120G, via the intermediate domain of BAG3. Peptides that inhibit this interaction in an in vitro binding assay indicate that two conserved Ile-Pro-Val regions of BAG3 are involved in the interaction with αB-crystallin, which is similar to results showing BAG3 binding to HspB8 and HspB6. BAG3 overexpression increased αB-crystallin R120G solubility and inhibited its intracellular aggregation in HEK293 cells. BAG3 suppressed cell death induced by αB-crystallin R120G overexpression in differentiating C2C12 mouse myoblast cells. Our findings indicate a novel function for BAG3 in inhibiting protein aggregation caused by the genetic mutation of CRYAB responsible for human myofibrillar myopathy.     

BAG3: a multifaceted protein that regulates major cell pathways

Bcl2-associated athanogene 3 (BAG3) protein is a member of BAG family of co-chaperones that interacts with the ATPase domain of the heat shock protein (Hsp) 70 through BAG domain (110–124 amino acids). BAG3 is the only member of the family to be induced by stressful stimuli, mainly through the activity of heat shock factor 1 on bag3 gene promoter. In addition to the BAG domain, BAG3 contains also a WW domain and a proline-rich (PXXP) repeat, that mediate binding to partners different from Hsp70. These multifaceted interactions underlie BAG3 ability to modulate major biological processes, that is, apoptosis, development, cytoskeleton organization and autophagy, thereby mediating cell adaptive responses to stressful stimuli. In normal cells, BAG3 is constitutively present in a very few cell types, including cardiomyocytes and skeletal muscle cells, in which the protein appears to contribute to cell resistance to mechanical stress. A growing body of evidence indicate that BAG3 is instead expressed in several tumor types. In different tumor contexts, BAG3 protein was reported to sustain cell survival, resistance to therapy, and/or motility and metastatization. In some tumor types, down-modulation of BAG3 levels was shown, as a proof-of-principle, to inhibit neoplastic cell growth in animal models. This review attempts to outline the emerging mechanisms that can underlie some of the biological activities of the protein, focusing on implications in tumor progression.     

Protein quality control during aging involves recruitment of the macroautophagy pathway by BAG3

The Hsc/Hsp70 co-chaperones of the BAG (Bcl-2-associated athanogene) protein family are modulators of protein quality control. We examined the specific roles of BAG1 and BAG3 in protein degradation during the aging process. We show that BAG1 and BAG3 regulate proteasomal and macroautophagic pathways, respectively, for the degradation of polyubiquitinated proteins. Moreover, using models of cellular aging, we find that a switch from BAG1 to BAG3 determines that aged cells use more intensively the macroautophagic system for turnover of polyubiquitinated proteins. This increased macroautophagic flux is regulated by BAG3 in concert with the ubiquitin-binding protein p62/SQSTM1. The BAG3/BAG1 ratio is also elevated in neurons during aging of the rodent brain, where, consistent with a higher macroautophagy activity, we find increased levels of the autophagosomal marker LC3-II as well as a higher cathepsin activity. We conclude that the BAG3-mediated recruitment of the macroautophagy pathway is an important adaptation of the protein quality control system to maintain protein homeostasis in the presence of an enhanced pro-oxidant and aggregation-prone milieu characteristic of aging.     

BAG5 inhibits parkin and enhances dopaminergic neuron degeneration

Loss-of-function mutations in the parkin gene, which encodes an E3 ubiquitin ligase, are the major cause of early-onset Parkinson's disease (PD). Decreases in parkin activity may also contribute to neurodegeneration in sporadic forms of PD. Here, we show that bcl-2-associated athanogene 5 (BAG5), a BAG family member, directly interacts with parkin and the chaperone Hsp70. Within this complex, BAG5 inhibits both parkin E3 ubiquitin ligase activity and Hsp70-mediated refolding of misfolded proteins. BAG5 enhances parkin sequestration within protein aggregates and mitigates parkin-dependent preservation of proteasome function. Finally, BAG5 enhances dopamine neuron death in an in vivo model of PD, whereas a mutant that inhibits BAG5 activity attenuates dopaminergic neurodegeneration. This contrasts with the antideath functions ascribed to BAG family members and suggests a potential role for BAG5 in promoting neurodegeneration in sporadic PD through its functional interactions with parkin and Hsp70.     

IGFBP3 and BAG1 enhance radiation-induced apoptosis in squamous esophageal cancer cells

Identification of reliable markers of radiosensitivity and the key molecules that enhance the susceptibility of esophageal cancer cells to anticancer treatments would be highly desirable. To identify molecules that confer radiosensitivity to esophageal squamous carcinoma cells, we assessed the radiosensitivities of the TE-5, TE-9 and TE-12 cloneA1 cell lines. TE-12 cloneA1 cells showed significantly greater susceptibility to radiotherapy at 5 and 10 Gy than either TE-5 or TE-9 cells. Consistent with that finding, 24 h after irradiation (5 Gy), TE-12 cloneA1 cells showed higher levels of caspase 3/7 activity than TE-5 or TE-9 cells. When we used DNA microarrays to compare the gene expression profiles of TE-5 and TE-12 cloneA1 cells, we found that the mRNA and protein expression of insulin-like growth factor binding protein 3 (IGFBP3) and Bcl-2-associated athanogene 1 (BAG1) was five or more times higher in TE-12 cloneA1 cells than TE-5 cells. Conversely, knocking down expression of IGFBP3 and BAG1 mRNA in TE-12 cloneA1 cells using small interfering RNA (siRNA) significantly reduced radiosensitivity. These data suggest that IGFBP3 and BAG1 may be key markers of radiosensitivity that enhance the susceptibility of squamous cell esophageal cancer to radiotherapy. IGFBP3 and BAG1 may thus be useful targets for improved and more individualized treatments for patients with esophageal squamous cell carcinoma.     

The Stress Protein BAG3 Stabilizes Mcl-1 Protein and Promotes Survival of Cancer Cells and Resistance to Antagonist ABT-737

Members of the Bcl-2 family of proteins are important inhibitors of apoptosis in human cancer and are targets for novel anticancer agents such as the Bcl-2 antagonists, ABT-263 (Navitoclax), and its analog ABT-737. Unlike Bcl-2, Mcl-1 is not antagonized by ABT-263 or ABT-737 and is considered to be a major factor in resistance. Also, Mcl-1 exhibits differential regulation when compared with other Bcl-2 family members and is a target for anticancer drug discovery. Here, we demonstrate that BAG3, an Hsp70 co-chaperone, protects Mcl-1 from proteasomal degradation, thereby promoting its antiapoptotic activity. Using neuroblastoma cell lines, with a defined Bcl-2 family dependence, we found that BAG3 expression correlated with Mcl-1 dependence and ABT-737 resistance. RNA silencing of BAG3 led to a marked reduction in Mcl-1 protein levels and overcame ABT-737 resistance in Mcl-1-dependent cells. In ABT-737-resistant cells, Mcl-1 co-immunoprecipitated with BAG3, and loss of Mcl-1 after BAG3 silencing was prevented by proteasome inhibition. BAG3 and Mcl-1 were co-expressed in a panel of diverse cancer cell lines resistant to ABT-737. Silencing BAG3 reduced Mcl-1 protein levels and overcame ABT-737 resistance in several of the cell lines, including triple-negative breast cancer (MDA-MB231) and androgen receptor-negative prostate cancer (PC3) cells. These studies identify BAG3-mediated Mcl-1 stabilization as a potential target for cancer drug discovery.     

Evidence for the impact of BAG3 on electrophysiological activity of primary culture of neonatal cardiomyocytes

Homeostasis of proteins involved in contractility of individual cardiomyocytes and those coupling adjacent cells is of critical importance as any abnormalities in cardiac electrical conduction may result in cardiac irregular activity and heart failure. Bcl2-associated athanogene 3 (BAG3) is a stress-induced protein whose role in stabilizing myofibril proteins as well as protein quality control pathways, especially in the cardiac tissue, has captured much attention. Mutations of BAG3 have been implicated in the pathogenesis of cardiac complications such as dilated cardiomyopathy. In this study, we have used an in vitro model of neonatal rat ventricular cardiomyocytes to investigate potential impacts of BAG3 on electrophysiological activity by employing the microelectrode array (MEA) technology. Our MEA data showed that BAG3 plays an important role in the cardiac signal generation as reduced levels of BAG3 led to lower signal frequency and amplitude. Our analysis also revealed that BAG3 is essential to the signal propagation throughout the myocardium, as the MEA data-based conduction velocity, connectivity degree, activation time, and synchrony were adversely affected by BAG3 knockdown. Moreover, BAG3 deficiency was demonstrated to be connected with the emergence of independently beating clusters of cardiomyocytes. On the other hand, BAG3 overexpression improved the activity of cardiomyocytes in terms of electrical signal amplitude and connectivity degree. Overall, by providing more in-depth analyses and characterization of electrophysiological parameters, this study reveals that BAG3 is of critical importance for electrical activity of neonatal cardiomyocytes.     

Co‐chaperone BAG3 and adenovirus penton base protein partnership

The BAG family of Hsp70/Hsc70 co‐chaperones is characterised by the presence of a conserved BAG domain at the carboxyl‐terminus. BAG3 protein is the only member of this family containing also the N‐terminally located WW domain. We describe here the identification of adenovirus (Ad) penton base protein as the first BAG3 partner recognising BAG3 WW domain. Ad penton base is the viral capsid constituent responsible for virus internalisation. It contains in the N‐terminal part two conserved PPxY motifs, known ligands of WW domains. In cells producing Ad penton base protein, cytoplasmic endogenous BAG3 interacts with it and co‐migrates to the nucleus. Preincubation of BAG3 with Ad base protein results in only slight modulation of BAG3 co‐chaperone activity, suggesting that this interaction is not related to the classical BAG3 co‐chaperone function. However, depletion of BAG3 impairs the cell entry of the virus and viral progeny production in Ad‐infected cells, suggesting that the interaction between virus penton base protein and cellular co‐chaperone BAG3 positively influences virus life cycle. These results thus demonstrate a novel host–pathogen interaction, which contributes to the successful infectious life cycle of adenoviruses. In addition, these data enrich our knowledge about the multifunctionality of the BAG3 co‐chaperone. J. Cell. Biochem. 111: 699–708, 2010. © 2010 Wiley‐Liss, Inc.     

BAG1L enhances trans-activation function of the vitamin D receptor

The vitamin D receptor (VDR) is a member of the steroid/retinoid receptor superfamily of nuclear receptors that has potential tumor-suppressive functions. We show here that VDR interacts with and is regulated by BAG1L, a nuclear protein that binds heat shock 70-kDa (Hsp70) family molecular chaperones. Endogenous BAG1L can be co-immunoprecipitated with VDR from prostate cancer cells (ALVA31; LNCaP) in a ligand-dependent manner. BAG1L, but not shorter non-nuclear isoforms of this protein (BAG1; BAG1M/Rap46), markedly enhanced, in a ligand-dependent manner, the ability of VDR to trans-activate reporter gene plasmids containing a vitamin D response element in transient transfection assays. Mutant BAG1L lacking the C-terminal Hsc70-binding domain suppressed (in a concentration-dependent fashion) VDR-mediated trans-activation of vitamin D response element-containing reporter gene plasmids, without altering levels of VDR or endogenous BAG1L protein, suggesting that it operates as a trans-dominant inhibitor of BAG1L. Gene transfer-mediated elevations in BAG1L protein levels in a prostate cancer cell line (PC3), which is moderately responsive to VDR ligands, increased the ability of natural (1alpha,25(OH)(2) vitamin D(3)) and synthetic (1alpha, 25-dihydroxy-19-nor-22(E)-vitamin D(3)) VDR ligands to induce expression of the VDR target gene, p21(Waf1), and suppress DNA synthesis. Thus, BAG1L is a direct regulator of VDR, which enhances its trans-activation function and improves tumor cell responses to growth-suppressive VDR ligands.