Bcl-2 localized at the nuclear compartment induces apoptosis after transient overexpression.
ABSTRACT Bcl-2 is the best characterized member of a large family of proteins that regulate apoptosis. Although it is established that Bcl-2 localized at the mitochondria functions as an anti-apoptotic protein, the function of Bcl-2 at the nucleus remains unclear. Recently we showed that nuclear compartment-associated Bcl-2 inhibits transcription factor activation. Based on this observation, we hypothesized that presence of Bcl-2 at the nucleus may induce rather than protect cells from apoptosis. Here we investigated the putative apoptotic role of nuclear compartment-associated Bcl-2. Additionally, we examined the role of the Bcl-2 BH4 domain in mediating binding to FKBP38, the Bcl-2 mitochondrial chaperone. Our results demonstrate a novel, pro-apoptotic function for nuclear Bcl-2 and identify the Bcl-2 BH4 domain as a key regulator in mediating Bcl-2/FKBP38 binding. These results indicate that Bcl-2 has a dual role as both a protector and a killer and that the ability to switch roles depends on Bcl-2 subcellular localization.
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ABSTRACT: Naturally occurring proteins in cellular networks often share peptide motifs. These motifs have been known to play a pivotal role in protein interactions among the components of a network. However, it remains unknown how these motifs have contributed to the evolution of the protein network. Here we addressed this issue by a synthetic biology approach. Through the motif programming method, we have constructed an artificial protein library by mixing four peptide motifs shared among the Bcl-2 family proteins that positively or negatively regulate the apoptosis networks. We found one strong pro-apoptotic protein, d29, and two proteins having moderate, but unambiguous anti-apoptotic functions, a10 and d16, from the 28 tested clones. Thus both the pro- and anti-apoptotic modulators were present in the library, demonstrating that functional proteins with opposing effects can emerge from a single pool prepared from common motifs. Motif programming studies have exhibited that the annotated function of the motifs were significantly influenced by the context that the motifs embedded. The results further revealed that reshuffling of a set of motifs realized the promiscuous state of protein, from which disparate functions could emerge. Our finding suggests that motifs contributed to the plastic evolvability of the protein network.Nucleic Acids Research 02/2007; 35(19):6357-66. · 8.03 Impact Factor
Article: Bcl-2 regulates HIF-1alpha protein stabilization in hypoxic melanoma cells via the molecular chaperone HSP90.[show abstract] [hide abstract]
ABSTRACT: Hypoxia-Inducible Factor 1 (HIF-1) is a transcription factor that is a critical mediator of the cellular response to hypoxia. Enhanced levels of HIF-1alpha, the oxygen-regulated subunit of HIF-1, is often associated with increased tumour angiogenesis, metastasis, therapeutic resistance and poor prognosis. It is in this context that we previously demonstrated that under hypoxia, bcl-2 protein promotes HIF-1/Vascular Endothelial Growth Factor (VEGF)-mediated tumour angiogenesis. By using human melanoma cell lines and their stable or transient derivative bcl-2 overexpressing cells, the current study identified HIF-1alpha protein stabilization as a key regulator for the induction of HIF-1 by bcl-2 under hypoxia. We also demonstrated that bcl-2-induced accumulation of HIF-1alpha protein during hypoxia was not due to an increased gene transcription or protein synthesis. In fact, it was related to a modulation of HIF-1alpha protein expression at a post-translational level, indeed its degradation rate was faster in the control lines than in bcl-2 transfectants. The bcl-2-induced HIF-1alpha stabilization in response to low oxygen tension conditions was achieved through the impairment of ubiquitin-dependent HIF-1alpha degradation involving the molecular chaperone HSP90, but it was not dependent on the prolyl hydroxylation of HIF-1alpha protein. We also showed that bcl-2, HIF-1alpha and HSP90 proteins form a tri-complex that may contribute to enhancing the stability of the HIF-1alpha protein in bcl-2 overexpressing clones under hypoxic conditions. Finally, by using genetic and pharmacological approaches we proved that HSP90 is involved in bcl-2-dependent stabilization of HIF-1alpha protein during hypoxia, and in particular the isoform HSP90beta is the main player in this phenomenon. We identified the stabilization of HIF-1alpha protein as a mechanism through which bcl-2 induces the activation of HIF-1 in hypoxic tumour cells involving the beta isoform of molecular chaperone HSP90.PLoS ONE 01/2010; 5(7):e11772. · 4.09 Impact Factor
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ABSTRACT: Viral replication efficiency is in large part governed by the ability of viruses to counteract pro-apoptotic signals induced by infection of the host cell. Human herpesvirus 8 (HHV-8) uses several strategies to block the host's innate antiviral defenses via interference with interferon and apoptotic signaling. Contributors include the four viral interferon regulatory factors (vIRFs 1-4), which function in dominant negative fashion to block cellular IRF activities in addition to targeting IRF signaling-induced proteins such as p53 and inhibiting other inducers of apoptosis such as TGFbeta receptor-activated Smad transcription factors. Here we identify direct targeting by vIRF-1 of BH3-only pro-apoptotic Bcl-2 family member Bim, a key negative regulator of HHV-8 replication, to effect its inactivation via nuclear translocation. vIRF-1-mediated relocalization of Bim was identified in transfected cells, by both immunofluorescence assay and western analysis of fractionated cell extracts. Also, co-localization of vIRF-1 and Bim was detected in nuclei of lytically infected endothelial cells. In vitro co-precipitation assays using purified vIRF-1 and Bim revealed direct interaction between the proteins, and Bim-binding residues of vIRF-1 were mapped by deletion and point mutagenesis. Generation and experimental utilization of Bim-refractory vIRF-1 variants revealed the importance of vIRF-1:Bim interaction, specifically, in pro-replication and anti-apoptotic activity of vIRF-1. Furthermore, blocking of the interaction with cell-permeable peptide corresponding to the Bim-binding region of vIRF-1 confirmed the relevance of vIRF-1:Bim association to vIRF-1 pro-replication activity. To our knowledge, this is the first report of an IRF protein that interacts with a Bcl-2 family member and of nuclear sequestration of Bim or any other member of the family as a means of inactivation. The data presented reveal a novel mechanism utilized by a virus to control replication-induced apoptosis and suggest that inhibitory targeting of vIRF-1:Bim interaction may provide an effective antiviral strategy.PLoS Pathogens 01/2010; 6(8):e1001031. · 9.13 Impact Factor