Analysis of interaction sites in homo- and heteromeric complexes containing Bcl-2 family members and the cellular prion proteinKurschner C; Morgan JI Brain Res Mol Brain Res 1996 Apr;37(1-2):249-58The cellular prion protein (PrP) binds to the C-terminus of Bcl-2 but not Bax. Therefore, we examined whether the C-terminus of Bcl-2 was important for other homomeric and heteromeric protein-protein interactions. Using the yeast two hybrid system and co-immunoprecipitation, three sites of homomeric interactions were identified within Bcl-2.
The carboxy terminal 37 amino acids selectively homodimerized. Two additional regions of Bcl-2 (residues 1-129 and 126-200) interacted with each other, but not themselves permitting both intra- and intermolecular association.
In addition, we analyzed heteromeric interactions of Bcl-2 with PrP and two Bcl-2 related proteins, Bax and A1. The domain requirements for binding of those three proteins to Bcl-2 were different from one another. Bax binding required almost the entire Bcl-2 molecule, while A1 bound to the amino terminal region (residues 1-82). PrP associated with the carboxy terminus of Bcl-2 (amino acids 200-236).
These data suggest configurational models for Bcl-2 containing complexes. First, Bcl-2 may exist as both heterodimers and heteromultimers. Second, molecules such as Bax and A1 may serve to cap chains of Bcl-2 homodimers by interacting with dimerization domains in the extramembrane region. PrP may disrupt chains of Bcl-2 molecules at the homomeric association site in the transmembrane region.
Structure-function analysis of Bcl-2 protein. Identification of conserved domains important for homodimerization with Bcl-2 and heterodimerization with Bax.Hanada M; Aime-Sempe C; Sato T; Reed JC J Biol Chem 270: 11962-9 (1995)The Bcl-2 protein is a suppressor of programmed cell death that homodimerizes with itself and forms heterodimers with a homologous protein Bax, a promoter of cell death. Functional analysis of deletion mutants of human Bcl-2 in yeast demonstrated the presence of at least three conserved domains that are required to suppress Bax-mediated cytotoxicity, termed domains A (amino acids 11-33), B (amino acids 138-154), and C (amino acids 188-196).
In vitro binding experiments using GST--2 fusion proteins demonstrated that Bcl-2(delta B) and -2(delta C) deletion mutants had a markedly impaired ability to heterodimerize with Bax but retained the ability to homodimerize with wild-type Bcl-2. In contrast, Bcl-2(delta A) and an NH2-terminal deletion mutant Bcl-2(delta 1-82) retained Bax binding activity in vitro but failed to suppress Bax-mediated cytotoxicity in yeast. Sequences downstream of domain C in the region 197-218 also were shown to be required for Bax-binding in vitro and anti-death function in yeast.
Analysis of Bcl-2/-2 homodimerization using both in vitro binding assays as well as a yeast two-hybrid method provided evidence in support of a head-to-tail model for Bcl-2/-2 homodimerization and revealed that sequences within the NH2-terminal A domain interact with a structure that requires the presence of both the carboxyl B and C domains in combination. In addition to further delineating structural features within Bcl-2 that are required for homo-dimerization, the findings reported here support the hypothesis that Bcl-2 promotes cell survival by binding directly to Bax but suggest that ability to bind Bax can be insufficient for anti-cell death function.
Dissection of functional domains in Bcl-2 alpha by site-directed mutagenesis.Borner C; Olivier R; Martinou I; Mattmann C; Tschopp J; Martinou JC Biochem Cell Biol 72: 463-9 (1994)-2 alpha is a mitochondrial or perinuclear-associated oncoprotein that prolongs the life span of a variety of cell types by interfering with programmed cell death. How Bcl-2 confers cell survival is unknown, although antioxidant and antiprotease functions have been proposed. In addition, protein structures of Bcl-2 that are crucial for its survival activity are still ill-defined. Bcl-2 can occur as Bcl-2 alpha or Bcl-2 beta, two alternatively spliced forms which solely differ in their carboxyl termini. The finding that Bcl-2 alpha is active and membrane bound, but Bcl-2 beta is inactive and cytosolic, indicates that the carboxyl terminus contributes to the survival activity of Bcl-2. This region contains two subdomains, a domain X with unknown function and a hydrophobic stretch reported to mediate membrane association of Bcl-2 alpha. Recently Bcl-2-related proteins have been identified. These include Bax that heterodimerizes with Bcl-2 and, when overexpressed, counteracts Bcl-2. Bax contains two highly conserved regions of sequence homology with Bcl-2, referred to as -2 homology 1 and 2 (BH1 and BH2) domains. Site-directed mutagenesis studies have revealed that both domains are not only novel dimerization motifs for the interaction of Bax with Bcl-2 but also crucial for the survival activity of Bcl-2. Interestingly, the C-terminal end of BH2 encompasses the Bcl-2 alpha/beta splice site, as well as part of domain X in Bcl-2 alpha.
A peptide sequence from Bax that converts Bcl-2 into an activator of apoptosis.Hunter JJ; Parslow TG J Biol Chem 271: 8521-4 (1996)Bcl-2 and Bax are members of a family of cytoplasmic proteins that regulate apoptosis. The two proteins have highly similar amino acid sequences but are functionally opposed: Bcl-2 acts to inhibit apoptosis, whereas Bax counteracts this effect. The antagonism appears to depend upon dimerization between Bcl-2 and Bax, but its mechanism is otherwise unknown.
Here we report that overexpressing Bax induces apoptosis in a mammalian fibroblast cell line, and we identify a novel, short "suicide domain" in Bax that is required for this effect. Inserting this domain in place of the corresponding, divergent sequence in Bcl-2 converts -2 from an inhibitor into an activator of cell death. These findings imply that a specific region in Bax confers an active propensity for apoptosis in mammalian cells and support the view that Bcl-2 may block death primarily by suppressing Bax activity.
Proapoptotic protein Bax heterodimerizes with Bcl-2 and homodimerizes with Bax via a novel domain (BH3) distinct from BH1 and BH2.Zha H; Aime-Sempe C; Sato T; Reed JC J Biol Chem 271: 7440-4 (1996)Most members of the Bcl-2 protein family of apoptosis regulating proteins contain two evolutionarily conserved domains, termed BH1 and BH2. Both BH1 and BH2 in the Bcl-2 protein are required for its function as an inhibitor of cell death and for heterodimerization with the proapoptotic protein Bax.
In this report, we mapped the region in Bax required for heterodimerization with Bcl-2 and homodimerization with Bax, using yeast two-hybrid and in vitro protein-protein interaction assays. Neither the BH1 nor the BH2 domain of Bax was required for binding to the wild-type Bcl-2 and Bax proteins. Moreover, Bax (deltaBH1) and Bax (deltaBH2) mutant proteins bound efficiently to themselves and each other, further confirming the lack of requirement for BH1 and BH2 for Bax/Bax homodimerization. Bax/Bax homodimerization was not dependent on the inclusion of the NH2-terminal 58 amino acids of the Bax protein in each dimerization partner, unlike Bcl-2/-2 homodimers which involve head-to-tail interactions between the region of Bcl-2 where BH1 and BH2 resides, and an NH2-terminal domain in Bcl-2 that contains another domain BH4 which is conserved among antiapoptotic members of the Bcl-2 family.
Similarly, heterodimerization with Bcl-2 occurred without the NH2-terminal domain of either Bax or Bcl-2, suggesting a tail-to-tail interaction. The essential region in Bax required for both homodimerization with Bax and heterodimerization with Bcl-2 was mapped to residues 59-101. This region in Bax contains a stretch of 15 amino acids that is highly homologous in several members of the Bcl-2 protein family, suggesting the existence of a novel functional domain which we have termed BH3. Deletion of this 15-amino acid region abolished the ability of Bax to dimerize with itself and to heterodimerize with Bcl-2. The findings suggest that the structural features of Bax and -2 that allow them to participate in homo-and heterodimerization phenomena are markedly different, despite their amino-acid sequence similarity.
Evolutionary conservation of function among mammalian, avian, and viral homologs of the Bcl-2 oncoprotein.Takayama S; et al DNA Cell Biol 13: 679-92 (1994)The Bcl-2 gene was originally cloned because of its involvement in B-cell lymphomas and encodes a 25-kD integral membrane protein that has been shown to inhibit programmed cell death (also termed apoptosis) in a wide variety of circumstances. The Epstein-Barr Virus (EBV) also has been implicated in B-cell malignancies and interestingly contains an open reading frame (BHRF-1) predicting a 19-kD protein with 22% homology to Bcl-2. these data demonstrate that despite marked differences in their predicted amino-acid sequences, the human, chicken, and EBV versions of Bcl-2 have retained the structural characteristics necessary to interface with pathways involved in the regulation of programmed cell death in murine cells.
Interactions among members of the Bcl-2 protein family analyzed with a yeast two-hybrid system.Sato T; Hanada M et al Proc Natl Acad Sci U S A 91: 9238-42 (1994)Interactions of the Bcl-2 protein with itself and other members of the Bcl-2 family, including -X-L, -X-S, Mcl-1, and Bax, were explored with a yeast two-hybrid system. Fusion proteins were created by linking Bcl-2 family proteins to a LexA DNA-binding domain or a B42 trans-activation domain.
Shift of the cellular oxidation-reduction potential in neural cells expressing Bcl-2.Ellerby LM; Ellerby HM etal J Neurochem 67: 1259-67 (1996)Expression of the protooncogene Bcl-2 inhibits both apoptotic and in some cases necrotic cell death in many cell types, including neural cells, and in response to a wide variety of inducers. The mechanism by which the Bcl-2 protein acts to prevent cell death remains elusive. One mechanism by which Bcl-2 has been proposed to act is by decreasing the net cellular generation of reactive oxygen species. To evaluate this proposal, we measured activities of antioxidant enzymes as well as levels of glutathione and pyridine nucleotides in control and Bcl-2 transfectants.
Bcl-2 protects macrophages from nitric oxide-induced apoptosis.Messmer UK; Reed UK; Brune B J Biol Chem 271: 20192-7 (1996)Endogenously generated or exogenously supplied nitric oxide (NO)-induced apoptotic cell death in the mouse macrophage cell line RAW 264.7. Apoptotic signaling caused an early accumulation of the tumor suppressor p53 prior to DNA fragmentation. Contrary to the notion of specific activating signals, inhibitory transduction mechanisms largely remain unknown. Therefore, RAW 264.7 macrophages were stably transfected with human Bcl-2, an anti-apoptotic protein. Bcl-2 transfectants showed substantial protection from cell death induced following the exposure to NO donors such as S-nitrosoglutathione (GSNO) and spermine-NO.