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Mutagenesis of the BH3 domain of BAX identifies residues critical for dimerization and killing 总被引:1,自引:0,他引:1
The BCL-2 family of proteins is comprised of proapoptotic as well as antiapoptotic members (S. N. Farrow and R. Brown, Curr. Opin. Genet. Dev. 6:45-49, 1996). A prominent death agonist, BAX, forms homodimers and heterodimerizes with multiple antiapoptotic members. Death agonists have an amphipathic alpha helix, called BH3; however, the initial assessment of BH3 in BAX has yielded conflicting results. Our BAX deletion constructs and minimal domain constructs indicated that the BH3 domain was required for BAX homodimerization and heterodimerization with BCL-2, BCL-XL, and MCL-1. An extensive site-directed mutagenesis of BH3 revealed that substitutions along the hydrophobic face of BH3, especially charged substitutions, had the greatest affects on dimerization patterns and death agonist activity. Particularly instructive was the BAX mutant mIII-1 (L63A, G67A, L70A, and M74A), which replaced the hydrophobic face of BH3 with alanines, preserving its amphipathic nature. BAXmIII-1 failed to form heterodimers or homodimers by yeast two-hybrid or immunoprecipitation analysis yet retained proapoptotic activity. This suggests that BAX's killing function reflects mechanisms beyond its binding to BCL-2 or BCL-XL to inhibit them or simply displace other protein partners. Notably, BAXmIII-1 was found predominantly in mitochondrial membranes, where it was homodimerized as assessed by homobifunctional cross-linkers. This characteristic of BAXmIII-1 correlates with its capacity to induce mitochondrial dysfunction, caspase activation, and apoptosis. These data are consistent with a model in which BAX death agonist activity may require an intramembranous conformation of this molecule that is not assessed accurately by classic binding assays. 相似文献
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MJ Tetel S Jung P Carbajo T Ladtkow DF Skafar DP Edwards 《Canadian Metallurgical Quarterly》1997,11(8):1114-1128
We and others have shown previously that progesterone receptors (PR) form homodimers in solution in the absence of DNA and that dimers are the preferential form of receptor that binds with high affinity to target DNA. To determine the sequence regions involved in solution homodimerization, wild type PR and truncated PR proteins were expressed in an insect baculovirus system. The expression constructs included the ligand-binding domain [LBD, amino acids (aa) 688-933], the LBD plus hinge (hLBD, aa 634-933), the hLBD plus the DNA-binding domain (DhLBD, aa 538-933), and the full- length A and B isoforms of PR. PR-PR interactions were detected by three methods, coimmunoprecipitation of the PR fragments with full-length PR-A, pull-down of PR-polypeptides with polyhistidine-tagged versions of the same polypeptides immobilized to metal affinity columns and cooperative ligand-binding assays (Hill coefficient, n(H) > 1 indicating PR-PR interaction). By all three assays, the LBD alone was not sufficient to mediate protein-protein interaction. However, the LBD did exhibit other properties ascribed to this domain, including binding to steroids with a relatively good affinity and specificity, ligand-induced conformational changes at the carboxyl terminus tail and binding of heat shock protein 90 and its dissociation in response to hormone. Thus, failure of the expressed LBD to mediate dimerization does not appear to be due to an extensively misfolded or unstable polypeptide. The minimal carboxyl-terminal fragment capable of mediating PR-PR interaction was the hLBD construct. However, by immobilized metal affinity chromatography assay, self-association of PR-A was 3.5-fold more efficient than that of either the DhLBD or hLBD constructs. An expressed amino-terminal domain (aa 165-535) lacking the DNA-binding domain, hinge, and LBD was found to physically associate with PR-A or with another amino-terminal fragment lacking the LBD, but retaining the DNA-binding domain. These results provide evidence for direct amino-terminal interactions in the more efficient PR-PR interaction exhibited by wild-type PR-A, as compared with DhLBD and hLBD constructs. The overall results of this paper are consistent with the conclusion that the carboxyl-terminal LBD is not sufficient for mediating PR dimerization and that multiple regions, including the hinge and amino-terminal sequences, contribute either directly or indirectly to homodimerization of PR. 相似文献
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A unique heterodimerization pathway involving orphan receptors TR2 and TR4 is demonstrated. TR2 and TR4 preferentially form heterodimers in solution as well as on DNA elements containing a direct repeat-5 (DR5). The in vitro interaction between TR2 and TR4 is demonstrated by the yeast and the mammalian two-hybrid interaction assays, the pull-down assay, and the gel mobility shift assay. The in vivo interaction is demonstrated by following the intracellular localization of fusion receptors tagged with a green fluorescent protein. The dimerization is mediated by the ligand binding domains, and the three leucine residues on helix 10 of TR2 are critical for this interaction. In addition, coexpression of these two receptors exerts a much stronger repressive activity on a DR5-containing reporter than expressing either receptor alone. In the developing testis, TR2 and TR4 are coexpressed in the same testicular cell populations and exhibit a parallel pattern of expression along development. The preferential heterodimerization between TR2 and TR4 and their coexistence in specific germ cell populations suggest a physiological role of TR2/TR4 heterodimers in germ cell development. 相似文献
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G Franzoso V Bours V Azarenko S Park M Tomita-Yamaguchi T Kanno K Brown U Siebenlist 《Canadian Metallurgical Quarterly》1993,12(10):3893-3901
Previously we have proposed a role for Bcl-3 in facilitating transactivation through kappa B sites by counteracting the inhibitory effects of bound, non-transactivating homodimers of the p50 subunit of NF-kappa B. Such homodimers are abundant for example in nuclei of unstimulated primary T cells. Here we extend the model and provide new evidence which fulfills a number of predictions. (i) Bcl-3 preferentially targets p50 homodimers over NF-kappa B heterodimers since the homodimers are completely dissociated from kappa B sites at concentrations of Bcl-3 which do not affect NF-kappa B. (ii) Select kappa B sites associate very strongly and stably with p50 homodimers, completely preventing binding by NF-kappa B. Such kappa B sites are likely candidates for regulation by p50 homodimers and Bcl-3. (iii) Bcl-3 and p50 can be co-localized in the nucleus, a requirement for active removal of homodimers from their binding sites in vivo. (iv) The ankyrin repeat domain of Bcl-3 is sufficient for the reversal of p50 homodimer-mediated inhibition, correlating with the ability of this domain alone to inhibit p50 binding to kappa B sites in vitro. Our data support the model that induction of nuclear Bcl-3 may be required during cellular stimulation to actively remove stably bound p50 homodimers from certain kappa B sites in order to allow transactivating NF-kappa B complexes to engage. This exact mechanism is demonstrated with in vitro experiments. 相似文献
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Transcriptional regulation by steroid hormones 总被引:2,自引:0,他引:2