Abstract
Bcl-2 is a cytoplasmic integral membrane protein with potent anti-apoptotic activity but whose mechanism of action is poorly understood. The purpose of this paper was to obtain large amounts of soluble Bcl-2 protein for structural and functional studies. Mouse Bcl-2(1-203) (missing the COOH-terminal hydrophobic tail) was produced in bacterial inclusion bodies, solubilized in guanidine, and refolded by dialysis. The resulting protein was monomeric in nondenaturing solution and was active in protecting mouse T hybridoma cells from glucocorticoid-induced apoptosis. Refolded Bcl-2(1-203) showed no tendency to homodimerize by gel filtration or analytical ultracentrifugation. Limited proteolysis experiments identified a region between the BH3 and BH4 homology domains of Bcl-2(1-203) which was extremely susceptible to digestion by several common proteases, but not by a cell extract known to contain CPP-32-like (interleukin-1beta-converting enzyme family) protease activity. The protease-sensitive sites were located within a 50-residue stretch that contained most of the nonconserved and proline residues of Bcl-2(1-203). Trypsin-cleaved Bcl-2(1-203) eluted in the same position as the undigested protein on gel filtration in nondenaturing solution, indicating that the two portions of the molecule connected by the protease-sensitive region associate stably and noncovalently. The solution properties of Bcl-2(1-203) suggest that it consists of two noncovalently associated domains connected by a long protease-sensitive linker and that its structure is similar to that of Bcl-xL, which has been determined by x-ray and NMR analysis.
Highlights
From the ‡Experimental Immunology Branch and §Laboratory of Immune Cell Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892-1360
Subsequent work indicated that overexpression of Bcl-2 prolonged cell survival (2) and inhibited programmed cell death induced by multiple, diverse stimuli including growth factor withdrawal, glucocorticoids, and ␥-irradiation (3, 4)
The flexible domain corresponds to a region of the Bcl-xL and Bcl-2 molecules which exhibits the greatest variability in amino acid sequence between molecules from different species and Bcl-2 family members
Summary
From the ‡Experimental Immunology Branch and §Laboratory of Immune Cell Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892-1360. Limited proteolysis experiments identified a region between the BH3 and BH4 homology domains of Bcl-2(1–203) which was extremely susceptible to digestion by several common proteases, but not by a cell extract known to contain CPP-32-like (interleukin-1-converting enzyme family) protease activity. Trypsincleaved Bcl-2(1–203) eluted in the same position as the undigested protein on gel filtration in nondenaturing solution, indicating that the two portions of the molecule connected by the protease-sensitive region associate stably and noncovalently. The solution properties of Bcl-2(1–203) suggest that it consists of two noncovalently associated domains connected by a long protease-sensitive linker and that its structure is similar to that of Bcl-xL, which has been determined by x-ray and NMR analysis. Bcl-2 heterodimerizes with the pro-apoptotic protein Bax, resulting in enhanced cell survival (9, 12). Unlike the BH1– 4 regions, the flexible domain can be deleted without loss of anti-apoptotic activity (7, 15)
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