Biophysical characterization of recombinant human Bcl-2 and its interactions with an inhibitory ligand, antimycin A.

Apoptosis is an essential physiological process, regulated by the family of Bcl-2-related proteins. However, the molecular mechanism by which Bcl-2 regulates apoptosis still remains elusive. Here we report the functional studies of recombinant human Bcl-2 with the deletion of 22 residues at the C-terminal membrane-anchoring region (rhBcl-2Delta22). Characterization of rhBcl-2Delta22 showed that the recombinant protein is homogeneous and monodisperse in nondenaturing solutions, stable at room temperature in the presence of a metal chelator, and an alpha-helical protein with unfolding of secondary structure at a T(m) of 62.8 degrees C. Optimal membrane pore formation by rhBcl-2Delta22 required negatively charged phospholipids. The existence of a hydrophobic groove in rhBcl-2Delta22 was demonstrated by the fluorescence enhancement of the hydrophobic ANS probe with which a pro-apoptotic Bak BH3 peptide competed. The respiratory inhibitor antimycin A also bound to the hydrophobic groove of rhBcl-2Delta22 with a K(d) of 0.82 microM. The optimal binding conformation of antimycin A was predicted from molecular docking of antimycin A with the hBcl-2 model created by homology modeling. Antimycin A selectively induces apoptosis in cells overexpressing Bcl-2, suggesting that hydrophobic groove-binding compounds may act as selective apoptotic triggers in tumor cells.

Antimycin A mimics a cell-death-inducing Bcl-2 homology domain 3.

The Bcl-2-related survival proteins confer cellular resistance to a wide range of agents. Bcl-xL-expressing hepatocyte cell lines are resistant to tumour necrosis factor and anti-cancer drugs, but are more sensitive than isogenic control cells to antimycin A, an inhibitor of mitochondrial electron transfer. Computational molecular docking analysis predicted that antimycin A interacts with the Bcl-2 homology domain 3 (BH3)-binding hydrophobic groove of Bcl-xL. We demonstrate that antimycin A and a Bak BH3 peptide bind competitively to recombinant Bcl-2. Antimycin A and BH3 peptide both induce mitochondrial swelling and loss of DeltaPsim on addition to mitochondria expressing Bcl-xL. The 2-methoxy derivative of antimycin A3 is inactive as an inhibitor of cellular respiration but still retains toxicity for Bcl-xL+ cells and mitochondria. Finally, antimycin A inhibits the pore-forming activity of Bcl-x L in synthetic liposomes, demonstrating that a small non-peptide ligand can directly inhibit the function of Bcl-2-related proteins.

The maize LAG1-O mutant suggests that reproductive cell lineages show unique gene expression patterns early in vegetative development.

Patterns of transposable element activity often provide useful information about how and when organisms regulate gene expression. The maize lowered Ac/Ds germinal reversion 1 (LAG1)-O mutation causes unusually low rates of germinal reversion by Ac/Ds-induced alleles even though these same alleles revert frequently and early in somatic development. LAG1-O suppresses Ds transposition at multiple, unlinked loci, and does not affect Spm elements, indicating that the mutation acts in trans and may be specific to Ac/Ds elements. Our data suggest that LAG1-O suppression gradually reduces Ac/Ds activity in the meristem and newly formed leaves until, by the floral transition, transposition is undetectable even with PCR-based assays. This suppression persists during tassel development and does not appear to be released until some point after meiosis. Competitive RT-PCR results show no difference in Ac transposase mRNA levels between LAG1-O and lag1(+) tassels, suggesting that suppression is post-transcriptional. The pattern of LAG1-O expression is consistent with a model in which at least some gene expression specific to those meristem cells that will ultimately give rise to floral tissue and therefore gametes begins very early in plant development, and then persists throughout development.