Biophysical Characterization of Recombinant Human Bcl-2 and Its Interactions with an Inhibitory Ligand, Antimycin A

Abstract

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.

Targeted therapies for epithelial cancers: In vivo efficacy of the BCL-2/BCL-XL inhibitor 2-MeAA

“…12 It was subsequently shown to have similar activity against Bcl-2. 13 Interestingly, in the antisense study, compensatory induction of Bcl-2 was observed with Bcl-x L antisense treatment of mesothelioma cell lines, while this was not observed in the present study, perhaps reflecting the bispecificity of 2-MeAA.…”

Bcl-2 Homodimerization Involves Two Distinct Binding Surfaces, a Topographic Arrangement That Provides an Effective Mechanism for Bcl-2 to Capture Activated Bax

“…Expression and purification of human Bcl-2 protein with a sequence of Leu-Gln-His 6 , replacing the C-terminal 22 residues (designated 6H-Bcl-2⌬TM) were carried out as described (34). A similar protocol was used to express and purify the 6H-Bcl-2⌬TM protein with mutation G145A, C158A, or C158A/P168C (designated 6H-Bcl-2⌬TM G145A, C0 or C168, respectively).…”

Recent advances in the development of anticancer agents targeting cell death inhibitors in the Bcl-2 protein family

“…176 Subsequently, molecular docking studies predicted the binding of antimycin A to the hydrophobic surface pocket in Bcl-X L and Bcl-2. 176,177 The binding to Bcl-2 has been verified in fluorescence spectroscopies assays. 176,177 Antimycin A3 (Figure 4), one of the subspecies of antimycin A, is an inhibitor of mitochondrial electron transport at complex III and has been reported to display toxicity toward Bcl-2 and Bcl-X L -overexpressing hepatocyte cell lines.…”

“…176,177 The binding to Bcl-2 has been verified in fluorescence spectroscopies assays. 176,177 Antimycin A3 (Figure 4), one of the subspecies of antimycin A, is an inhibitor of mitochondrial electron transport at complex III and has been reported to display toxicity toward Bcl-2 and Bcl-X L -overexpressing hepatocyte cell lines. 176,177 Cell death resulting from antimycin A 3 occurs independently from inhibition of cellular respiration since 2-methoxy antimycin A 3 , an antimycin A 3 derivative lacking these inhibitory properties, retains the ability to kill cells.…”

“…176,177 Antimycin A3 (Figure 4), one of the subspecies of antimycin A, is an inhibitor of mitochondrial electron transport at complex III and has been reported to display toxicity toward Bcl-2 and Bcl-X L -overexpressing hepatocyte cell lines. 176,177 Cell death resulting from antimycin A 3 occurs independently from inhibition of cellular respiration since 2-methoxy antimycin A 3 , an antimycin A 3 derivative lacking these inhibitory properties, retains the ability to kill cells. 176 Strikingly, Bcl-X L -overexpressing murine hepatocytes, cells that are normally resistant to doxorubicin, cisplatin, and TNF, were shown to be hypersensitive to antimycin A 3 treatment.…”