The carboxyl-terminal sequence of bim enables bax activation and killing of unprimed cells

Abstract

The Bcl-2 family BH3 protein Bim promotes apoptosis at mitochondria by activating the pore-forming proteins Bax and Bak and by inhibiting the anti-apoptotic proteins Bcl-XL, Bcl-2 and Mcl-1. Bim binds to these proteins via its BH3 domain and to the mitochondrial membrane by a carboxyl-terminal sequence (CTS). In cells killed by Bim, the expression of a Bim mutant in which the CTS was deleted (BimL-dCTS) triggered apoptosis that correlated with inhibition of anti-apoptotic proteins being sufficient to permeabilize mitochondria isolated from the same cells. Detailed analysis of the molecular mechanism demonstrated that BimL-dCTS inhibited Bcl-XL but did not activate Bax. Examination of additional point mutants unexpectedly revealed that the CTS of Bim directly interacts with Bax, is required for physiological concentrations of Bim to activate Bax and that different residues in the CTS enable Bax activation and binding to membranes.

Bim escapes displacement by BH3-mimetic anti-cancer drugs by double-bolt locking both Bcl-XL and Bcl-2

“…Bim not only inhibits anti-apoptotic proteins it also activates the executioner proteins Bax and Bak, potentially confounding analysis. As this mechanism may be relevant in multiple cell lines it is examined in more detail elsewhere ( Chi et al, 2019 ). Second, MCF-7 cells depend on MCL-1 for survival ( Figure 3E ).…”

“…Purified Bcl-XL and BimL protein binding in the presence of different concentrations of ABT-263 quantified in vitro using purified full-length proteins with ( D ) and without ( E ) liposomes. Control experiments demonstrating efficient binding of BimL to liposomes and Bcl-XL binding data for the Bim mutants are presented in the companion paper ( Chi et al, 2019 ). Data are mean ±SD (n = 3 independent experiments).…”

“…Together the two anti-apoptotic protein binding sequences in Bim confer resistance of complexes to the dual Bcl-2, Bcl-XL inhibitor ABT-263 and to the new generation Bcl-XL inhibitors in clinical development. Because the CTS of Bim also functions to activate the pro-apoptotic proteins Bax and Bak, ( Chi et al, 2019 ) our results suggest that BH3-mimetics may have unpredictable results in cells depending on the Bcl-2 family proteins expressed and that targeting both binding sites on Bcl-XL and/or Bcl-2 may be required to kill some cancer cells that have bypassed apoptosis by inhibiting Bim.…”

Neuronal cell life, death, and axonal degeneration as regulated by the BCL-2 family proteins

“…Additionally, primary cultures of murine hippocampal neurons taken at the embryonic stage develop resistance to apoptotic stimuli as they mature in vitro [21]. Furthermore, we have observed that mature cultures of primary cortical neurons resist death in response to the expression of truncated BIM, a protein with reduced pro-apoptotic activity [22]. However, these cultures of mature neurons remain sensitive to the expression of full-length BIM, demonstrating that mature neurons are resistant, but not entirely refractory to apoptotic stimuli [22].…”

“…Furthermore, we have observed that mature cultures of primary cortical neurons resist death in response to the expression of truncated BIM, a protein with reduced pro-apoptotic activity [22]. However, these cultures of mature neurons remain sensitive to the expression of full-length BIM, demonstrating that mature neurons are resistant, but not entirely refractory to apoptotic stimuli [22]. Resistance to apoptotic stimuli correlating with the maturation of neurons has also been shown in vitro with cultures of sympathetic neurons of the PNS [23][24][25].…”