Abstract
Tumor initiation, progression and resistance to chemotherapy rely on cancer cells bypassing programmed cell death by apoptosis. We report that unlike other pro-apoptotic proteins, Bim contains two distinct binding sites for the anti-apoptotic proteins Bcl-XL and Bcl-2. These include the BH3 sequence shared with other pro-apoptotic proteins and an unexpected sequence located near the Bim carboxyl-terminus (residues 181-192). Using automated Fluorescence Lifetime Imaging Microscopy - Fluorescence Resonance Energy Transfer (FLIM-FRET) we show that the two binding interfaces enable Bim to double-bolt lock Bcl-XL and Bcl-2 in complexes resistant to displacement by BH3-mimetic drugs currently in use or being evaluated for cancer therapy. Quantifying in live cells the contributions of individual amino acids revealed that residue L185 previously thought involved in binding Bim to membranes, instead contributes to binding to anti-apoptotic proteins. This double-bolt lock mechanism has profound implications for the utility of BH3-mimetics as drugs. .
Highlights
A major function of apoptosis is to inhibit tumor initiation and progression while its inhibition can result in cancer cell survival and resistance to chemotherapy (Kirkin et al, 2004)
Because the C-terminal sequence (CTS) of Bim 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
New instrumentation for automated time correlated single photon counting (TCSPC) from two fluorescence proteins simultaneously enabled automation of FLIM-Forster Resonance Energy Transfer (FRET) measurements to evaluate the effects of drugs on the binding of Bcl-2 family proteins fused to mCerulean3 and Venus fluorescence proteins
Summary
A major function of apoptosis is to inhibit tumor initiation and progression while its inhibition can result in cancer cell survival and resistance to chemotherapy (Kirkin et al, 2004). The surprising lack of activity for BH3 mimetics on displacement of Bim could significantly reduce their efficacy in some cancer cells (Pecot et al, 2016) Understanding this mechanism of resistance may suggest ways to release specific sequestered BH3-proteins from Bcl-XL and Bcl-2 to kill cancer cells more selectively than broadly inhibiting anti-apoptotic proteins (Goldsmith et al, 2006; Ni Chonghaile and Letai, 2008). Because the CTS of Bim 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
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