The carboxyl-terminal sequence of PUMA binds to both anti-apoptotic proteins and membranes

• Published in: eLife Vol. 12
• Main Authors: Pemberton, James M, Nguyen, Dang, Osterlund, Elizabeth J, Schormann, Wiebke, Pogmore, Justin P, Hirmiz, Nehad, Leber, Brian, Andrews, David W
• Format: Journal Article
• Language: English
• Published: England eLife Sciences Publications Ltd 20.04.2023; eLife Sciences Publications, Ltd
• Subjects: Amino acids; anti-apoptotic proteins; Apoptosis; Apoptosis Regulatory Proteins - metabolism; Bcl-2 protein; Bcl-2-Like Protein 11 - genetics; Bcl-x protein; bcl-X Protein - chemistry; Bcl-XL; BH3-proteins; BIM protein; Biochemistry and Chemical Biology; Cancer therapies; Cell Biology; Cell death; Cell survival; Chemotherapy; double bolt lock mechanism; Endoplasmic reticulum; Humans; Leukemia; Localization; Mcl-1 protein; Membranes; Mitochondria; Molecular modelling; Neoplasms; programmed cell death; Proteins; Proto-Oncogene Proteins c-bcl-2 - metabolism; Tumorigenesis; Bcl-XL; cell biology; chemical biology; biochemistry; anti-apoptotic proteins; apoptosis; none; programmed cell death; BH3-proteins; double bolt lock mechanism
• ISSN: 2050-084X; 2050-084X
• DOI: 10.7554/eLife.88329

Anti-apoptotic proteins such as BCL-X promote cell survival by sequestering pro-apoptotic BCL-2 family members, an activity that frequently contributes to tumorigenesis. Thus, the development of small-molecule inhibitors for anti-apoptotic proteins, termed BH3-mimetics, is revolutionizing how we treat cancer. BH3 mimetics kill cells by displacing sequestered pro-apoptotic proteins to initiate tumor-cell death. Recent evidence has demonstrated that in live cells the BH3-only proteins PUMA and BIM resist displacement by BH3-mimetics, while others like tBID do not. Analysis of the molecular mechanism by which PUMA resists BH3-mimetic mediated displacement from full-length anti-apoptotic proteins (BCL-X , BCL-2, BCL-W, and MCL-1) reveals that both the BH3-motif and a novel binding site within the carboxyl-terminal sequence (CTS) of PUMA contribute to binding. Together these sequences bind to anti-apoptotic proteins, which effectively 'double-bolt locks' the proteins to resist BH3-mimetic displacement. The pro-apoptotic protein BIM has also been shown to double-bolt lock to anti-apoptotic proteins however, the novel binding sequence in PUMA is unrelated to that in the CTS of BIM and functions independent of PUMA binding to membranes. Moreover, contrary to previous reports, we find that when exogenously expressed, the CTS of PUMA directs the protein primarily to the endoplasmic reticulum (ER) rather than mitochondria and that residues I175 and P180 within the CTS are required for both ER localization and BH3-mimetic resistance. Understanding how PUMA resists BH3-mimetic displacement will be useful in designing more efficacious small-molecule inhibitors of anti-apoptotic BCL-2 proteins.