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

  1. James M Pemberton
  2. Dang Nguyen
  3. Elizabeth J Osterlund
  4. Wiebke Schormann
  5. Justin P Pogmore
  6. Nehad Hirmiz
  7. Brian Leber
  8. David W Andrews  Is a corresponding author
  1. Sunnybrook Health Science Centre, Canada
  2. University of Toronto, Canada
  3. McMaster University, Canada

Abstract

Anti-apoptotic proteins such as BCL-XL 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-XL, 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.

Data availability

We provide the MATLAB data analysis package on DataVerse (https://doi.org/10.5683/SP3/ZKXQW8)

Article and author information

Author details

  1. James M Pemberton

    Biological Sciences Platform, Sunnybrook Health Science Centre, Toronto, Canada
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8386-1081
  2. Dang Nguyen

    Department of Medical Biophysics, University of Toronto, Toronto, Canada
    Competing interests
    The authors declare that no competing interests exist.
  3. Elizabeth J Osterlund

    Biological Sciences Platform, Sunnybrook Health Science Centre, Toronto, Canada
    Competing interests
    The authors declare that no competing interests exist.
  4. Wiebke Schormann

    Biological Sciences Platform, Sunnybrook Health Science Centre, Toronto, Canada
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3055-2706
  5. Justin P Pogmore

    Biological Sciences Platform, Sunnybrook Health Science Centre, Toronto, Canada
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4198-2779
  6. Nehad Hirmiz

    Biological Sciences Platform, Sunnybrook Health Science Centre, Toronto, Canada
    Competing interests
    The authors declare that no competing interests exist.
  7. Brian Leber

    Department of Medicine, McMaster University, Hamilton, Canada
    Competing interests
    The authors declare that no competing interests exist.
  8. David W Andrews

    Department of Medical Biophysics, University of Toronto, Toronto, Canada
    For correspondence
    david.andrews@sunnybrook.ca
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9266-7157

Funding

Canadian Institutes of Health Research (FDN143312)

  • David W Andrews

Canada Research Chairs (Tier 1)

  • David W Andrews

Canada Foundation for Innovation

  • David W Andrews

Ontario Ministry of Research and Innovation

  • David W Andrews

CQDM

  • David W Andrews

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Volker Dötsch, Goethe University, Germany

Version history

  1. Preprint posted: April 2, 2023 (view preprint)
  2. Received: April 2, 2023
  3. Accepted: April 3, 2023
  4. Accepted Manuscript published: April 20, 2023 (version 1)
  5. Version of Record published: May 15, 2023 (version 2)

Copyright

© 2023, Pemberton et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 708
    views
  • 127
    downloads
  • 1
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. James M Pemberton
  2. Dang Nguyen
  3. Elizabeth J Osterlund
  4. Wiebke Schormann
  5. Justin P Pogmore
  6. Nehad Hirmiz
  7. Brian Leber
  8. David W Andrews
(2023)
The carboxyl-terminal sequence of PUMA binds to both anti-apoptotic proteins and membranes
eLife 12:e88329.
https://doi.org/10.7554/eLife.88329

Share this article

https://doi.org/10.7554/eLife.88329

Further reading

    1. Cancer Biology
    2. Cell Biology
    Qian Liu, Elizabeth J Osterlund ... David William Andrews
    Research Article

    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. ​

    1. Biochemistry and Chemical Biology
    2. Plant Biology
    Henning Mühlenbeck, Yuko Tsutsui ... Cyril Zipfel
    Research Article

    Transmembrane signaling by plant receptor kinases (RKs) has long been thought to involve reciprocal trans-phosphorylation of their intracellular kinase domains. The fact that many of these are pseudokinase domains, however, suggests that additional mechanisms must govern RK signaling activation. Non-catalytic signaling mechanisms of protein kinase domains have been described in metazoans, but information is scarce for plants. Recently, a non-catalytic function was reported for the leucine-rich repeat (LRR)-RK subfamily XIIa member EFR (elongation factor Tu receptor) and phosphorylation-dependent conformational changes were proposed to regulate signaling of RKs with non-RD kinase domains. Here, using EFR as a model, we describe a non-catalytic activation mechanism for LRR-RKs with non-RD kinase domains. EFR is an active kinase, but a kinase-dead variant retains the ability to enhance catalytic activity of its co-receptor kinase BAK1/SERK3 (brassinosteroid insensitive 1-associated kinase 1/somatic embryogenesis receptor kinase 3). Applying hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis and designing homology-based intragenic suppressor mutations, we provide evidence that the EFR kinase domain must adopt its active conformation in order to activate BAK1 allosterically, likely by supporting αC-helix positioning in BAK1. Our results suggest a conformational toggle model for signaling, in which BAK1 first phosphorylates EFR in the activation loop to stabilize its active conformation, allowing EFR in turn to allosterically activate BAK1.