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Pore-forming proteins as drivers of membrane permeabilization in cell death pathways

Nature Reviews Molecular Cell Biology volume 24, pages 312–333 (2023)Cite this article

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Abstract

Regulated cell death (RCD) relies on activation and recruitment of pore-forming proteins (PFPs) that function as executioners of specific cell death pathways: apoptosis regulator BAX (BAX), BCL-2 homologous antagonist/killer (BAK) and BCL-2-related ovarian killer protein (BOK) for apoptosis, gasdermins (GSDMs) for pyroptosis and mixed lineage kinase domain-like protein (MLKL) for necroptosis. Inactive precursors of PFPs are converted into pore-forming entities through activation, membrane recruitment, membrane insertion and oligomerization. These mechanisms involve protein–protein and protein–lipid interactions, proteolytic processing and phosphorylation. In this Review, we discuss the structural rearrangements incurred by RCD-related PFPs and describe the mechanisms that manifest conversion from autoinhibited to membrane-embedded molecular states. We further discuss the formation and maturation of membrane pores formed by BAX/BAK/BOK, GSDMs and MLKL, leading to diverse pore architectures. Lastly, we highlight commonalities and differences of PFP mechanisms involving BAX/BAK/BOK, GSDMs and MLKL and conclude with a discussion on how, in a population of challenged cells, the coexistence of cell death modalities may have profound physiological and pathophysiological implications.

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Fig. 1: Cell death modalities and pore-forming proteins involved in cell death.
Fig. 2: Regulation of BAX/BAK activation, oligomerization and pore formation.
Fig. 3: Structural rearrangements and models of pore formation of BAX/BAK.
Fig. 4: Structural mechanisms for the activation of BAK, BAX, GSDMs and MLKL to mediate membrane permeabilization.

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Acknowledgements

Research in the Vandenabeele group is supported by Excellence of Science (EOS MODEL-IDI, 30826052; EOS CD-INFLADIS, 40007512), FWO research grants (G.0C76.18N, G.0B71.18N, G.0B96.20N and G.0A93.22N), Methusalem (BOF16/MET_V/007), iBOF20/IBF/039 ATLANTIS, the Foundation against Cancer (FAF-F/2016/865 and F/2020/1505), the CRIG and GIGG consortia, and VIB. Research in the Bultynck group is supported by FWO research grants (G.0901.18N, G.0E75.20N, G.0818.21N and G094522N), the Research Council–KU Leuven (C14/19/99 and AKUL/19/34), the Central European Leuven Strategic Alliance (CELSA/18/040), the Foundation Alzheimer Research (SAO IP3 RECEPTOR) and the Eye Hope Foundation, King Baudouin Foundation (grantbuitenoproep_FondsEyeHope_J1160630_Bultynck). Research in the Savvides group is supported by FWO research grants (G0B4918N, G049820N and G0H1222N), the CRIG consortium and VIB.

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Author notes

  1. These authors contributed equally: Peter Vandenabeele, Geert Bultynck, Savvas N. Savvides.

Authors and Affiliations

  1. VIB Center for Inflammation Research (IRC), Ghent, Belgium

    Peter Vandenabeele & Savvas N. Savvides

  2. Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium

    Peter Vandenabeele

  3. Cancer Research Institute Ghent, Ghent, Belgium

    Peter Vandenabeele & Savvas N. Savvides

  4. Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium

    Geert Bultynck

  5. Leuven Kankerinstitut, Leuven, Belgium

    Geert Bultynck

  6. Department of Biochemistry and Microbiology, Ghent University, Ghent, Belgium

    Savvas N. Savvides

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Glossary

Apoptosis-driven secondary necrosis

A necrotic cell death modality in which apoptotic dying cells in the absence of efferocytosis undergo plasma membrane permeabilization.

Barrel-stave protein–protein pore

A model for membrane disruption and perforation whereby multiple copies of interacting protein segments with amphipathic properties insert themselves into a lipid bilayer such that their hydrophobic surface faces the lipid bilayer and their polar surface faces the pore opening to interact with solvent, forming a pore continuous lining of protein–protein interactions.

BH3-mimetic compounds

Small-molecule antagonists of anti-apoptotic BCL-2-family members that mimic the properties of the BCL-2 homology 3 (BH3) domain of sensitizer BH3-only proteins and thus occupy the hydrophobic groove of anti-apoptotic BCL-2-family members, thereby evoking the release of BH3-only proteins that were sequestered by these anti-apoptotic BCL-2 proteins.

Carpet protein–lipid pores

A model for membrane disruption and perforation whereby amphipathic discrete protein segments extensively cover the lipid membrane by binding to the polar groups of the lipid layer via electrostatic interactions and binding of lipids through hydrophobic interactions.

Chemical stapling

Chemically modified peptides in which the α-helical structure of the peptide is stabilized or enforced by chemically bonding the side chains of two amino acids that are on the same face of the helix (that is, typically four or seven amino acids apart from each other in the sequence of the peptide).

Chimeric antigen receptor (CAR) T cells

T cells that have been genetically engineered to produce an artificial T cell receptor against tumour-associated antigens for use in immunotherapy.

Damage-associated molecular patterns

Molecules released from necrotic or stressed cells that function as alarm signals for the innate immune system and contribute to inflammation.

Death receptors

Membrane-associated members of the tumour necrosis factor receptor superfamily that feature a cytosolic ‘death domain’ that mediates cytotoxic signal transduction upon engagement of the receptor by cognate ligands.

Endoplasmic reticulum-associated degradation

The process responsible for proteasomal degradation of misfolded endoplasmic reticulum (ER)-resident proteins in the cytosol, which first are recognized by cytoplasmic and ER-luminal chaperones and are subsequently retrotranslocated from the ER into the cytosol, where they become ubiquitylated and targeted to the proteasome for degradation.

ESCRT-III machinery

A protein complex consisting of long filaments that coil around the site of membrane constriction just before membrane cleavage during membrane abscission and viral budding.

Exosomes

Extracellular vesicles produced by endosomal multivesicular bodies that originate from intraluminal vesicles that bud inwards into the endosomal lumen. If the multivesicular body fuses with the plasma membrane, these intraluminal vesicles are released as exosomes.

Inflammasome-driven pyroptosis

A form of necrotic regulated cell death involving the caspases 1/11/4/5-mediated proteolytic cleavage of gasdermin D (GSDMD) initiated by a cytosolic sensor complex following detection of damage-associated molecular patterns or pathogen-associated molecular patterns (canonical inflammasome pyroptosis by caspase 1) or initiated by direct sensing of lipopolysaccharide or Gram-negative bacteria by caspases 11/4/5 (non-canonical inflammasome).

Line tension

Interfacial energy per unit length of interface at the edge of a (nascent) membrane pore (for instance, due to the bending of lipids at the rim of the pore by a positive curvature) that opposes membrane tension.

Membrane tension

A biophysical characteristic of the membrane relating to the in-plane force required to stretch or compress the membrane. It is dictated by the elastic properties of the membrane as a function of lipid composition and insertion of transmembrane proteins.

Necroptosis

A form of necrotic regulated cell death that is driven by RIPK3 and the subsequent activation of mixed lineage kinase domain-like pseudokinase (MLKL), a plasma membrane-permeabilizing protein.

Neutrophil extracellular trap cell death

(NETosis). A form of necrotic regulated cell death characterized by the release of decondensed chromatin and granular contents to the extracellular space involving proteolytic activation of gasdermin D (GSDMD) by neutrophil elastase (ELANE) and cathepsin G.

Perforin

Glycoprotein released by granules from cytotoxic T lymphocytes and natural killer cells that causes lytic cell death by oligomeric pore formation at the outer exit of the plasma membrane.

Phospholipid nanodiscs

A reconstituted native-like membrane-mimetic environment that is constructed by using α-helical amphipathic membrane proteins or lipoproteins that wrap around a small portion of a lipid bilayer.

Programmed cell death 1 ligand 1

(PDL1). A ligand expressed on tumour cells and tumour-associated immune cells that interacts with the immune checkpoint receptor programmed cell death 1 (PD1), mainly expressed on activated T cells, B cells, dendritic cells, natural killer cells and regulatory T cells, resulting in immunosuppression.

Regulated cell death

(RCD). Evolutionarily conserved cell death modalities, including apoptosis, necroptosis, pyroptosis and ferroptosis, induced by ligand binding of a membrane receptor, a cytosolic sensor or perturbation of redox metabolism resulting in the activation of RCD pore-forming proteins or the formation of lipid pores by phospholipid peroxidation.

Supported lipid bilayers

In vitro planar bilayers formed by synthetic or natural lipids on a solid support, thereby enabling the study of biological membranes or the role of lipids and membranes in controlling the function and activity of proteins in a simplified and well-controlled context.

Toroidal protein–lipid pores

A model for membrane disruption and perforation whereby lipid molecules bend inwards to form a toroidal protein–lipid pore lined by alternating discrete protein segments and lipid moieties.

Voltage-dependent anion-selective channel protein 2

(VDAC2). A 19-stranded β-barrel protein belonging to the family of large-conductance VDAC channels, which mediate the transport of metabolites and ions across the outer mitochondrial membrane. Among the three VDAC isoforms, VDAC2 proteins have unique roles in controlling the translocation and activity of BCL-2-associated X (BAX)/BCL-2 antagonist killer (BAK) proteins at the outer mitochondrial membrane.

Z-RNA

Left-handed conformation of double-stranded RNA that is particularly favoured in RNA molecules rich in CG base repeats.

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Vandenabeele, P., Bultynck, G. & Savvides, S.N. Pore-forming proteins as drivers of membrane permeabilization in cell death pathways. Nat Rev Mol Cell Biol 24, 312–333 (2023). https://doi.org/10.1038/s41580-022-00564-w

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