Pore formation in regulated cell death

Abstract

The discovery of alternative signaling pathways that regulate cell death has revealed multiple strategies for promoting cell death with diverse consequences at the tissue and organism level. Despite the divergence in the molecular components involved, membrane permeabilization is a common theme in the execution of regulated cell death. In apoptosis, the permeabilization of the outer mitochondrial membrane by BAX and BAK releases apoptotic factors that initiate the caspase cascade and is considered the point of no return in cell death commitment. Pyroptosis and necroptosis also require the perforation of the plasma membrane at the execution step, which involves Gasdermins in pyroptosis, and MLKL in the case of necroptosis. Although BAX/BAK, Gasdermins and MLKL share certain molecular features like oligomerization, they form pores in different cellular membranes via distinct mechanisms. Here, we compare and contrast how BAX/BAK, Gasdermins, and MLKL alter membrane permeability from a structural and biophysical perspective and discuss the general principles of membrane permeabilization in the execution of regulated cell death.

Keywords: apoptosis; cell death; membrane pores; necroptosis; pyroptosis.

Figure 1. Mechanism of pore formation by cationic amphipathic peptides to exemplify the formation and stabilization of a toroidal pore

(i) Pore‐forming peptides bind avidly to the accessible interface of the lipid bilayer and occupy a volume only in the interfacial region, which causes asymmetric stretching and membrane thinning (∆h). As a consequence, the membrane is stressed and destabilized, so that defects in the lipid bilayer become more likely and eventually a pore is formed (ii). Once the pore is open, a line tension appears at the pore edge due to the extra energy cost associated with the reorientation of the lipids into a highly curved boundary. This tension increases with the pore perimeter and is therefore a line tension. The initial pore grows quickly as long as the membrane tension dominates. But as the pore size grows, so does the counterbalancing line tension too. Furthermore, with the open pore, the peptides redistribute in the membrane by diffusion through the pore to the other monolayer, which reduces the membrane tension due to asymmetric distribution of the peptides. At a certain moment, the line tension becomes predominant and the pore size starts to decrease. However, if the pore‐forming peptides bound near the pore rim are able to reduce the line tension, an equilibrium can be reached with a smaller but stable pore (iii). R = radius, ∆h = change in the thickness of the membrane (absence vs. presence of the protein/peptide mass). Adapted from (Fuertes et al, 2011).

Figure 2. BAX/BAK toroidal pore

(A) Schematic representations of the protein/lipid model shown as a 3D view cut through the membrane pore. Gray layers represent lipid headgroups of the bilayer, the acyl chains are shown in red and protein helices by dark cylinders (top). The corresponding normalized electron density distributions of acyl chains in lipid bilayers containing BAX α5 (bottom). Note that, unlike in a protein channel, in a toroidal pore: (i) the surface of the pore is lined by lipid headgroups, (ii) membrane monolayers are bent at the pore edge, and (iii) the two leaflets of the bilayer become continuous. Taken from (Qian et al, 2008). (B) Structural representation of membrane‐embedded BAX in the context of a toroidal pore based on (Bleicken et al, 2014; Bleicken et al, 2018). BAX is represented with nine cylinders corresponding to its nine helices. BH3 domain and C‐terminal/tail anchoring domain are depicted in orange and green, respectively. One monomer is shown in gray (1–9) and the other is depicted in white (1ʹ–9ʹ). The relative orientation of the helices α9 remains unresolved. (C) BAX oligomers are organized into line, arc, and rings. Each panel shows the schematic representation (left) and the AFM images (right) of BAX assemblies. Both arcs and rings but not lines, reveal a circular depression (black) that spans the lipid membrane (dark orange). BAX molecules around the pore rim (yellow) protrude above the membrane plane, as confirmed by the height cross‐sections shown below each image (corresponding to the white line in the AFM images). The topography of the arc structure reveals a pore only partially surrounded by BAX molecules, while lipids alone form the rest of the pore rim. Based on (Salvador‐Gallego et al, 2016). (D) Model for the temporal control of content release during MOMP. Upon apoptotic stimuli, BAX and BAK permeabilize the MOM and induce the release of apoptotic factors, for example, cyt C. The consequent MIM permeabilization and the widening of BAX/BAK pores induce the release of mtDNA in the cytosol. In absence of caspase activity, this leads to the activation of the cGAS/STING signaling pathway. Based on (Cosentino & Garcia‐Saez, 2018).

Figure 3. GSDMs pores evolve from toroidal to barrel structures

(A) Crystal structure of GSDMA3 in its auto‐inhibited form (PDB: 5B5R). The GSDMA3^NT and GSDMA3^CT domains are colored pink and blue, respectively. Inter‐domain interactions between the GSDMA3^NT and the GSDMA3^CT keep the protein in an auto‐inhibited state (Ding et al, 2016). (B) GSMDs involves the form arc‐, slit, and ring‐shaped GSDMD^NT oligomers as imaged using time‐resolved AFM (Mulvihill et al, 2018). (C) Cryo‐EM structure of the GSDMA3 membrane pore (PDB: 6CB8). Atomic model of the 27‐fold symmetric GSDMA3 pore at 3.8 Å resolution (Ruan et al, 2018). (D) Model of pore formation by GSDMs. After cleavage, monomers of GSDM^NT translocate to the inner leaflet of the plasma membrane and then self‐associate into arcs or slit structures that resemble toroidal pores and later evolve into ring‐shape protein‐lined pores with a β‐barrel configuration.

Figure 4. MLKL induces pores by a still unclear mechanism

(A) Crystal structure of mouse MLKL (PDB: 4BTF). The 4HB domain, the brace region, and the psK domain are colored blue, orange, and gray, respectively. (B) Alternative models proposed for the mechanism how MLKL mediates plasma membrane permeabilization. (1) indirect mechanism via activation of endogenous ion channels, (2) partial insertion into the lipid bilayer, or (3) formation of defined channels or pores.

Figure 5. Alternative models of pore formation by the amphipathic α‐helices of MLKL

(A) Hydropathy profiles of the 4HB of MLKL, the HeLo‐like N domain‐containing protein from Chaetomium globosum and BAX. Profiles were built with the program Protscale (https://web.expasy.org/protscale/), using the Eisenberg scale with a window of 9 amino acid residues. Segments above 0 are predicted as hydrophobic and below 0 as hydrophilic. Predicted transmembrane segments are highlighted in red. Predictions were made using two different softwares: TMHMM (http://www.cbs.dtu.dk/services/TMHMM) and TCDB (http://www.tcdb.org/progs/?tool=hydro). (B) Amphipathic nature of the α‐helices of the 4HB of MLKL. Left: Wheel projections of the α‐helices of the 4HB of human (top) or mouse (bottom) MLKL. Arrows point toward the hydrophobic face of the α‐helices. Projections were built using the server Heliquest (https://heliquest.ipmc.cnrs.fr/). Right: μ of the amphipathic α‐helices of the 4HB of MLKL, the pore‐forming domain of the α‐PFT sticholysin II and the lytic peptides melittin, LL37 and PSMα3. (C) Amphipathic α‐helices of MLKL could alter membrane integrity by two alterative models. In a carpet model, MLKL would act as a surfactant on the membrane surface, while in the toroidal pore, MLKL would induce membrane curvature and lipid–protein pores. Cylinders indicate individual amphipathic helices of the 4HB of MLKL. Hydrophobic surfaces are depicted in red and hydrophilic in blue.