A critical role for palmitoylation in pyroptosis

Abstract

Three recent publications by Du et al.,¹ Balasubramanian et al.,² and Zhang et al.³ identified palmitoylation on cysteine 191/192 in gasdermin D as a key determinant of gasdermin D membrane translocation and oligomerization, ensuring efficient plasma membrane permeabilization during pyroptosis.

Pyroptosis is a lytic form of programmed cell death that is characterized by the formation of large plasma-membrane pores and non-vesicular secretion of proinflammatory cytokines such as interleukin (IL)-1β and IL-18. Plasma-membrane pore formation during pyroptosis is mediated by gasdermin (GSDM) family proteins. Among the 6 GSDM proteins (GSDMA, GSDMB, GSDMC, GSDMD, GSDME, and DFNB59) encoded in the human genome, GSDMD is critically required for pyroptotic cell death upon inflammasome activation. Despite intensive research in the last decade, the dynamic process of GSDMD pore assembly remains scarcely understood. Du et al.,¹ Balasubramanian et al.,² and Zhang et al.³ now report that the palmitoylation of cysteine 191 in human GSDMD (Cys192 in mouse GSDMD) plays an important role in GSDMD activation, its membrane translocation, and the ensuing cell lysis. Blocking GSDMD palmitoylation strongly suppresses pyroptosis and prevents lipopolysaccharide (LPS)-induced lethal sepsis.

Under resting conditions, the pore-forming activity of the GSDMD N-terminal domain (GSDMD-NT) is restrained by its C-terminal domain (GSDMD-CT) via an intramolecular interaction. Proteolytic cleavage in the linker region between GSDMD-NT and GSDMD-CT at site Asp275 in human GSDMD (Asp276 in mouse) releases GSDMD-NT from this autoinhibition. Proteolytic cleavage of GSDMD is mediated by inflammatory caspases, including caspases-1/4/5 in humans and caspases-1/11 in mice. While caspase-1 is typically activated by canonical inflammasomes, caspases-4/5/11 are activated upon engaging cytosolic LPS, a process known as noncanonical inflammasome signaling. In addition, hyperactivated caspase-8 during Yersinia infection can also cleave and activate GSDMD.

The released GSDMD-NT fragments translocate to the plasma membrane, where 31–34 GSDMD-NT molecules oligomerize into a large intermolecular β-barrel pore with an inner diameter of 215 Å.⁴ GSDMD-NT oligomerization involves a concerted structural rearrangement of multiple subunits from a globular shape to an extended amphiphilic β-sheet during membrane insertion. The initial GSDMD-NT oligomers may be in a “slit” or “arc” shape, which further rearranges into large transmembrane pores. A GSDMD “prepore” structure was also documented in cryoelectron microscopy (cryo-EM) in which GSDMD-NT subunits adopt an autoinhibited conformation. Hence, a mechanism that targets GSDMD-NT monomers or the putative prepore to the plasma membrane could facilitate GSDMD pore formation. This mechanism—as now uncovered in these three studies—can be fulfilled by the palmitoylation of Cys191/192 (Figure 1).

Figure 1. Model of GSDMD palmitoylation in pyroptosis.

Inflammasome activation upregulates the activity of palmitoyltransferases ZDHHC5,7,9, possibly through ROS signaling by unknown mechanisms. ZDHHC5,7,9 mediate palmitoylation of Cys191/192 in the N-terminal domain of GSDMD, which may be counteracted by APT2-mediated depalmitoylation. Palmitoylation may enhance caspase-1-mediated cleavage of GSDMD under certain conditions. The palmitoyl group at Cys191/192 then anchors the released GSDMD N-terminus to the plasma membrane and facilitates its membrane insertion, oligomerization and pore formation.

Cys191/192 is located on an accessible loop in the inactive GSDMD structure and on the hydrophobic rim of the GSDMD-NT pore at the edge of outer leaflet of the plasma membrane.⁵ Therefore, the palmitoyl group initially anchors a GSDMD-NT monomer on the inner leaflet of the plasma membrane and potentially guides the membrane-insertion process. Several palmitoyltransferases, including ZDHHC5, ZDHHC9,^1,2 and ZDHHC7,³ can mediate GSDMD palmitoylation, likely due to the high accessibility of Cys191/192 and low substrate specificity of ZDHHCs. Various stimuli targeting NLRP3 (LPS and/or Nigericin), NLRC4 (LFn-Rod), NLRP1 (Val-boroPro) can all induce palmitoylation on full-length GSDMD as well as on GSDMD-NT, suggesting that these ZDHHCs may be activated by a general stress response. One mediator appears to be reactive oxygen species (ROS), which may be induced by GSDMD-mediated mitochondrial damage.⁶ How cytosolic GSDMD comes into contact with the membrane-associated ZDHHCs remains unclear. In this regard, palmitoylation may function as a kinetic trap to retain GSDMD-NT once it is on the membrane. Of note, a GSDMD C192A mutant maintains attenuated cytotoxic activity likely due to its intrinsic lipid-binding capacity.³

The critical role of C191/192 in GSDMD activity has been previously documented. Earlier work revealed that GSDMD activation correlated with the formation of GSDMD-NT aggregates in non-reducing SDS-PAGE, and mutation of Cys192 to alanine blocked this aggregate formation.⁷ However, no disulfide bond could be conclusively assigned in the cryo-EM structure of the GSDMD pore.⁴ It was later proposed that oxidation of Cys191/192 may promote GSDMD oligomerization downstream of ROS.⁸ Several thiol-reactive compounds, including disulfiram, necrosulfonamide, NU6300, and fumarate, conjugate to Cys191/192 and inhibit GSDMD-NT oligomerization. Besides blocking palmitoylation, these covalent modifications may simply prevent GSDMD-NT from membrane insertion (especially for the highly charged succination).

Although all three reports reach a consensus on the importance of C191/192 palmitoylation in GSDMD membrane translocation, several discrepancies between their models are worth discussing.

First, does Cys191/192 palmitoylation affect caspase binding and cleavage? Zhang et al.³ found in bone-marrow-derived macrophages that GSDMD C192A mutation or palmitoyltransferase inhibition strongly repressed GSDMD cleavage by caspase-1, a phenomenon not observed by Du et al.¹ or Balasubramanian et al.² While the modification of Cys191/192 by succination or by NU6300 prevents GSDMD binding to caspase-1 and the subsequent cleavage, necrosulfonamide or disulfiram do not affect caspase-1-mediated GSDMD cleavage. The potential effects of palmitoylation or succination on caspase-1 binding cannot be structurally explained since in the caspase-1-bound GSDMD structures the unstructured loop bearing Cys192 is not resolved and is unlikely to be involved in caspase-1 binding.⁹ Possibly, palmitoylation can target GSDMD to certain membrane compartments in proximity with the upstream caspases. Whether palmitoylation can also influence GSDMD cleavage by caspase-8 or caspases-4/5/11 in noncanonical inflammasome signaling remains unclear.

Second, while GSDMD-NT palmitoylation correlates with the formation of SDS-resistant oligomers in Du et al.¹ and Balasubramanian et al.,² Zhang et al.³ propose that GSDMD-NT needs to be depalmitoylated by acyl-protein thioesterase 2 (APT2) upon reaching the plasma membrane to oligomerize. In general, targeting either ZDHHCs or APT2 will certainly affect palmitoylation of many other proteins with a consequence of altered plasma membrane property, which may indirectly influence GSDMD-induced cell death. Hence, biophysical analysis is needed to clarify the impact of Cys191/192 palmitoylation on GSDMD-NT oligomerization.

Finally, Du et al.¹ suggest that intact GSDMD can be partially activated by palmitoylation without cleavage. They also show that the non-cleavable GSDMD mutant can from a pore-like structure. However, previous studies generating mice with a non-cleavable GSDMD molecule (D276A) suggest that inflammasome-dependent cell death and cytokine release in vitro require GSDMD cleavage and that LPS-induced cytokine release in vivo and acute septic shock are also GSDMD cleavage dependent.¹⁰ Thus, it remains to be determined under which conditions cleavage-independent GSDMD activation plays a physiologically relevant role.

In conclusion, palmitoylation at GSDMD Cys191/192 is clearly a key determinant of GSDMD membrane translocation during pyroptosis. Future work is needed to elucidate its role upstream of caspase-mediated GSDMD cleavage, oligomerization within the plasma membrane, and cleavage-independent activation. Furthermore, it is conceivable that lipidation on other GSDM family proteins may similarly affect their trafficking and membrane insertion.