Nature Cell Biology丨棕榈酰化-去棕榈酰化通过 GSDMD 控制焦亡_百 ...

Chestnut Studying

摘要

Gasdermin D (GSDMD) plays a crucial role in pyroptosis, a host defense mechanism against pathogens. Following activation, caspase-mediated cleavage of GSDMD results in the release of its amino-terminal fragment (GSDMD-NT), which oligomerizes and forms pores in the plasma membrane, causing cell death and release of proinflammatory cytokines. The precise regulation of this process in cells remains unclear. This study identifies GSDMD as a substrate for reversible S-palmitoylation on C192 during pyroptosis. The palmitoyl acyltransferase DHHC7 palmitoylates GSDMD, directing its cleavage by caspases. Subsequently, palmitoylation of GSDMD-NT facilitates its translocation to the plasma membrane, where APT2 depalmitoylates GSDMD-NT, exposing the C192 residue and promoting its oligomerization. Disruption of either palmitoylation or depalmitoylation suppresses pyroptosis, leading to enhanced survival in mice with lipopolysaccharide-induced lethal septic shock and increased sensitivity to bacterial infection.

实验结果1

LPS stimulation results in S-palmitoylation of GSDMD at C192

LPS, a component of the outer membrane of gram-negative bacteria, activates immune cells and triggers a robust pro-inflammatory response. The authors aimed to determine if GSDMD is a substrate of LPS-mediated palmitoylation. Using 15-hexadecylhexadiyne (Alk-14), an alkynyl palmitate analog that can undergo click chemistry with TAMRA-azide, to label GSDMD metabolically, they observed that Alk-14 binds to GSDMD in bone marrow-derived macrophages (BMDMs) stimulated with LPS, as evidenced by gel electrophoretic fluorescence. This demonstrates that palmitoylation occurs at lysine or cysteine residues, with only S-palmitoylation being sensitive to HAM. Inhibiting HAM completely eliminated the palmitoylation signal on GSDMD, indicating that palmitoylation occurs at cysteine residues. Moreover, the presence of 2-Bromo-12-hydroxy-12-oxo-4,6-dioctadecenoic acid (2-BP), a common DHHC PAT inhibitor, prevented palmitoylation, as it irreversibly modifies catalytic cysteines. In contrast, direct labeling of GSDMD with click chemistry probe 16C-BYA did not show 2-BP modification. Further, an acylation biotinylation exchange (ABE) assay showed that only the HAM-treated immunoprecipitated GSDMD was acylated, while 2-BP reduced the acylated GSDMD. GSDMD also underwent palmitoylation in LPS-stimulated BMDMs infected with Salmonella Typhimurium, with 2-BP inhibiting this process without affecting the number of bacteria taken up by the cells. Notably, in the absence of LPS stimulation, ectopically expressed GSDMD also showed palmitoylation.

实验结果2

Palmitoylation promotes GSDMD-mediated pyroptosis

The authors then investigated the palmitoylation status of GSDMD in BMDMs treated with LPS and Nigericin (Nig), a mature inducer of NLRP3 inflammasome activation and caspase-1-dependent pyroptosis. GSDMD-FL and its cleaved fragment, GSDMD-NT, were both palmitoylated in LPS/Nig-stimulated BMDMs, and this effect was blocked by 2-BP. Treatment with 2-BP significantly reduced LPS/Nig-induced pyroptosis and IL-1β release. In primary BMDMs, this effect was also observed after LPS/Nig stimulation. After acute LPS stimulation followed by ATP treatment (LPS/ATP), which activates the NLRP3 inflammasome and causes pyroptosis in macrophages, 2-BP significantly decreased cell death and lactate dehydrogenase (LDH) release. In BMDMs infected with Salmonella typhimurium, a broad range of pyroptosis and IL-1β secretion was observed due to activation of the NLRC4 inflammasome and Dectin-1. However

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