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1.
Cell ; 2024 Sep 02.
Artículo en Inglés | MEDLINE | ID: mdl-39243763

RESUMEN

Gasdermin-mediated inflammatory cell death (pyroptosis) can activate protective immunity in immunologically cold tumors. Here, we performed a high-throughput screen for compounds that could activate gasdermin D (GSDMD), which is expressed widely in tumors. We identified 6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline (DMB) as a direct and selective GSDMD agonist that activates GSDMD pore formation and pyroptosis without cleaving GSDMD. In mouse tumor models, pulsed and low-level pyroptosis induced by DMB suppresses tumor growth without harming GSDMD-expressing immune cells. Protection is immune-mediated and abrogated in mice lacking lymphocytes. Vaccination with DMB-treated cancer cells protects mice from secondary tumor challenge, indicating that immunogenic cell death is induced. DMB treatment synergizes with anti-PD-1. DMB treatment does not alter circulating proinflammatory cytokine or leukocyte numbers or cause weight loss. Thus, our studies reveal a strategy that relies on a low level of tumor cell pyroptosis to induce antitumor immunity and raise the possibility of exploiting pyroptosis without causing overt toxicity.

2.
Nature ; 630(8016): 437-446, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38599239

RESUMEN

Gasdermin D (GSDMD) is the common effector for cytokine secretion and pyroptosis downstream of inflammasome activation and was previously shown to form large transmembrane pores after cleavage by inflammatory caspases to generate the GSDMD N-terminal domain (GSDMD-NT)1-10. Here we report that GSDMD Cys191 is S-palmitoylated and that palmitoylation is required for pore formation. S-palmitoylation, which does not affect GSDMD cleavage, is augmented by mitochondria-generated reactive oxygen species (ROS). Cleavage-deficient GSDMD (D275A) is also palmitoylated after inflammasome stimulation or treatment with ROS activators and causes pyroptosis, although less efficiently than palmitoylated GSDMD-NT. Palmitoylated, but not unpalmitoylated, full-length GSDMD induces liposome leakage and forms a pore similar in structure to GSDMD-NT pores shown by cryogenic electron microscopy. ZDHHC5 and ZDHHC9 are the major palmitoyltransferases that mediate GSDMD palmitoylation, and their expression is upregulated by inflammasome activation and ROS. The other human gasdermins are also palmitoylated at their N termini. These data challenge the concept that cleavage is the only trigger for GSDMD activation. They suggest that reversible palmitoylation is a checkpoint for pore formation by both GSDMD-NT and intact GSDMD that functions as a general switch for the activation of this pore-forming family.


Asunto(s)
Gasderminas , Lipoilación , Proteínas de Unión a Fosfato , Especies Reactivas de Oxígeno , Animales , Femenino , Humanos , Masculino , Ratones , Aciltransferasas/metabolismo , Microscopía por Crioelectrón , Cisteína/metabolismo , Gasderminas/química , Gasderminas/metabolismo , Inflamasomas/metabolismo , Liposomas/metabolismo , Liposomas/química , Mitocondrias/metabolismo , Proteínas de Unión a Fosfato/química , Proteínas de Unión a Fosfato/metabolismo , Piroptosis , Especies Reactivas de Oxígeno/metabolismo , Células THP-1
3.
Trends Immunol ; 44(8): 571-573, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-37414717

RESUMEN

In a recent article, He et al. report that, in response to dietary protein antigens, mouse intestinal epithelial cells (IECs) accumulate a newfound 13-kDa N terminus of gasdermin D (GSDMD-N13), cleaved by caspase-3/7. Unlike the pyroptotic 30-kDa fragment, GSDMD-N13 translocates to the nucleus, inducing CIITA and major histocompatibility complex class II (MHCII) expression to promote type 1 regulatory T (T1r) cell development, thus revealing its role in balancing immunity and food tolerance.


Asunto(s)
Péptidos y Proteínas de Señalización Intracelular , Piroptosis , Animales , Ratones
4.
J Biol Chem ; 297(4): 101112, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34428449

RESUMEN

S-acylation, also known as palmitoylation, is the most widely prevalent form of protein lipidation, whereby long-chain fatty acids get attached to cysteine residues facing the cytosol. In humans, 23 members of the zDHHC family of integral membrane enzymes catalyze this modification. S-acylation is critical for the life cycle of many enveloped viruses. The Spike protein of SARS-CoV-2, the causative agent of COVID-19, has the most cysteine-rich cytoplasmic tail among known human pathogens in the closely related family of ß-coronaviruses; however, it is unclear which of the cytoplasmic cysteines are S-acylated, and what the impact of this modification is on viral infectivity. Here we identify specific cysteine clusters in the Spike protein of SARS-CoV-2 that are targets of S-acylation. Interestingly, when we investigated the effect of the cysteine clusters using pseudotyped virus, mutation of the same three clusters of cysteines severely compromised viral infectivity. We developed a library of expression constructs of human zDHHC enzymes and used them to identify zDHHC enzymes that can S-acylate SARS-CoV-2 Spike protein. Finally, we reconstituted S-acylation of SARS-CoV-2 Spike protein in vitro using purified zDHHC enzymes. We observe a striking heterogeneity in the S-acylation status of the different cysteines in our in cellulo experiments, which, remarkably, was recapitulated by the in vitro assay. Altogether, these results bolster our understanding of a poorly understood posttranslational modification integral to the SARS-CoV-2 Spike protein. This study opens up avenues for further mechanistic dissection and lays the groundwork toward developing future strategies that could aid in the identification of targeted small-molecule modulators.


Asunto(s)
COVID-19/patología , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Acilación , Aciltransferasas/genética , Aciltransferasas/metabolismo , Secuencia de Aminoácidos , COVID-19/virología , Cisteína/metabolismo , Células HEK293 , Humanos , Lipoilación , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , SARS-CoV-2/aislamiento & purificación , Alineación de Secuencia , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Internalización del Virus
5.
bioRxiv ; 2023 Mar 07.
Artículo en Inglés | MEDLINE | ID: mdl-36945424

RESUMEN

Gasdermin D (GSDMD) is the common effector for cytokine secretion and pyroptosis downstream of inflammasome activation by forming large transmembrane pores upon cleavage by inflammatory caspases. Here we report the surprising finding that GSDMD cleavage is not sufficient for its pore formation. Instead, GSDMD is lipidated by S-palmitoylation at Cys191 upon inflammasome activation, and only palmitoylated GSDMD N-terminal domain (GSDMD-NT) is capable of membrane translocation and pore formation, suggesting that palmitoylation licenses GSDMD activation. Treatment by the palmitoylation inhibitor 2-bromopalmitate and alanine mutation of Cys191 abrogate GSDMD membrane localization, cytokine secretion, and cell death, without affecting GSDMD cleavage. Because palmitoylation is formed by a reversible thioester bond sensitive to free thiols, we tested if GSDMD palmitoylation is regulated by cellular redox state. Lipopolysaccharide (LPS) mildly and LPS plus the NLRP3 inflammasome activator nigericin markedly elevate reactive oxygen species (ROS) and GSDMD palmitoylation, suggesting that these two processes are coupled. Manipulation of cellular ROS by its activators and quenchers augment and abolish, respectively, GSDMD palmitoylation, GSDMD pore formation and cell death. We discover that zDHHC5 and zDHHC9 are the major palmitoyl transferases that mediate GSDMD palmitoylation, and when cleaved, recombinant and partly palmitoylated GSDMD is 10-fold more active in pore formation than bacterially expressed, unpalmitoylated GSDMD, evidenced by liposome leakage assay. Finally, other GSDM family members are also palmitoylated, suggesting that ROS stress and palmitoylation may be a general switch for the activation of this pore-forming family. One-Sentence Summary: GSDMD palmitoylation is induced by ROS and required for pore formation.

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