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1.
Cell Rep ; 42(1): 112008, 2023 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-36662620

RESUMO

Reactive oxygen species (ROS) regulate the activities of inflammasomes, which are innate immune signaling organelles that induce pyroptosis. The mechanisms by which ROS control inflammasome activities are unclear and may be multifaceted. Herein, we report that the protein gasdermin D (GSDMD), which forms membrane pores upon cleavage by inflammasome-associated caspases, is a direct target of ROS. Exogenous and endogenous sources of ROS, and ROS-inducing stimuli that prime cells for pyroptosis induction, promote oligomerization of cleaved GSDMD, leading to membrane rupture and cell death. We find that ROS enhance GSDMD activities through oxidative modification of cysteine 192 (C192). Within macrophages, GSDMD mutants lacking C192 show impaired ability to form membrane pores and induce pyroptosis. Reciprocal mutagenesis studies reveal that C192 is the only cysteine within GSDMD that mediates ROS responsiveness. Cellular redox state is therefore a key determinant of GSDMD activities.


Assuntos
Inflamassomos , Peptídeos e Proteínas de Sinalização Intracelular , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Inflamassomos/metabolismo , Gasderminas , Espécies Reativas de Oxigênio/metabolismo , Cisteína/metabolismo , Proteínas de Neoplasias/metabolismo , Oxirredução
2.
Cell ; 184(17): 4495-4511.e19, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34289345

RESUMO

The process of pyroptosis is mediated by inflammasomes and a downstream effector known as gasdermin D (GSDMD). Upon cleavage by inflammasome-associated caspases, the N-terminal domain of GSDMD forms membrane pores that promote cytolysis. Numerous proteins promote GSDMD cleavage, but none are known to be required for pore formation after GSDMD cleavage. Herein, we report a forward genetic screen that identified the Ragulator-Rag complex as being necessary for GSDMD pore formation and pyroptosis in macrophages. Mechanistic analysis revealed that Ragulator-Rag is not required for GSDMD cleavage upon inflammasome activation but rather promotes GSDMD oligomerization in the plasma membrane. Defects in GSDMD oligomerization and pore formation can be rescued by mitochondrial poisons that stimulate reactive oxygen species (ROS) production, and ROS modulation impacts the ability of inflammasome pathways to promote pore formation downstream of GSDMD cleavage. These findings reveal an unexpected link between key regulators of immunity (inflammasome-GSDMD) and metabolism (Ragulator-Rag).


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Multimerização Proteica , Piroptose , Transdução de Sinais , Aminoácidos/metabolismo , Animais , Moléculas de Adesão Celular Neuronais/metabolismo , Linhagem Celular , Testes Genéticos , Humanos , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Macrófagos/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Fatores de Crescimento Neural/metabolismo , Proteínas de Ligação a Fosfato/química , Domínios Proteicos , RNA Guia de Cinetoplastídeos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Serina-Treonina Quinases TOR/metabolismo
3.
Science ; 359(6375): 545-550, 2018 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-29301962

RESUMO

Oligosaccharyltransferase (OST) is an essential membrane protein complex in the endoplasmic reticulum, where it transfers an oligosaccharide from a dolichol-pyrophosphate-activated donor to glycosylation sites of secretory proteins. Here we describe the atomic structure of yeast OST determined by cryo-electron microscopy, revealing a conserved subunit arrangement. The active site of the catalytic STT3 subunit points away from the center of the complex, allowing unhindered access to substrates. The dolichol-pyrophosphate moiety binds to a lipid-exposed groove of STT3, whereas two noncatalytic subunits and an ordered N-glycan form a membrane-proximal pocket for the oligosaccharide. The acceptor polypeptide site faces an oxidoreductase domain in stand-alone OST complexes or is immediately adjacent to the translocon, suggesting how eukaryotic OSTs efficiently glycosylate a large number of polypeptides before their folding.


Assuntos
Hexosiltransferases/química , Proteínas de Membrana/química , Complexos Multienzimáticos/química , Proteínas de Saccharomyces cerevisiae/química , Domínio Catalítico , Sequência Conservada , Microscopia Crioeletrônica , Glicosilação , Hexosiltransferases/ultraestrutura , Proteínas de Membrana/ultraestrutura , Complexos Multienzimáticos/ultraestrutura , Oxirredução , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Especificidade por Substrato
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