RESUMO
Inflammatory caspases cleave the gasdermin D (GSDMD) protein to trigger pyroptosis, a lytic form of cell death that is crucial for immune defences and diseases. GSDMD contains a functionally important gasdermin-N domain that is shared in the gasdermin family. The functional mechanism of action of gasdermin proteins is unknown. Here we show that the gasdermin-N domains of the gasdermin proteins GSDMD, GSDMA3 and GSDMA can bind membrane lipids, phosphoinositides and cardiolipin, and exhibit membrane-disrupting cytotoxicity in mammalian cells and artificially transformed bacteria. Gasdermin-N moved to the plasma membrane during pyroptosis. Purified gasdermin-N efficiently lysed phosphoinositide/cardiolipin-containing liposomes and formed pores on membranes made of artificial or natural phospholipid mixtures. Most gasdermin pores had an inner diameter of 1014 nm and contained 16 symmetric protomers. The crystal structure of GSDMA3 showed an autoinhibited two-domain architecture that is conserved in the gasdermin family. Structure-guided mutagenesis demonstrated that the liposome-leakage and pore-forming activities of the gasdermin-N domain are required for pyroptosis. These findings reveal the mechanism for pyroptosis and provide insights into the roles of the gasdermin family in necrosis, immunity and diseases.
Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Proteínas/antagonistas & inibidores , Proteínas/química , Piroptose , Animais , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/química , Biomarcadores Tumorais/metabolismo , Cardiolipinas/metabolismo , Caspases/metabolismo , Linhagem Celular , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cristalografia por Raios X , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lipossomos , Lipídeos de Membrana/metabolismo , Camundongos , Modelos Moleculares , Necrose , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Proteínas de Ligação a Fosfato , Fosfatidilinositóis/metabolismo , Porosidade/efeitos dos fármacos , Estrutura Terciária de Proteína , Proteínas/metabolismo , Proteínas/farmacologia , Piroptose/efeitos dos fármacos , Piroptose/imunologiaRESUMO
Novel "pairs" of drugs possessing pharmacological synergies could be encapsulated into polymeric micelles and exert superb therapeutic effects in vivo upon intravenous administration, with the prerequisite that the micelles remain stable. NADP(H) quinone oxidoreductase 1 (NQO1) inhibitors, such as ß-lapachone (LPC) and tanshinone IIA (THA), are structurally and pharmacologically similar molecules that are poorly water-soluble, crystallize extremely fast, and demonstrate synergistic anticancer effect when used together with paclitaxel (PTX). However, when coencapsulated with PTX in poly(ethylene glycol)-b-poly(d,l-lactic acid) (PEG-PLA) micelles, only PTX/LPC but not the PTX/THA pair yields satisfactory colloidal stability. To reveal the molecular mechanism contributing to the colloidal stability of the coencapsulated micelles, we investigated the molecular interactions of PTX/LPC and PTX/THA, through both experimental methods (crystallization kinetics, 13C NMR) and molecular dynamic simulation. We observed that PTX was capable of inhibiting LPC but not THA crystallization both in an aqueous environment and in the solid state, which could be attributed to the strong hetero-intermolecular interactions (π-π, H-bonding) between LPC and PTX, which disrupted the homo-intermolecular interactions between LPC molecules and thus formed a favorable miscible binary system. In comparison, the lack of a strong PTX/THA interaction left the strong THA/THA stacking interaction undisturbed and the fast THA crystallization tendency unrestrained. We conclude that the intermolecular interactions, i.e., the "pharmaceutical synergy", between the coencapsulated drugs critically control the colloidal stability of polymeric micelles and, therefore, should be evaluated when coencapsulated drug delivery systems are designed for optimal therapeutic benefits.