RESUMEN
Bacterial lipopolysaccharide (LPS) is implicated in disrupting the blood-brain barrier (BBB). In a recent issue of Nature, Wei et al. now show that LPS activates the inflammatory caspases (4, 5, and 11) and gasdermin D (GSDMD) in brain endothelial cells, which triggers their pyroptotic cell death and disrupts the BBB.
Asunto(s)
Barrera Hematoencefálica , Células Endoteliales , Lipopolisacáridos , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/inmunología , Animales , Humanos , Células Endoteliales/metabolismo , Células Endoteliales/inmunología , Lipopolisacáridos/inmunología , Caspasas/metabolismo , Piroptosis , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Unión a Fosfato/metabolismo , RatonesRESUMEN
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-1RESUMEN
Phosphatidic acid (PA) and reactive oxygen species (ROS) are crucial cellular messengers mediating diverse signaling processes in metazoans and plants. How PA homeostasis is tightly regulated and intertwined with ROS signaling upon immune elicitation remains elusive. We report here that Arabidopsis diacylglycerol kinase 5 (DGK5) regulates plant pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). The pattern recognition receptor (PRR)-associated kinase BIK1 phosphorylates DGK5 at Ser-506, leading to a rapid PA burst and activation of plant immunity, whereas PRR-activated intracellular MPK4 phosphorylates DGK5 at Thr-446, which subsequently suppresses DGK5 activity and PA production, resulting in attenuated plant immunity. PA binds and stabilizes the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD), regulating ROS production in plant PTI and ETI, and their potentiation. Our data indicate that distinct phosphorylation of DGK5 by PRR-activated BIK1 and MPK4 balances the homeostasis of cellular PA burst that regulates ROS generation in coordinating two branches of plant immunity.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Diacilglicerol Quinasa , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Diacilglicerol Quinasa/metabolismo , NADPH Oxidasas/metabolismo , Ácidos Fosfatidicos/metabolismo , Fosforilación , Inmunidad de la Planta , Proteínas Serina-Treonina Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores de Reconocimiento de Patrones/metabolismoRESUMEN
Natural killer (NK) cell kill infected, transformed and stressed cells when an activating NK cell receptor is triggered1. Most NK cells and some innate lymphoid cells express the activating receptor NKp46, encoded by NCR1, the most evolutionarily ancient NK cell receptor2,3. Blockage of NKp46 inhibits NK killing of many cancer targets4. Although a few infectious NKp46 ligands have been identified, the endogenous NKp46 cell surface ligand is unknown. Here we show that NKp46 recognizes externalized calreticulin (ecto-CRT), which translocates from the endoplasmic reticulum (ER) to the cell membrane during ER stress. ER stress and ecto-CRT are hallmarks of chemotherapy-induced immunogenic cell death5,6, flavivirus infection and senescence. NKp46 recognition of the P domain of ecto-CRT triggers NK cell signalling and NKp46 caps with ecto-CRT in NK immune synapses. NKp46-mediated killing is inhibited by knockout or knockdown of CALR, the gene encoding CRT, or CRT antibodies, and is enhanced by ectopic expression of glycosylphosphatidylinositol-anchored CRT. NCR1)-deficient human (and Nrc1-deficient mouse) NK cells are impaired in the killing of ZIKV-infected, ER-stressed and senescent cells and ecto-CRT-expressing cancer cells. Importantly, NKp46 recognition of ecto-CRT controls mouse B16 melanoma and RAS-driven lung cancers and enhances tumour-infiltrating NK cell degranulation and cytokine secretion. Thus, NKp46 recognition of ecto-CRT as a danger-associated molecular pattern eliminates ER-stressed cells.
Asunto(s)
Calreticulina , Estrés del Retículo Endoplásmico , Células Asesinas Naturales , Receptor 1 Gatillante de la Citotoxidad Natural , Animales , Humanos , Ratones , Alarminas/metabolismo , Calreticulina/inmunología , Calreticulina/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Inmunidad Innata , Sinapsis Inmunológicas , Células Asesinas Naturales/metabolismo , Neoplasias Pulmonares/metabolismo , Melanoma Experimental/metabolismo , Receptor 1 Gatillante de la Citotoxidad Natural/metabolismo , Virus Zika/fisiologíaRESUMEN
Protein ubiquitylation profoundly expands proteome functionality and diversifies cellular signaling processes, with recent studies providing ample evidence for its importance to plant immunity. To gain a proteome-wide appreciation of ubiquitylome dynamics during immune recognition, we employed a two-step affinity enrichment protocol based on a 6His-tagged ubiquitin (Ub) variant coupled with high sensitivity mass spectrometry to identify Arabidopsis proteins rapidly ubiquitylated upon plant perception of the microbe-associated molecular pattern (MAMP) peptide flg22. The catalog from 2-week-old seedlings treated for 30 min with flg22 contained 690 conjugates, 64 Ub footprints, and all seven types of Ub linkages, and included previously uncharacterized conjugates of immune components. In vivo ubiquitylation assays confirmed modification of several candidates upon immune elicitation, and revealed distinct modification patterns and dynamics for key immune components, including poly- and monoubiquitylation, as well as induced or reduced levels of ubiquitylation. Gene ontology and network analyses of the collection also uncovered rapid modification of the Ub-proteasome system itself, suggesting a critical auto-regulatory loop necessary for an effective MAMP-triggered immune response and subsequent disease resistance. Included targets were UBIQUITIN-CONJUGATING ENZYME 13 (UBC13) and proteasome component REGULATORY PARTICLE NON-ATPASE SUBUNIT 8b (RPN8b), whose subsequent biochemical and genetic analyses implied negative roles in immune elicitation. Collectively, our proteomic analyses further strengthened the connection between ubiquitylation and flg22-based immune signaling, identified components and pathways regulating plant immunity, and increased the database of ubiquitylated substrates in plants.
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Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Inmunidad Innata/fisiología , Inmunidad de la Planta/fisiología , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Ubiquitinación/genética , Ubiquitinación/fisiología , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , ProteómicaRESUMEN
Recognition of microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) triggers the first line of inducible defence against invading pathogens1-3. Receptor-like cytoplasmic kinases (RLCKs) are convergent regulators that associate with multiple PRRs in plants4. The mechanisms that underlie the activation of RLCKs are unclear. Here we show that when MAMPs are detected, the RLCK BOTRYTIS-INDUCED KINASE 1 (BIK1) is monoubiquitinated following phosphorylation, then released from the flagellin receptor FLAGELLIN SENSING 2 (FLS2)-BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) complex, and internalized dynamically into endocytic compartments. The Arabidopsis E3 ubiquitin ligases RING-H2 FINGER A3A (RHA3A) and RHA3B mediate the monoubiquitination of BIK1, which is essential for the subsequent release of BIK1 from the FLS2-BAK1 complex and activation of immune signalling. Ligand-induced monoubiquitination and endosomal puncta of BIK1 exhibit spatial and temporal dynamics that are distinct from those of the PRR FLS2. Our study reveals the intertwined regulation of PRR-RLCK complex activation by protein phosphorylation and ubiquitination, and shows that ligand-induced monoubiquitination contributes to the release of BIK1 family RLCKs from the PRR complex and activation of PRR signalling.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/inmunología , Arabidopsis/metabolismo , Inmunidad de la Planta/inmunología , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Reconocimiento de Patrones/inmunología , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Arabidopsis/enzimología , Endocitosis , Ligandos , Moléculas de Patrón Molecular Asociado a Patógenos/inmunología , Fosforilación , Proteínas Quinasas/metabolismoRESUMEN
Pattern recognition receptors (PRRs) sense microbial patterns and activate innate immunity against attempted microbial invasions. The leucine-rich repeat receptor kinases (LRR-RK) FLS2 and EFR, and the LRR receptor protein (LRR-RP) receptors RLP23 and RLP42, respectively, represent prototypical members of these two prominent and closely related PRR families. We conducted a survey of Arabidopsis thaliana immune signaling mediated by these receptors to address the question of commonalities and differences between LRR-RK and LRR-RP signaling. Quantitative differences in timing and amplitude were observed for several early immune responses, with RP-mediated responses typically being slower and more prolonged than those mediated by RKs. Activation of RLP23, but not FLS2, induced the production of camalexin. Transcriptomic analysis revealed that RLP23-regulated genes represent only a fraction of those genes differentially expressed upon FLS2 activation. Several positive and negative regulators of FLS2-signaling play similar roles in RLP23 signaling. Intriguingly, the cytoplasmic receptor kinase BIK1, a positive regulator of RK signaling, acts as a negative regulator of RP-type immune receptors in a manner dependent on BIK1 kinase activity. Our study unveiled unexpected differences in two closely related receptor systems and reports a new negative role of BIK1 in plant immunity.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Inmunidad de la Planta , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Reconocimiento de Patrones/metabolismo , Transducción de Señal , Flagelina/farmacología , Genotipo , Péptidos/farmacología , Fosforilación , Reguladores del Crecimiento de las Plantas/biosíntesis , Inmunidad de la Planta/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Ácido Salicílico/farmacología , Sesquiterpenos/metabolismo , Transducción de Señal/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , FitoalexinasRESUMEN
Plants employ cell-surface pattern recognition receptors (PRRs) to detect pathogens. Although phytohormones produced during PRR signaling play an essential role in innate immunity, a direct link between PRR activation and hormone regulation is unknown. EFR is a PRR that recognizes bacterial EF-Tu and activates immune signaling. Here we report that EFR regulates the phytohormone jasmonic acid (JA) through direct phosphorylation of a receptor-like cytoplasmic kinase, BIK1. The BIK1 structure revealed that the EFR-phosphorylated sites reside on a uniquely extended loop away from the BIK1 kinase core domain. Phosphomimetic mutations of these sites resulted in increased phytohormones and enhanced resistance to bacterial infections. In addition to its documented plasma membrane localization, BIK1 also localizes to the nucleus and interacts directly with WRKY transcription factors involved in the JA and salicylic acid (SA) regulation. These findings demonstrate the mechanistic basis of signal transduction from PRR to phytohormones, mediated through a PRR-BIK1-WRKY axis.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Reguladores del Crecimiento de las Plantas/biosíntesis , Inmunidad de la Planta , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Reconocimiento de Patrones/metabolismo , Factores de Transcripción/metabolismo , Arabidopsis/fisiología , Infecciones Bacterianas/inmunología , Ciclopentanos/metabolismo , Inmunidad Innata , Oxilipinas/metabolismo , Fosforilación , Enfermedades de las Plantas/inmunología , Unión Proteica , Procesamiento Proteico-Postraduccional , Transducción de SeñalRESUMEN
Plants largely rely on plasma membrane (PM)-resident receptor-like kinases (RLKs) to sense extracellular and intracellular stimuli and coordinate cell differentiation, growth, and immunity. Several RLKs have been shown to undergo internalization through the endocytic pathway with a poorly understood mechanism. Here, we show that endocytosis and protein abundance of the Arabidopsis brassinosteroid (BR) receptor, BR INSENSITIVE1 (BRI1), are regulated by plant U-box (PUB) E3 ubiquitin ligase PUB12- and PUB13-mediated ubiquitination. BR perception promotes BRI1 ubiquitination and association with PUB12 and PUB13 through phosphorylation at serine 344 residue. Loss of PUB12 and PUB13 results in reduced BRI1 ubiquitination and internalization accompanied with a prolonged BRI1 PM-residence time, indicating that ubiquitination of BRI1 by PUB12 and PUB13 is a key step in BRI1 endocytosis. Our studies provide a molecular link between BRI1 ubiquitination and internalization and reveal a unique mechanism of E3 ligase-substrate association regulated by phosphorylation.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Endocitosis , Proteínas Quinasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Brasinoesteroides/metabolismo , Proteínas Quinasas/genética , Transducción de Señal , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Plants have evolved a large number of cell surface-resident receptor-like kinases (RLKs) and receptor-like proteins (RLPs), many of which are implicated in sensing extrinsic and intrinsic signals, and govern diverse cellular responses. The signaling pathways mediated by RLKs and RLPs converge at a small group of RLKs, somatic embryogenesis receptor kinases (SERKs), via ligand-induced heterodimerization and transphosphorylation. As shared coreceptors in diverse signaling receptorsomes, SERKs exhibit functional plasticity yet maintain a high degree of signaling specificity. Here, we review recent advances in newly identified SERK functions in plant cell differentiation, growth, and immunity; discuss the regulation and activation mechanisms of SERK-associated receptorsomes; and provide insights into how SERKs maintain signaling specificity as convergent hubs in various signaling pathways.
Asunto(s)
Proteínas de Plantas/metabolismo , Transducción de Señal/fisiología , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/genéticaRESUMEN
Maintaining active growth and effective immune responses is often costly for a living organism to survive. Fine-tuning the shared cross-regulators is crucial for metazoans and plants to make a trade-off between growth and immunity. The Arabidopsis regulatory receptor-like kinase BAK1 complexes with the receptor kinases FLS2 in bacterial flagellin-triggered immunity and BRI1 in brassinosteroid (BR)-mediated growth. BR homeostasis and signaling unidirectionally modulate FLS2-mediated immune responses at multiple levels. We have shown previously that BIK1, a receptor-like cytoplasmic kinase, is directly phosphorylated by BAK1 and associates with FLS2/BAK1 complex in transducing flagellin signaling. In contrast to its positive role in plant immunity, we report here that BIK1 acts as a negative regulator in BR signaling. The bik1 mutant displays various BR hypersensitive phenotypes accompanied with increased accumulation of de-phosphorylated BES1 proteins and transcriptional regulation of BZR1 and BES1 target genes. BIK1 associates with BRI1, and is released from BRI1 receptor upon BR treatment, which is reminiscent of FLS2-BIK1 complex dynamics in flagellin signaling. The ligand-induced release of BIK1 from receptor complexes is associated with BIK1 phosphorylation. However, in contrast to BAK1-dependent FLS2-BIK1 dissociation, BAK1 is dispensable for BRI1-BIK1 dissociation. Unlike FLS2 signaling which depends on BAK1 to phosphorylate BIK1, BRI1 directly phosphorylates BIK1 to transduce BR signaling. Thus, BIK1 relays the signaling in plant immunity and BR-mediated growth via distinct phosphorylation by BAK1 and BRI1, respectively. Our studies indicate that BIK1 mediates inverse functions in plant immunity and development via dynamic association with specific receptor complexes and differential phosphorylation events.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/inmunología , Brasinoesteroides/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/fisiología , Western Blotting , Cartilla de ADN/genética , Fluorescencia , Inmunoprecipitación , Fosforilación , Plantas Modificadas Genéticamente , Proteínas Quinasas/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Plants have evolved a large number of receptor-like cytoplasmic kinases (RLCKs) that often functionally and physically associate with receptor-like kinases (RLKs) to modulate plant growth, development and immune responses. Without any apparent extracellular domain, RLCKs relay intracellular signaling often via RLK complex-mediated transphosphorylation events. Recent advances have suggested essential roles of diverse RLCKs in concert with RLKs in regulating various cellular and physiological responses. We summarize here the complex roles of RLCKs in mediating plant immune responses and growth regulation, and discuss specific and overlapping functions of RLCKs in transducing diverse signaling pathways. [Figure: see text] Ping He (Corresponding author).