RESUMEN
Although hydroxychloroquine (HCQ) has long been used to treat autoimmune diseases, its mechanism of action remains poorly understood. In CD4 T-cells, we found that a clinically relevant concentration of HCQ inhibited the mitochondrial antioxidant system triggered by TCR crosslinking, leading to increased mitochondrial superoxide, impaired activation-induced autophagic flux, and reduced proliferation of CD4 T-cells. In antigen-presenting cells, HCQ also reduced constitutive activation of the endo-lysosomal protease legumain and toll-like receptor 9, thereby reducing cytokine production, but it had little apparent impact on constitutive antigen processing and peptide presentation. HCQ's effects did not require endo-lysosomal pH change, nor impaired autophagosome-lysosome fusion. We explored the clinical relevance of these findings in patients with celiac disease-a prototypic CD4 T-cell-mediated disease-and found that HCQ limits ex vivo antigen-specific T cell responses. We report a T-cell-intrinsic immunomodulatory effect from HCQ and suggest potential re-purposing of HCQ for celiac disease.
RESUMEN
BACKGROUND: Acute rheumatic fever (ARF) and rheumatic heart disease are autoimmune consequences of group A streptococcus infection and remain major causes of cardiovascular morbidity and mortality around the world. Improved treatment has been stymied by gaps in understanding key steps in the immunopathogenesis of ARF and rheumatic heart disease. This study aimed to identify (1) effector T cell cytokine(s) that might be dysregulated in the autoimmune response of patients with ARF by group A streptococcus, and (2) an immunomodulatory agent that suppresses this response and could be clinically translatable to high-risk patients with ARF. METHODS: The immune response to group A streptococcus was analyzed in peripheral blood mononuclear cells from an Australian Aboriginal ARF cohort by a combination of multiplex cytokine array, flow cytometric analysis, and global gene expression analysis by RNA sequencing. The immunomodulatory drug hydroxychloroquine was tested for effects on this response. RESULTS: We found a dysregulated interleukin-1ß-granulocyte-macrophage colony-stimulating factor (GM-CSF) cytokine axis in ARF peripheral blood mononuclear cells exposed to group A streptococcus in vitro, whereby persistent interleukin-1ß production is coupled to overproduction of GM-CSF and selective expansion of CXCR3+CCR4-CCR6- CD4 T cells. CXCR3+CCR4-CCR6- CD4 T cells are the major source of GM-CSF in human CD4 T cells and CXCL10, a CXCR3 ligand and potent T helper 1 chemoattractant, was elevated in sera from patients with ARF. GM-CSF has recently emerged as a key T cell-derived effector cytokine in numerous autoimmune diseases, including myocarditis, and the production of CXCL10 may explain selective trafficking of these cells to the heart. We provide evidence that interleukin-1ß amplifies the expansion of GM-CSF-expressing CD4 T cells, which is effectively suppressed by hydroxychloroquine. RNA sequencing showed shifts in gene expression profiles and differentially expressed genes in peripheral blood mononuclear cells derived from patients at different clinical stages of ARF. CONCLUSIONS: Given the safety profile of hydroxychloroquine and its clinical pedigree in treating autoimmune diseases such as rheumatoid arthritis, where GM-CSF plays a pivotal role, we propose that hydroxychloroquine could be repurposed to reduce the risk of rheumatic heart disease after ARF.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Hidroxicloroquina/farmacología , Interleucina-1beta/metabolismo , Fiebre Reumática/patología , Adolescente , Adulto , Proteína C-Reactiva/análisis , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Diferenciación Celular , Niño , Citocinas/análisis , Citocinas/genética , Citocinas/metabolismo , Femenino , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Masculino , ARN/química , ARN/aislamiento & purificación , ARN/metabolismo , Fiebre Reumática/metabolismo , Streptococcus pyogenes/inmunología , Células TH1/citología , Células TH1/inmunología , Células TH1/metabolismo , Adulto JovenRESUMEN
Anti-microbial signaling pathways are normally triggered by innate immune receptors when detecting pathogenic microbes to provide protective immunity. Here we show that the inflammasome sensor Nlrp1 aggravates DSS-induced experimental mouse colitis by limiting beneficial, butyrate-producing Clostridiales in the gut. The colitis-protective effects of Nlrp1 deficiency are thus reversed by vancomycin treatment, but recapitulated with butyrate supplementation in wild-type mice. Moreover, an activating mutation in Nlrp1a increases IL-18 and IFNγ production, and decreases colonic butyrate to exacerbate colitis. We also show that, in patients with ulcerative colitis, increased NLRP1 in inflamed regions of the colon is associated with increased IFN-γ. In this context, NLRP1, IL-18 or IFN-γ expression negatively correlates with the abundance of Clostridiales in human rectal mucosal biopsies. Our data identify the NLRP1 inflammasome to be a key negative regulator of protective, butyrate-producing commensals, which therefore promotes inflammatory bowel disease.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Butiratos/metabolismo , Clostridiales , Enfermedades Inflamatorias del Intestino/metabolismo , Interferón gamma/metabolismo , Interleucina-18/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas Reguladoras de la Apoptosis/genética , Colitis/metabolismo , Colon/patología , Femenino , Microbioma Gastrointestinal , Eliminación de Gen , Humanos , Inflamasomas , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Mucosa Intestinal/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas NLR , Recto/metabolismo , Transducción de Señal , Linfocitos T/citología , Vancomicina/farmacologíaRESUMEN
Gain-of-function mutations that activate the innate immune system can cause systemic autoinflammatory diseases associated with increased IL-1ß production. This cytokine is activated identically to IL-18 by an intracellular protein complex known as the inflammasome; however, IL-18 has not yet been specifically implicated in the pathogenesis of hereditary autoinflammatory disorders. We have now identified an autoinflammatory disease in mice driven by IL-18, but not IL-1ß, resulting from an inactivating mutation of the actin-depolymerizing cofactor Wdr1. This perturbation of actin polymerization leads to systemic autoinflammation that is reduced when IL-18 is deleted but not when IL-1 signaling is removed. Remarkably, inflammasome activation in mature macrophages is unaltered, but IL-18 production from monocytes is greatly exaggerated, and depletion of monocytes in vivo prevents the disease. Small-molecule inhibition of actin polymerization can remove potential danger signals from the system and prevents monocyte IL-18 production. Finally, we show that the inflammasome sensor of actin dynamics in this system requires caspase-1, apoptosis-associated speck-like protein containing a caspase recruitment domain, and the innate immune receptor pyrin. Previously, perturbation of actin polymerization by pathogens was shown to activate the pyrin inflammasome, so our data now extend this guard hypothesis to host-regulated actin-dependent processes and autoinflammatory disease.
Asunto(s)
Actinas/fisiología , Proteínas del Citoesqueleto/metabolismo , Enfermedades Autoinflamatorias Hereditarias/metabolismo , Inflamación/metabolismo , Interleucina-1beta/metabolismo , Proteínas de Microfilamentos/metabolismo , Actinas/química , Animales , Células de la Médula Ósea/citología , Caspasa 1/metabolismo , Caspasas/metabolismo , Ácido Clodrónico/química , Cruzamientos Genéticos , Medios de Cultivo Condicionados/química , Ensayo de Inmunoadsorción Enzimática , Interleucina-18/metabolismo , Lipopolisacáridos/metabolismo , Liposomas/química , Hígado/embriología , Factor Estimulante de Colonias de Macrófagos/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Fluorescente , Monocitos/citología , Pirina , Transducción de SeñalRESUMEN
In addition to several other extracellular substances, phagocytosis of amyloid-forming peptides can perturb cellular homeostasis, leading to activation of the cytoplasmic innate immune receptor NLRP3. Once triggered, NLRP3 forms an inflammasome complex that ultimately cleaves pro-IL-1ß and pro-IL-18 into their mature, secreted forms. Here we describe a protocol by which one type of amyloidogenic peptide, islet amyloid polypeptide (IAPP, otherwise known as amylin) can be prepared and used to stimulate myeloid cells in vitro to engage the NLRP3 inflammasome. Methods for measuring the ensuing inflammasome activation are also described. Although initially soluble, IAPP monomers rapidly aggregate in solution to form oligomers and subsequently insoluble amyloid fibrils. More work is required to examine how this transition influences inflammasome activation for different types of amyloid. The course of amyloid formation and corresponding inflammatory capacity of these pre-fibrillar species following uptake also requires further examination, and we hope that our protocols are useful in these endeavors. While these protocols are restricted to examination of synthetic IAPP, isolation of IAPP aggregates from human and transgenic mouse pancreas will be required to definitively determine the proinflammatory effects of endogenous IAPP oligomers and fibrils.
Asunto(s)
Proteínas Portadoras/metabolismo , Inflamasomas/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/farmacología , Animales , Caspasa 1/metabolismo , Activación Enzimática , Ensayo de Inmunoadsorción Enzimática , Humanos , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Microscopía Fluorescente , Proteína con Dominio Pirina 3 de la Familia NLRRESUMEN
BACKGROUND: Nuclear factor-κB (NF-κB) is a transcription factor that regulates the transcription of genes involved in a variety of biological processes, including innate and adaptive immunity, stress responses and cell proliferation. Constitutive or excessive NF-κB activity has been associated with inflammatory disorders and higher risk of cancer. In contrast to the mechanisms controlling inducible activation, the regulation of basal NF-κB activation is not well understood. Here we test whether clathrin heavy chain (CHC) contributes to the regulation of basal NF-κB activity in epithelial cells. METHODOLOGY: Using RNA interference to reduce endogenous CHC expression, we found that CHC is required to prevent constitutive activation of NF-κB and gene expression. Immunofluorescence staining showed constitutive nuclear localization of the NF-κB subunit p65 in absence of stimulation after CHC knockdown. Elevated basal p65 nuclear localization is caused by constitutive phosphorylation and degradation of inhibitor of NF-κB alpha (IκBα) through an IκB kinase α (IKKα)-dependent mechanism. The role of CHC in NF-κB signaling is functionally relevant as constitutive expression of the proinflammatory chemokine interleukin-8 (IL-8), whose expression is regulated by NF-κB, was found after CHC knockdown. Disruption of clathrin-mediated endocytosis by chemical inhibition or depletion of the µ2-subunit of the endocytosis adaptor protein AP-2, and knockdown of clathrin light chain a (CHLa), failed to induce constitutive NF-κB activation and IL-8 expression, showing that CHC acts on NF-κB independently of endocytosis and CLCa. CONCLUSIONS: We conclude that CHC functions as a built-in molecular brake that ensures a tight control of basal NF-κB activation and gene expression in unstimulated cells. Furthermore, our data suggest a potential link between a defect in CHC expression and chronic inflammation disorder and cancer.
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Cadenas Pesadas de Clatrina/metabolismo , Cadenas Pesadas de Clatrina/fisiología , Endocitosis/fisiología , FN-kappa B/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/genética , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Cadenas Pesadas de Clatrina/antagonistas & inhibidores , Cadenas Pesadas de Clatrina/genética , Endocitosis/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Células HeLa , Humanos , Quinasa I-kappa B/metabolismo , Quinasa I-kappa B/fisiología , Interleucina-8/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Procesamiento Proteico-Postraduccional/genética , ARN Interferente Pequeño/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Células Tumorales CultivadasRESUMEN
The enteroinvasive bacterium Shigella flexneri uses multiple secreted effector proteins to downregulate interleukin-8 (IL-8) expression in infected epithelial cells. Yet, massive IL-8 secretion is observed in Shigellosis. Here we report a host mechanism of cell-cell communication that circumvents the effector proteins and strongly amplifies IL-8 expression during bacterial infection. By monitoring proinflammatory signals at the single-cell level, we found that the activation of the transcription factor NF-κB and the MAP kinases JNK, ERK, and p38 rapidly propagated from infected to uninfected adjacent cells, leading to IL-8 production by uninfected bystander cells. Bystander IL-8 production was also observed during Listeria monocytogenes and Salmonella typhimurium infection. This response could be triggered by recognition of peptidoglycan and is mediated by gap junctions. Thus, we have identified a mechanism of cell-cell communication that amplifies innate immunity against bacterial infection by rapidly spreading proinflammatory signals via gap junctions to yet uninfected cells.
Asunto(s)
Disentería Bacilar/inmunología , Inmunidad Innata , Sistema de Señalización de MAP Quinasas/inmunología , Proteínas Quinasas Activadas por Mitógenos/inmunología , FN-kappa B/inmunología , Shigella flexneri/inmunología , Células CACO-2 , Comunicación Celular/inmunología , Proliferación Celular , Disentería Bacilar/enzimología , Uniones Comunicantes/inmunología , Uniones Comunicantes/microbiología , Células HeLa , Humanos , Interleucina-8/análisis , Interleucina-8/inmunología , Listeria monocytogenes/inmunología , Listeriosis/enzimología , Listeriosis/inmunología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Peptidoglicano/inmunología , Shigella flexneri/enzimologíaRESUMEN
BACKGROUND: During pathogen infection, innate immunity is initiated via the recognition of microbial products by pattern recognition receptors and the subsequent activation of transcription factors that upregulate proinflammatory genes. By controlling the expression of cytokines, chemokines, anti-bacterial peptides and adhesion molecules, the transcription factor nuclear factor-kappa B (NF-κB) has a central function in this process. In a typical model of NF-κB activation, the recognition of pathogen associated molecules triggers the canonical NF-κB pathway that depends on the phosphorylation of Inhibitor of NF-κB (IκB) by the catalytic subunit IκB kinase ß (IKKß), its degradation and the nuclear translocation of NF-κB dimers. METHODOLOGY: Here, we performed an RNA interference (RNAi) screen on Shigella flexneri-induced NF-κB activation to identify new factors involved in the regulation of NF-κB following infection of epithelial cells by invasive bacteria. By targeting a subset of the human signaling proteome, we found that the catalytic subunit IKKα is also required for complete NF-κB activation during infection. Depletion of IKKα by RNAi strongly reduces the nuclear translocation of NF-κB p65 during S. flexneri infection as well as the expression of the proinflammatory chemokine interleukin-8. Similar to IKKß, IKKα contributes to the phosphorylation of IκBα on serines 32 and 36, and to its degradation. Experiments performed with the synthetic Nod1 ligand L-Ala-D-γ-Glu-meso-diaminopimelic acid confirmed that IKKα is involved in NF-κB activation triggered downstream of Nod1-mediated peptidoglycan recognition. CONCLUSIONS: Taken together, these results demonstrate the unexpected role of IKKα in the canonical NF-κB pathway triggered by peptidoglycan recognition during bacterial infection. In addition, they suggest that IKKα may be an important drug target for the development of treatments that aim at limiting inflammation in bacterial infection.
Asunto(s)
Quinasa I-kappa B/metabolismo , FN-kappa B/metabolismo , Proteína Adaptadora de Señalización NOD1/metabolismo , Peptidoglicano/metabolismo , Secuencia de Bases , Western Blotting , Cartilla de ADN , Ensayo de Inmunoadsorción Enzimática , Células HeLa , Humanos , Interferencia de ARNRESUMEN
Zinc finger-homeodomain proteins (ZF-HDs) have been identified in many plant species. In soybean (Glycine max), GmZF-HD1 functions as a transcription factor that activates the soybean calmodulin isoform-4 (GmCaM-4) gene in response to pathogens. Recently, we reported specific binding of GmZF-HD1 to a 30-nt A/T-rich cis-element which constitutes two repeats of a conserved homeodomain binding site, ATTA, within -1207 to -1128bp of the GmCaM-4 promoter. Herein, homeodomain sequences of the GmZF-HD1 protein were compared to those of other homeodomain proteins and characterized the specificity of DNA sequences in the interaction of the GmCaM-4 promoter with GmZF-HD1 protein. Considering the conservation of homeodomains in plants, the AG sequence within a 30-nt A/T-rich cis-element is required for binding of the GmZF-HD1 protein. Approximately 25-bp of A/T-rich DNA sequences containing an AG sequence is necessary for effective binding to the GmZF-HD1 protein. Taken together, the results support the notion that the GmZF-HD1 protein specifically functions in plant stress signalling by interacting with the promoter of GmCaM-4.
Asunto(s)
Proteínas de Unión al ADN/genética , Glycine max/genética , Proteínas de Homeodominio/genética , Proteínas de Soja/genética , Factores de Transcripción/genética , Dedos de Zinc/genética , Secuencia de Bases , Calmodulina/química , Calmodulina/genética , Calmodulina/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio/química , Proteínas de Homeodominio/metabolismo , Isoformas de Proteínas , Proteínas de Soja/química , Proteínas de Soja/metabolismo , Glycine max/química , Glycine max/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The transcription of soybean (Glycine max) calmodulin isoform-4 (GmCaM-4) is dramatically induced within 0.5 h of exposure to pathogen or NaCl. Core cis-acting elements that regulate the expression of the GmCaM-4 gene in response to pathogen and salt stress were previously identified, between -1,207 and -1,128 bp, and between -858 and -728 bp, in the GmCaM-4 promoter. Here, we characterized the properties of the DNA-binding complexes that form at the two core cis-acting elements of the GmCaM-4 promoter in pathogen-treated nuclear extracts. We generated GUS reporter constructs harboring various deletions of approximately 1.3-kb GmCaM-4 promoter, and analyzed GUS expression in tobacco plants transformed with these constructs. The GUS expression analysis suggested that the two previously identified core regions are involved in inducing GmCaM-4 expression in the heterologous system. Finally, a transient expression assay of Arabidopsis protoplasts showed that the GmCaM-4 promoter produced greater levels of GUS activity than did the CaMV35S promoter after pathogen or NaCl treatments, suggesting that the GmCaM-4 promoter may be useful in the production of conditional gene expression systems.
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Calmodulina/genética , Glycine max/genética , Nicotiana/genética , Sitios de Unión , Calmodulina/biosíntesis , Proteínas de Unión al ADN , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , Isoformas de Proteínas , Análisis de Secuencia de ADN , Glycine max/metabolismo , Estrés Fisiológico , Nicotiana/metabolismo , Activación TranscripcionalRESUMEN
TOR (Target of Rapamycin) is a highly conserved protein kinase and a central controller of cell growth. TOR is found in two functionally and structurally distinct multiprotein complexes termed TOR complex 1 (TORC1) and TOR complex 2 (TORC2). In the present study, we developed a two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS) based proteomic strategy to identify new mammalian TOR (mTOR) binding proteins. We report the identification of Proline-rich Akt substrate (PRAS40) and the hypothetical protein Q6MZQ0/FLJ14213/CAE45978 as new mTOR binding proteins. PRAS40 binds mTORC1 via Raptor, and is an mTOR phosphorylation substrate. PRAS40 inhibits mTORC1 autophosphorylation and mTORC1 kinase activity toward eIF-4E binding protein (4E-BP) and PRAS40 itself. HeLa cells in which PRAS40 was knocked down were protected against induction of apoptosis by TNFalpha and cycloheximide. Rapamycin failed to mimic the pro-apoptotic effect of PRAS40, suggesting that PRAS40 mediates apoptosis independently of its inhibitory effect on mTORC1. Q6MZQ0 is structurally similar to proline rich protein 5 (PRR5) and was therefore named PRR5-Like (PRR5L). PRR5L binds specifically to mTORC2, via Rictor and/or SIN1. Unlike other mTORC2 members, PRR5L is not required for mTORC2 integrity or kinase activity, but dissociates from mTORC2 upon knock down of tuberous sclerosis complex 1 (TSC1) and TSC2. Hyperactivation of mTOR by TSC1/2 knock down enhanced apoptosis whereas PRR5L knock down reduced apoptosis. PRR5L knock down reduced apoptosis also in mTORC2 deficient cells. The above suggests that mTORC2-dissociated PRR5L may promote apoptosis when mTOR is hyperactive. Thus, PRAS40 and PRR5L are novel mTOR-associated proteins that control the balance between cell growth and cell death.
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Apoptosis/fisiología , Proteínas Portadoras/fisiología , Fosfoproteínas/fisiología , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Apoptosis/efectos de los fármacos , Línea Celular , Cromatografía Liquida/métodos , Cicloheximida/farmacología , Humanos , Péptidos y Proteínas de Señalización Intracelular , Diana Mecanicista del Complejo 1 de la Rapamicina , Complejos Multiproteicos , Fosfoproteínas/metabolismo , Fosforilación , Unión Proteica , Proteínas , Interferencia de ARN , Serina-Treonina Quinasas TOR , Espectrometría de Masas en Tándem , Factores de Transcripción/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
BACKGROUND: Iron uptake via endocytosis of iron-transferrin-transferrin receptor complexes is a rate-limiting step for cell growth, viability and proliferation in tumor cells as well as non-transformed cells such as activated lymphocytes. Signaling pathways that regulate transferrin uptake have not yet been identified. RESULTS: We surveyed the human signaling proteome for regulators that increase or decrease transferrin uptake by screening 1,804 dicer-generated signaling small interfering RNAs using automated quantitative imaging. In addition to known transport proteins, we identified 11 signaling proteins that included a striking signature set for the phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3)-target of rapamycin (mTOR) signaling pathway. We show that the PI3K-mTOR signaling pathway is a positive regulator of transferrin uptake that increases the number of transferrin receptors per endocytic vesicle without affecting endocytosis or recycling rates. CONCLUSION: Our study identifies the PtdIns(3,4,5)P3-mTOR signaling pathway as a new regulator of iron-transferrin uptake and serves as a proof-of-concept that targeted RNA interference screens of the signaling proteome provide a powerful and unbiased approach to discover or rank signaling pathways that regulate a particular cell function.
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Fosfatos de Fosfatidilinositol/fisiología , Proteínas Quinasas/fisiología , Proteoma/fisiología , Proteómica/métodos , Transferrina/metabolismo , Endocitosis/efectos de los fármacos , Humanos , Proteínas Quinasas/genética , Transporte de Proteínas/efectos de los fármacos , Proteoma/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Transducción de Señal , Serina-Treonina Quinasas TOR , TransfecciónRESUMEN
Calmodulin (CaM) is involved in defense responses in plants. In soybean (Glycine max), transcription of calmodulin isoform 4 (GmCaM4) is rapidly induced within 30 min after pathogen stimulation, but regulation of the GmCaM4 gene in response to pathogen is poorly understood. Here, we used the yeast one-hybrid system to isolate two cDNA clones encoding proteins that bind to a 30-nt A/T-rich sequence in the GmCaM4 promoter, a region that contains two repeats of a conserved homeodomain binding site, ATTA. The two proteins, GmZF-HD1 and GmZF-HD2, belong to the zinc finger homeodomain (ZF-HD) transcription factor family. Domain deletion analysis showed that a homeodomain motif can bind to the 30-nt GmCaM4 promoter sequence, whereas the two zinc finger domains cannot. Critically, the formation of super-shifted complexes by an anti-GmZF-HD1 antibody incubated with nuclear extracts from pathogen-treated cells suggests that the interaction between GmZF-HD1 and two homeodomain binding site repeats is regulated by pathogen stimulation. Finally, a transient expression assay with Arabidopsis protoplasts confirmed that GmZF-HD1 can activate the expression of GmCaM4 by specifically interacting with the two repeats. These results suggest that the GmZF-HD1 and -2 proteins function as ZF-HD transcription factors to activate GmCaM4 gene expression in response to pathogen.
Asunto(s)
Calmodulina/genética , Glycine max/genética , Proteínas de Homeodominio/metabolismo , Proteínas de Plantas/metabolismo , Regiones Promotoras Genéticas , Secuencia de Aminoácidos , Sitios de Unión , ADN Complementario/química , ADN Complementario/aislamiento & purificación , Regulación de la Expresión Génica de las Plantas , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Alineación de Secuencia , Glycine max/metabolismo , Glycine max/microbiología , Secuencias Repetidas en Tándem , Activación Transcripcional , Dedos de ZincRESUMEN
Many signaling, cytoskeletal, and transport proteins have to be localized to the plasma membrane (PM) in order to carry out their function. We surveyed PM-targeting mechanisms by imaging the subcellular localization of 125 fluorescent protein-conjugated Ras, Rab, Arf, and Rho proteins. Out of 48 proteins that were PM-localized, 37 contained clusters of positively charged amino acids. To test whether these polybasic clusters bind negatively charged phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] lipids, we developed a chemical phosphatase activation method to deplete PM PI(4,5)P2. Unexpectedly, proteins with polybasic clusters dissociated from the PM only when both PI(4,5)P2 and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] were depleted, arguing that both lipid second messengers jointly regulate PM targeting.
Asunto(s)
Membrana Celular/metabolismo , GTP Fosfohidrolasas/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Factores de Ribosilacion-ADP/química , Factores de Ribosilacion-ADP/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , GTP Fosfohidrolasas/química , Células HeLa , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Ratones , Datos de Secuencia Molecular , Células 3T3 NIH , Sistemas de Mensajero Secundario , Transducción de Señal , Electricidad Estática , Proteínas de Unión al GTP rab/química , Proteínas de Unión al GTP rab/metabolismo , Proteínas ras/química , Proteínas ras/metabolismo , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Ca(2+) signaling in nonexcitable cells is typically initiated by receptor-triggered production of inositol-1,4,5-trisphosphate and the release of Ca(2+) from intracellular stores. An elusive signaling process senses the Ca(2+) store depletion and triggers the opening of plasma membrane Ca(2+) channels. The resulting sustained Ca(2+) signals are required for many physiological responses, such as T cell activation and differentiation. Here, we monitored receptor-triggered Ca(2+) signals in cells transfected with siRNAs against 2,304 human signaling proteins, and we identified two proteins required for Ca(2+)-store-depletion-mediated Ca(2+) influx, STIM1 and STIM2. These proteins have a single transmembrane region with a putative Ca(2+) binding domain in the lumen of the endoplasmic reticulum. Ca(2+) store depletion led to a rapid translocation of STIM1 into puncta that accumulated near the plasma membrane. Introducing a point mutation in the STIM1 Ca(2+) binding domain resulted in prelocalization of the protein in puncta, and this mutant failed to respond to store depletion. Our study suggests that STIM proteins function as Ca(2+) store sensors in the signaling pathway connecting Ca(2+) store depletion to Ca(2+) influx.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Transducción de Señal/fisiología , Moléculas de Adhesión Celular , Retículo Endoplásmico/metabolismo , Fluorescencia , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Células Jurkat , Proteínas de la Membrana/genética , Mutación/genética , Proteínas de Neoplasias/genética , ARN Interferente Pequeño/genética , Molécula de Interacción Estromal 1 , Molécula de Interacción Estromal 2RESUMEN
The Ca(2+)-binding protein calmodulin mediates cellular Ca(2+) signals in response to a wide array of stimuli in higher eukaryotes. Plants express numerous CaM isoforms. Transcription of one soybean (Glycine max) CaM isoform, SCaM-4, is dramatically induced within 30 min of pathogen or NaCl stresses. To characterize the cis-acting element(s) of this gene, we isolated an approximately 2-kb promoter sequence of the gene. Deletion analysis of the promoter revealed that a 130-bp region located between nucleotide positions -858 and -728 is required for the stressors to induce expression of SCaM-4. A hexameric DNA sequence within this region, GAAAAA (GT-1 cis-element), was identified as a core cis-acting element for the induction of the SCaM-4 gene. The GT-1 cis-element interacts with an Arabidopsis GT-1-like transcription factor, AtGT-3b, in vitro and in a yeast selection system. Transcription of AtGT-3b is also rapidly induced within 30 min after pathogen and NaCl treatment. These results suggest that an interaction between a GT-1 cis-element and a GT-1-like transcription factor plays a role in pathogen- and salt-induced SCaM-4 gene expression in both soybean and Arabidopsis.
Asunto(s)
Proteínas de Unión al Calcio/genética , Glycine max/genética , Regiones Promotoras Genéticas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN , Eliminación de Gen , Datos de Secuencia Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , ARN de Planta/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Glycine max/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Hundreds of proteins involved in signaling pathways contain a Ca(2+)-dependent membrane-binding motif called the C2-domain. However, no small C2-domain proteins consisting of a single C2-domain have been reported in animal cells. We have isolated two cDNA clones, OsERG1a and OsERG1b, that encode two small C2-domain proteins of 156 and 159 amino acids, respectively, from a fungal elicitor-treated rice cDNA library. The clones are believed to have originated from a single gene by alternative splicing. Transcript levels of the OsERG1 gene are dramatically elevated by a fungal elicitor prepared from Magnaporthe grisea or by Ca(2+) ions. The OsERG1 protein produced in Escherichia coli binds to phospholipid vesicles in a Ca(2+)-dependent manner and is translocated to the plasma membrane of plant cells by treatment with either a fungal elicitor or a Ca(2+) ionophore. These results suggest that OsERG1 proteins containing a single C2-domain are involved in plant defense signaling systems.