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1.
J Biol Chem ; 296: 100611, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33798552

RESUMEN

Human macrophage migration inhibitory factor (MIF) is an atypical chemokine implicated in intercellular signaling and innate immunity. MIF orthologs (MIF/D-DT-like proteins, MDLs) are present throughout the plant kingdom, but remain experimentally unexplored in these organisms. Here, we provide an in planta characterization and functional analysis of the three-member gene/protein MDL family in Arabidopsis thaliana. Subcellular localization experiments indicated a nucleo-cytoplasmic distribution of MDL1 and MDL2, while MDL3 is localized to peroxisomes. Protein-protein interaction assays revealed the in vivo formation of MDL1, MDL2, and MDL3 homo-oligomers, as well as the formation of MDL1-MDL2 hetero-oligomers. Functionally, Arabidopsismdl mutants exhibited a delayed transition from vegetative to reproductive growth (flowering) under long-day conditions, but not in a short-day environment. In addition, mdl mutants were more resistant to colonization by the bacterial pathogen Pseudomonas syringae pv. maculicola. The latter phenotype was compromised by the additional mutation of SALICYLIC ACID INDUCTION DEFICIENT 2 (SID2), a gene implicated in the defense-induced biosynthesis of the key signaling molecule salicylic acid. However, the enhanced antibacterial immunity was not associated with any constitutive or pathogen-induced alterations in the levels of characteristic phytohormones or defense-associated metabolites. Interestingly, bacterial infection triggered relocalization and accumulation of MDL1 and MDL2 at the peripheral lobes of leaf epidermal cells. Collectively, our data indicate redundant functionality and a complex interplay between the three chemokine-like Arabidopsis MDL proteins in the regulation of both developmental and immune-related processes. These insights expand the comparative cross-kingdom analysis of MIF/MDL signaling in human and plant systems.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/inmunología , Quimiocinas/metabolismo , Flores/inmunología , Inmunidad Innata/inmunología , Enfermedades de las Plantas/inmunología , Pseudomonas syringae/fisiología , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Flores/crecimiento & desarrollo , Flores/metabolismo , Flores/microbiología , Regulación de la Expresión Génica de las Plantas , Enfermedades de las Plantas/microbiología
2.
J Biol Chem ; 295(3): 850-867, 2020 01 17.
Artículo en Inglés | MEDLINE | ID: mdl-31811089

RESUMEN

Human macrophage migration-inhibitory factor (MIF) is an evolutionarily-conserved protein that has both extracellular immune-modulating and intracellular cell-regulatory functions. MIF plays a role in various diseases, including inflammatory diseases, atherosclerosis, autoimmunity, and cancer. It serves as an inflammatory cytokine and chemokine, but also exhibits enzymatic activity. Secreted MIF binds to cell-surface immune receptors such as CD74 and CXCR4. Plants possess MIF orthologs but lack the associated receptors, suggesting functional diversification across kingdoms. Here, we characterized three MIF orthologs (termed MIF/d-dopachrome tautomerase-like proteins or MDLs) of the model plant Arabidopsis thaliana Recombinant Arabidopsis MDLs (AtMDLs) share similar secondary structure characteristics with human MIF, yet only have minimal residual tautomerase activity using either p-hydroxyphenylpyruvate or dopachrome methyl ester as substrate. Site-specific mutagenesis suggests that this is due to a distinct amino acid difference at the catalytic cavity-defining residue Asn-98. Surprisingly, AtMDLs bind to the human MIF receptors CD74 and CXCR4. Moreover, they activate CXCR4-dependent signaling in a receptor-specific yeast reporter system and in CXCR4-expressing human HEK293 transfectants. Notably, plant MDLs exert dose-dependent chemotactic activity toward human monocytes and T cells. A small molecule MIF inhibitor and an allosteric CXCR4 inhibitor counteract this function, revealing its specificity. Our results indicate cross-kingdom conservation of the receptor signaling and leukocyte recruitment capacities of human MIF by its plant orthologs. This may point toward a previously unrecognized interplay between plant proteins and the human innate immune system.


Asunto(s)
Antígenos de Diferenciación de Linfocitos B/genética , Antígenos de Histocompatibilidad Clase II/genética , Inmunidad Innata/genética , Oxidorreductasas Intramoleculares/genética , Factores Inhibidores de la Migración de Macrófagos/genética , Receptores CXCR4/genética , Antígenos de Diferenciación de Linfocitos B/química , Arabidopsis/genética , Arabidopsis/inmunología , Quimiotaxis/genética , Quimiotaxis/inmunología , Secuencia Conservada/genética , Secuencia Conservada/inmunología , Citocinas/genética , Citocinas/inmunología , Células HEK293 , Antígenos de Histocompatibilidad Clase II/química , Humanos , Oxidorreductasas Intramoleculares/química , Oxidorreductasas Intramoleculares/inmunología , Factores Inhibidores de la Migración de Macrófagos/química , Factores Inhibidores de la Migración de Macrófagos/inmunología , Monocitos/química , Monocitos/metabolismo , Unión Proteica/genética , Receptores CXCR4/química , Homología de Secuencia , Linfocitos T/química , Linfocitos T/metabolismo
3.
Chembiochem ; 22(6): 1012-1019, 2021 03 16.
Artículo en Inglés | MEDLINE | ID: mdl-33125165

RESUMEN

Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine and atypical chemokine with a key role in inflammatory diseases including atherosclerosis. Key atherogenic functions of MIF are mediated by noncognate interaction with the chemokine receptor CXCR2. The MIF N-like loop comprising the sequence 47-56 is an important structural determinant of the MIF/CXCR2 interface and MIF(47-56) blocks atherogenic MIF activities. However, the mechanism and critical structure-activity information within this sequence have remained elusive. Here, we show that MIF(47-56) directly binds to CXCR2 to compete with MIF receptor activation. By using alanine scanning, essential and dispensable residues were identified. Moreover, MIF(cyclo10), a designed cyclized variant of MIF(47-56), inhibited key inflammatory and atherogenic MIF activities in vitro and in vivo/ex vivo, and exhibited strongly improved resistance to proteolytic degradation in human plasma in vitro, thus suggesting that it could serve as a promising basis for MIF-derived anti-atherosclerotic peptides.


Asunto(s)
Factores Inhibidores de la Migración de Macrófagos/química , Péptidos Cíclicos/metabolismo , Receptores de Interleucina-8B/metabolismo , Secuencia de Aminoácidos , Animales , Adhesión Celular , Fluoresceínas/química , Células HEK293 , Humanos , Leucocitos/química , Leucocitos/citología , Leucocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Péptidos Cíclicos/sangre , Péptidos Cíclicos/química , Unión Proteica , Estabilidad Proteica , Receptores de Interleucina-8B/antagonistas & inhibidores , Espectrometría de Fluorescencia , Ácidos Sulfónicos/química
4.
Proc Natl Acad Sci U S A ; 114(13): E2766-E2775, 2017 03 28.
Artículo en Inglés | MEDLINE | ID: mdl-28292897

RESUMEN

Constitutive photomorphogenesis 9 (COP9) signalosome 5 (CSN5), an isopeptidase that removes neural precursor cell-expressed, developmentally down-regulated 8 (NEDD8) moieties from cullins (thus termed "deNEDDylase") and a subunit of the cullin-RING E3 ligase-regulating COP9 signalosome complex, attenuates proinflammatory NF-κB signaling. We previously showed that CSN5 is up-regulated in human atherosclerotic arteries. Here, we investigated the role of CSN5 in atherogenesis in vivo by using mice with myeloid-specific Csn5 deletion. Genetic deletion of Csn5 in Apoe-/- mice markedly exacerbated atherosclerotic lesion formation. This was broadly observed in aortic root, arch, and total aorta of male mice, whereas the effect was less pronounced and site-specific in females. Mechanistically, Csn5 KO potentiated NF-κB signaling and proinflammatory cytokine expression in macrophages, whereas HIF-1α levels were reduced. Inversely, inhibition of NEDDylation by MLN4924 blocked proinflammatory gene expression and NF-κB activation while enhancing HIF-1α levels and the expression of M2 marker Arginase 1 in inflammatory-elicited macrophages. MLN4924 further attenuated the expression of chemokines and adhesion molecules in endothelial cells and reduced NF-κB activation and monocyte arrest on activated endothelium in vitro. In vivo, MLN4924 reduced LPS-induced inflammation, favored an antiinflammatory macrophage phenotype, and decreased the progression of early atherosclerotic lesions in mice. On the contrary, MLN4924 treatment increased neutrophil and monocyte counts in blood and had no net effect on the progression of more advanced lesions. Our data show that CSN5 is atheroprotective. We conclude that MLN4924 may be useful in preventing early atherogenesis, whereas selectively promoting CSN5-mediated deNEDDylation may be beneficial in all stages of atherosclerosis.


Asunto(s)
Aterosclerosis/enzimología , Complejo del Señalosoma COP9/metabolismo , Péptido Hidrolasas/metabolismo , Animales , Aorta/metabolismo , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerosis/genética , Aterosclerosis/metabolismo , Complejo del Señalosoma COP9/genética , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Células Endoteliales/enzimología , Células Endoteliales/metabolismo , Femenino , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Macrófagos/enzimología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína NEDD8/genética , Proteína NEDD8/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Péptido Hidrolasas/genética
5.
Int J Med Microbiol ; 303(8): 635-44, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24120365

RESUMEN

The vascular endothelium provides the critical barrier during hematogenous spreading of bacteria, a phenomenon that might contribute to severe diseases in humans including endocarditis and sepsis as known from infections by Staphylococcus aureus. Here we aimed to uncover early responses of the endothelium to S. aureus infection with respect to (a) inflammatory reactions such as paracellular endothelial barrier function and expression of cell adhesion molecule-1 (ICAM-1) and (b) translocation through the endothelium. After infection of the cultured endothelium with 22 different clinical isolates of S. aureus and two well-characterized lab strains a diverse and strain-specific change in para- and transcellular endothelial barrier function was observed. Bayesian data analysis revealed positive correlation of paracellular barrier function decrease followed by expression of ICAM-1 while these parameters negatively correlated with transcellular bacterial translocation. Translocating bacteria largely blocked TNFα-induced ICAM-1 expression indicating an active anti-inflammatory effect mediated by those strains probably due to intracellularly released virulence factors. Furthermore, the underlying background of barrier function decrease was investigated in more detail using two well-characterized lab strains, ls 8325-4 and ls 6850 and respective mutants. Barrier function decrease was found to be independent of early cell death and early release of virulence factors into the medium, but require internalization of live bacteria. The data show for the first time that endothelial cells respond diversely to infection with different strains of S. aureus and that translocating strains downregulate inflammatory response of the endothelium. Furthermore, data indicate that S. aureus-mediated activation of the endothelium reduces bacterial translocation.


Asunto(s)
Traslocación Bacteriana , Permeabilidad Capilar , Células Endoteliales/microbiología , Células Endoteliales/fisiología , Interacciones Huésped-Patógeno , Staphylococcus aureus/fisiología , Células Cultivadas , Expresión Génica , Humanos , Molécula 1 de Adhesión Intercelular/biosíntesis
6.
Sci Signal ; 16(812): eadg2621, 2023 11 21.
Artículo en Inglés | MEDLINE | ID: mdl-37988455

RESUMEN

Mammalian macrophage migration inhibitory factor (MIF) and its paralog, D-dopachrome tautomerase, are multifunctional inflammatory cytokines. Plants have orthologous MIF and D-dopachrome tautomerase-like (MDL) proteins that mimic some of the effects of MIF on immune cells in vitro. We explored the structural and functional similarities between the three Arabidopsis thaliana MDLs and MIF. X-ray crystallography of the MDLs revealed high structural similarity between MDL and MIF homotrimers and suggested a potential explanation for the lack of tautomerase activity in the MDLs. MDL1 and MDL2 interacted with each other and with MIF in vitro, in yeast, and in plant leaves and formed hetero-oligomeric complexes with MIF in vitro. The MDLs stimulated signaling through the MIF receptors CXCR2 or CXCR4 and enhanced the responses to MIF in a yeast reporter system, in human neutrophils, and in human lung epithelial cells. Pharmacological inhibitors that disrupted MIF activity or prevented the formation of MIF-MDL hetero-oligomers blocked the observed synergism. These findings demonstrate that MDLs can enhance cellular responses to MIF, which may have functional implications in tissues exposed to MDLs from the diet or environment.


Asunto(s)
Factores Inhibidores de la Migración de Macrófagos , Animales , Humanos , Factores Inhibidores de la Migración de Macrófagos/genética , Factores Inhibidores de la Migración de Macrófagos/química , Proteínas de Plantas , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Saccharomyces cerevisiae/metabolismo , Neutrófilos/metabolismo , Mamíferos/metabolismo , Oxidorreductasas Intramoleculares/genética
7.
Exp Hematol ; 115: 30-43, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-36096455

RESUMEN

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of small, mature CD5+ B lymphocytes in the blood, marrow, and lymphoid organs. Cell survival depends on interaction with the leukemic microenvironment. However, the mechanisms controlling CLL cell survival are still incompletely understood. Macrophage migration-inhibitory factor (MIF), a pro-inflammatory and immunoregulatory chemokine-like cytokine, interacts with CXCR4, a major chemokine receptor, as well as with CD74/invariant chain, a single-pass type II receptor. In this study, we analyzed the roles of CXCR4, CD74, and MIF in CLL. Mononuclear cells from patients with hematological malignancies were analyzed for coexpression of CXCR4 and CD74 by flow cytometry. Strong co- and overexpression of CXCR4 and CD74 were observed on B cells of CLL patients (n = 10). Survival and chemotaxis assays indicated that CXCR4 and CD74 work together to enhance the survival and migration of malignant cells in CLL. Blockade of the receptors, either individually or in combination, promoted cell death and led to an abrogation of MIF-driven migration responses in murine and human CLL cells, suggesting that joint activation of both receptors is crucial for CLL cell survival and mobility. These findings indicate that the MIF/CXCR4/CD74 axis represents a novel therapeutic target in CLL.


Asunto(s)
Leucemia Linfocítica Crónica de Células B , Humanos , Ratones , Animales , Leucemia Linfocítica Crónica de Células B/patología , Supervivencia Celular , Antígenos de Diferenciación de Linfocitos B/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Macrófagos/metabolismo , Microambiente Tumoral
8.
Methods Mol Biol ; 2080: 249-261, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31745887

RESUMEN

Human macrophage migration inhibitory factor (MIF) is an inflammatory cytokine with chemokine-like characteristics and an upstream regulator of host innate immunity. It is a critical mediator of a variety of human diseases, such as acute and chronic inflammatory diseases, autoimmunity, atherosclerosis, and cancer. MIF is an atypical chemokine that not only signals through its cognate receptor CD74, but also interacts with the classical chemokine receptors CXCR2 and CXCR4. MIF and its homolog D-dopachrome tautomerase (D-DT)/MIF-2 are structurally unique proteins that are conserved across kingdoms and that share a remarkable homology with bacterial tautomerases/isomerases, albeit the relevance of the tautomerase activity in mammalian systems has remained unclear. Intriguingly, in silico analysis also predicts MIF orthologs in plants such as in the model plant Arabidopsis thaliana. There are three predicted MIF orthologs in A. thaliana, which have been termed A. thaliana MIF/D-DT-like proteins (AtMDLs). Anticipating that there will be a future research interest in studying AtMDLs or other plant MDLs, here we describe methods how to clone, recombinantly express and purify AtMDL proteins, taking into account codon usage differences between plant and mammalian cell systems.


Asunto(s)
Factores Inhibidores de la Migración de Macrófagos/genética , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/genética , Plantas/metabolismo , Arabidopsis/genética , Arabidopsis/inmunología , Arabidopsis/metabolismo , Clonación Molecular , Regulación de la Expresión Génica de las Plantas , Orden Génico , Inmunidad Innata , Plantas/inmunología , Plásmidos/genética
9.
Nat Commun ; 11(1): 5981, 2020 11 25.
Artículo en Inglés | MEDLINE | ID: mdl-33239628

RESUMEN

Targeting a specific chemokine/receptor axis in atherosclerosis remains challenging. Soluble receptor-based strategies are not established for chemokine receptors due to their discontinuous architecture. Macrophage migration-inhibitory factor (MIF) is an atypical chemokine that promotes atherosclerosis through CXC-motif chemokine receptor-4 (CXCR4). However, CXCR4/CXCL12 interactions also mediate atheroprotection. Here, we show that constrained 31-residue-peptides ('msR4Ms') designed to mimic the CXCR4-binding site to MIF, selectively bind MIF with nanomolar affinity and block MIF/CXCR4 without affecting CXCL12/CXCR4. We identify msR4M-L1, which blocks MIF- but not CXCL12-elicited CXCR4 vascular cell activities. Its potency compares well with established MIF inhibitors, whereas msR4M-L1 does not interfere with cardioprotective MIF/CD74 signaling. In vivo-administered msR4M-L1 enriches in atherosclerotic plaques, blocks arterial leukocyte adhesion, and inhibits atherosclerosis and inflammation in hyperlipidemic Apoe-/- mice in vivo. Finally, msR4M-L1 binds to MIF in plaques from human carotid-endarterectomy specimens. Together, we establish an engineered GPCR-ectodomain-based mimicry principle that differentiates between disease-exacerbating and -protective pathways and chemokine-selectively interferes with atherosclerosis.


Asunto(s)
Aterosclerosis/tratamiento farmacológico , Oxidorreductasas Intramoleculares/antagonistas & inhibidores , Factores Inhibidores de la Migración de Macrófagos/antagonistas & inhibidores , Fragmentos de Péptidos/farmacología , Receptores CXCR4/metabolismo , Anciano , Animales , Antígenos CD/metabolismo , Aterosclerosis/genética , Aterosclerosis/patología , Aterosclerosis/cirugía , Sitios de Unión , Arteria Carótida Común/patología , Arteria Carótida Común/cirugía , Quimiocina CXCL12/metabolismo , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Endarterectomía Carotidea , Femenino , Humanos , Oxidorreductasas Intramoleculares/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Masculino , Ratones , Ratones Noqueados para ApoE , Persona de Mediana Edad , Fragmentos de Péptidos/uso terapéutico , Receptores CXCR4/química , Receptores CXCR4/ultraestructura , Sialiltransferasas/metabolismo , Transducción de Señal/efectos de los fármacos
10.
Thromb Haemost ; 119(4): 553-566, 2019 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-30716779

RESUMEN

Chemokines orchestrate leukocyte recruitment in atherosclerosis and their blockade is a promising anti-atherosclerotic strategy, but few chemokine-based approaches have advanced into clinical trials, in part owing to the complexity and redundancy of the chemokine network. Macrophage migration inhibitory factor (MIF) is a pivotal mediator of atherosclerotic lesion formation. It has been characterized as an inflammatory cytokine and atypical chemokine that promotes atherogenic leukocyte recruitment and lesional inflammation through interactions with the chemokine receptors CXCR2 and CXCR4, but also exhibits phase-specific CD74-mediated cardioprotective activity. The unique structural properties of MIF and its homologue MIF-2/D-DT offer intriguing therapeutic opportunities including small molecule-, antibody- and peptide-based approaches that may hold promise as inhibitors of atherosclerosis, while sparing tissue-protective classical chemokine pathways. In this review, we summarize the pros and cons of anti-MIF protein strategies and discuss their molecular characteristics and receptor specificities with a focus on cardiovascular disease.


Asunto(s)
Aterosclerosis/metabolismo , Quimiocinas/metabolismo , Inflamación/metabolismo , Oxidorreductasas Intramoleculares/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Animales , Anticuerpos Monoclonales Humanizados/uso terapéutico , Antígenos de Diferenciación de Linfocitos B/metabolismo , Aterosclerosis/tratamiento farmacológico , Enfermedades Cardiovasculares/metabolismo , Diseño de Fármacos , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Inflamación/tratamiento farmacológico , Leucocitos/metabolismo , Péptidos/química , Unión Proteica , Receptores CXCR4/metabolismo , Receptores de Interleucina-8B/metabolismo , Transducción de Señal
11.
Sci Rep ; 8(1): 5171, 2018 03 26.
Artículo en Inglés | MEDLINE | ID: mdl-29581527

RESUMEN

MIF is a chemokine-like cytokine that plays a role in the pathogenesis of inflammatory and cardiovascular disorders. It binds to the chemokine-receptors CXCR2/CXCR4 to trigger atherogenic leukocyte migration albeit lacking canonical chemokine structures. We recently characterized an N-like-loop and the Pro-2-residue of MIF as critical molecular determinants of the CXCR4/MIF binding-site and identified allosteric agonism as a mechanism that distinguishes CXCR4-binding to MIF from that to the cognate ligand CXCL12. By using peptide spot-array technology, site-directed mutagenesis, structure-activity-relationships, and molecular docking, we identified the Arg-Leu-Arg (RLR) sequence-region 87-89 that - in three-dimensional space - 'extends' the N-like-loop to control site-1-binding to CXCR4. Contrary to wildtype MIF, mutant R87A-L88A-R89A-MIF fails to bind to the N-terminal of CXCR4 and the contribution of RLR to the MIF/CXCR4-interaction is underpinned by an ablation of MIF/CXCR4-specific signaling and reduction in CXCR4-dependent chemotactic leukocyte migration of the RLR-mutant of MIF. Alanine-scanning, functional competition by RLR-containing peptides, and molecular docking indicate that the RLR residues directly participate in contacts between MIF and CXCR4 and highlight the importance of charge-interactions at this interface. Identification of the RLR region adds important structural information to the MIF/CXCR4 binding-site that distinguishes this interface from CXCR4/CXCL12 and will help to design MIF-specific drug-targeting approaches.


Asunto(s)
Oxidorreductasas Intramoleculares/genética , Factores Inhibidores de la Migración de Macrófagos/genética , Unión Proteica/genética , Receptores CXCR4/genética , Relación Estructura-Actividad , Sitios de Unión , Enfermedades Cardiovasculares/genética , Enfermedades Cardiovasculares/patología , Quimiotaxis/genética , Humanos , Inflamación/genética , Inflamación/patología , Oxidorreductasas Intramoleculares/química , Ligandos , Factores Inhibidores de la Migración de Macrófagos/química , Simulación del Acoplamiento Molecular , Péptidos/química , Péptidos/genética , Receptores CXCR4/química , Receptores de Interleucina-8B/química , Receptores de Interleucina-8B/genética
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