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1.
Immunity ; 43(4): 715-26, 2015 Oct 20.
Artículo en Inglés | MEDLINE | ID: mdl-26488816

RESUMEN

CARD9 is a central component of anti-fungal innate immune signaling via C-type lectin receptors, and several immune-related disorders are associated with CARD9 alterations. Here, we used a rare CARD9 variant that confers protection against inflammatory bowel disease as an entry point to investigating CARD9 regulation. We showed that the protective variant of CARD9, which is C-terminally truncated, acted in a dominant-negative manner for CARD9-mediated cytokine production, indicating an important role for the C terminus in CARD9 signaling. We identified TRIM62 as a CARD9 binding partner and showed that TRIM62 facilitated K27-linked poly-ubiquitination of CARD9. We identified K125 as the ubiquitinated residue on CARD9 and demonstrated that this ubiquitination was essential for CARD9 activity. Furthermore, we showed that similar to Card9-deficient mice, Trim62-deficient mice had increased susceptibility to fungal infection. In this study, we utilized a rare protective allele to uncover a TRIM62-mediated mechanism for regulation of CARD9 activation.


Asunto(s)
Proteínas Adaptadoras de Señalización CARD/fisiología , Candidiasis Invasiva/inmunología , Receptores de Angiotensina/fisiología , Receptores de Endotelina/fisiología , Ubiquitina-Proteína Ligasas/fisiología , Adyuvantes Inmunológicos/farmacología , Animales , Proteínas Adaptadoras de Señalización CARD/química , Proteínas Adaptadoras de Señalización CARD/deficiencia , Proteínas Adaptadoras de Señalización CARD/genética , Candidiasis Invasiva/genética , Colitis/inducido químicamente , Colitis/genética , Colitis/prevención & control , Citocinas/biosíntesis , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Genes Dominantes , Predisposición Genética a la Enfermedad , Células HEK293 , Células HeLa , Humanos , Enfermedades Inflamatorias del Intestino/genética , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Mapeo de Interacción de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Receptores de Angiotensina/química , Receptores de Angiotensina/deficiencia , Receptores de Endotelina/química , Receptores de Endotelina/deficiencia , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Organismos Libres de Patógenos Específicos , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas/química , Ubiquitinación
2.
Proc Natl Acad Sci U S A ; 111(21): 7741-6, 2014 May 27.
Artículo en Inglés | MEDLINE | ID: mdl-24821797

RESUMEN

A coding polymorphism (Thr300Ala) in the essential autophagy gene, autophagy related 16-like 1 (ATG16L1), confers increased risk for the development of Crohn disease, although the mechanisms by which single disease-associated polymorphisms contribute to pathogenesis have been difficult to dissect given that environmental factors likely influence disease initiation in these patients. Here we introduce a knock-in mouse model expressing the Atg16L1 T300A variant. Consistent with the human polymorphism, T300A knock-in mice do not develop spontaneous intestinal inflammation, but exhibit morphological defects in Paneth and goblet cells. Selective autophagy is reduced in multiple cell types from T300A knock-in mice compared with WT mice. The T300A polymorphism significantly increases caspase 3- and caspase 7-mediated cleavage of Atg16L1, resulting in lower levels of full-length Atg16Ll T300A protein. Moreover, Atg16L1 T300A is associated with decreased antibacterial autophagy and increased IL-1ß production in primary cells and in vivo. Quantitative proteomics for protein interactors of ATG16L1 identified previously unknown nonoverlapping sets of proteins involved in ATG16L1-dependent antibacterial autophagy or IL-1ß production. These findings demonstrate how the T300A polymorphism leads to cell type- and pathway-specific disruptions of selective autophagy and suggest a mechanism by which this polymorphism contributes to disease.


Asunto(s)
Proteínas Portadoras/genética , Enfermedad de Crohn/inmunología , Células de Paneth/patología , Polimorfismo de Nucleótido Simple/genética , Infecciones por Salmonella/inmunología , Animales , Autofagia/genética , Proteínas Relacionadas con la Autofagia , Western Blotting , Cromatografía Liquida , Enfermedad de Crohn/genética , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Técnicas de Sustitución del Gen , Células Caliciformes/patología , Ratones , Proteómica , Reacción en Cadena en Tiempo Real de la Polimerasa , Espectrometría de Masas en Tándem
3.
Proc Natl Acad Sci U S A ; 108 Suppl 1: 4631-8, 2011 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-20616063

RESUMEN

In innate immune sensing, the detection of pathogen-associated molecular patterns by recognition receptors typically involve leucine-rich repeats (LRRs). We provide a categorization of 375 human LRR-containing proteins, almost half of which lack other identifiable functional domains. We clustered human LRR proteins by first assigning LRRs to LRR classes and then grouping the proteins based on these class assignments, revealing several of the resulting protein groups containing a large number of proteins with certain non-LRR functional domains. In particular, a statistically significant number of LRR proteins in the typical (T) and bacterial + typical (S+T) categories have transmembrane domains, whereas most of the LRR proteins in the cysteine-containing (CC) category contain an F-box domain (which mediates interactions with the E3 ubiquitin ligase complex). Furthermore, by examining the evolutionary profiles of the LRR proteins, we identified a subset of LRR proteins exhibiting strong conservation in fungi and an enrichment for "nucleic acid-binding" function. Expression analysis of LRR genes identifies a subset of pathogen-responsive genes in human primary macrophages infected with pathogenic bacteria. Using functional RNAi, we show that MFHAS1 regulates Toll-like receptor (TLR)-dependent signaling. By using protein interaction network analysis followed by functional RNAi, we identified LRSAM1 as a component of the antibacterial autophagic response.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Evolución Molecular , Inmunidad Innata/genética , Proteínas Oncogénicas/metabolismo , Proteínas/genética , Proteínas/inmunología , Transducción de Señal/genética , Análisis por Conglomerados , Biología Computacional/métodos , Perfilación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Humanos , Inmunidad Innata/inmunología , Proteínas Repetidas Ricas en Leucina , Macrófagos/metabolismo , Macrófagos/microbiología , Proteínas/clasificación , Interferencia de ARN , Receptores Toll-Like/metabolismo
4.
PLoS Pathog ; 7(4): e1001332, 2011 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-21490959

RESUMEN

Infection of host cells by pathogenic microbes triggers signal transduction pathways leading to a multitude of host cell responses including actin cytoskeletal re-arrangements and transcriptional programs. The diarrheagenic pathogens Enteropathogenic E. coli (EPEC) and the related Enterohemorrhagic E. coli (EHEC) subvert the host-cell actin cytoskeleton to form attaching and effacing lesions on the surface of intestinal epithelial cells by injecting effector proteins via a type III secretion system. Here we use a MAL translocation assay to establish the effect of bacterial pathogens on host cell signaling to transcription factor activation. MAL is a cofactor of Serum response factor (SRF), a transcription factor with important roles in the regulation of the actin cytoskeleton. We show that EPEC induces nuclear accumulation of MAL-GFP. The translocated intimin receptor is essential for this process and phosphorylation of Tyrosine residues 454 and 474 is important. Using an expression screen we identify FLRT3, C22orf28 and TESK1 as novel activators of SRF. Importantly we demonstrate that ABRA (actin-binding Rho-activating protein, also known as STARS) is necessary for EPEC-induced nuclear accumulation of MAL and the novel SRF activator FLRT3, is a component of this pathway. We further demonstrate that ABRA is important for structural maintenance of EPEC pedestals. Our results uncover novel components in pathogen-activated cytoskeleton signalling to MAL activation.


Asunto(s)
Escherichia coli Enteropatógena/patogenicidad , Infecciones por Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas de la Mielina/metabolismo , Proteolípidos/metabolismo , Receptores de Superficie Celular/metabolismo , Actinas/metabolismo , Animales , Adhesión Bacteriana , Células COS , Chlorocebus aethiops , Citoesqueleto/metabolismo , Escherichia coli Enteropatógena/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Infecciones por Escherichia coli/patología , Proteínas de Escherichia coli/genética , Mucosa Intestinal/metabolismo , Intestinos/citología , Intestinos/microbiología , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas Proteolipídicas Asociadas a Mielina y Linfocito , Fosforilación , Unión Proteica , Transporte de Proteínas , Receptores de Superficie Celular/genética , Transducción de Señal , Factores de Transcripción/metabolismo , Tirosina/metabolismo
5.
Curr Opin Gastroenterol ; 25(6): 512-20, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19826372

RESUMEN

PURPOSE OF REVIEW: To give an overview of autophagy and its effects on innate and adaptive immunity and touch on some of the roles of autophagy in disease. RECENT FINDINGS: Precise regulation of autophagy is necessary to maintain metabolic equilibrium, immune homeostasis, delineate cell fate and influence host cell responses to cytosolic pathogens. A growing number of studies have implicated that inactivation of autophagy-selective responses contributes to inflammatory disorders, neurodegeneration and cancer, but the precise steps at which disease-associated autophagy-related (ATG) genes affect autophagy pathways is unknown at present. SUMMARY: In eukaryotic cells autophagy is constitutively active at low levels, whereas significant up-regulation occurs in response to a multitude of stresses. Autophagy has achieved notoriety as a perturbed biological process in many disease states and an exponential increase of studies attribute roles for autophagy in innate and adaptive immunity. Understanding how individual disease-associated ATG genes function will lead to a better understanding of and potentially novel therapies for treating the diseases in which they are involved.


Asunto(s)
Autofagia/inmunología , Enfermedad de Crohn/inmunología , Inmunidad Innata/inmunología , Adaptación Biológica , Animales , Presentación de Antígeno/inmunología , Autofagia/genética , Proteínas Relacionadas con la Autofagia , Proteínas Portadoras/inmunología , Citoprotección/inmunología , Células Eucariotas/citología , Proteínas de Unión al GTP/inmunología , Homeostasis/inmunología , Humanos
6.
Cell Rep ; 17(9): 2183-2194, 2016 11 22.
Artículo en Inglés | MEDLINE | ID: mdl-27880896

RESUMEN

Xenophagy is a form of selective autophagy that involves the targeting and elimination of intracellular pathogens through several recognition, recruitment, and ubiquitination events. E3 ubiquitin ligases control substrate selectivity in the ubiquitination cascade; however, systematic approaches to map the role of E3 ligases in antibacterial autophagy have been lacking. We screened more than 600 putative human E3 ligases, identifying E3 ligases that are required for adaptor protein recruitment and LC3-bacteria colocalization, critical steps in antibacterial autophagy. An unbiased informatics approach pinpointed RNF166 as a key gene that interacts with the autophagy network and controls the recruitment of ubiquitin as well as the autophagy adaptors p62 and NDP52 to bacteria. Mechanistic studies demonstrated that RNF166 catalyzes K29- and K33-linked polyubiquitination of p62 at residues K91 and K189. Thus, our study expands the catalog of E3 ligases that mediate antibacterial autophagy and identifies a critical role for RNF166 in this process.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antibacterianos/farmacología , Autofagia/efectos de los fármacos , Ubiquitina-Proteína Ligasas/metabolismo , Células HEK293 , Células HeLa , Humanos , Listeria/efectos de los fármacos , Listeria/crecimiento & desarrollo , Lisina/metabolismo , Unión Proteica , ARN Interferente Pequeño/metabolismo , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/crecimiento & desarrollo , Salmonella typhimurium/metabolismo , Proteína Sequestosoma-1/metabolismo , Ubiquitinación
7.
Cell Rep ; 11(12): 1905-18, 2015 Jun 30.
Artículo en Inglés | MEDLINE | ID: mdl-26095365

RESUMEN

The polymorphism ATG16L1 T300A, associated with increased risk of Crohn's disease, impairs pathogen defense mechanisms including selective autophagy, but specific pathway interactions altered by the risk allele remain unknown. Here, we use perturbational profiling of human peripheral blood cells to reveal that CLEC12A is regulated in an ATG16L1-T300A-dependent manner. Antibacterial autophagy is impaired in CLEC12A-deficient cells, and this effect is exacerbated in the presence of the ATG16L1(∗)300A risk allele. Clec12a(-/-) mice are more susceptible to Salmonella infection, supporting a role for CLEC12A in antibacterial defense pathways in vivo. CLEC12A is recruited to sites of bacterial entry, bacteria-autophagosome complexes, and sites of sterile membrane damage. Integrated genomics identified a functional interaction between CLEC12A and an E3-ubiquitin ligase complex that functions in antibacterial autophagy. These data identify CLEC12A as early adaptor molecule for antibacterial autophagy and highlight perturbational profiling as a method to elucidate defense pathways in complex genetic disease.


Asunto(s)
Proteínas Portadoras/genética , Enfermedad de Crohn/genética , Lectinas Tipo C/genética , Receptores Mitogénicos/genética , Infecciones por Salmonella/genética , Alelos , Animales , Autofagia/genética , Proteínas Relacionadas con la Autofagia , Enfermedad de Crohn/microbiología , Enfermedad de Crohn/patología , Predisposición Genética a la Enfermedad , Genómica , Humanos , Lectinas Tipo C/biosíntesis , Ratones , Receptores Mitogénicos/biosíntesis , Factores de Riesgo , Salmonella/patogenicidad , Infecciones por Salmonella/microbiología
8.
Cell Host Microbe ; 12(6): 778-90, 2012 Dec 13.
Artículo en Inglés | MEDLINE | ID: mdl-23245322

RESUMEN

Several species of pathogenic bacteria replicate within an intracellular vacuolar niche. Bacteria that escape into the cytosol are captured by the autophagic pathway and targeted for lysosomal degradation, representing a defense against bacterial exploitation of the host cytosol. Autophagic capture of Salmonella Typhimurium occurs predominantly via generation of a polyubiquitin signal around cytosolic bacteria, binding of adaptor proteins, and recruitment of autophagic machinery. However, the components mediating bacterial target selection and ubiquitination remain obscure. We identify LRSAM1 as the E3 ligase responsible for anti-Salmonella autophagy-associated ubiquitination. LRSAM1 localizes to several intracellular bacterial pathogens and generates the bacteria-associated ubiquitin signal; these functions require LRSAM1's leucine-rich repeat and RING domains, respectively. Using cells from LRSAM1-deficient individuals, we confirm that LRSAM1 is required for ubiquitination associated with intracellular bacteria but dispensable for ubiquitination of aggregated proteins. LRSAM1 is therefore a bacterial recognition protein and ubiquitin ligase that defends the cytoplasm from invasive pathogens.


Asunto(s)
Autofagia , Salmonella typhimurium/inmunología , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Técnicas de Inactivación de Genes , Células HeLa , Humanos , Estructura Terciaria de Proteína , Ubiquitina-Proteína Ligasas/genética
9.
J Vis Exp ; (53)2011 Jul 28.
Artículo en Inglés | MEDLINE | ID: mdl-21841755

RESUMEN

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system(1, 2); however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis(3) have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture. Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions(4). Radiation pressure was first observed and applied to optical tweezer systems in 1970(5, 6), and was first used to control biological specimens in 1987(7). Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena(8-13). We describe a method(14) that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals(15, 16) (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.


Asunto(s)
Interacciones Huésped-Patógeno/fisiología , Microscopía Confocal/instrumentación , Pinzas Ópticas , Animales , Aspergillus fumigatus , Candida albicans , Humanos , Macrófagos/inmunología , Macrófagos/microbiología , Ratones , Microscopía Confocal/métodos , Linfocitos T/inmunología , Linfocitos T/microbiología
10.
PLoS One ; 5(12): e15215, 2010 Dec 31.
Artículo en Inglés | MEDLINE | ID: mdl-21217821

RESUMEN

The application of live cell imaging allows direct visualization of the dynamic interactions between cells of the immune system. Some preliminary observations challenge long-held beliefs about immune responses to microorganisms; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. This paper outlines a method that advances live cell imaging by integrating a spinning disk confocal microscope with an optical trap, also known as an optical tweezer, in order to provide exquisite spatial and temporal control of pathogenic organisms and place them in proximity to host cells, as determined by the operator. Polymeric beads and live, pathogenic organisms (Candida albicans and Aspergillus fumigatus) were optically trapped using non-destructive forces and moved adjacent to living cells, which subsequently phagocytosed the trapped particle. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability of this method to immunological studies, anti-CD3 polymeric beads were also trapped and manipulated to form synapses with T cells in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.


Asunto(s)
Microscopía/instrumentación , Microscopía/métodos , Pinzas Ópticas , Linfocitos T/microbiología , Animales , Aspergillus fumigatus/metabolismo , Complejo CD3/metabolismo , Candida albicans/metabolismo , Comunicación Celular , Células Cultivadas , Humanos , Células Jurkat , Ratones , Microscopía Confocal/métodos , Modelos Biológicos , Fagocitosis , Polímeros/química , Linfocitos T/citología
11.
J Cell Sci ; 121(Pt 7): 1054-64, 2008 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-18334553

RESUMEN

PCH family proteins are fundamentally important proteins, linking membrane curvature events with cytoskeletal reorganisation. One group, the MEGAPs (also called srGAPs and WRPs) contain RhoGAP domains in addition to the F-BAR domain. We disrupted MEGAP1 and MEGAP2 in Dictyostelium both singly and in combination. We found a strong cytoskeletal phenotype in MEGAP1(-) cells and a subtle phototaxis defect in MEGAP2(-) slugs. MEGAP1(-)/2(-) cells have an overabundance of filopodia and slug motility and function are affected. The most dramatic changes, however, are on contractile vacuoles. MEGAP1(-)/2(-) cells empty their contractile vacuoles less efficiently than normal and consequently have three times the usual number. GFP-tagged MEGAP1 localises to tubules of the contractile vacuole network and when vacuoles start to empty they recruit cytosolic GFP-MEGAP1. Mutants in the Saccharomyces homologues RGD1 and RGD2 also show abnormal vacuoles, implying that this role is conserved. Thus, MEGAP is an important regulator of the contractile vacuole network, and we propose that tubulation of the contractile vacuole by MEGAP1 represents a novel mechanism for driving vacuole emptying.


Asunto(s)
Membrana Celular/metabolismo , Dictyostelium/metabolismo , Proteínas Protozoarias/metabolismo , Vacuolas/metabolismo , Secuencia de Aminoácidos , Animales , Dictyostelium/citología , Dictyostelium/genética , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Microscopía Confocal , Modelos Biológicos , Datos de Secuencia Molecular , Mutación , Proteínas Protozoarias/genética , Proteínas Protozoarias/fisiología , Análisis de Secuencia de Proteína
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