RESUMEN
T follicular helper (Tfh) cells promote affinity maturation of B cells in germinal centers (GCs), whereas T follicular regulatory (Tfr) cells limit the GC reaction. Store-operated Ca(2+) entry (SOCE) through Ca(2+) release-activated Ca(2+) (CRAC) channels mediated by STIM and ORAI proteins is a fundamental signaling pathway in T lymphocytes. Conditional deletion of Stim1 and Stim2 genes in T cells abolished SOCE and strongly reduced antibody-mediated immune responses following viral infection caused by impaired differentiation and function of Tfh cells. Conversely, aging Stim1Stim2-deficient mice developed humoral autoimmunity with spontaneous autoantibody production due to abolished Tfr cell differentiation in the presence of residual Tfh cells. Mechanistically, SOCE controlled Tfr and Tfh cell differentiation through NFAT-mediated IRF4, BATF, and Bcl-6 transcription-factor expression. SOCE had a dual role in controlling the GC reaction by regulating both Tfh and Tfr cell differentiation, thus enabling protective B cell responses and preventing humoral autoimmunity.
Asunto(s)
Autoinmunidad , Linfocitos B/inmunología , Centro Germinal/inmunología , Inmunidad Humoral , Molécula de Interacción Estromal 1/metabolismo , Molécula de Interacción Estromal 2/metabolismo , Linfocitos T/inmunología , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Canales de Calcio Activados por la Liberación de Calcio/metabolismo , Señalización del Calcio , Células Cultivadas , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factores de Transcripción NFATC/metabolismo , Proteína ORAI1/metabolismo , Proteínas Proto-Oncogénicas c-bcl-6/genética , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 2/genéticaRESUMEN
Missense mutations in the nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain containing family of gene 12 (Nlrp12) are associated with periodic fever syndromes and atopic dermatitis in humans. Here, we have demonstrated a crucial role for NLRP12 in negatively regulating pathogenic T cell responses. Nlrp12(-/-) mice responded to antigen immunization with hyperinflammatory T cell responses. Furthermore, transfer of CD4(+)CD45RB(hi)Nlrp12(-/-) T cells into immunodeficient mice led to more severe colitis and atopic dermatitis. NLRP12 deficiency did not, however, cause exacerbated ascending paralysis during experimental autoimmune encephalomyelitis (EAE); instead, Nlrp12(-/-) mice developed atypical neuroinflammatory symptoms that were characterized by ataxia and loss of balance. Enhanced T-cell-mediated interleukin-4 (IL-4) production promotes the development of atypical EAE disease in Nlrp12(-/-) mice. These results define an unexpected role for NLRP12 as an intrinsic negative regulator of T-cell-mediated immunity and identify altered NF-κB regulation and IL-4 production as key mediators of NLRP12-associated disease.
Asunto(s)
Ataxia/inmunología , Colitis/inmunología , Dermatitis Atópica/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Inmunidad Celular , Interleucina-4/inmunología , Péptidos y Proteínas de Señalización Intracelular/inmunología , Animales , Ataxia/genética , Ataxia/patología , Autoinmunidad , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Colitis/genética , Colitis/patología , Células Dendríticas/inmunología , Células Dendríticas/patología , Dermatitis Atópica/genética , Dermatitis Atópica/patología , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/patología , Regulación de la Expresión Génica , Interleucina-4/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Macrófagos/inmunología , Macrófagos/patología , Ratones , Ratones Noqueados , FN-kappa B/genética , FN-kappa B/inmunología , Transducción de SeñalRESUMEN
The adaptor ASC contributes to innate immunity through the assembly of inflammasome complexes that activate the cysteine protease caspase-1. Here we demonstrate that ASC has an inflammasome-independent, cell-intrinsic role in cells of the adaptive immune response. ASC-deficient mice showed defective antigen presentation by dendritic cells (DCs) and lymphocyte migration due to impaired actin polymerization mediated by the small GTPase Rac. Genome-wide analysis showed that ASC, but not the cytoplasmic receptor NLRP3 or caspase-1, controlled the mRNA stability and expression of Dock2, a guanine nucleotide-exchange factor that mediates Rac-dependent signaling in cells of the immune response. Dock2-deficient DCs showed defective antigen uptake similar to that of ASC-deficient cells. Ectopic expression of Dock2 in ASC-deficient cells restored Rac-mediated actin polymerization, antigen uptake and chemotaxis. Thus, ASC shapes adaptive immunity independently of inflammasomes by modulating Dock2-dependent Rac activation and actin polymerization in DCs and lymphocytes.
Asunto(s)
Actinas/química , Proteínas del Citoesqueleto/fisiología , Proteínas Activadoras de GTPasa/fisiología , Inflamasomas/fisiología , Proteínas de Unión al GTP rac/metabolismo , Actinas/metabolismo , Inmunidad Adaptativa , Animales , Presentación de Antígeno , Proteínas Reguladoras de la Apoptosis , Proteínas Adaptadoras de Señalización CARD , Movimiento Celular , Quimiotaxis de Leucocito , Células Dendríticas/inmunología , Proteínas Activadoras de GTPasa/genética , Factores de Intercambio de Guanina Nucleótido , Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Polimerizacion , Estabilidad del ARNRESUMEN
Peripheral peptidolgycan (PGN) is present within antigen-presenting cells in the central nervous system (CNS) of multiple sclerosis (MS) patients, possibly playing a role in neuroinflammation. Accordingly, PGN is linked with disease progression in the animal model of MS, experimental autoimmune encephalomyelitis (EAE), but the role of specific PGN-sensing proteins is unknown. Here we report that the progression of EAE was dependent on the intracellular PGN sensors NOD1 and NOD2 and their common downstream adaptor molecule, receptor interacting protein 2 (RIP2; also known as RIPK2 and RICK). We found that RIP2, but not toll-like receptor 2 (TLR2), played a critical role in the activation of CNS-infiltrating dendritic cells. Our results suggest that PGN in the CNS is involved in the pathogenesis of EAE through the activation of infiltrating dendritic cells via NOD1-, NOD2-, and RIP2-mediated pathways.
Asunto(s)
Sistema Nervioso Central/inmunología , Células Dendríticas/metabolismo , Encefalomielitis Autoinmune Experimental/inmunología , Esclerosis Múltiple/inmunología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Animales , Movimiento Celular/genética , Células Cultivadas , Células Dendríticas/inmunología , Células Dendríticas/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Encefalomielitis Autoinmune Experimental/fisiopatología , Humanos , Inflamación , Ratones , Ratones Noqueados , Proteína Adaptadora de Señalización NOD1/genética , Proteína Adaptadora de Señalización NOD1/inmunología , Proteína Adaptadora de Señalización NOD1/metabolismo , Proteína Adaptadora de Señalización NOD2/genética , Proteína Adaptadora de Señalización NOD2/inmunología , Proteína Adaptadora de Señalización NOD2/metabolismo , Prostaglandinas/inmunología , Prostaglandinas/metabolismo , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/inmunología , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
A hexanucleotide repeat expansion (HRE), (GGGGCC)n, in C9orf72 is the most common genetic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we identify a molecular mechanism by which structural polymorphism of the HRE leads to ALS/FTD pathology and defects. The HRE forms DNA and RNA G-quadruplexes with distinct structures and promotes RNAâ¢DNA hybrids (R-loops). The structural polymorphism causes a repeat-length-dependent accumulation of transcripts aborted in the HRE region. These transcribed repeats bind to ribonucleoproteins in a conformation-dependent manner. Specifically, nucleolin, an essential nucleolar protein, preferentially binds the HRE G-quadruplex, and patient cells show evidence of nucleolar stress. Our results demonstrate that distinct C9orf72 HRE structural polymorphism at both DNA and RNA levels initiates molecular cascades leading to ALS/FTD pathologies, and provide the basis for a mechanistic model for repeat-associated neurodegenerative diseases.
Asunto(s)
Expansión de las Repeticiones de ADN/genética , Sistemas de Lectura Abierta/genética , Esclerosis Amiotrófica Lateral/genética , Linfocitos B , Secuencia de Bases , Nucléolo Celular/genética , Nucléolo Celular/patología , ADN/genética , ADN/metabolismo , Demencia Frontotemporal/genética , G-Cuádruplex , Células HEK293 , Humanos , Modelos Moleculares , Neuronas , Fosfoproteínas/metabolismo , ARN/biosíntesis , ARN/química , ARN/genética , ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/metabolismo , Estrés Fisiológico , Transcripción Genética/genética , NucleolinaRESUMEN
The availability of human genome sequence has transformed biomedical research over the past decade. However, an equivalent map for the human proteome with direct measurements of proteins and peptides does not exist yet. Here we present a draft map of the human proteome using high-resolution Fourier-transform mass spectrometry. In-depth proteomic profiling of 30 histologically normal human samples, including 17 adult tissues, 7 fetal tissues and 6 purified primary haematopoietic cells, resulted in identification of proteins encoded by 17,294 genes accounting for approximately 84% of the total annotated protein-coding genes in humans. A unique and comprehensive strategy for proteogenomic analysis enabled us to discover a number of novel protein-coding regions, which includes translated pseudogenes, non-coding RNAs and upstream open reading frames. This large human proteome catalogue (available as an interactive web-based resource at http://www.humanproteomemap.org) will complement available human genome and transcriptome data to accelerate biomedical research in health and disease.
Asunto(s)
Proteoma/metabolismo , Proteómica , Adulto , Células Cultivadas , Bases de Datos de Proteínas , Feto/metabolismo , Análisis de Fourier , Perfilación de la Expresión Génica , Genoma Humano/genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Internet , Espectrometría de Masas , Anotación de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Especificidad de Órganos , Biosíntesis de Proteínas , Isoformas de Proteínas/análisis , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Señales de Clasificación de Proteína , Transporte de Proteínas , Proteoma/análisis , Proteoma/química , Proteoma/genética , Seudogenes/genética , ARN no Traducido/genética , Reproducibilidad de los Resultados , Regiones no Traducidas/genéticaAsunto(s)
Proteínas del Citoesqueleto/inmunología , Proteínas Activadoras de GTPasa/biosíntesis , Proteínas Activadoras de GTPasa/inmunología , Animales , Proteínas Reguladoras de la Apoptosis , Proteínas Adaptadoras de Señalización CARD , Línea Celular , Proteínas del Citoesqueleto/genética , Factores de Intercambio de Guanina Nucleótido , Inflamasomas/inmunología , Inflamasomas/metabolismo , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos BALB C , Ratones Endogámicos C57BLRESUMEN
The function of CD4(+) T cells is dependent on Ca(2+) influx through Ca(2+) release-activated Ca(2+) (CRAC) channels formed by ORAI proteins. To investigate the role of ORAI1 in proinflammatory Th1 and Th17 cells and autoimmune diseases, we genetically and pharmacologically modulated ORAI1 function. Immunization of mice lacking Orai1 in T cells with MOG peptide resulted in attenuated severity of experimental autoimmune encephalomyelitis (EAE). The numbers of T cells and innate immune cells in the CNS of ORAI1-deficient animals were strongly reduced along with almost completely abolished production of IL-17A, IFN-γ, and GM-CSF despite only partially reduced Ca(2+) influx. In Th1 and Th17 cells differentiated in vitro, ORAI1 was required for cytokine production but not the expression of Th1- and Th17-specific transcription factors T-bet and RORγt. The differentiation and function of induced regulatory T cells, by contrast, was independent of ORAI1. Importantly, induced genetic deletion of Orai1 in adoptively transferred, MOG-specific T cells was able to halt EAE progression after disease onset. Likewise, treatment of wild-type mice with a selective CRAC channel inhibitor after EAE onset ameliorated disease. Genetic deletion of Orai1 and pharmacological ORAI1 inhibition reduced the leukocyte numbers in the CNS and attenuated Th1/Th17 cell-mediated cytokine production. In human CD4(+) T cells, CRAC channel inhibition reduced the expression of IL-17A, IFN-γ, and other cytokines in a dose-dependent manner. Taken together, these findings support the conclusion that Th1 and Th17 cell function is particularly dependent on CRAC channels, which could be exploited as a therapeutic approach to T cell-mediated autoimmune diseases.
Asunto(s)
Canales de Calcio/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Subgrupos de Linfocitos T/inmunología , Células TH1/inmunología , Células Th17/inmunología , Animales , Separación Celular , Cromatografía Liquida , Encefalomielitis Autoinmune Experimental/patología , Citometría de Flujo , Humanos , Ratones , Ratones Transgénicos , Proteína ORAI1 , Reacción en Cadena en Tiempo Real de la Polimerasa , Médula Espinal/inmunología , Médula Espinal/patología , Linfocitos T Reguladores/inmunología , Espectrometría de Masas en TándemRESUMEN
Epstein-Barr virus (EBV) is etiologically linked to infectious mononucleosis and several human cancers. EBV encodes a conserved protein kinase BGLF4 that plays a key role in the viral life cycle. To provide new insight into the host proteins regulated by BGLF4, we utilized stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics to compare site-specific phosphorylation in BGLF4-expressing Akata B cells. Our analysis revealed BGLF4-mediated hyperphosphorylation of 3,046 unique sites corresponding to 1,328 proteins. Frequency analysis of these phosphosites revealed a proline-rich motif signature downstream of BGLF4, indicating a broader substrate recognition for BGLF4 than its cellular ortholog cyclin-dependent kinase 1 (CDK1). Further, motif analysis of the hyperphosphorylated sites revealed enrichment in ATM, ATR and Aurora kinase substrates while functional analyses revealed significant enrichment of pathways related to the DNA damage response (DDR), mitosis and cell cycle. Phosphorylation of proteins associated with the mitotic spindle assembly checkpoint (SAC) indicated checkpoint activation, an event that inactivates the anaphase promoting complex/cyclosome, APC/C. Furthermore, we demonstrated that BGLF4 binds to and directly phosphorylates the key cellular proteins PP1, MPS1 and CDC20 that lie upstream of SAC activation and APC/C inhibition. Consistent with APC/C inactivation, we found that BGLF4 stabilizes the expression of many known APC/C substrates. We also noted hyperphosphorylation of 22 proteins associated the nuclear pore complex, which may contribute to nuclear pore disassembly and SAC activation. A drug that inhibits mitotic checkpoint activation also suppressed the accumulation of extracellular EBV virus. Taken together, our data reveal that, in addition to the DDR, manipulation of mitotic kinase signaling and SAC activation are mechanisms associated with lytic EBV replication. All MS data have been deposited in the ProteomeXchange with identifier PXD002411 (http://proteomecentral.proteomexchange.org/dataset/PXD002411).
Asunto(s)
Daño del ADN/fisiología , Infecciones por Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Mitosis/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Virales/metabolismo , Replicación Viral/fisiología , Secuencia de Aminoácidos , Línea Celular , Cromatografía Liquida , Regulación Viral de la Expresión Génica , Humanos , Immunoblotting , Datos de Secuencia Molecular , Fosforilación , Proteómica/métodos , Transducción de Señal/fisiología , Espectrometría de Masas en TándemRESUMEN
Store-operated Ca(2+) entry (SOCE) through Ca(2+) release-activated Ca(2+) (CRAC) channels is essential for immunity to infection. CRAC channels are formed by ORAI1 proteins in the plasma membrane and activated by stromal interaction molecule (STIM)1 and STIM2 in the endoplasmic reticulum. Mutations in ORAI1 and STIM1 genes that abolish SOCE cause severe immunodeficiency with recurrent infections due to impaired T cell function. SOCE has also been observed in cells of the innate immune system such as macrophages and dendritic cells (DCs) and may provide Ca(2+) signals required for their function. The specific role of SOCE in macrophage and DC function, as well as its contribution to innate immunity, however, is not well defined. We found that nonselective inhibition of Ca(2+) signaling strongly impairs many effector functions of bone marrow-derived macrophages and bone marrow-derived DCs, including phagocytosis, inflammasome activation, and priming of T cells. Surprisingly, however, macrophages and DCs from mice with conditional deletion of Stim1 and Stim2 genes, and therefore complete inhibition of SOCE, showed no major functional defects. Their differentiation, FcR-dependent and -independent phagocytosis, phagolysosome fusion, cytokine production, NLRP3 inflammasome activation, and their ability to present Ags to activate T cells were preserved. Our findings demonstrate that STIM1, STIM2, and SOCE are dispensable for many critical effector functions of macrophages and DCs, which has important implications for CRAC channel inhibition as a therapeutic strategy to suppress pathogenic T cells while not interfering with myeloid cell functions required for innate immunity.
Asunto(s)
Canales de Calcio/metabolismo , Señalización del Calcio/inmunología , Células Dendríticas/inmunología , Macrófagos/inmunología , Glicoproteínas de Membrana/metabolismo , Animales , Presentación de Antígeno/genética , Presentación de Antígeno/inmunología , Proteínas Reguladoras de la Apoptosis/inmunología , Calcio/metabolismo , Canales de Calcio/genética , Proteínas de Unión al Calcio/inmunología , Proteínas Portadoras/inmunología , Diferenciación Celular/inmunología , Células Dendríticas/citología , Retículo Endoplásmico/metabolismo , Humanos , Inmunidad Innata/inmunología , Inflamasomas/inmunología , Activación de Linfocitos/inmunología , Macrófagos/citología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteína con Dominio Pirina 3 de la Familia NLR , Proteína ORAI1 , Fagocitosis/inmunología , Inmunodeficiencia Combinada Grave/genética , Molécula de Interacción Estromal 1 , Molécula de Interacción Estromal 2 , Linfocitos T/inmunologíaRESUMEN
Plasma Proteome Database (PPD; http://www.plasmaproteomedatabase.org/) was initially described in the year 2005 as a part of Human Proteome Organization's (HUPO's) pilot initiative on Human Plasma Proteome Project. Since then, improvements in proteomic technologies and increased throughput have led to identification of a large number of novel plasma proteins. To keep up with this increase in data, we have significantly enriched the proteomic information in PPD. This database currently contains information on 10,546 proteins detected in serum/plasma of which 3784 have been reported in two or more studies. The latest version of the database also incorporates mass spectrometry-derived data including experimentally verified proteotypic peptides used for multiple reaction monitoring assays. Other novel features include published plasma/serum concentrations for 1278 proteins along with a separate category of plasma-derived extracellular vesicle proteins. As plasma proteins have become a major thrust in the field of biomarkers, we have enabled a batch-based query designated Plasma Proteome Explorer, which will permit the users in screening a list of proteins or peptides against known plasma proteins to assess novelty of their data set. We believe that PPD will facilitate both clinical and basic research by serving as a comprehensive reference of plasma proteins in humans and accelerate biomarker discovery and translation efforts.
Asunto(s)
Proteínas Sanguíneas/análisis , Bases de Datos de Proteínas , Proteoma/análisis , Humanos , Internet , Proteómica , Vesículas Secretoras/químicaRESUMEN
Mutations in the epidermal growth factor receptor (EGFR) kinase domain occur in 10-30% of lung adenocarcinoma and are associated with tyrosine kinase inhibitor (TKI) sensitivity. We sought to identify the immediate direct and indirect phosphorylation targets of mutant EGFRs in lung adenocarcinoma. We undertook SILAC strategy, phosphopeptide enrichment, and quantitative MS to identify dynamic changes of phosphorylation downstream of mutant EGFRs in lung adenocarcinoma cells harboring EGFR(L858R) and EGFR(L858R/T790M) , the TKI-sensitive, and TKI-resistant mutations, respectively. Top canonical pathways that were inhibited upon erlotinib treatment in sensitive cells, but not in the resistant cells include EGFR, insulin receptor, hepatocyte growth factor, mitogen-activated protein kinase, mechanistic target of rapamycin, ribosomal protein S6 kinase beta 1, and Janus kinase/signal transducer and activator of transcription signaling. We identified phosphosites in proteins of the autophagy network, such as ULK1 (S623) that is constitutively phosphorylated in these lung adenocarcinoma cells; phosphorylation is inhibited upon erlotinib treatment in sensitive cells, but not in resistant cells. Finally, kinase-substrate prediction analysis from our data indicated that substrates of basophilic kinases from, AGC and Calcium and calmodulin-dependent kinase groups, as well as STE group kinases were significantly enriched and those of proline-directed kinases from, CMGC and Casein kinase groups were significantly depleted among substrates that exhibited increased phosphorylation upon EGF stimulation and reduced phosphorylation upon TKI inhibition. This is the first study to date to examine global phosphorylation changes upon erlotinib treatment of lung adenocarcinoma cells and results from this study provide new insights into signaling downstream of mutant EGFRs in lung adenocarcinoma. All MS data have been deposited in the ProteomeXchange with identifier PXD001101 (http://proteomecentral.proteomexchange.org/dataset/PXD001101).
Asunto(s)
Adenocarcinoma/metabolismo , Receptores ErbB/metabolismo , Neoplasias Pulmonares/metabolismo , Proteómica , Transducción de Señal , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Clorhidrato de Erlotinib , Humanos , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Espectrometría de Masas , Fosfopéptidos/análisis , Fosfopéptidos/metabolismo , Fosforilación/efectos de los fármacos , Mutación Puntual , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
Mercury (Hg) is a global contaminant of aquatic food chains. Aquatic birds, such as the osprey (Pandion haliaetus), with migratory populations breeding in Canada and the northern United States and wintering in the Central and South America, can be exposed to mercury on both the breeding and wintering ranges. We examined Hg levels in 14 fish taxa from 24 osprey wintering sites identified from satellite telemetry. Our main goal was to determine whether fish species that feature in the diet of overwintering and resident fish-eating birds reached toxicity thresholds for Hg. Mean Hg levels in fish whole carcasses ranged from a high of 0.18 µg g(-1) (wet weight) in Scomberomorus sierra to a low of 0.009 µg g(-1) in Catostomidae. Average Hg levels were within published toxicity threshold values in forage fish for only two sites in Mexico (Puerto Vallarta and San Blas Estuary), and all were marine species, such as mackerel (Scomberomorus sierra), sea catfish (Ariopus spp.), and sardinas species (Centropomus spp.). Except for one sample from Nicaragua, sea catfish from Puerto Morazan, none of the fish from sites in Central America had Hg levels which exceeded the thresholds. Nonmetric multidimensional scaling revealed geographical differences in Hg levels with significant pairwise differences between sites along the Pacific Ocean (Mexico) versus the Bay of Campeche, partly due to differences in species composition of sampled fish (and species distributions). Hg increased with trophic level, as assessed by nitrogen stable isotope ratios (δ(15)N but not δ(13)C), in freshwater and marine, but not estuarine, environments. Hg concentrations in forage fish do not account for the elevated Hg reported for many osprey populations on the breeding grounds, thus primary sources of contamination appear to be in the north.
Asunto(s)
Peces/metabolismo , Cadena Alimentaria , Mercurio/metabolismo , Contaminantes Químicos del Agua/metabolismo , Animales , Aves , América Central , México , Perciformes , Medición de Riesgo , Estaciones del AñoRESUMEN
Entry of lymphocytes into secondary lymphoid organs (SLOs) involves intravascular arrest and intracellular calcium ion ([Ca(2+)]i) elevation. TCR activation triggers increased [Ca(2+)]i and can arrest T-cell motility in vitro. However, the requirement for [Ca(2+)]i elevation in arresting T cells in vivo has not been tested. Here, we have manipulated the Ca(2+) release-activated Ca(2+) (CRAC) channel pathway required for [Ca(2+)]i elevation in T cells through genetic deletion of stromal interaction molecule (STIM) 1 or by expression of a dominant-negative ORAI1 channel subunit (ORAI1-DN). Interestingly, the absence of CRAC did not interfere with homing of naïve CD4(+) T cells to SLOs and only moderately reduced crawling speeds in vivo. T cells expressing ORAI1-DN lacked TCR activation induced [Ca(2+)]i elevation, yet arrested motility similar to control T cells in vitro. In contrast, antigen-specific ORAI1-DN T cells had a twofold delayed onset of arrest following injection of OVA peptide in vivo. CRAC channel function is not required for homing to SLOs, but enhances spatiotemporal coordination of TCR signaling and motility arrest.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Canales de Calcio/inmunología , Calcio/inmunología , Glicoproteínas de Membrana/inmunología , Animales , Canales de Calcio/genética , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , Proteína ORAI1 , Péptidos/inmunología , Péptidos/farmacología , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Molécula de Interacción Estromal 1RESUMEN
Polychlorinated biphenyl (PCB) and polybrominated diphenyl ether (PBDE) concentrations and profiles in paired sediment-plankton samples were determined along a 500 km transect in coastal British Columbia, Canada. PCB and PBDE levels in sediment were both greater in the industrialized Strait of Georgia than in remote northern sites and exhibited parallel spatial trends. In plankton, recent-use PBDE levels were higher near-source, while levels of legacy PCBs were uniform across sites. Principal component analysis of 95 PCB congeners illustrated the influence of proximity to source (i.e., latitude) on congener patterns for both matrices (sediment, r(2) = 0.52, p = 0.012; plankton, r(2) = 0.59, p = 0.016). The PCB pattern in plankton grew lighter with latitude, but the opposite pattern in sediments suggested that temperature-related fractionation, sediment processes, and basin-wide oceanography had divergent effects on each matrix. Biota-sediment accumulation factors (BSAFs) were greater for PBDEs than PCBs, but spatial profiles were similar; PCBs and PBDEs were near equilibrium in remote atmospherically driven sites (BSAF = 1.7 and 1.3) but accumulated preferentially in sediments at source-driven sites (BSAF = 0.2 and 0.4). The influences of particle-binding and hydrophobicity on the aquatic fate of PCBs and PBDEs was evident by the strong influence of log KOW on congener-specific BSAFs (PCBs, r(2) = 0.18 p < 0.001; PBDEs, r(2) = 0.61 p < 0.001). While biotic uptake of PCBs has become spatially uniform in coastal BC because of dilution over time, biomagnification of PBDEs remains higher in industrialized waters.
Asunto(s)
Sedimentos Geológicos/química , Éteres Difenilos Halogenados/metabolismo , Plancton/metabolismo , Bifenilos Policlorados/metabolismo , Agua de Mar/química , Contaminantes Químicos del Agua/metabolismo , Biota , Colombia Británica , Geografía , Análisis de Componente PrincipalRESUMEN
Calcium (Ca(2+)) influx is required for the activation and function of all cells in the immune system. It is mediated mainly by store-operated Ca(2+) entry (SOCE) through Ca(2+) release-activated Ca(2+) (CRAC) channels located in the plasma membrane. CRAC channels are composed of ORAI proteins that form the channel pore and are activated by stromal interaction molecules (STIM) 1 and 2. Located in the membrane of the endoplasmic reticulum, STIM1 and STIM2 have the dual function of sensing the intraluminal Ca(2+) concentration in the ER and to activate CRAC channels. A decrease in the ER's Ca(2+) concentration induces STIM multimerization and translocation into puncta close to the plasma membrane where they bind to and activate ORAI channels. Since the identification of ORAI and STIM genes as the principal mediators of CRAC channel function, substantial advances have been achieved in understanding the molecular regulation and physiological role of CRAC channels in cells of the immune system and other organs. In this review, we discuss the mechanisms that regulate CRAC channel function and SOCE, the role of recently identified proteins and mechanisms that modulate the activation of ORAI/STIM proteins and the consequences of CRAC channel dysregulation for lymphocyte function and immunity.
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Canales de Calcio/metabolismo , Calcio/metabolismo , Moléculas de Adhesión Celular/metabolismo , Linfocitos/fisiología , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Proteínas de Neoplasias/metabolismo , Conformación Proteica , Canales de Calcio/genética , Moléculas de Adhesión Celular/genética , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/genética , Proteínas de Neoplasias/genética , Proteína ORAI1 , Oxidación-Reducción , Fosforilación , Polimerizacion , Procesamiento Proteico-Postraduccional/fisiología , Molécula de Interacción Estromal 1 , Molécula de Interacción Estromal 2 , UbiquitinaciónRESUMEN
Store-operated Ca(2+) entry (SOCE) in cells of the immune system is mediated by Ca(2+) release-activated Ca(2+) (CRAC) channels that are formed by ORAI1 and its homologues ORAI2 and ORAI3. They are activated by stromal interaction molecules (STIM) 1 and 2 in response to depletion of endoplasmic reticulum Ca(2+) stores. Loss-of-function mutations in the human ORAI1 and STIM1 genes abolish CRAC channel function and SOCE in a variety of non-excitable cells including lymphocytes and other immune cells, resulting in a unique clinical syndrome termed CRAC channelopathy. It is dominated by severe immunodeficiency and autoimmunity due to impaired SOCE and defects in the function of several lymphocyte subsets. These include CD8(+) T cells, CD4(+) effector and regulatory T cells, natural killer (NK) cells and B cells. This review provides a concise discussion of the role of CRAC channels in these lymphocyte populations and the regulation of adaptive immune responses to infection, in autoimmunity and inflammation.
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Autoinmunidad , Canales de Calcio/inmunología , Canales de Calcio/metabolismo , Infecciones/inmunología , Infecciones/metabolismo , Linfocitos/inmunología , Linfocitos/metabolismo , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Animales , Canales de Calcio/genética , Señalización del Calcio , Humanos , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/inmunología , Síndromes de Inmunodeficiencia/metabolismo , Glicoproteínas de Membrana/genética , Modelos Biológicos , MutaciónRESUMEN
The Nlrp3 inflammasome has been proposed to play an important role in antifungal host defense. However, studies exploring the role of the inflammasome in antifungal host defense have been limited to the direct effects on IL-1ß processing. Although IL-1ß has important direct effects on the innate immune response, important effects of the caspase-1-dependent cytokines IL-1ß and IL-18 are exerted on the initiation of the adaptive Th1 and Th17 cellular responses. No studies have been employed to assess the impact of the inflammasome on the Th1/Th17 defense mechanisms in vivo during candidiasis. In the present study, we demonstrate an essential role for caspase-1 and ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) in disseminated candidiasis through regulating antifungal Th1 and Th17 responses. Caspase-1(-/-) and ASC(-/-) mice display diminished Th1/Th17 responses, followed by increased fungal outgrowth and lower survival. These observations identify a critical role for the inflammasome in controlling protective adaptive immune responses during invasive fungal infection.
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Candidiasis/inmunología , Inflamasomas/inmunología , Células TH1/inmunología , Células Th17/inmunología , Animales , Proteínas Reguladoras de la Apoptosis , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Proteínas Adaptadoras de Señalización CARD , Candida albicans/inmunología , Candida albicans/fisiología , Candidiasis/microbiología , Caspasa 1/genética , Caspasa 1/inmunología , Caspasa 1/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/inmunología , Proteínas del Citoesqueleto/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Predisposición Genética a la Enfermedad , Interacciones Huésped-Patógeno/inmunología , Inmunidad Celular/inmunología , Inflamasomas/genética , Inflamasomas/metabolismo , Interleucina-18/genética , Interleucina-18/inmunología , Interleucina-18/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Interleucina-1beta/metabolismo , Riñón/inmunología , Riñón/microbiología , Riñón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células TH1/metabolismo , Células Th17/metabolismo , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Abnormal activities of histone lysine demethylases (KDMs) and lysine deacetylases (HDACs) are associated with aberrant gene expression in breast cancer development. However, the precise molecular mechanisms underlying the crosstalk between KDMs and HDACs in chromatin remodeling and regulation of gene transcription are still elusive. In this study, we showed that treatment of human breast cancer cells with inhibitors targeting the zinc cofactor dependent class I/II HDAC, but not NAD(+) dependent class III HDAC, led to significant increase of H3K4me2 which is a specific substrate of histone lysine-specific demethylase 1 (LSD1) and a key chromatin mark promoting transcriptional activation. We also demonstrated that inhibition of LSD1 activity by a pharmacological inhibitor, pargyline, or siRNA resulted in increased acetylation of H3K9 (AcH3K9). However, siRNA knockdown of LSD2, a homolog of LSD1, failed to alter the level of AcH3K9, suggesting that LSD2 activity may not be functionally connected with HDAC activity. Combined treatment with LSD1 and HDAC inhibitors resulted in enhanced levels of H3K4me2 and AcH3K9, and exhibited synergistic growth inhibition of breast cancer cells. Finally, microarray screening identified a unique subset of genes whose expression was significantly changed by combination treatment with inhibitors of LSD1 and HDAC. Our study suggests that LSD1 intimately interacts with histone deacetylases in human breast cancer cells. Inhibition of histone demethylation and deacetylation exhibits cooperation and synergy in regulating gene expression and growth inhibition, and may represent a promising and novel approach for epigenetic therapy of breast cancer.
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Neoplasias de la Mama/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Histona Demetilasas/antagonistas & inhibidores , Histonas/metabolismo , Acetilación/efectos de los fármacos , Neoplasias de la Mama/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Biología Computacional/métodos , Sinergismo Farmacológico , Femenino , Perfilación de la Expresión Génica , Humanos , Metilación/efectos de los fármacosRESUMEN
Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, is a potent inhibitor of experimental mammary carcinogenesis and may be an effective, safe chemopreventive agent for use in humans. SFN acts in part on the Keap1/Nrf2 pathway to regulate a battery of cytoprotective genes. In this study, transcriptomic and proteomic changes in the estrogen receptor negative, non-tumorigenic human breast epithelial MCF10A cell line were analyzed following SFN treatment or KEAP1 knockdown with siRNA using microarray and stable isotopic labeling with amino acids in culture (SILAC), respectively. Changes in selected transcripts and proteins were confirmed by PCR and Western blot in MCF10A and MCF12A cells. There was strong correlation between the transcriptomic and proteomic responses in both the SFN treatment (R = 0.679, P < 0.05) and KEAP1 knockdown (R = 0.853, P < 0.05) experiments. Common pathways for SFN treatment and KEAP1 knockdown were xenobiotic metabolism and antioxidants, glutathione metabolism, carbohydrate metabolism, and NADH/NADPH regeneration. Moreover, these pathways were most prominent in both the transcriptomic and the proteomic analyses. The aldo-keto reductase family members, AKR1B10, AKR1C1, AKR1C2 and AKR1C3, as well as NQO1 and ALDH3A1, were highly upregulated at both the transcriptomic and the proteomic levels. Collectively, these studies served to identify potential biomarkers that can be used in clinical trials to investigate the initial pharmacodynamic action of SFN in the breast.