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1.
Hum Mol Genet ; 28(18): 3072-3090, 2019 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-31174210

RESUMEN

X-linked juvenile retinoschisis (XLRS) is an early-onset inherited condition that affects primarily males and is characterized by cystic lesions of the inner retina, decreased visual acuity and contrast sensitivity and a selective reduction of the electroretinogram (ERG) b-wave. Although XLRS is genetically heterogeneous, all mouse models developed to date involve engineered or spontaneous null mutations. In the present study, we have studied three new Rs1 mutant mouse models: (1) a knockout with inserted lacZ reporter gene; (2) a C59S point mutant substitution and (3) an R141C point mutant substitution. Mice were studied from postnatal day (P15) to 28 weeks by spectral domain optical coherence tomography and ERG. Retinas of P21-22 mice were examined using biochemistry, single cell electrophysiology of retinal ganglion cells (RGCs) and by immunohistochemistry. Each model developed intraretinal schisis and reductions in the ERG that were greater for the b-wave than the a-wave. The phenotype of the C59S mutant appeared less severe than the other mutants by ERG at adult ages. RGC electrophysiology demonstrated elevated activity in the absence of a visual stimulus and reduced signal-to-noise ratios in response to light stimuli. Immunohistochemical analysis documented early abnormalities in all cells of the outer retina. Together, these results provide significant insight into the early events of XLRS pathophysiology, from phenotype differences between disease-causing variants to common mechanistic events that may play critical roles in disease presentation and progression.


Asunto(s)
Genes Ligados a X , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Genotipo , Fenotipo , Retinosquisis/genética , Retinosquisis/patología , Animales , Biomarcadores , Moléculas de Adhesión Celular/genética , Modelos Animales de Enfermedad , Electrorretinografía , Proteínas del Ojo/genética , Estudios de Asociación Genética/métodos , Inmunohistoquímica , Ratones , Mutación , Estimulación Luminosa , Retinosquisis/diagnóstico , Índice de Severidad de la Enfermedad , Tomografía de Coherencia Óptica
2.
Am J Hum Genet ; 101(6): 985-994, 2017 Dec 07.
Artículo en Inglés | MEDLINE | ID: mdl-29198724

RESUMEN

Bone morphogenetic protein 2 (BMP2) in chromosomal region 20p12 belongs to a gene superfamily encoding TGF-ß-signaling proteins involved in bone and cartilage biology. Monoallelic deletions of 20p12 are variably associated with cleft palate, short stature, and developmental delay. Here, we report a cranioskeletal phenotype due to monoallelic truncating and frameshift BMP2 variants and deletions in 12 individuals from eight unrelated families that share features of short stature, a recognizable craniofacial gestalt, skeletal anomalies, and congenital heart disease. De novo occurrence and autosomal-dominant inheritance of variants, including paternal mosaicism in two affected sisters who inherited a BMP2 splice-altering variant, were observed across all reported families. Additionally, we observed similarity to the human phenotype of short stature and skeletal anomalies in a heterozygous Bmp2-knockout mouse model, suggesting that haploinsufficiency of BMP2 could be the primary phenotypic determinant in individuals with predicted truncating variants and deletions encompassing BMP2. These findings demonstrate the important role of BMP2 in human craniofacial, skeletal, and cardiac development and confirm that individuals heterozygous for BMP2 truncating sequence variants or deletions display a consistent distinct phenotype characterized by short stature and skeletal and cardiac anomalies without neurological deficits.


Asunto(s)
Proteína Morfogenética Ósea 2/genética , Anomalías Craneofaciales/genética , Discapacidades del Desarrollo/genética , Enanismo/genética , Haploinsuficiencia/genética , Cardiopatías Congénitas/genética , Animales , Huesos/embriología , Niño , Preescolar , Cromosomas Humanos Par 20/genética , Fisura del Paladar/genética , Modelos Animales de Enfermedad , Femenino , Corazón/embriología , Humanos , Lactante , Masculino , Ratones , Ratones Noqueados , Factor de Crecimiento Transformador beta/genética
3.
Immunity ; 30(6): 875-87, 2009 Jun 19.
Artículo en Inglés | MEDLINE | ID: mdl-19501000

RESUMEN

NLRP3 nucleates the inflammasome, a protein complex responsible for cleavage of prointerleukin-1beta (IL-1beta) to its active form. Mutations in the NLRP3 gene cause the autoinflammatory disease spectrum cryopyrin-associated periodic syndromes (CAPS). The central role of IL-1beta in CAPS is supported by the response to IL-1-targeted therapy. We developed two Nlrp3 mutant knockin mouse strains to model CAPS to examine the role of other inflammatory mediators and adaptive immune responses in an innate immune-driven disease. These mice had systemic inflammation and poor growth, similar to some human CAPS patients, and demonstrated early mortality, primarily mediated by myeloid cells. Mating these mutant mice to various gene mutant backgrounds showed that the mouse disease phenotype required an intact inflammasome, was only partially dependent on IL-1beta, and was independent of T cells. These data suggest that CAPS are true inflammasome-mediated diseases and provide insight for more common inflammatory disorders.


Asunto(s)
Proteínas Portadoras/metabolismo , Citocinas/metabolismo , Inmunidad Innata/genética , Inflamación/genética , Interleucina-1beta/metabolismo , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Citocinas/inmunología , Modelos Animales de Enfermedad , Técnicas de Sustitución del Gen , Inmunidad Activa , Inflamación/inmunología , Interleucina-18 , Interleucina-1beta/inmunología , Ratones , Ratones Mutantes , Mutación/genética , Mutación/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR , Linfocitos T/inmunología , Linfocitos T/metabolismo
4.
J Immunol ; 189(6): 2707-11, 2012 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-22904305

RESUMEN

IL-6 is a known downstream target of IL-1ß and is consistently increased in serum from patients with NLRP3 inflammasome-mediated conditions. Therefore, IL-6 could be a therapeutic target in the treatment of IL-1ß-provoked inflammation. IL-6 was increased in serum with accompanying neutrophilia in tissues of an inducible mouse model of Muckle-Wells syndrome. However, an IL-6-null background failed to provide phenotypic rescue and did not significantly impact inflammatory cytokine levels. In a second model of IL-1ß-driven inflammation, NLRP3 activation by monosodium urate crystals similarly increased IL-6. Consistent with our Muckle-Wells syndrome model, ablation of IL-6 did not impact an acute neutrophilic response in this in vivo evaluation of gouty arthritis. Taken together, our results indicate that IL-6 is a reliable marker of inflammation, with no direct role in inflammasome-mediated disease.


Asunto(s)
Proteínas Portadoras , Modelos Animales de Enfermedad , Inflamasomas , Mediadores de Inflamación/metabolismo , Interleucina-6/metabolismo , Animales , Biomarcadores/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Portadoras/fisiología , Síndromes Periódicos Asociados a Criopirina/inmunología , Síndromes Periódicos Asociados a Criopirina/metabolismo , Síndromes Periódicos Asociados a Criopirina/terapia , Técnicas de Sustitución del Gen , Marcación de Gen , Inmunofenotipificación , Inflamasomas/genética , Inflamasomas/metabolismo , Inflamasomas/fisiología , Mediadores de Inflamación/fisiología , Interleucina-1beta/genética , Interleucina-1beta/fisiología , Interleucina-6/deficiencia , Interleucina-6/fisiología , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteína con Dominio Pirina 3 de la Familia NLR , Reproducibilidad de los Resultados
5.
J Clin Invest ; 132(12)2022 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-35511419

RESUMEN

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder whose most debilitating pathology is progressive and cumulative heterotopic ossification (HO) of skeletal muscles, ligaments, tendons, and fascia. FOP is caused by mutations in the type I BMP receptor gene ACVR1, which enable ACVR1 to utilize its natural antagonist, activin A, as an agonistic ligand. The physiological relevance of this property is underscored by the fact that HO in FOP is exquisitely dependent on activation of FOP-mutant ACVR1 by activin A, an effect countered by inhibition of anti-activin A via monoclonal antibody treatment. Hence, we surmised that anti-ACVR1 antibodies that block activation of ACVR1 by ligands should also inhibit HO in FOP and provide an additional therapeutic option for this condition. Therefore, we generated anti-ACVR1 monoclonal antibodies that block ACVR1's activation by its ligands. Surprisingly, in vivo, these anti-ACVR1 antibodies stimulated HO and activated signaling of FOP-mutant ACVR1. This property was restricted to FOP-mutant ACVR1 and resulted from anti-ACVR1 antibody-mediated dimerization of ACVR1. Conversely, wild-type ACVR1 was inhibited by anti-ACVR1 antibodies. These results uncover an additional property of FOP-mutant ACVR1 and indicate that anti-ACVR1 antibodies should not be considered as therapeutics for FOP.


Asunto(s)
Miositis Osificante , Osificación Heterotópica , Receptores de Activinas Tipo I/genética , Receptores de Activinas Tipo I/farmacología , Anticuerpos/inmunología , Humanos , Ligandos , Mutación , Miositis Osificante/genética , Osificación Heterotópica/genética , Osificación Heterotópica/patología , Transducción de Señal/genética
6.
Bone ; 138: 115473, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32553795

RESUMEN

Heterotopic ossification (HO), the formation of ectopic bone in soft tissues, has been extensively studied in its two primary forms: post-traumatic HO (tHO) typically found in patients who have experienced musculoskeletal or neurogenic injury and in fibrodysplasia ossificans progressiva (FOP), where it is genetically driven. Given that in both diseases HO arises via endochondral ossification, the molecular mechanisms behind both diseases have been postulated to be manifestations of similar pathways including those activated by BMP/TGFß superfamily ligands. A significant step towards understanding the molecular mechanism by which HO arises in FOP was the discovery that FOP causing ACVR1 variants trigger HO in response to activin A, a ligand that does not activate signaling from wild type ACVR1, and that is not inherently osteogenic in wild type settings. The physiological significance of this finding was demonstrated by showing that activin A neutralizing antibodies stop HO in two different genetically accurate mouse models of FOP. In order to explore the role of activin A in tHO, we performed single cell RNA sequencing and compared the expression of activin A as well as other BMP pathway genes in tHO and FOP HO. We show that activin A is expressed in response to injury in both settings, but by different types of cells. Given that wild type ACVR1 does not transduce signal when engaged by activin A, we hypothesized that inhibition of activin A will not block tHO. Nonetheless, as activin A was expressed in tHO lesions, we tested its inhibition and compared it with inhibition of BMPs. We show here that anti-activin A does not block tHO, whereas agents such as antibodies that neutralize ACVR1 or ALK3-Fc (which blocks osteogenic BMPs) are beneficial, though not completely curative. These results demonstrate that inhibition of activin A should not be considered as a therapeutic strategy for ameliorating tHO.


Asunto(s)
Miositis Osificante , Osificación Heterotópica , Receptores de Activinas Tipo I/genética , Activinas , Animales , Humanos , Ratones , Miositis Osificante/genética
7.
Sci Immunol ; 5(54)2020 12 18.
Artículo en Inglés | MEDLINE | ID: mdl-33443029

RESUMEN

Deficiency in interleukin-36R (IL-36R) antagonist caused by loss-of-function mutations in IL-36RN leads to DITRA (deficiency of IL-36 receptor antagonist), a rare inflammatory human disease that belongs to a subgroup of generalized pustular psoriasis (GPP). We report a functional genetic mouse model of DITRA with enhanced IL-36R signaling analogous to that observed in patients with DITRA, which provides new insight into our understanding of the IL-36 family of molecules in regulating barrier integrity across multiple tissues. Humanized DITRA-like mice displayed increased skin inflammation in a preclinical model of psoriasis, and in vivo blockade of IL-36R pathway using anti-human IL-36R antibody ameliorated imiquimod-induced skin pathology as both prophylactic and therapeutic treatments. Deeper characterization of the humanized DITRA-like mice revealed that deregulated IL-36R signaling promoted tissue pathology during intestinal injury and led to impairment in mucosal restoration in the repair phase of chronic dextran sulfate sodium (DSS)-induced colitis. Blockade of IL-36R pathway significantly ameliorated DSS-induced intestinal inflammation and rescued the inability of DITRA-like mice to recover from mucosal damage in vivo. Our results indicate a central role for IL-36 in regulating proinflammatory responses in the skin and epithelial barrier function in the intestine, suggesting a new therapeutic potential for targeting the IL-36R axis in psoriasis and at the later stages of intestinal pathology in inflammatory bowel disease.


Asunto(s)
Dermatitis/etiología , Dermatitis/metabolismo , Gastroenteritis/etiología , Gastroenteritis/metabolismo , Receptores de Interleucina-1/metabolismo , Transducción de Señal , Animales , Biomarcadores , Dermatitis/patología , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Gastroenteritis/patología , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Ratones , Piel/metabolismo , Piel/patología
8.
Biol Cell ; 100(4): 253-64, 2008 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-17995454

RESUMEN

BACKGROUND INFORMATION: Accurate sorting of proteins to the three types of secretory granules in Toxoplasma gondii is crucial for successful cell invasion by this obligate intracellular parasite. As in other eukaryotic systems, propeptide sequences are a common yet poorly understood feature of proteins destined for regulated secretion, which for Toxoplasma occurs through two distinct invasion organelles, rhoptries and micronemes. Microneme discharge during parasite apical attachment plays a pivotal role in cell invasion by delivering adhesive proteins for host receptor engagement. RESULTS: We show here that the small micronemal proprotein MIC5 (microneme protein-5) undergoes proteolytic maturation at a site beyond the Golgi, and only the processed form of MIC5 is secreted via the micronemes. Proper cleavage of the MIC5 propeptide relies on an arginine residue in the P1' position, although P1' mutants are still cleaved to a lesser extent at an alternative site downstream of the primary site. Nonetheless, this aberrantly cleaved species still correctly traffics to the micronemes, indicating that correct cleavage is not necessary for micronemal targeting. In contrast, a deletion mutant lacking the propeptide was retained within the secretory system, principally in the ER (endoplasmic reticulum). The MIC5 propeptide also supported correct trafficking when exchanged for the M2AP propeptide, which was recently shown to also be required for micronemal trafficking of the TgMIC2 (T. gondii MIC2)-M2AP complex [Harper, Huynh, Coppens, Parussini, Moreno and Carruthers (2006) Mol. Biol. Cell 17, 4551-4563]. CONCLUSION: Our results illuminate common and unique features of micronemal propeptides in their role as trafficking facilitators.


Asunto(s)
Orgánulos/metabolismo , Proteínas Protozoarias/metabolismo , Toxoplasma/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Quelantes/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Aparato de Golgi/metabolismo , Humanos , Microscopía Electrónica , Datos de Secuencia Molecular , Plásmidos , Pliegue de Proteína , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Proteínas Protozoarias/análisis , Proteínas Protozoarias/genética , Proteínas Protozoarias/aislamiento & purificación , Vesículas Secretoras/metabolismo , Toxoplasma/ultraestructura
9.
Nat Commun ; 10(1): 3644, 2019 08 13.
Artículo en Inglés | MEDLINE | ID: mdl-31409799

RESUMEN

B cell development is a highly regulated process involving multiple differentiation steps, yet many details regarding this pathway remain unknown. Sequencing of patients with B cell-restricted immunodeficiency reveals autosomal dominant mutations in TOP2B. TOP2B encodes a type II topoisomerase, an essential gene required to alleviate topological stress during DNA replication and gene transcription, with no previously known role in B cell development. We use Saccharomyces cerevisiae, and knockin and knockout murine models, to demonstrate that patient mutations in TOP2B have a dominant negative effect on enzyme function, resulting in defective proliferation, survival of B-2 cells, causing a block in B cell development, and impair humoral function in response to immunization.


Asunto(s)
Linfocitos B/inmunología , ADN-Topoisomerasas de Tipo II/genética , Enfermedades de Inmunodeficiencia Primaria/enzimología , Animales , Diferenciación Celular , ADN-Topoisomerasas de Tipo II/inmunología , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Mutación , Enfermedades de Inmunodeficiencia Primaria/genética , Enfermedades de Inmunodeficiencia Primaria/inmunología , Enfermedades de Inmunodeficiencia Primaria/fisiopatología , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética
10.
Sci Immunol ; 3(29)2018 11 02.
Artículo en Inglés | MEDLINE | ID: mdl-30389797

RESUMEN

Most patients with cancer do not develop durable antitumor responses after programmed cell death protein 1 (PD-1) or programmed cell death ligand 1(PD-L1) checkpoint inhibition monotherapy because of an ephemeral reversal of T cell dysfunction and failure to promote long-lasting immunological T cell memory. Activating costimulatory pathways to induce stronger T cell activation may improve the efficacy of checkpoint inhibition and lead to durable antitumor responses. We performed single-cell RNA sequencing of more than 2000 tumor-infiltrating CD8+ T cells in mice receiving both PD-1 and GITR (glucocorticoid-induced tumor necrosis factor receptor-related protein) antibodies and found that this combination synergistically enhanced the effector function of expanded CD8+ T cells by restoring the balance of key homeostatic regulators CD226 and T cell immunoreceptor with Ig and ITIM domains (TIGIT), leading to a robust survival benefit. Combination therapy decreased CD8+ T cell dysfunction and induced a highly proliferative precursor effector memory T cell phenotype in a CD226-dependent manner. PD-1 inhibition rescued CD226 activity by preventing PD-1-Src homology region 2 (SHP2) dephosphophorylation of the CD226 intracellular domain, whereas GITR agonism decreased TIGIT expression. Unmasking the molecular pathways driving durable antitumor responses will be essential to the development of rational approaches to optimizing cancer immunotherapy.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Proteína Relacionada con TNFR Inducida por Glucocorticoide/inmunología , Memoria Inmunológica/inmunología , Inmunoterapia , Neoplasias/terapia , Receptor de Muerte Celular Programada 1/inmunología , Animales , Antígenos de Diferenciación de Linfocitos T/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/patología , Femenino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias/inmunología , Fenotipo
12.
Sci Rep ; 6: 23204, 2016 Mar 16.
Artículo en Inglés | MEDLINE | ID: mdl-26979938

RESUMEN

The expansion of a hexanucleotide (GGGGCC) repeat in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Both the function of C9ORF72 and the mechanism by which the repeat expansion drives neuropathology are unknown. To examine whether C9ORF72 haploinsufficiency induces neurological disease, we created a C9orf72-deficient mouse line. Null mice developed a robust immune phenotype characterized by myeloid expansion, T cell activation, and increased plasma cells. Mice also presented with elevated autoantibodies and evidence of immune-mediated glomerulonephropathy. Collectively, our data suggest that C9orf72 regulates immune homeostasis and an autoimmune response reminiscent of systemic lupus erythematosus (SLE) occurs in its absence. We further imply that haploinsufficiency is unlikely to be the causative factor in C9ALS/FTD pathology.


Asunto(s)
Autoanticuerpos/biosíntesis , Autoinmunidad , Glomerulonefritis Membranoproliferativa/genética , Factores de Intercambio de Guanina Nucleótido/genética , Animales , Autoanticuerpos/sangre , Proteína C9orf72 , Citocinas/sangre , Femenino , Glomerulonefritis Membranoproliferativa/sangre , Glomerulonefritis Membranoproliferativa/inmunología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Activación de Linfocitos , Tejido Linfoide/patología , Macrófagos/inmunología , Masculino , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Células Plasmáticas/inmunología , Análisis de Secuencia de ARN , Transcriptoma
13.
Int J Parasitol ; 34(6): 693-701, 2004 May.
Artículo en Inglés | MEDLINE | ID: mdl-15111091

RESUMEN

Serine protease inhibitors have been implicated in viral and parasite pathogenesis through their ability to inhibit apoptosis, provide protection against digestive enzymes in the gut and dictate host range specificity. Two Kazal family serine protease inhibitors from the obligate intracellular parasite Toxoplasma gondii (TgPI-1 and TgPI-2) have been characterised previously. Here, we describe the identification and initial characterisation of a novel Kazal inhibitor, NcPI-S, from a closely related apicomplexan parasite, Neospora caninum. Unlike the multidomain inhibitors identified in T. gondii, NcPI-S is a single domain inhibitor bearing a methionine in the position (P1) that typically dictates specificity for target proteases. Based on this, NcPI-S was predicted to inhibit elastase, chymotrypsin and subtilisin. However, we found that recombinant NcPI-S inhibited subtilisin very well, with little or no activity against elastase or chymotrypsin. NcPI-S localises to the dense granules and is secreted into the parasitophorous vacuole. Finally, antibodies raised against recombinant NcPI-S recognise two polypeptides in an N. caninum lysate, one with a molecular mass approximately 11 kDa and another at approximately 20 kDa. This, along with mass spectrometry analysis of recombinant NcPI-S, suggests that the inhibitor is expressed as a dimer in the parasite.


Asunto(s)
Neospora/metabolismo , Proteínas Protozoarias/análisis , Inhibidores de Serina Proteinasa/análisis , Secuencia de Aminoácidos , Animales , Anticuerpos Antiprotozoarios/inmunología , Quimotripsina/antagonistas & inhibidores , Peso Molecular , Péptidos/inmunología , Proteínas Recombinantes/análisis , Proteínas Recombinantes/inmunología , Subtilisina/antagonistas & inhibidores , Inhibidores de Tripsina/análisis , Vacuolas/química
14.
J Clin Invest ; 123(11): 4695-705, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-24084736

RESUMEN

The inflammasome is a cytoplasmic multiprotein complex that promotes proinflammatory cytokine maturation in response to host- and pathogen-derived signals. Missense mutations in cryopyrin (NLRP3) result in a hyperactive inflammasome that drives overproduction of the proinflammatory cytokines IL-1ß and IL-18, leading to the cryopyrin-associated periodic syndromes (CAPS) disease spectrum. Mouse lines harboring CAPS-associated mutations in Nlrp3 have elevated levels of IL-1ß and IL-18 and closely mimic human disease. To examine the role of inflammasome-driven IL-18 in murine CAPS, we bred Nlrp3 mutations onto an Il18r-null background. Deletion of Il18r resulted in partial phenotypic rescue that abolished skin and visceral disease in young mice and normalized serum cytokines to a greater extent than breeding to Il1r-null mice. Significant systemic inflammation developed in aging Nlrp3 mutant Il18r-null mice, indicating that IL-1 and IL-18 drive pathology at different stages of the disease process. Ongoing inflammation in double-cytokine knockout CAPS mice implicated a role for caspase-1-mediated pyroptosis and confirmed that CAPS is inflammasome dependent. Our results have important implications for patients with CAPS and residual disease, emphasizing the need to explore other NLRP3-mediated pathways and the potential for inflammasome-targeted therapy.


Asunto(s)
Proteínas Portadoras/genética , Síndromes Periódicos Asociados a Criopirina/genética , Síndromes Periódicos Asociados a Criopirina/inmunología , Inflamasomas/genética , Inflamasomas/inmunología , Interleucina-18/biosíntesis , Interleucina-1beta/biosíntesis , Animales , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/patología , Caspasa 1/deficiencia , Caspasa 1/genética , Caspasa 1/metabolismo , Muerte Celular , Síndromes Periódicos Asociados a Criopirina/patología , Modelos Animales de Enfermedad , Humanos , Interleucina-18/sangre , Interleucina-1beta/sangre , Hígado/inmunología , Hígado/patología , Ratones , Ratones Noqueados , Ratones Mutantes , Proteínas Mutantes/genética , Mutación , Proteína con Dominio Pirina 3 de la Familia NLR , Receptores de Interleucina-1/deficiencia , Receptores de Interleucina-1/genética , Receptores de Interleucina-18/deficiencia , Receptores de Interleucina-18/genética , Piel/inmunología , Piel/patología
15.
PLoS One ; 7(4): e35979, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22558291

RESUMEN

The NLRP3 inflammasome complex is responsible for maturation of the pro-inflammatory cytokine, IL-1ß. Mutations in NLRP3 are responsible for the cryopyrinopathies, a spectrum of conditions including neonatal-onset multisystem inflammatory disease (NOMID). While excessive production of IL-1ß and systemic inflammation are common to all cryopyrinopathy disorders, skeletal abnormalities, prominently in the knees, and low bone mass are unique features of patients with NOMID. To gain insights into the mechanisms underlying skeletal abnormalities in NOMID, we generated knock-in mice globally expressing the D301N NLRP3 mutation (ortholog of D303N in human NLRP3). NOMID mice exhibit neutrophilia in blood and many tissues, including knee joints, and high levels of serum inflammatory mediators. They also exhibit growth retardation and severe postnatal osteopenia stemming at least in part from abnormally accelerated bone resorption, attended by increased osteoclastogenesis. Histologic analysis of knee joints revealed abnormal growth plates, with loss of chondrocytes and growth arrest in the central region of the epiphyses. Most strikingly, a tissue "spike" was observed in the mid-region of the growth plate in the long bones of all NOMID mice that may be the precursor to more severe deformations analogous to those observed in NOMID patients. These findings provide direct evidence linking a NOMID-associated NLRP3-activating mutation to abnormalities of postnatal skeletal growth and bone remodeling.


Asunto(s)
Desarrollo Óseo , Huesos/anomalías , Huesos/metabolismo , Proteínas Portadoras/metabolismo , Inflamación/patología , Animales , Médula Ósea/metabolismo , Médula Ósea/patología , Resorción Ósea/complicaciones , Resorción Ósea/patología , Huesos/patología , Diferenciación Celular , Fraccionamiento Celular , Linaje de la Célula , Proliferación Celular , Colágeno Tipo II/metabolismo , Síndromes Periódicos Asociados a Criopirina/complicaciones , Síndromes Periódicos Asociados a Criopirina/patología , Placa de Crecimiento/anomalías , Inflamasomas , Inflamación/complicaciones , Mediadores de Inflamación/metabolismo , Articulaciones/patología , Leucocitosis/complicaciones , Leucocitosis/patología , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR , Tamaño de los Órganos , Osteoblastos/metabolismo , Osteoblastos/patología , Osteoclastos/metabolismo , Osteoclastos/patología , Coloración y Etiquetado , Análisis de Supervivencia
16.
J Leukoc Biol ; 90(1): 37-47, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21330349

RESUMEN

Autoinflammatory syndromes are a clinically heterogeneous collection of diseases characterized by dysregulation of the innate immune system. The hereditary recurrent fever disorders were the first to be defined as autoinflammatory. Several of the responsible genes are now known to encode proteins forming multimeric complexes called inflammasomes, which are intracellular "danger sensors" that respond to a variety of different signals by activating caspase-1, responsible for cleavage and subsequent release of bioactive IL-1ß. This discovery of the causative link between autoinflammation and IL-1ß maturation has led to a significantly improved understanding of the mechanisms of innate immunity, as well as life-altering treatments for patients. Targeting IL-1ß for the treatment of autoinflammatory syndromes is an excellent example of the power of translational research. Given the central role of inflammation in many complex multigenic diseases, these treatments may benefit larger numbers of patients in the future. Here, we review current treatment strategies of autoinflammatory diseases with a focus on IL-1 antagonism.


Asunto(s)
Enfermedades Autoinflamatorias Hereditarias/inmunología , Inflamasomas/inmunología , Interleucina-1beta/inmunología , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Proteínas Portadoras/metabolismo , Enfermedades Autoinflamatorias Hereditarias/genética , Enfermedades Autoinflamatorias Hereditarias/patología , Humanos , Inflamasomas/genética , Inflamasomas/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR
17.
PLoS One ; 4(7): e6147, 2009 Jul 07.
Artículo en Inglés | MEDLINE | ID: mdl-19584923

RESUMEN

PSTPIP1 is a cytoskeleton-associated adaptor protein that links PEST-type phosphatases to their substrates. Mutations in PSTPIP1 cause PAPA syndrome (Pyogenic sterile Arthritis, Pyoderma gangrenosum, and Acne), an autoinflammatory disease. PSTPIP1 binds to pyrin and mutations in pyrin result in familial Mediterranean fever (FMF), a related autoinflammatory disorder. Since disease-associated mutations in PSTPIP1 enhance pyrin binding, PAPA syndrome and FMF are thought to share a common pathoetiology. The studies outlined here describe several new aspects of PSTPIP1 and pyrin biology. We document that PSTPIP1, which has homology to membrane-deforming BAR proteins, forms homodimers and generates membrane-associated filaments in native and transfected cells. An extended FCH (Fes-Cip4 homology) domain in PSTPIP1 is necessary and sufficient for its self-aggregation. We further show that the PSTPIP1 filament network is dependent upon an intact tubulin cytoskeleton and that the distribution of this network can be modulated by pyrin, indicating that this is a dynamic structure. Finally, we demonstrate that pyrin can recruit PSTPIP1 into aggregations (specks) of ASC, another pyrin binding protein. ASC specks are associated with inflammasome activity. PSTPIP1 molecules with PAPA-associated mutations are recruited by pyrin to ASC specks with particularly high efficiency, suggesting a unique mechanism underlying the robust inflammatory phenotype of PAPA syndrome.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/fisiología , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Células COS , Chlorocebus aethiops , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Modelos Moleculares , Mutación , Pirina
18.
Eukaryot Cell ; 5(12): 2174-83, 2006 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-16980407

RESUMEN

Limited proteolysis of proteins transiently expressed on the surface of the opportunistic pathogen Toxoplasma gondii accompanies cell invasion and facilitates parasite migration across cell barriers during infection. However, little is known about what factors influence this specialized proteolysis or how these proteolytic events are regulated. Here we show that genetic ablation of the micronemal protein MIC5 enhances the normal proteolytic processing of several micronemal proteins secreted by Toxoplasma tachyzoites. Restoring MIC5 expression by genetic complementation reversed this phenotype, as did treatment with the protease inhibitor ALLN, which was previously shown to block the activity of a hypothetical parasite surface protease called MPP2. We show that, despite its lack of obvious membrane association signals, MIC5 occupies the parasite surface during invasion in the vicinity of the proteins affected by enhanced processing. Proteolysis of other secretory proteins, including GRA1, was also enhanced in MIC5 knockout parasites, indicating that the phenotype is not strictly limited to proteins derived from micronemes. Together, our findings suggest that MIC5 either directly regulates MPP2 activity or it influences MPP2's ability to access substrate cleavage sites on the parasite surface.


Asunto(s)
Epítopos Inmunodominantes/genética , Epítopos Inmunodominantes/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Toxoplasma/genética , Toxoplasma/metabolismo , Animales , Secuencia de Bases , ADN Protozoario/genética , Eliminación de Gen , Genes Protozoarios , Prueba de Complementación Genética , Toxoplasma/patogenicidad , Virulencia/genética
19.
J Cell Sci ; 116(Pt 22): 4675-85, 2003 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-14576360

RESUMEN

Proteins destined for the mitochondria travel an intricate pathway through two membranes, each with its own receptors and channels. These proteins interact with receptors via N-terminal presequences that form amphipathic helices. Generally, these helices contain abundant positive charges on one face and hydrophobic residues on the other, but share little primary sequence homology. While extensive research on mitochondrial import has been done in yeast and mammalian cells, little is known about import or contents of the single mitochondrion of Toxoplasma gondii, a parasite in the phylum Apicomplexa. We describe here the characterization of TgSODB2, a novel, mitochondrial superoxide dismutase in T. gondii with an unusual targeting sequence consisting of a hydrophobic segment resembling a signal peptide, followed by a presequence. We show that although the hydrophobic segment is competent to target a reporter protein to the secretory system, it is prevented from directing ER translocation when coupled with the presequence. When we mutated the only charged residue in the hydrophobic sequence, ER translocation is restored and the reporter targeted to the apicoplast, a chloroplast-like organelle found in most apicomplexans. The presequence that follows is predicted to form an amphipathic helix, but targeted the cytoplasm when the hydrophobic peptide is removed. In addition to having an unusual targeting sequence, TgSODB2 is only the second mitochondrially imported, iron-containing SOD to be described.


Asunto(s)
Mitocondrias/metabolismo , Superóxido Dismutasa/metabolismo , Toxoplasma/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico , Células Cultivadas , Clonación Molecular , Retículo Endoplásmico/metabolismo , Epidermis/metabolismo , Etiquetas de Secuencia Expresada , Fibroblastos/metabolismo , Humanos , Datos de Secuencia Molecular , Mutación , Orgánulos/metabolismo , Unión Proteica , Señales de Clasificación de Proteína/genética , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Superóxido Dismutasa/genética
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