RESUMO
Sialic acid acetylesterase (SIAE) is an enzyme that negatively regulates B lymphocyte antigen receptor signalling and is required for the maintenance of immunological tolerance in mice. Heterozygous loss-of-function germline rare variants and a homozygous defective polymorphic variant of SIAE were identified in 24/923 subjects of European origin with relatively common autoimmune disorders and in 2/648 controls of European origin. All heterozygous loss-of-function SIAE mutations tested were capable of functioning in a dominant negative manner. A homozygous secretion-defective polymorphic variant of SIAE was catalytically active, lacked the ability to function in a dominant negative manner, and was seen in eight autoimmune subjects but in no control subjects. The odds ratio for inheriting defective SIAE alleles was 8.6 in all autoimmune subjects, 8.3 in subjects with rheumatoid arthritis, and 7.9 in subjects with type I diabetes. Functionally defective SIAE rare and polymorphic variants represent a strong genetic link to susceptibility in relatively common human autoimmune disorders.
Assuntos
Acetilesterase/genética , Doenças Autoimunes/enzimologia , Doenças Autoimunes/genética , Autoimunidade/genética , Hidrolases de Éster Carboxílico/genética , Predisposição Genética para Doença/genética , Mutação em Linhagem Germinativa/genética , Ácido N-Acetilneuramínico/metabolismo , Acetilação , Acetilesterase/metabolismo , Alelos , Animais , Anticorpos Antinucleares/sangue , Artrite Reumatoide/enzimologia , Artrite Reumatoide/genética , Linfócitos B/metabolismo , Biocatálise , Hidrolases de Éster Carboxílico/metabolismo , Estudos de Casos e Controles , Linhagem Celular , Diabetes Mellitus Tipo 1/enzimologia , Diabetes Mellitus Tipo 1/genética , Europa (Continente)/etnologia , Éxons/genética , Humanos , Camundongos , Razão de Chances , Polimorfismo de Nucleotídeo Único/genética , Tamanho da Amostra , Análise de Sequência de DNARESUMO
Somatic mutations altering lysine 171 of the IKBKB gene that encodes (IKKß), the critical activating kinase in canonical (NFκB) signaling, have been described in splenic marginal zone lymphomas and multiple myeloma. Lysine 171 forms part of a cationic pocket that interacts with the activation loop phosphate in the activated wild type kinase. We show here that K171E IKKß and K171T IKKß represent kinases that are constitutively active even in the absence of activation loop phosphorylation. Predictive modeling and biochemical studies establish why mutations in a positively charged residue in the cationic pocket of an activation loop phosphorylation-dependent kinase result in constitutive activation. Transcription activator-like effector nuclease-based knock-in mutagenesis provides evidence from a B lymphoid context that K171E IKKß contributes to lymphomagenesis.
Assuntos
Quinase I-kappa B , Linfoma , Mutação de Sentido Incorreto , NF-kappa B , Proteínas de Neoplasias , Transdução de Sinais/genética , Substituição de Aminoácidos , Células HeLa , Humanos , Quinase I-kappa B/imunologia , Quinase I-kappa B/metabolismo , Linfoma/genética , Linfoma/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fosforilação/genéticaRESUMO
The autosomal-recessive form of popliteal pterygium syndrome, also known as Bartsocas-Papas syndrome, is a rare, but frequently lethal disorder characterized by marked popliteal pterygium associated with multiple congenital malformations. Using Affymetrix 250K SNP array genotyping and homozygosity mapping, we mapped this malformation syndrome to chromosomal region 21q22.3. Direct sequencing of RIPK4 (receptor-interacting serine/threonine kinase protein 4) showed a homozygous transversion (c.362T>A) that causes substitution of a conserved isoleucine with asparagine at amino acid position 121 (p.Ile121Asn) in the serine/threonine kinase domain of the protein. Additional pathogenic mutations-a homozygous transition (c.551C>T) that leads to a missense substitution (p.Thr184Ile) at a conserved position and a homozygous one base-pair insertion mutation (c.777_778insA) predicted to lead to a premature stop codon (p.Arg260ThrfsX14) within the kinase domain-were observed in two families. Molecular modeling of the kinase domain showed that both the Ile121 and Thr184 positions are critical for the protein's stability and kinase activity. Luciferase reporter assays also demonstrated that these mutations are critical for the catalytic activity of RIPK4. RIPK4 mediates activation of the nuclear factor-κB (NF-κB) signaling pathway and is required for keratinocyte differentiation and craniofacial and limb development. The phenotype of Ripk4(-/-) mice is consistent with the human phenotype presented herein. Additionally, the spectrum of malformations observed in the presented families is similar, but less severe than the conserved helix-loop-helix ubiquitous kinase (CHUK)-deficient human fetus phenotype; known as Cocoon syndrome; this similarity indicates that RIPK4 and CHUK might function via closely related pathways to promote keratinocyte differentiation and epithelial growth.
Assuntos
Cromossomos Humanos Par 21/genética , Fenda Labial/genética , Fissura Palatina/genética , Mutação , Proteínas Serina-Treonina Quinases/genética , Pterígio/congênito , Adolescente , Animais , Sequência de Bases , Análise Mutacional de DNA , Feminino , Genes Recessivos , Humanos , Lactente , Recém-Nascido , Masculino , Camundongos , Dados de Sequência Molecular , NF-kappa B/metabolismo , Polimorfismo de Nucleotídeo Único , Pterígio/genética , Anormalidades da PeleRESUMO
Aptamers, due to their small size, strong target affinity, and ease of chemical modification, are ideally suited for molecular detection technologies. Here, we describe successful use of aptamer technology in a consumer device for the detection of peanut antigen in food. The novel aptamer-based protein detection method is robust across a wide variety of food matrices and sensitive to peanut protein at concentrations as low as 12.5 ppm (37.5 µg peanut protein in the sample). Integration of the assay into a sensitive, stable, and consumer friendly portable device will empower users to easily and quickly assess the presence of peanut allergens in foods before eating. With many food reactions occurring outside the home, the type of technology described here has significant potential to improve lives for children and families.
Assuntos
Alérgenos/análise , Alérgenos/imunologia , Antígenos de Plantas/análise , Antígenos de Plantas/imunologia , Aptâmeros de Nucleotídeos/metabolismo , Arachis/química , Análise de Alimentos/métodos , Hipersensibilidade Alimentar/prevenção & controle , Proteínas de Membrana/análise , Proteínas de Membrana/imunologia , Proteínas de Plantas/análise , Proteínas de Plantas/imunologia , Testes Imediatos , Humanos , Ligação Proteica , Sensibilidade e EspecificidadeRESUMO
Catalytically defective rare variants of Sialic acid Acetyl Esterase (SIAE) have previously been linked to autoimmunity. Studies presented here confirm that the M89V SIAE protein and all other products of common variant alleles of SIAE are catalytically normal. Although overexpressing transfected non-lymphoid cells secrete small amounts of SIAE that can associate with the cell surface, normal human lymphocytes do not exhibit cell surface SIAE, supporting genetic evidence in mice that indicates that this protein functions in a lymphocyte intrinsic manner. Analyses of the plasma proteome also indicate that SIAE is not secreted in vivo. A re-analysis exclusively of catalytically defective rare variant alleles of SIAE in subjects in which this gene was completely sequenced confirmed an association of SIAE with autoimmunity. A subset of catalytically defective rare variant SIAE alleles has previously been typed in a large genotyping study comparing a diverse group of disease subjects and controls; our re-analysis of this data shows that catalytically defective alleles are enriched in disease subjects. These data suggest that SIAE may be associated with autoimmunity and that further study of catalytically defective rare variant SIAE alleles in terms of autoimmune disease susceptibility is strongly warranted.