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
The clinical efficacy of selective kinase inhibitors suggests that some cancer cells may become dependent on a single oncogene for survival. RNAi has been increasingly used to understand such "oncogene addiction" and validate new therapeutic targets. However, RNAi approaches suffer from significant off-target effects that limit their utility. Here, we combine carefully titrated lentiviral-mediated short hairpin RNA knockdown of the epidermal growth factor receptor (EGFR) with heterologous reconstitution by EGFR mutants to rigorously analyze the structural features and signaling activities that determine addiction to the mutationally activated EGFR in human lung cancer cells. EGFR dependence is differentially rescued by distinct EGFR variants and oncogenic mutants, is critically dependent on its heterodimerization partner ErbB-3, and surprisingly, does not require autophosphorylation sites in the cytoplasmic domain. Quantitative "oncogene rescue" analysis allows mechanistic dissection of oncogene addiction, and, when broadly applied, may provide functional validation for potential therapeutic targets identified through large-scale RNAi screens.
Assuntos
Receptores ErbB/antagonistas & inibidores , Neoplasias Pulmonares/genética , Modelos Biológicos , Oncogenes/fisiologia , Interferência de RNA , Linhagem Celular Tumoral , Receptores ErbB/genética , Humanos , Neoplasias Pulmonares/patologia , Mutação , Oncogenes/genética , RNA Interferente Pequeno/farmacologia , Receptor ErbB-3RESUMO
Neural induction is the first fundamental step in nervous system formation. During development, a tightly regulated niche modulates transient extracellular signals to influence neural lineage commitment. To date, however, the cascade of molecular events that sustain these signals in humans is not well understood. Here we show that NPTX1, a secreted protein, is rapidly upregulated during neural induction from human pluripotent stem cells (hPSCs). By manipulating its expression, we were able to reduce or initiate neural lineage commitment. A time-course transcriptome analysis and functional assays show that NPTX1 acts in part by binding the Nodal receptor cofactor TDGF1, reducing both Nodal and BMP signaling. Our findings identify one of the earliest genes expressed upon neural induction and provide insight into human neural lineage specification.
Assuntos
Proteína C-Reativa/metabolismo , Linhagem da Célula , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Proteína C-Reativa/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas do Tecido Nervoso/genética , Células-Tronco Neurais/citologia , Neurogênese , Ligação Proteica , Transcriptoma , Regulação para CimaRESUMO
An essential aspect of stem cell culture is the successful maintenance of the undifferentiated state. Many types of stem cells are FGF2 dependent, and pluripotent stem cells are maintained by replacing FGF2-containing media daily, while tissue-specific stem cells are typically fed every 3rd day. Frequent feeding, however, results in significant variation in growth factor levels due to FGF2 instability, which limits effective maintenance due to spontaneous differentiation. We report that stabilization of FGF2 levels using controlled release PLGA microspheres improves expression of stem cell markers, increases stem cell numbers and decreases spontaneous differentiation. The controlled release FGF2 additive reduces the frequency of media changes needed to maintain stem cell cultures, so that human embryonic stem cells and induced pluripotent stem cells can be maintained successfully with biweekly feedings.