RESUMO
The role of the autoimmune regulator (Aire) in central immune tolerance and thymic self-representation was first described more than 20 years ago, but fascinating new insights into its biology continue to emerge, particularly in the era of advanced single-cell genomics. We briefly describe the role of human genetics in the discovery of Aire, as well as insights into its function gained from genotype-phenotype correlations and the spectrum of Aire-associated autoimmunity-including insights from patients with Aire mutations with broad and diverse implications for human health. We then highlight emerging trends in Aire biology, focusing on three topic areas. First, we discuss medullary thymic epithelial diversity and the role of Aire in thymic epithelial development. Second, we highlight recent developments regarding the molecular mechanisms of Aire and its binding partners. Finally, we describe the rapidly evolving biology of the identity and function of extrathymic Aire-expressing cells (eTACs), and a novel eTAC subset called Janus cells, as well as their potential roles in immune homeostasis.
Assuntos
Proteína AIRE , Autoimunidade , Fatores de Transcrição , Humanos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Animais , Timo/imunologia , Timo/metabolismo , Mutação , Tolerância Imunológica , Células Epiteliais/metabolismo , Células Epiteliais/imunologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/genética , Doenças Autoimunes/metabolismoRESUMO
Mutations in the gene encoding the zinc-finger transcription factor Ikaros (IKZF1) are found in patients with immunodeficiency, leukemia, and autoimmunity. Although Ikaros has a well-established function in modulating gene expression programs important for hematopoietic development, its role in other cell types is less well defined. Here, we uncover functions for Ikaros in thymic epithelial lineage development in mice and show that Ikzf1 expression in medullary thymic epithelial cells (mTECs) is required for both autoimmune regulator-positive (Aire+) mTEC development and tissue-specific antigen (TSA) gene expression. Accordingly, TEC-specific deletion of Ikzf1 in mice results in a profound decrease in Aire+ mTECs, a global loss of TSA gene expression, and the development of autoimmunity. Moreover, Ikaros shapes thymic mimetic cell diversity, and its deletion results in a marked expansion of thymic tuft cells and muscle-like mTECs and a loss of other Aire-dependent mimetic populations. Single-cell analysis reveals that Ikaros modulates core transcriptional programs in TECs that correlate with the observed cellular changes. Our findings highlight a previously undescribed role for Ikaros in regulating epithelial lineage development and function and suggest that failed thymic central tolerance could contribute to the autoimmunity seen in humans with IKZF1 mutations.
Assuntos
Tolerância Central , Timo , Humanos , Camundongos , Animais , Diferenciação Celular , Fatores de Transcrição , Regulação da Expressão GênicaRESUMO
BACKGROUND: Eight human catalytic phosphoinositide 3-kinase (PI3K) isoforms exist which are subdivided into three classes. While class I isoforms have been well-studied in cancer, little is known about the functions of class II PI3Ks. MATERIALS AND METHODS: The expression pattern and functions of the class II PI3KC2ß isoform were investigated in a panel of tumour samples and cell lines. RESULTS: Overexpression of PI3KC2ß was found in subsets of tumours and cell lines from acute myeloid leukemia (AML), glioblastoma multiforme (GBM), medulloblastoma (MB), neuroblastoma (NB), and small cell lung cancer (SCLC). Specific pharmacological inhibitors of PI3KC2ß or RNA interference impaired proliferation of a panel of human cancer cell lines and primary cultures. Inhibition of PI3KC2ß also induced apoptosis and sensitised the cancer cells to chemotherapeutic agents. CONCLUSION: Together, these data show that PI3KC2ß contributes to proliferation and survival in AML, brain tumours and neuroendocrine tumours, and may represent a novel target in these malignancies.
Assuntos
Neoplasias Encefálicas , Neoplasias Cerebelares , Leucemia Mieloide Aguda , Tumores Neuroendócrinos , Doença Aguda , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Cerebelares/tratamento farmacológico , Neoplasias Cerebelares/genética , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Neoplasias Pulmonares , Tumores Neuroendócrinos/tratamento farmacológico , Tumores Neuroendócrinos/genética , Fosfatidilinositol 3-Quinases/metabolismoRESUMO
CD8+ T cells are critical for the immune response to pathogens and tumors, and CD8+ T cell memory protects against repeat infections. In this study, we identify the activating transcription factor 7 interacting protein (ATF7ip) as a critical regulator of CD8+ T cell immune responses. Mice with a T cell-specific deletion of ATF7ip have a CD8+ T cell-intrinsic enhancement of Il7r expression and Il2 expression leading to enhanced effector and memory responses. Chromatin immunoprecipitation sequencing studies identified ATF7ip as a repressor of Il7r and Il2 gene expression through the deposition of the repressive histone mark H3K9me3 at the Il7r gene and Il2-Il21 intergenic region. Interestingly, ATF7ip targeted transposable elements for H3K9me3 deposition at both the IL7r locus and the Il2-Il21 intergenic region, indicating that ATF7ip silencing of transposable elements is important for regulating CD8+ T cell function. These results demonstrate a new epigenetic pathway by which IL-7R and IL-2 production are constrained in CD8+ T cells, and this may open up new avenues for modulating their production.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Memória Imunológica/imunologia , Interleucina-2/biossíntese , Receptores de Interleucina-7/biossíntese , Proteínas Repressoras/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Células Cultivadas , Imunoprecipitação da Cromatina , Elementos de DNA Transponíveis/genética , Deleção de Genes , Inativação Gênica , Histonas/genética , Humanos , Interleucina-2/metabolismo , Listeria monocytogenes/imunologia , Listeriose/imunologia , Listeriose/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Interleucina-7/genética , Receptores de Interleucina-7/metabolismo , Proteínas Repressoras/genéticaRESUMO
OBJECTIVE: We conducted a comprehensive gene expression meta-analysis in dermatomyositis (DM) muscle and skin tissues to identify shared disease-relevant genes and pathways across tissues. METHODS: Six publicly available data sets from DM muscle and two from skin were identified. Meta-analysis was performed by first processing data sets individually then cross-study normalization and merging creating tissue-specific gene expression matrices for subsequent analysis. Complementary single-gene and network analyses using Significance Analysis of Microarrays (SAM) and Weighted Gene Co-expression Network Analysis (WGCNA) were conducted to identify genes significantly associated with DM. Cell-type enrichment was performed using xCell. RESULTS: There were 544 differentially expressed genes (FC ≥ 1.3, q < 0.05) in muscle and 300 in skin. There were 94 shared upregulated genes across tissues enriched in type I and II interferon (IFN) signaling and major histocompatibility complex (MHC) class I antigen-processing pathways. In a network analysis, we identified eight significant gene modules in muscle and seven in skin. The most highly correlated modules were enriched in pathways consistent with the single-gene analysis. Additional pathways uncovered by WGCNA included T-cell activation and T-cell receptor signaling. In the cell-type enrichment analysis, both tissues were highly enriched in activated dendritic cells and M1 macrophages. CONCLUSION: There is striking similarity in gene expression across DM target tissues with enrichment of type I and II IFN pathways, MHC class I antigen-processing, T-cell activation, and antigen-presenting cells. These results suggest IFN-γ may contribute to the global IFN signature in DM, and altered auto-antigen presentation through the class I MHC pathway may be important in disease pathogenesis.
RESUMO
T helper 17 cells (Th17) are critical for fighting infections at mucosal surfaces; however, they have also been found to contribute to the pathogenesis of multiple autoimmune diseases and have been targeted therapeutically. Due to the role of Th17 cells in autoimmune pathogenesis, it is important to understand the factors that control Th17 development. Here we identify the activating transcription factor 7 interacting protein (ATF7ip) as a critical regulator of Th17 differentiation. Mice with T cell-specific deletion of Atf7ip have impaired Th17 differentiation secondary to the aberrant overproduction of IL-2 with T cell receptor (TCR) stimulation and are resistant to colitis in vivo. ChIP-seq studies identified ATF7ip as an inhibitor of Il2 gene expression through the deposition of the repressive histone mark H3K9me3 in the Il2-Il21 intergenic region. These results demonstrate a new epigenetic pathway by which IL-2 production is constrained, and this may open up new avenues for modulating its production.
Assuntos
Epigênese Genética , Interleucina-2/metabolismo , Proteínas Repressoras/metabolismo , Células Th17/imunologia , Animais , Diferenciação Celular , Colite/imunologia , Colite/patologia , DNA Intergênico/genética , Histonas/metabolismo , Lisina/metabolismo , Metilação , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Repressoras/deficiência , Células Th17/citologiaRESUMO
The COPA syndrome is a monogenic, autoimmune lung and joint disorder first identified in 2015. This study sought to define the main pulmonary features of the COPA syndrome in an international cohort of patients, analyse patient responses to treatment and highlight when genetic testing should be considered. We established a cohort of subjects (N=14) with COPA syndrome seen at multiple centres including the University of California, San Francisco, CA, USA. All subjects had one of the previously established mutations in the COPA gene, and had clinically apparent lung disease and arthritis. We analysed cohort characteristics using descriptive statistics. All subjects manifested symptoms before the age of 12â years, had a family history of disease, and developed diffuse parenchymal lung disease and arthritis. 50% had diffuse alveolar haemorrhage. The most common pulmonary findings included cysts on chest computed tomography and evidence of follicular bronchiolitis on lung biopsy. All subjects were positive for anti-neutrophil cytoplasmic antibody, anti-nuclear antibody or both and 71% of subjects had rheumatoid factor positivity. All subjects received immunosuppressive therapy. COPA syndrome is an autoimmune disorder defined by diffuse parenchymal lung disease and arthritis. We analysed an international cohort of subjects with genetically confirmed COPA syndrome and found that common pulmonary features included cysts, follicular bronchiolitis and diffuse alveolar haemorrhage. Common extrapulmonary features included early age of onset, family history of disease, autoantibody positivity and arthritis. Longitudinal data demonstrated improvement on chest radiology but an overall decline in pulmonary function despite chronic treatment.
RESUMO
Thymic central tolerance is essential to preventing autoimmunity. In medullary thymic epithelial cells (mTECs), the Autoimmune regulator (Aire) gene plays an essential role in this process by driving the expression of a diverse set of tissue-specific antigens (TSAs), which are presented and help tolerize self-reactive thymocytes. Interestingly, Aire has a highly tissue-restricted pattern of expression, with only mTECs and peripheral extrathymic Aire-expressing cells (eTACs) known to express detectable levels in adults. Despite this high level of tissue specificity, the cis-regulatory elements that control Aire expression have remained obscure. Here, we identify a highly conserved noncoding DNA element that is essential for Aire expression. This element shows enrichment of enhancer-associated histone marks in mTECs and also has characteristics of being an NF-κB-responsive element. Finally, we find that this element is essential for Aire expression in vivo and necessary to prevent spontaneous autoimmunity, reflecting the importance of this regulatory DNA element in promoting immune tolerance.
Assuntos
DNA/imunologia , Tolerância Imunológica/imunologia , Sequências Reguladoras de Ácido Nucleico/imunologia , Fatores de Transcrição/imunologia , Animais , Sequência de Bases , DNA/genética , DNA/metabolismo , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células HEK293 , Humanos , Tolerância Imunológica/genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência , Dados de Sequência Molecular , NF-kappa B/imunologia , NF-kappa B/metabolismo , Ligação Proteica/imunologia , Sequências Reguladoras de Ácido Nucleico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico , Timo/citologia , Timo/imunologia , Timo/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcriptoma/genética , Transcriptoma/imunologia , Proteína AIRERESUMO
Unbiased genetic studies have uncovered surprising molecular mechanisms in human cellular immunity and autoimmunity. We performed whole-exome sequencing and targeted sequencing in five families with an apparent mendelian syndrome of autoimmunity characterized by high-titer autoantibodies, inflammatory arthritis and interstitial lung disease. We identified four unique deleterious variants in the COPA gene (encoding coatomer subunit α) affecting the same functional domain. Hypothesizing that mutant COPA leads to defective intracellular transport via coat protein complex I (COPI), we show that COPA variants impair binding to proteins targeted for retrograde Golgi-to-ER transport. Additionally, expression of mutant COPA results in ER stress and the upregulation of cytokines priming for a T helper type 17 (TH17) response. Patient-derived CD4(+) T cells also demonstrate significant skewing toward a TH17 phenotype that is implicated in autoimmunity. Our findings uncover an unexpected molecular link between a vesicular transport protein and a syndrome of autoimmunity manifested by lung and joint disease.
Assuntos
Artrite/genética , Doenças Autoimunes/genética , Proteína Coatomer/genética , Complexo de Golgi/metabolismo , Doenças Pulmonares Intersticiais/genética , Sequência de Aminoácidos , Pré-Escolar , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Células HEK293 , Humanos , Lactente , Escore Lod , Masculino , Dados de Sequência Molecular , Linhagem , Transporte ProteicoRESUMO
The maintenance of immunological tolerance requires the deletion of self-reactive T cells in the thymus. The expression of genes encoding tissue-specific antigens (TSAs) by thymic epithelial cells is critical for this process and depends on activity of the transcriptional regulator Aire; however, the molecular mechanisms Aire uses to target loci encoding TSAs are unknown. Here we identified two Aire-interacting proteins known to be involved in gene repression, ATF7ip and MBD1, that were required for Aire's targeting of loci encoding TSAs. Moreover, Mbd1(-/-) mice developed pathological autoimmunity and had a defect in Aire-dependent thymic expression of genes encoding TSAs, which underscores the importance of Aire's interaction with the ATF7ip-MBD1 protein complex in maintaining central tolerance.
Assuntos
Tolerância Central/imunologia , Proteínas de Ligação a DNA/imunologia , Regulação da Expressão Gênica/imunologia , Tolerância Imunológica , Proteínas Repressoras/imunologia , Fatores de Transcrição/imunologia , Animais , Autoantígenos/imunologia , Tolerância Central/genética , Proteínas de Ligação a DNA/genética , Citometria de Fluxo , Células HEK293 , Humanos , Immunoblotting , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Proteína AIRERESUMO
In Swiss 3T3 fibroblasts, long-term stimulation with PDGF, but not insulin-like growth factor 1 (IGF-1) or EGF, results in the establishment of an elongated migratory phenotype, characterized by the formation of retractile dendritic protrusions and absence of actin stress fibers and focal adhesion complexes. To identify receptor tyrosine kinase-specific reorganization of the Swiss 3T3 proteome during phenotypic differentiation, we compared changes in the pattern of protein synthesis and phosphorylation during long-term exposure to PDGF, IGF-1, EGF, and their combinations using 2DE-based proteomics after (35)S- and (33)P-metabolic labeling. One hundred and five differentially regulated proteins were identified by mass spectrometry and some of these extensively validated. PDGF stimulation produced the highest overall rate of protein synthesis at any given time and induced the most sustained phospho-signaling. Simultaneous activation with two or three of the growth factors revealed both synergistic and antagonistic effects on protein synthesis and expression levels with PDGF showing dominance over both IGF-1 and EGF in generating distinct proteome compositions. Using signaling pathway inhibitors, PI3K was identified as an early site for signal diversification, with sustained activity of the PI3K/AKT pathway critical for regulating late protein synthesis and phosphorylation of target proteins and required for maintaining the PDGF-dependent motile phenotype. Several proteins were identified with novel PI3K/Akt-dependent synthesis and phosphorylations including eEF2, PRS7, RACK-1, acidic calponin, NAP1L1, Hsp73, and fascin. The data also reveal induction/suppression of key F-actin and actomyosin regulators and chaperonins that enable PDGFR to direct the assembly of a motile cytoskeleton, despite simultaneous antagonistic signaling activities. Together, the study demonstrates that long-term exposure to different growth factors results in receptor tyrosine kinase-specific regulation of relatively small subproteomes, and implies that the strength and longevity of receptor tyrosine kinase-specific signals are critical in defining the composition and functional activity of the resulting proteome.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteoma/análise , Células 3T3 , Animais , Benzamidas/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Linhagem Celular , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Fibroblastos , Flavonoides/farmacologia , Marcação por Isótopo , Camundongos , Morfolinas/farmacologia , Proteína 1 de Modelagem do Nucleossomo/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Eight human catalytic phosphoinositide 3-kinase (PI3K) isoforms exist which are subdivided into three classes. While class I isoforms have been well-studied in cancer, little is known about the functions of class II PI3Ks. MATERIALS AND METHODS: The expression pattern and functions of the class II PI3KC2ß isoform were investigated in a panel of tumour samples and cell lines. RESULTS: Overexpression of PI3KC2ß was found in subsets of tumours and cell lines from acute myeloid leukemia (AML), glioblastoma multiforme (GBM), medulloblastoma (MB), neuroblastoma (NB), and small cell lung cancer (SCLC). Specific pharmacological inhibitors of PI3KC2ß or RNA interference impaired proliferation of a panel of human cancer cell lines and primary cultures. Inhibition of PI3KC2ß also induced apoptosis and sensitised the cancer cells to chemotherapeutic agents. CONCLUSION: Together, these data show that PI3KC2ß contributes to proliferation and survival in AML, brain tumours and neuroendocrine tumours, and may represent a novel target in these malignancies.
Assuntos
Neoplasias Encefálicas/patologia , Proliferação de Células , Isoenzimas/metabolismo , Leucemia Mieloide Aguda/patologia , Tumores Neuroendócrinos/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Taxa de Sobrevida , Animais , Linhagem Celular Tumoral , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Humanos , Concentração Inibidora 50 , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Inibidores de Fosfoinositídeo-3 QuinaseRESUMO
Autoimmune disease affects a significant proportion of the population. The etiology of most autoimmune diseases is largely unknown, but it is thought to be multifactorial with both environmental and genetic influences. Rare monogenic autoimmune diseases, however, offer an invaluable window into potential disease mechanisms. In this review, we will discuss the autoimmune polyglandular syndrome (APS1), the immunedysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX), and autoimmune lymphoproliferative syndrome (ALPS). Significantly, the information gained from the study of these diseases has provided new insights into more common autoimmune disease and have yielded new diagnostics and therapeutic opportunities.
Assuntos
Doenças Autoimunes/genética , Doenças Genéticas Inatas/genética , Tolerância Imunológica/genética , Doenças Autoimunes/diagnóstico , Doenças Autoimunes/imunologia , Doenças Autoimunes/terapia , Doenças Genéticas Inatas/diagnóstico , Doenças Genéticas Inatas/imunologia , Doenças Genéticas Inatas/terapia , Humanos , SíndromeRESUMO
A growing body of evidence suggests that reactive oxygen species are critical components of cell signaling pathways, in particular regulating protein phosphorylation events. Here, we show that oxidative stress in response to hydrogen peroxide treatment of human epithelial cells induces robust tyrosine phosphorylation on multiple proteins. Using an anti-phosphotyrosine purification and liquid chromatography-tandem mass spectrometry approach, we have identified many of these H(2)O(2)-induced tyrosine-phosphorylated proteins. Importantly, we show that epidermal growth factor receptor (EGFR) and Src are the primary upstream kinases mediating these events through their redox activation. The finding that many of the identified proteins have functions in cell adhesion, cell-cell junctions, and the actin cytoskeleton prompted us to examine stress-induced changes in adhesion. Immunofluorescence analysis showed that H(2)O(2) alters cell adhesion structures and the actin cytoskeleton causing loss of adhesion and apoptosis. Remarkably, these cellular changes could be attenuated by inhibition of EGFR and Src, identifying these kinases as targets to block oxidative damage. In summary, our data demonstrate that EGFR and Src together play a central role in oxidative stress-induced phosphorylation, which in turn results in loss of adhesion, morphological changes, and cell damage in epithelial cells. These data also provide a general model for redox signaling in other cell systems.
Assuntos
Apoptose/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Receptores ErbB/metabolismo , Peróxido de Hidrogênio/farmacologia , Quinases da Família src/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida , Células Epiteliais/efeitos dos fármacos , Humanos , Immunoblotting , Imunoprecipitação , Microscopia Confocal , Estresse Oxidativo/fisiologia , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Espectrometria de Massas em TandemRESUMO
The mTOR (mammalian target of rapamycin) promotes growth in response to nutrients and growth factors and is deregulated in numerous pathologies, including cancer. The mechanisms by which mTOR senses and regulates energy metabolism and cell growth are relatively well understood, whereas the molecular events underlining how it mediates survival and proliferation remain to be elucidated. Here, we describe the existence of the mTOR splicing isoform, TOR beta, which, in contrast to the full-length protein (mTOR alpha), has the potential to regulate the G(1) phase of the cell cycle and to stimulate cell proliferation. mTOR beta is an active protein kinase that mediates downstream signaling through complexing with Rictor and Raptor proteins. Remarkably, overexpression of mTOR beta transforms immortal cells and is tumorigenic in nude mice and therefore could be a proto-oncogene.
Assuntos
Proliferação de Células , Transformação Celular Neoplásica , Proteínas Quinases/metabolismo , Splicing de RNA , Animais , Ciclo Celular , Linhagem Celular , Humanos , Camundongos , Camundongos Nus , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Quinases/genética , Transporte Proteico , Proto-Oncogene Mas , Serina-Treonina Quinases TORRESUMO
A series of small molecule, ATP-competitive phosphoinositide 3-kinase inhibitors have been examined in homology models of the four class I isoforms, p110alpha, p110beta, p110delta and p110gamma. This analysis provided an insight into the mode of binding of these inhibitors to the hinge and to other key regions of the ATP binding site in each of the four subtypes. Significantly, residues were identified that differ between these proteins, and which help explain the isoform-selective inhibition profiles of the compounds.
Assuntos
Trifosfato de Adenosina/química , Aminoácidos/química , Modelos Químicos , Modelos Moleculares , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/ultraestrutura , Sítios de Ligação , Simulação por Computador , Ativação Enzimática , Inibidores Enzimáticos/química , Isoenzimas/química , Ligação Proteica , Conformação Proteica , Especificidade por SubstratoRESUMO
Phosphoinositides have crucial roles in cellular controls, many of which have been established through the use of small-molecule inhibitors. Here, we describe YM201636, a potent inhibitor of the mammalian class III phosphatidylinositol phosphate kinase PIKfyve, which synthesizes phosphatidylinositol 3,5-bisphosphate. Acute treatment of cells with YM201636 shows that the PIKfyve pathway is involved in the sorting of endosomal transport, with inhibition leading to the accumulation of a late endosomal compartment and blockade of retroviral exit. Inhibitor specificity is shown by the use of short interfering RNA against the target, as well as by rescue with the drug-resistant yeast orthologue Fab1. We concluded that the phosphatidylinositol 3,5-bisphosphate pathway is integral to endosome formation, determining morphology and cargo flux.
Assuntos
Aminopiridinas/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Inibidores Enzimáticos/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Fosfatos de Fosfatidilinositol/biossíntese , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Retroviridae/efeitos dos fármacos , Retroviridae/metabolismo , Aminopiridinas/química , Animais , Transporte Biológico/efeitos dos fármacos , Biomarcadores/metabolismo , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Inibidores Enzimáticos/química , Compostos Heterocíclicos com 3 Anéis/química , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , Células NIH 3T3RESUMO
We have previously reported the imidazo[1,2-a]pyridine derivative 4 as a novel p110alpha inhibitor; however, although 4 is a potent inhibitor of p110alpha enzymatic activity and tumor cell proliferation in vitro, it is unstable in solution and ineffective in vivo. To increase stability the pyrazole of 4 was replaced with a hydrazone and a moderately potent p110alpha inhibitor 7a was obtained. Subsequent optimization of 7a afforded exceptionally potent p110alpha inhibitors, including 8c and 8h, with IC(50) values of 0.30 nM and 0.26 nM, respectively; to the best of our knowledge, these compounds are the most potent PI3K p110alpha inhibitors reported to date. Compound 8c was also stable in solution and exhibited significant anti-tumor effectiveness in vivo.
Assuntos
Hidrazonas/química , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Piridinas/síntese química , Piridinas/farmacologia , Enxofre/química , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Humanos , Imidazóis/química , Concentração Inibidora 50 , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Camundongos , Estrutura Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/química , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/metabolismo , Piridinas/química , Relação Estrutura-Atividade , Temperatura , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
During animal cell chemotaxis, signalling at the plasma membrane induces actin polymerisation to drive forward cell movement. Since the cellular pool of actin is limited, efficient protrusion formation also requires the coordinated disassembly of pre-existing actin filaments. To search for proteins that can monitor filamentous and globular actin levels to maintain the balance of polymerisation and disassembly, we followed changes in the proteome induced by RNA interference (RNAi)-mediated alterations in actin signalling. This unbiased approach revealed an increase in the levels of an inactive, phosphorylated form of the actin-severing protein cofilin in cells unable to generate actin-based lamellipodia. Conversely, an increase in F-actin levels induced the dephosphorylation and activation of cofilin via activation of the Ssh phosphatase. Similarly, in the context of acute phosphoinositide 3-kinase (PI3K) signalling, dynamic changes in cofilin phosphorylation were found to depend on the Ssh phosphatase and on changes in lamellipodial F-actin. These results indicate that changes in the extent of cofilin phosphorylation are regulated by Ssh in response to changes in the levels and/or organisation of F-actin. Together with the recent finding that Ssh phosphatase activity is augmented by F-actin binding, these results identify Ssh-dependent regulation of phosphorylated cofilin levels as an important feedback control mechanism that maintains actin filament homeostasis during actin signalling.