RESUMO
Ptpn6 is a cytoplasmic phosphatase that functions to prevent autoimmune and interleukin-1 (IL-1) receptor-dependent, caspase-1-independent inflammatory disease. Conditional deletion of Ptpn6 in neutrophils (Ptpn6∆PMN) is sufficient to initiate IL-1 receptor-dependent cutaneous inflammatory disease, but the source of IL-1 and the mechanisms behind IL-1 release remain unclear. Here, we investigate the mechanisms controlling IL-1α/ß release from neutrophils by inhibiting caspase-8-dependent apoptosis and Ripk1-Ripk3-Mlkl-regulated necroptosis. Loss of Ripk1 accelerated disease onset, whereas combined deletion of caspase-8 and either Ripk3 or Mlkl strongly protected Ptpn6∆PMN mice. Ptpn6∆PMN neutrophils displayed increased p38 mitogen-activated protein kinase-dependent Ripk1-independent IL-1 and tumor necrosis factor production, and were prone to cell death. Together, these data emphasize dual functions for Ptpn6 in the negative regulation of p38 mitogen-activated protein kinase activation to control tumor necrosis factor and IL-1α/ß expression, and in maintaining Ripk1 function to prevent caspase-8- and Ripk3-Mlkl-dependent cell death and concomitant IL-1α/ß release.
Assuntos
Apoptose/imunologia , Caspase 8/imunologia , Neutrófilos/imunologia , Proteínas Quinases/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/imunologia , Animais , Caspase 8/genética , Células Cultivadas , Deleção de Genes , Inflamação/imunologia , Interleucina-1/imunologia , Interleucina-1alfa/metabolismo , Interleucina-1beta/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Receptores Tipo I de Interleucina-1/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Signal transducer and activator of transcription (STAT) proteins act downstream of cytokine receptors to facilitate changes in gene expression that impact a range of developmental and homeostatic processes. Patients harbouring loss-of-function (LOF) STAT5B mutations exhibit postnatal growth failure due to lack of responsiveness to growth hormone as well as immune perturbation, a disorder called growth hormone insensitivity syndrome with immune dysregulation 1 (GHISID1). This study aimed to generate a zebrafish model of this disease by targeting the stat5.1 gene using CRISPR/Cas9 and characterising the effects on growth and immunity. The zebrafish Stat5.1 mutants were smaller, but exhibited increased adiposity, with concomitant dysregulation of growth and lipid metabolism genes. The mutants also displayed impaired lymphopoiesis with reduced T cells throughout the lifespan, along with broader disruption of the lymphoid compartment in adulthood, including evidence of T cell activation. Collectively, these findings confirm that zebrafish Stat5.1 mutants mimic the clinical impacts of human STAT5B LOF mutations, establishing them as a model of GHISID1.
Assuntos
Síndrome de Laron , Peixe-Zebra , Animais , Humanos , Peixe-Zebra/genética , Fator de Transcrição STAT5/genética , Síndrome de Laron/genética , Mutação , Hormônio do Crescimento/genéticaRESUMO
A current paradigm proposes that mitochondrial damage is a critical determinant of NLRP3 inflammasome activation. Here, we genetically assess whether mitochondrial signalling represents a unified mechanism to explain how NLRP3 is activated by divergent stimuli. Neither co-deletion of the essential executioners of mitochondrial apoptosis BAK and BAX, nor removal of the mitochondrial permeability transition pore component cyclophilin D, nor loss of the mitophagy regulator Parkin, nor deficiency in MAVS affects NLRP3 inflammasome function. In contrast, caspase-8, a caspase essential for death-receptor-mediated apoptosis, is required for efficient Toll-like-receptor-induced inflammasome priming and cytokine production. Collectively, these results demonstrate that mitochondrial apoptosis is not required for NLRP3 activation, and highlight an important non-apoptotic role for caspase-8 in regulating inflammasome activation and pro-inflammatory cytokine levels.
Assuntos
Proteínas de Transporte/biossíntese , Caspase 8/biossíntese , Inflamassomos/metabolismo , Mitocôndrias/metabolismo , Apoptose/genética , Autofagia/genética , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Proteínas de Transporte/genética , Caspase 8/genética , Células Cultivadas , Peptidil-Prolil Isomerase F , Ciclofilinas/antagonistas & inibidores , Ciclofilinas/genética , Humanos , Interleucina-1beta/biossíntese , Mitocôndrias/patologia , Mitofagia/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR , Receptores Toll-Like/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Cytokine-inducible SH2 domain-containing protein (CISH), a member of the suppressor of cytokine signaling family of negative feedback regulators, is induced by cytokines that activate STAT5 and can inhibit STAT5 signaling in vitro. However, demonstration of a definitive in vivo role for CISH during development has remained elusive. This study employed expression analysis and morpholino-mediated knockdown in zebrafish in concert with bioinformatics and biochemical approaches to investigate CISH function. Two zebrafish CISH paralogs were identified, cish.a and cish.b, with high overall conservation (43-46% identity) with their mammalian counterparts. The cish.a gene was maternally derived, with transcripts present throughout embryogenesis, and increasing at 4-5 d after fertilization, whereas cish.b expression commenced at 8 h after fertilization. Expression of cish.a was regulated by the JAK2/STAT5 pathway via conserved tetrameric STAT5 binding sites (TTCN3GAA) in its promoter. Injection of morpholinos targeting cish.a, but not cish.b or control morpholinos, resulted in enhanced embryonic erythropoiesis, myelopoiesis, and lymphopoiesis, including a 2- 3-fold increase in erythrocytic markers. This occurred concomitantly with increased activation of STAT5. This study indicates that CISH functions as a conserved in vivo target and regulator of STAT5 in the control of embryonic hematopoiesis.
Assuntos
Embrião não Mamífero/imunologia , Hematopoese/imunologia , Fator de Transcrição STAT5/imunologia , Proteínas Supressoras da Sinalização de Citocina/imunologia , Proteínas de Peixe-Zebra/imunologia , Peixe-Zebra/imunologia , Animais , Sequência de Bases , Hematopoese/genética , Janus Quinase 2/genética , Janus Quinase 2/imunologia , Dados de Sequência Molecular , Fator de Transcrição STAT5/genética , Proteínas Supressoras da Sinalização de Citocina/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Suppressor of cytokine signaling 1 (SOCS1) has been shown to play important roles in the immune system. It acts as a key negative regulator of signaling via receptors for IFNs and other cytokines controlling T cell development, as well as Toll receptor signaling in macrophages and other immune cells. To gain further insight into SOCS1, we have identified and characterized the zebrafish socs1 gene, which exhibited sequence and functional conservation with its mammalian counterparts. Initially maternally derived, the socs1 gene showed early zygotic expression in mesodermal structures, including the posterior intermediate cell mass, a site of primitive hematopoiesis. At later time points, expression was seen in a broad anterior domain, liver, notochord, and intersegmental vesicles. Morpholino-mediated knockdown of socs1 resulted in perturbation of specific hematopoietic populations prior to the commencement of lymphopoiesis, ruling out T cell involvement. However, socs1 knockdown also lead to a reduction in the size of the developing thymus later in embryogenesis. Zebrafish SOCS1 was shown to be able to interact with both zebrafish Jak2a and Stat5.1 in vitro and in vivo. These studies demonstrate a conserved role for SOCS1 in T cell development and suggest a novel T cell-independent function in embryonic myelopoiesis mediated, at least in part, via its effects on receptors using the Jak2-Stat5 pathway.
Assuntos
Mielopoese , Proteínas Supressoras da Sinalização de Citocina/genética , Linfócitos T/metabolismo , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Clonagem Molecular , Embrião não Mamífero/irrigação sanguínea , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Hibridização In Situ , Dados de Sequência Molecular , Filogenia , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Tirosina Quinases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT5/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina/classificação , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/classificação , Proteínas de Peixe-Zebra/metabolismoRESUMO
The regulation of neutrophil recruitment, activation, and disposal is pivotal for circumscribed inflammation. SHP1(Y208N/Y208N) mutant mice develop severe cutaneous inflammatory disease that is IL-1R dependent. Genetic reduction in neutrophil numbers and neutrophilic responses to infection is sufficient to prevent the spontaneous initiation of this disease. Neutrophils from SHP1(Y208N/Y208N) mice display increased pro-IL-1ß production due to altered responses to MyD88-dependent and MyD88-independent signals. The IL-1R-dependent inflammatory disease in SHP1(Y208N/Y208N) mice develops independently of caspase 1 and proteinase 3 and neutrophil elastase. In response to Fas ligand, a caspase 1-independent inducer of IL-1ß production, neutrophils from SHP1(Y208N/Y208N) mice produce elevated levels of IL-1ß but display reduced caspase 3 and caspase 7 activation. In neutrophils deficient in SHP1, IL-1ß induces high levels of pro-IL-1ß suggesting the presence of a paracrine IL-1ß loop. These data indicate that the neutrophil- and IL-1-dependent disease in SHP1(Y208N/Y208N) mice is a consequence of loss of negative regulation of TLR and IL-1R signaling.
Assuntos
Mediadores da Inflamação/fisiologia , Interleucina-1beta/biossíntese , Neutrófilos/imunologia , Neutrófilos/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/fisiologia , Dermatopatias/patologia , Dermatopatias/prevenção & controle , Animais , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Doenças Autoimunes/prevenção & controle , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Humanos , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/metabolismo , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Neutrófilos/metabolismo , Comunicação Parácrina/genética , Comunicação Parácrina/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/deficiência , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Índice de Gravidade de Doença , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Dermatopatias/imunologia , Receptores Toll-Like/antagonistas & inibidores , Receptores Toll-Like/fisiologiaRESUMO
The mammalian innate immune system has evolved to recognize foreign molecules derived from pathogens via the TLRs. TLR3 and TLR4 can signal via the TIR domain-containing adapter inducing IFN-ß (TRIF), which results in the transcription of a small array of genes, including IFN-ß. Inducible NO synthase (iNOS), which catalyzes the production of NO, is induced by a range of stimuli, including cytokines and microbes. NO is a potent source of reactive nitrogen species that play an important role in killing intracellular pathogens and forms a crucial component of host defense. We have recently identified iNOS as a target of the mammalian SPSB2 protein. The SOCS box is a peptide motif, which, in conjunction with elongins B and C, recruits cullin-5 and Rbx-2 to form an active E3 ubiquitin ligase complex. In this study, we show that SPSB1 is the only SPSB family member to be regulated by the same TLR pathways that induce iNOS expression and characterize the interaction between SPSB1 and iNOS. Through the use of SPSB1 transgenic mouse macrophages and short hairpin RNA knockdown of SPSB1, we show that SPSB1 controls both the induction of iNOS and the subsequent production of NO downstream of TLR3 and TLR4. Further, we demonstrate that regulation of iNOS by SPSB1 is dependent on the proteasome. These results suggest that SPSB1 acts through a negative-feedback loop that, together with SPSB2, controls the extent of iNOS induction and NO production.
Assuntos
Regulação da Expressão Gênica/imunologia , Macrófagos/metabolismo , Óxido Nítrico Sintase Tipo II/biossíntese , Transdução de Sinais/imunologia , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Receptores Toll-Like/metabolismo , Animais , Western Blotting , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Imunoprecipitação , Macrófagos/imunologia , Camundongos , Camundongos Transgênicos , Óxido Nítrico/biossíntese , Óxido Nítrico/imunologia , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/imunologia , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/imunologia , Receptores Toll-Like/genética , Receptores Toll-Like/imunologiaRESUMO
Most severe congenital neutropenia (SCN) cases possess constitutive neutrophil elastase mutations; a smaller cohort has acquired mutations truncating the granulocyte colony-stimulating factor receptor (G-CSF-R). We have described a case with constitutive extracellular G-CSF-R mutation hyporesponsive to ligand. Here we report two independent acquired G-CSF-R truncation mutations and a novel constitutive neutrophil elastase mutation in this patient. Co-expression of a truncated receptor chain restored STAT5 signalling responses of the extracellular G-CSF-R mutant, while constitutively-active STAT5 enhanced its proliferative capacity. These data add to our knowledge of SCN and further highlight the importance of STAT5 in mediating proliferative responses to G-CSF.
Assuntos
Elastase de Leucócito/genética , Mutação/genética , Neutropenia/congênito , Receptores de Fator Estimulador de Colônias de Granulócitos/genética , Criança , Análise Mutacional de DNA , Humanos , Neutropenia/enzimologia , Neutropenia/genética , Reação em Cadeia da Polimerase , Receptores de Fator Estimulador de Colônias de Granulócitos/metabolismo , Fator de Transcrição STAT5/genética , Serina Endopeptidases/genéticaRESUMO
The complexity of multicellular organisms is dependent on systems enabling cells to respond to specific stimuli. Cytokines and their receptors are one such system, whose perturbation can lead to a variety of disease states. This review represents an overview of our current understanding of the cytokine receptors, Janus kinases (Jaks), Signal transducers and activators of transcription (Stats) and Suppressors of cytokine signaling (Socs), focussing on their contribution to diseases of an immune or hematologic nature.
Assuntos
Doenças Hematológicas/imunologia , Doenças do Sistema Imunitário/imunologia , Janus Quinases/metabolismo , Receptores de Citocinas/metabolismo , Fatores de Transcrição STAT/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Animais , Humanos , Transdução de SinaisRESUMO
OBJECTIVE: Constitutive activation of Stat5 has been observed in a variety of malignancies, particularly myeloid leukemias. To directly investigate the in vivo consequences of Stat5 perturbation, we expressed constitutively active forms in zebrafish. METHODS: We generated mutants of the zebrafish stat5.1 protein (N646H, H298R/N714F, and N714F) based on previously identified constitutively active mutants of murine Stat5a. The in vitro properties of these mutants were determined using phosphorylation-specific antibodies and luciferase reporter assays, and their in vivo effects were analyzed through microinjection of zebrafish embryos. RESULTS: Two of these stat5.1 mutants (N646H and H298R/N714F) showed increased tyrosine phosphorylation and transactivation activity compared to the wild-type protein. Expression of either mutant led to a range of hematological perturbations, which were more pronounced for the H298R/N714F mutant. Interestingly, expression of wild-type also produced generally similar phenotypes. Further analysis showed that expression of the H298R/N714F mutant led to increased numbers of early and late myeloid cells, erythrocytes, and B cells. Some nonhematopoietic developmental perturbations were also observed, but these were equally prominent with wild-type or mutant forms. CONCLUSION: These data implicate Stat5 activity as a direct critical regulator of hematological cell proliferation, suggesting a causal role for constitutively-active Stat5 in the etiology of hematological malignancies.
Assuntos
Doenças Hematológicas/genética , Células-Tronco Hematopoéticas/metabolismo , Fator de Transcrição STAT5/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Substituição de Aminoácidos , Animais , Linhagem Celular , Linhagem da Célula/fisiologia , Proliferação de Células/efeitos dos fármacos , Doenças Hematológicas/patologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Técnicas In Vitro , Mutagênese Sítio-Dirigida , Mutação , Fosforilação , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/farmacologia , Tirosina/efeitos dos fármacos , Tirosina/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/farmacologiaRESUMO
There are seven mammalian signal transducer and activator of transcription (Stat) proteins that act downstream of cytokine and growth factor receptors to mediate rapid changes in gene expression. The mammalian Stat5a and Stat5b genes show high sequence identity and lie adjacent in a head-to-head configuration next to the Stat3 gene, apparently the result of a relatively recent mammal-specific gene duplication event. We have identified and characterized two stat5 homologues that are expressed in zebrafish, named stat5.1 and stat5.2. The stat5.1 gene shows a high level of conservation with the single stat5 gene found in other teleosts and lies next to the stat3 gene, in the same relative orientation as the mammalian Stat5b gene. In contrast, the stat5.2 gene lies on a different chromosome to stat5.1 and stat3, and has diverged from the stat5 genes of other teleosts, with no apparent orthologue. Together, these data suggest that the ancestral Stat5 gene has undergone two independent gene duplication events to generate a stat5.2 paralogue in zebrafish and a Stat5a paralogue in mammals.
Assuntos
Proteínas de Ligação a DNA/genética , Duplicação Gênica , Variação Genética , Proteínas do Leite/genética , Transativadores/genética , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Sequência Conservada/genética , DNA/química , DNA/genética , DNA/isolamento & purificação , DNA Complementar/química , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Éxons , Ordem dos Genes , Genes/genética , Íntrons , Dados de Sequência Molecular , Filogenia , Mapeamento de Híbridos Radioativos , Fator de Transcrição STAT5 , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Takifugu/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
The mammalian SPRY domain- and SOCS box-containing proteins, SPSB1 to SPSB4, belong to the SOCS box family of E3 ubiquitin ligases. Substrate recognition sites for the SPRY domain are identified only for human Par-4 (ELNNNL) and for the Drosophila orthologue GUSTAVUS binding to the DEAD-box RNA helicase VASA (DINNNN). To further investigate this consensus motif, we determined the crystal structures of SPSB1, SPSB2, and SPSB4, as well as their binding modes and affinities for both Par-4 and VASA. Mutation of each of the three Asn residues in Par-4 abrogated binding to all three SPSB proteins, while changing EL to DI enhanced binding. By comparison to SPSB1 and SPSB4, the more divergent protein SPSB2 showed only weak binding to Par-4 and was hypersensitive to DI substitution. Par-4((59-77)) binding perturbed NMR resonances from a number of SPSB2 residues flanking the ELNNN binding site, including loop D, which binds the EL/DI sequence. Although interactions with the consensus peptide motif were conserved in all structures, flanking sites in SPSB2 were identified as sites of structural change. These structural changes limit high-affinity interactions for SPSB2 to aspartate-containing sequences, whereas SPSB1 and SPSB4 bind strongly to both Par-4 and VASA peptides.
Assuntos
RNA Helicases DEAD-box/química , Receptores de Trombina/química , Proteínas Supressoras da Sinalização de Citocina/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Sequência Consenso , Cristalografia por Raios X , RNA Helicases DEAD-box/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Ligação Proteica , Conformação Proteica , Receptores de Trombina/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismoRESUMO
Inducible nitric oxide (NO) synthase (iNOS; NOS2) produces NO and related reactive nitrogen species, which are critical effectors of the innate host response and are required for the intracellular killing of pathogens such as Mycobacterium tuberculosis and Leishmania major. We have identified SPRY domain-containing SOCS (suppressor of cytokine signaling) box protein 2 (SPSB2) as a novel negative regulator that recruits an E3 ubiquitin ligase complex to polyubiquitinate iNOS, resulting in its proteasomal degradation. SPSB2 interacts with the N-terminal region of iNOS via a binding interface on SPSB2 that has been mapped by nuclear magnetic resonance spectroscopy and mutational analyses. SPSB2-deficient macrophages showed prolonged iNOS expression, resulting in a corresponding increase in NO production and enhanced killing of L. major parasites. These results lay the foundation for the development of small molecule inhibitors that could disrupt the SPSB-iNOS interaction and thus prolong the intracellular lifetime of iNOS, which may be beneficial in chronic and persistent infections.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Leishmania major , Leishmaniose Cutânea/metabolismo , Macrófagos/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Regulação Enzimológica da Expressão Gênica/genética , Leishmaniose Cutânea/tratamento farmacológico , Leishmaniose Cutânea/genética , Macrófagos/parasitologia , Camundongos , Camundongos Knockout , Mycobacterium tuberculosis , Óxido Nítrico Sintase Tipo II/genética , Complexo de Endopeptidases do Proteassoma/genética , Estrutura Terciária de Proteína , Proteínas Supressoras da Sinalização de Citocina/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/genéticaRESUMO
The four mammalian SPRY (a sequence repeat in dual-specificity kinase splA and ryanodine receptors) domain-containing suppressor of cytokine signalling (SOCS) box proteins (SSB-1 to -4) are characterised by a C-terminal SOCS box and a central SPRY domain. The latter is a protein interaction module found in over 1600 proteins, with more than 70 encoded in the human genome. Here we report the crystal structure of the SPRY domain of murine SSB-2 and compare it with the SSB-2 solution structure and crystal structures of other B30.2/SPRY domain-containing family proteins. The structure is a bent beta-sandwich, consisting of two seven-stranded beta-sheets wrapped around a long loop that extends from the centre strands of the inner or concave beta-sheet; it closely matches those of GUSTAVUS and SSB-4. The structure is also similar to those of two recently determined Neuralized homology repeat (NHR) domains (also known as NEUZ domains), with detailed comparisons, suggesting that the NEUZ/NHR domains form a subclass of SPRY domains. The binding site on SSB-2 for the prostate apoptosis response-4 (Par-4) protein has been mapped in finer detail using mutational analyses. Moreover, SSB-1 was shown to have a Par-4 binding surface similar to that identified for SSB-2. Structural perturbations of SSB-2 induced by mutations affecting its interaction with Par-4 and/or c-Met have been characterised by NMR. These comparisons, in conjunction with previously published dynamics data from NMR relaxation studies and coarse-grained dynamics simulation using normal mode analysis, further refine our understanding of the structural basis for protein recognition of SPRY domain-containing proteins.
Assuntos
Proteínas de Ligação a DNA/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Análise Mutacional de DNA , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Receptores Ativados por Proteinase/metabolismo , Alinhamento de SequênciaRESUMO
OBJECTIVE: The Janus kinase 2 (JAK2) is important for embryonic primitive hematopoiesis. A gain-of-function JAK2 (JAK2(V617F)) mutation in human is pathogenetically linked to polycythemia vera (PV). In this study, we generated a zebrafish ortholog of human JAK2(V617F) (referred herewith jak2a(V581F)) by site-directed mutagenesis and examined its relevance as a model of human PV. MATERIALS AND METHODS: Zebrafish embryos at one-cell stage were injected with jak2a(V581F) mRNA (200pg/embryo). In some experiments, the embryos were treated with a specific JAK2 inhibitor, TG101209. The effects of jak2a stimulation on hematopoiesis, jak/stat signaling, and erythropoietin signaling were evaluated at 18-somites. RESULTS: Injection with jak2a(V581F) mRNA significantly increased erythropoiesis, as enumerated by flow cytometry based on gfp(+) population in dissociated Tg(gata1:gfp) embryos. The response was reduced by stat5.1 morpholino coinjection (control: 4.37% +/- 0.08%; jak2a(V581F) injected: 5.71% +/- 0.07%, coinjecting jak2a(V581F) mRNA and stat5.1 morpholino: 4.66% +/- 0.13%; p<0.01). jak2a(V581F) mRNA also upregulated gata1 (1.83 +/- 0.08 fold; p=0.005), embryonic alpha-hemoglobin (1.61 +/- 0.12 fold; p=0.049), and beta-hemoglobin gene expression (1.65 +/- 0.13-fold; p=0.026) and increased stat5 phosphorylation. These responses were also ameliorated by stat5.1 morpholino coinjection or treatment with a specific JAK2 inhibitor, TG101209. jak2a(V581F) mRNA significantly reduced erythropoietin gene (0.24 +/- 0.03 fold; p=0.006) and protein expression (control: 0.633+/-0.11; jak2a(V581F) mRNA: 0.222+/-0.07 mIU/mL; p=0.019). CONCLUSION: The zebrafish jak2a(V581F) model shared many features with human PV and might provide us with mechanistic insights of this disease.
Assuntos
Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Mutação , Policitemia Vera/patologia , Proteínas Tirosina Quinases/genética , Proteínas de Peixe-Zebra/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Modelos Animais de Doenças , Embrião não Mamífero/enzimologia , Embrião não Mamífero/metabolismo , Eritropoese/genética , Eritropoetina/genética , Eritropoetina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Janus Quinase 2/antagonistas & inibidores , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Policitemia Vera/enzimologia , Policitemia Vera/genética , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Pirimidinas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Homologia de Sequência de Aminoácidos , Sulfonamidas/farmacologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/metabolismoRESUMO
The Jak-Stat-Socs pathway is an important component of cytokine receptor signaling. Not surprisingly, perturbation of this pathway is implicated in diseases of hematopoietic and immune origin, including leukemia, lymphoma and immune deficiencies. This review examines the role of a key component of this pathway, Stat5. This has been shown to be activated in a variety of leukemias and myeloproliferative disorders, including downstream of a range of key oncogenes where it has been shown to play an important role in mediating their effects. Therefore, Stat5 represents a useful pan-leukemia/myeloproliferative disorder diagnostic marker and key therapeutic end point, as well as representing an attractive therapeutic target for these disorders.
Assuntos
Biomarcadores Tumorais/metabolismo , Leucemia/diagnóstico , Fator de Transcrição STAT5/metabolismo , HumanosRESUMO
The human RBMX gene was discovered recently through its homology to the spermatogenesis candidate gene RBMY. Its position on the human X chromosome suggests that it may be involved in X-linked mental retardation syndromes. However, to date there is scant information on the in vivo role of RBMX. To address this issue, we have isolated a zebrafish rbmx orthologue and characterized its embryonic expression pattern. Zebrafish rbmx is maternally expressed and then widely expressed in the embryo up to 24 hr postfertilization. In later stages of embryonic development, rbmx transcripts are localized predominantly in the brain, branchial arches, and liver primordium. The function of rbmx during embryonic development was examined by the use of an antisense morpholino targeting rbmx. The rbmx-morphants displayed an underdeveloped head and eyes, reduced body size, defective somite patterning, and absence of jaws. Furthermore, in the absence of functional rbmx, expression of specific markers for the fore- and hindbrain (otx2, krox20) was severely reduced. These studies demonstrate for the first time that rbmx is required for normal embryonic development, in particular of the brain, consistent with a role in X-linked mental retardation.