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This study reports the following: (1) frequency of TP53 comutation within each component of the European LeukemiaNet 2022 acute myeloid leukemia risk classification, (2) relevance of TP53 mutated variant allelic fraction <10%, (3) prognostic impact of -7, -5/del(5q), -17/abn(17p), complex karyotype/monosomal karyotype, or myelodysplasia-related gene mutations with/without mutated TP53.
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Leucemia Mieloide Aguda , Síndromes Mielodisplásicos , Humanos , Leucemia Mieloide Aguda/genética , Mutación , Cariotipo Anormal , Síndromes Mielodisplásicos/genética , Pronóstico , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Randomized trials in acute myeloid leukemia (AML) have demonstrated improved survival by the BCL-2 inhibitor venetoclax combined with azacitidine in older patients, and clinical trials are actively exploring the role of venetoclax in combination with intensive chemotherapy in fitter patients with AML. As most patients still develop recurrent disease, improved understanding of relapse mechanisms is needed. We find that 17% of patients relapsing after venetoclax-based therapy for AML have acquired inactivating missense or frameshift/nonsense mutations in the apoptosis effector gene BAX. In contrast, such variants were rare after genotoxic chemotherapy. BAX variants arose within either leukemic or preleukemic compartments, with multiple mutations observed in some patients. In vitro, AML cells with mutated BAX were competitively selected during prolonged exposure to BCL-2 antagonists. In model systems, AML cells rendered deficient for BAX, but not its close relative BAK, displayed resistance to BCL-2 targeting, whereas sensitivity to conventional chemotherapy was variable. Acquired mutations in BAX during venetoclax-based therapy represent a novel mechanism of resistance to BH3-mimetics and a potential barrier to the long-term efficacy of drugs targeting BCL-2 in AML.
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Leucemia Mieloide Aguda , Proteínas Proto-Oncogénicas c-bcl-2 , Humanos , Anciano , Proteína X Asociada a bcl-2/genética , Línea Celular Tumoral , Proteínas Proto-Oncogénicas c-bcl-2/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Apoptosis , MutaciónRESUMEN
Microbial dysbiosis has emerged as a modulator of oncogenesis and response to therapy, particularly in lung cancer. Here, we investigate the evolution of the gut and lung microbiomes following exposure to a tobacco carcinogen. We performed 16S rRNA-Seq of fecal and lung samples collected prior to and at several timepoints following (nicotine-specific nitrosamine ketone/NNK) exposure in Gprc5a-/- mice that were previously shown to exhibit accelerated lung adenocarcinoma (LUAD) development following NNK exposure. We found significant progressive changes in human-relevant gut and lung microbiome members (e.g., Odoribacter, Alistipes, Akkermansia, and Ruminococus) that are closely associated with the phenotypic development of LUAD and immunotherapeutic response in human lung cancer patients. These changes were associated with decreased short-chain fatty acids (propionic acid and butyric acid) following exposure to NNK. We next sought to study the impact of Lcn2 expression, a bacterial growth inhibitor, given our previous findings on its protective role in LUAD development. Indeed, we found that the loss of Lcn2 was associated with widespread gut and lung microbiome changes at all timepoints, distinct from those observed in our Gprc5a-/- mouse model, including a decrease in abundance and diversity. Our overall findings apprise novel cues implicating microbial phenotypes in the development of tobacco-associated LUAD.
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Adenocarcinoma del Pulmón , Adenocarcinoma , Neoplasias Pulmonares , Microbiota , Nitrosaminas , Adenocarcinoma/genética , Animales , Butiratos , Carcinógenos , Disbiosis/microbiología , Inhibidores de Crecimiento , Humanos , Cetonas , Pulmón/patología , Neoplasias Pulmonares/metabolismo , Ratones , Nicotina , Propionatos , ARN Ribosómico 16S/genética , Receptores Acoplados a Proteínas G , Nicotiana/genéticaRESUMEN
Interleukin 6 is the classical member of the IL-6 family of cytokines which triggers activation of the JAK/STAT signaling cascade in cells. IL-6 is a pleiotropic cytokine that acts on many cell types and plays a critical role in immune responses, inflammation, and haematopoiesis. Our understanding of the molecular mechanisms governing IL-6 signaling has been aided by numerous studies of this signal transduction pathway, including those utilising the M1 cell line. Here we discuss the studies that we and others have undertaken using the M1 line to examine IL-6 inducible genes, particularly those targets that acts as negative regulators of signaling. Finally, we present a model for the current understanding of the IL-6 signaling pathway at a structural and mechanistic level.
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Interleucina-6/metabolismo , Macrófagos/metabolismo , Transducción de Señal , Animales , Línea Celular , Regulación de la Expresión Génica , Humanos , Modelos Biológicos , Proteínas Supresoras de la Señalización de Citocinas/metabolismoRESUMEN
Modern ischaemic stroke management involves intravenous thrombolysis followed by mechanical thrombectomy, which allows markedly higher rates of recanalization and penumbral salvage than thrombolysis alone. However, <50% of treated patients eventually enjoy independent life. It is therefore important to identify complementary therapeutic targets. In rodent models, the salvaged penumbra is consistently affected by selective neuronal loss, which may hinder recovery by interfering with plastic processes, as well as by microglial activation, which may exacerbate neuronal death. However, whether the salvaged penumbra in man is similarly affected is still unclear. Here we determined whether these two processes affect the non-infarcted penumbra in man and, if so, whether they are inter-related. We prospectively recruited patients with (i) acute middle-cerebral artery stroke; (ii) penumbra present on CT perfusion obtained <4.5 h of stroke onset; and (iii) early neurological recovery as a marker of penumbral salvage. PET with 11C-flumazenil and 11C-PK11195, as well as MRI to map the final infarct, were obtained at predefined follow-up times. The presence of selective neuronal loss and microglial activation was determined voxel-wise within the MRI normal-appearing ipsilateral non-infarcted zone and surviving penumbra masks, and their inter-relationship was assessed both across and within patients. Dilated infarct contours were consistently excluded to control for partial volume effects. Across the 16 recruited patients, there was reduced 11C-flumazenil and increased 11C-PK11195 binding in the whole ipsilateral non-infarcted zone (P = 0.04 and 0.02, respectively). Within the non-infarcted penumbra, 11C-flumazenil was also reduced (P = 0.001), but without clear increase in 11C-PK11195 (P = 0.18). There was no significant correlation between 11C-flumazenil and 11C-PK11195 in either compartment. This mechanistic study provides direct evidence for the presence of both neuronal loss and microglial activation in the ipsilateral non-infarcted zone. Further, we demonstrate the presence of neuronal loss affecting the surviving penumbra, with no or only mild microglial activation, and no significant relationship between these two processes. Thus, microglial activation may not contribute to penumbral neuronal loss in man, and its presence in the ipsilateral hemisphere may merely reflect secondary remote degeneration. Selective neuronal loss in the surviving penumbra may represent a novel therapeutic target as an adjunct to penumbral salvage to further improve functional outcome. However, microglial activation may not stand as the primary therapeutic approach. Protecting the penumbra by acutely improving perfusion and oxygenation in conjunction with thrombectomy for example, may be a better approach. 11C-flumazenil PET would be useful to monitor the effects of such therapies.
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Infarto de la Arteria Cerebral Media/fisiopatología , Microglía/fisiología , Neuronas/fisiología , Anciano , Apoptosis , Isquemia Encefálica/complicaciones , Isquemia Encefálica/diagnóstico por imagen , Femenino , Humanos , Infarto de la Arteria Cerebral Media/complicaciones , Infarto de la Arteria Cerebral Media/diagnóstico por imagen , Activación de Macrófagos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Malformaciones del Sistema Nervioso , Tomografía de Emisión de Positrones/métodosRESUMEN
PURPOSE: Mapping the ischaemic penumbra in acute stroke is of considerable clinical interest. For this purpose, mapping tissue hypoxia with (18)F-misonidazole (FMISO) PET is attractive, and is straightforward compared to (15)O PET. Given the current emphasis on penumbra imaging using diffusion/perfusion MR or CT perfusion, investigating the relationships between FMISO uptake and abnormalities with these modalities is important. METHODS: According to a prospective design, three patients (age 54-81 years; admission NIH stroke scale scores 16-22) with an anterior circulation stroke and extensive penumbra on CT- or MR-based perfusion imaging successfully completed FMISO PET, diffusion-weighted imaging and MR angiography 6-26 h after stroke onset, and follow-up FLAIR to map the final infarction. All had persistent proximal occlusion and a poor outcome despite thrombolysis. Significant FMISO trapping was defined voxel-wise relative to ten age-matched controls and mapped onto coregistered maps of the penumbra and irreversibly damaged ischaemic core. RESULTS: FMISO trapping was present in all patients (volume range 18-119 ml) and overlapped mainly with the penumbra but also with the core in each patient. There was a significant (p ≤ 0.001) correlation in the expected direction between FMISO uptake and perfusion, with a sharp FMISO uptake bend around the expected penumbra threshold. CONCLUSION: FMISO uptake had the expected overlap with the penumbra and relationship with local perfusion. However, consistent with recent animal data, our study suggests FMISO trapping may not be specific to the penumbra. If confirmed in larger samples, this preliminary finding would have potential implications for the clinical application of FMISO PET in acute ischaemic stroke.
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Isquemia Encefálica/diagnóstico por imagen , Imagen de Difusión por Resonancia Magnética , Angiografía por Resonancia Magnética , Misonidazol/análogos & derivados , Tomografía de Emisión de Positrones , Radiofármacos , Accidente Cerebrovascular/diagnóstico por imagen , Anciano , Anciano de 80 o más Años , Isquemia Encefálica/diagnóstico , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proyectos Piloto , Accidente Cerebrovascular/diagnósticoRESUMEN
After stroke, penumbral salvage determines clinical recovery. However, the rescued penumbra may be affected by selective neuronal loss, as documented both histopathologically in animals and using the validated in vivo positron emission tomography marker (11)C-flumazenil in humans. However, whether the non-infarcted penumbra is capable of neuronal activation, and how selective neuronal loss may interfere, is unknown. Here we prospectively mapped the topographical relationships between functional magnetic resonance imaging responses and non-infarcted penumbra, and tested the hypothesis that the former do take place in the latter, but only in its subsets spared selective neuronal loss. Seven patients (mean age 74 years; three thrombolysed) with first-ever acute anterior circulation stroke, presence of penumbra on computed tomography perfusion performed within 6 h of onset, and substantial deficit on admission but good outcome at 1-3 months (National Institute of Health Stroke Score range 6-13 and 0-1, respectively, P = 0.001), were studied. At follow-up, patients underwent structural magnetic resonance imaging to map the infarct, functional magnetic resonance imaging (three tasks selected to probe the right or left hemisphere), and (11)C-flumazenil positron emission tomography generating binding potential maps. Patients with significant carotid or middle-cerebral artery disease or impaired vasoreactivity were excluded. Following image coregistration, the non-infarcted penumbra comprised all acutely ischaemic voxels (identified on acute computed tomography perfusion using previously validated thresholds) not part of the final infarct. To test our hypotheses, the overlap between functional magnetic resonance imaging activation clusters and non-infarcted penumbra was mapped, and binding potential values then computed both within and outside this overlap. In addition, the overlap between functional magnetic resonance imaging activation clusters and areas of significantly reduced binding potential (determined using Statistical Parametric Mapping against 16 age-matched control subjects) was assessed in each patient. An overlap between non-infarcted penumbra and functional magnetic resonance imaging clusters was present in seven of seven patients, substantial in four. Binding potential was significantly reduced in the whole non-infarcted penumbra (P < 0.01) but not within the functional magnetic resonance imaging overlap. Clusters with significantly reduced binding potential showed virtually no overlap with functional magnetic resonance imaging activation compared with 12 age-matched controls (P = 0.04).The results from this proof of principle study suggest that 1-3 months after stroke the non-infarcted penumbra is capable of neuronal activation, consistent with its established role in recovery of neurological functions. However, although the non-infarcted penumbra as a whole was affected by selective neuronal loss, activations tended to occur within portions spared selective neuronal loss, suggesting the latter impedes neuronal activation. Although its clinical correlates are still elusive, selective neuronal loss may represent a novel therapeutic target in the aftermath of ischaemic stroke.
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Neuronas/metabolismo , Neuronas/patología , Desempeño Psicomotor/fisiología , Accidente Cerebrovascular/diagnóstico , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/patología , Estimulación Acústica/métodos , Anciano , Anciano de 80 o más Años , Recuento de Células/métodos , Muerte Celular , Femenino , Estudios de Seguimiento , Humanos , Imagen por Resonancia Magnética/métodos , Masculino , Persona de Mediana Edad , Estudios ProspectivosRESUMEN
Management of continuous focal seizure activity provides a unique challenge in palliative medicine. In cases where the patient is conscious and would prefer to remain so, we need to balance seizure-control with the sedating side effects of anti-seizure medications. Here we present a case in which subcutaneous lacosamide was given as part of a multi-modal treatment regime for a patient with continuous focal seizure activity. Lacosamide is a relatively new anti-seizure medication, that, unlike some anticonvulsants, has few drug interactions, is relatively non-sedating, and has physicochemical properties compatible with administration by the subcutaneous route. This case report adds to the very limited existing literature on the administration of lacosamide by the subcutaneous route. We conclude that lacosamide potentially provides an attractive option to contribute to the individualised care of this group of patients, and it may also have a role in the management of neuropathic pain where the enteral route is not available.
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TP53-mutant blood cancers remain a clinical challenge. BH3-mimetic drugs inhibit BCL-2 pro-survival proteins, inducing cancer cell apoptosis. Despite acting downstream of p53, functional p53 is required for maximal cancer cell killing by BH3-mimetics through an unknown mechanism. Here, we report p53 is activated following BH3-mimetic induced mitochondrial outer membrane permeabilization, leading to BH3-only protein induction and thereby potentiating the pro-apoptotic signal. TP53-deficient lymphomas lack this feedforward loop, providing opportunities for survival and disease relapse after BH3-mimetic treatment. The therapeutic barrier imposed by defects in TP53 can be overcome by direct activation of the cGAS/STING pathway, which promotes apoptosis of blood cancer cells through p53-independent BH3-only protein upregulation. Combining clinically relevant STING agonists with BH3-mimetic drugs efficiently kills TRP53/TP53-mutant mouse B lymphoma, human NK/T lymphoma, and acute myeloid leukemia cells. This represents a promising therapy regime that can be fast-tracked to tackle TP53-mutant blood cancers in the clinic.
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Apoptosis , Proteínas de la Membrana , Proteína p53 Supresora de Tumor , Proteína p53 Supresora de Tumor/genética , Humanos , Animales , Ratones , Proteínas de la Membrana/genética , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Mutación , Neoplasias Hematológicas/tratamiento farmacológico , Neoplasias Hematológicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Fragmentos de Péptidos/farmacología , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Transducción de Señal/efectos de los fármacos , Proteínas Proto-Oncogénicas/genéticaRESUMEN
Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease with a clear genetic component. While most SLE patients carry rare gene variants in lupus risk genes, little is known about their contribution to disease pathogenesis. Amongst them, SH2B3-a negative regulator of cytokine and growth factor receptor signaling-harbors rare coding variants in over 5% of SLE patients. Here, we show that unlike the variant found exclusively in healthy controls, SH2B3 rare variants found in lupus patients are predominantly hypomorphic alleles, failing to suppress IFNGR signaling via JAK2-STAT1. The generation of two mouse lines carrying patients' variants revealed that SH2B3 is important in limiting the number of immature and transitional B cells. Furthermore, hypomorphic SH2B3 was shown to impair the negative selection of immature/transitional self-reactive B cells and accelerate autoimmunity in sensitized mice, at least in part due to increased IL-4R signaling and BAFF-R expression. This work identifies a previously unappreciated role for SH2B3 in human B cell tolerance and lupus risk.
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Autoinmunidad , Lupus Eritematoso Sistémico , Animales , Humanos , Ratones , Autoinmunidad/genética , Factor Activador de Células B/metabolismo , Linfocitos B , Lupus Eritematoso Sistémico/genética , Células Precursoras de Linfocitos BRESUMEN
Protein Tyrosine Phosphatase 1B (PTP1B) is the prototypical protein tyrosine phosphatase and plays an essential role in the regulation of several kinase-driven signalling pathways. PTP1B displays a preference for bisphosphorylated substrates. Here we identify PTP1B as an inhibitor of IL-6 and show that, in vitro, it can dephosphorylate all four members of the JAK family. In order to gain a detailed understanding of the molecular mechanism of JAK dephosphorylation, we undertook a structural and biochemical analysis of the dephosphorylation reaction. We identified a product-trapping PTP1B mutant that allowed visualisation of the tyrosine and phosphate products of the reaction and a substrate-trapping mutant with a vastly decreased off-rate compared to those previously described. The latter mutant was used to determine the structure of bisphosphorylated JAK peptides bound to the enzyme active site. These structures revealed that the downstream phosphotyrosine preferentially engaged the active site, in contrast to the analogous region of IRK. Biochemical analysis confirmed this preference. In this binding mode, the previously identified second aryl binding site remains unoccupied and the non-substrate phosphotyrosine engages Arg47. Mutation of this arginine disrupts the preference for the downstream phosphotyrosine. This study reveals a previously unappreciated plasticity in how PTP1B interacts with different substrates.
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Quinasas Janus , Monoéster Fosfórico Hidrolasas , Fosfotirosina , Arginina , Sitios de UniónRESUMEN
There is considerable intersubject variability in early neurological course after anterior circulation stroke, yet the pathophysiology underlying this variability is not fully understood. Here, we hypothesize that, although not predicted by current pathophysiological models, infarction of 'non-core-non-penumbral' (i.e. clinically silent) brain tissue may nevertheless occur, and negatively influence clinical course over and above the established positive impact of penumbral salvage. In order to test this hypothesis, non-core-non-penumbral tissue was identified in two independent prospectively recruited cohorts, using computed tomography perfusion, and magnetic resonance perfusion- and diffusion-weighted imaging, respectively. Follow-up structural magnetic resonance imaging was obtained about 1 month later in all patients to map the final infarct. The volumes of both the acutely silent but eventually infarcted tissue, and the eventually non-infarcted penumbra, were determined by performing voxel-wise analysis of the acute and follow-up image sets, using previously validated perfusion thresholds. Early neurological course was expressed as change in National Institutes of Health Stroke Scale scores between the acute and 1-month assessments, relative to the acute score. The relationship between the acutely silent but eventually infarcted tissue volume and early neurological course was tested using a multivariate regression model that included the volume of non-infarcted penumbra. Thirty-four and 58 patients were recruited in the computed tomography perfusion and magnetic resonance perfusion cohorts, respectively (mean onset-to-imaging time: 136 and 156 min; 27 and 42 patients received intravenous thrombolysis, respectively). Infarction of acutely silent tissue was identified in most patients in both cohorts. Although its volume (median 0.2 and 2 ml, respectively) was much smaller than that of salvaged penumbra (59.3 and 93 ml, respectively), it was substantial in â¼10% of patients. As expected, salvaged penumbra strongly positively influenced early neurological course. Even after correcting for the latter effect in the multivariate model, infarction of acutely silent tissue independently negatively influenced early neurological course in both cohorts (P=0.018 and 0.031, respectively). This is the first systematic study to document infarction of acutely silent tissue after anterior circulation stroke, and to show that it affects a sizeable fraction of patients and has the predicted negative impact on clinical course. These findings were replicated in two independent cohorts, regardless of the perfusion imaging modality used. Preventing infarction of the tissue not initially at risk should have direct clinical benefit.
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Infarto Encefálico/etiología , Infarto Encefálico/patología , Mapeo Encefálico , Accidente Cerebrovascular/complicaciones , Anciano , Análisis de Varianza , Infarto Encefálico/diagnóstico por imagen , Estudios de Cohortes , Imagen de Difusión por Resonancia Magnética , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Modelos Estadísticos , Imagen de Perfusión , Valor Predictivo de las Pruebas , Índice de Severidad de la Enfermedad , Factores de TiempoRESUMEN
Almost all interactions and reactions that occur in living organisms involve proteins. The various biological roles of proteins include, but are not limited to, signal transduction, gene transcription, cell death, immune function, structural support, and catalysis of all the chemical reactions that enable organisms to survive. The varied roles of proteins have led to them being dubbed 'the workhorses of all living organisms'. This article discusses the functions of proteins and how protein function is studied in a laboratory setting. In this article, we begin by examining the functions of protein domains, followed by a discussion of some of the major classes of proteins based on their function. We consider protein binding in detail, which is central to protein function. We then examine how protein function can be altered through various mechanisms including post-translational modification, and changes to environment, oligomerisation and mutations. Finally, we consider a handful of the techniques employed in the laboratory to understand and measure the function of proteins.
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Procesamiento Proteico-Postraduccional , Proteínas , Unión Proteica , Proteínas/metabolismo , Transducción de SeñalRESUMEN
LNK is a member of the SH2B family of adaptor proteins and is a non-redundant regulator of cytokine signalling. Cytokines are secreted intercellular messengers that bind to specific receptors on the surface of target cells to activate the Janus Kinase-Signal Transducer and Activator of Transcription (JAK-STAT) signalling pathway. Activation of the JAK-STAT pathway leads to proliferative and often inflammatory effects, and so the amplitude and duration of signalling are tightly controlled. LNK binds phosphotyrosine residues to signalling proteins downstream of cytokines and constrains JAK-STAT signalling. Mutations in LNK have been identified in a range of haematological and inflammatory diseases due to increased signalling following the loss of LNK function. Here, we review the regulation of JAK-STAT signalling via the adaptor protein LNK and discuss the role of LNK in haematological diseases.
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The SH2B family of adaptor proteins, SH2-B, APS, and LNK are key modulators of cellular signalling pathways. Whilst SH2-B and APS have been partially structurally and biochemically characterised, to date there has been no such characterisation of LNK. Here we present two crystal structures of the LNK substrate recognition domain, the SH2 domain, bound to phosphorylated motifs from JAK2 and EPOR, and biochemically define the basis for target recognition. The LNK SH2 domain adopts a canonical SH2 domain fold with an additional N-terminal helix. Targeted analysis of binding to phosphosites in signalling pathways indicated that specificity is conferred by amino acids one- and three-residues downstream of the phosphotyrosine. Several mutations in LNK showed impaired target binding in vitro and a reduced ability to inhibit signalling, allowing an understanding of the molecular basis of LNK dysfunction in variants identified in patients with myeloproliferative disease.
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Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencias de Aminoácidos , Animales , Sitios de Unión , Cristalografía por Rayos X , Humanos , Janus Quinasa 2/química , Janus Quinasa 2/metabolismo , Janus Quinasa 3/química , Janus Quinasa 3/metabolismo , Ratones , Mutación , Trastornos Mieloproliferativos/genética , Fosfotirosina , Unión Proteica , Proteínas Proto-Oncogénicas c-kit/química , Proteínas Proto-Oncogénicas c-kit/metabolismo , Receptores de Eritropoyetina/química , Receptores de Eritropoyetina/metabolismo , Transducción de Señal , Tirosina Quinasa 3 Similar a fms/química , Tirosina Quinasa 3 Similar a fms/metabolismo , Dominios Homologos srcRESUMEN
Janus kinases (JAKs) are found constitutively associated with cytokine receptors and are present in an inactive state prior to cytokine exposure. Activating mutations of JAKs are causative for a number of leukemias, lymphomas, and myeloproliferative diseases. In particular, the JAK2V617F mutant is found in most human cases of polycythemia vera, a disease characterized by over-production of erythrocytes. The V617F mutation is found in the pseudokinase domain of JAK2 and it leads to cytokine-independent activation of the kinase, as does the orthologous mutation in other JAK-family members. The mechanism whereby this mutation hyperactivates these kinases is not well understood, primarily due to the fact that the full-length JAK proteins are difficult to produce for structural and kinetic studies. Here we have overcome this limitation to perform a series of enzymatic analyses on full-length JAK1 and its constitutively active mutant form (JAK1V658F). Consistent with previous studies, we show that the presence of the pseudokinase domain leads to a dramatic decrease in enzymatic activity with no further decrease from the presence of the FERM or SH2 domains. However, we find that the mutant kinase, in vitro, is indistinguishable from the wild-type enzyme in every measurable parameter tested: KM (ATP), KM (substrate), kcat, receptor binding, thermal stability, activation rate, dephosphorylation rate, and inhibitor affinity. These results show that the V658F mutation does not enhance the intrinsic enzymatic activity of JAK. Rather this data is more consistent with a model in which there are cellular processes and interactions that prevent JAK from being activated in the absence of cytokine and it is these constraints that are affected by disease-causing mutations.
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More than 50 cytokines signal via the JAK/STAT pathway to orchestrate hematopoiesis, induce inflammation and control the immune response. Cytokines are secreted glycoproteins that act as intercellular messengers, inducing proliferation, differentiation, growth, or apoptosis of their target cells. They act by binding to specific receptors on the surface of target cells and switching on a phosphotyrosine-based intracellular signaling cascade initiated by kinases then propagated and effected by SH2 domain-containing transcription factors. As cytokine signaling is proliferative and often inflammatory, it is tightly regulated in terms of both amplitude and duration. Here we review molecular details of the cytokine-induced signaling cascade and describe the architectures of the proteins involved, including the receptors, kinases, and transcription factors that initiate and propagate signaling and the regulatory proteins that control it.
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Citocinas/metabolismo , Quinasas Janus/metabolismo , Factores de Transcripción STAT/metabolismo , Transducción de Señal , Animales , Humanos , Modelos MolecularesRESUMEN
INTRODUCTION: Stroke is the third most common cause of death in most developed countries. It is a worldwide problem; about 4.5 million people die from stroke each year. Stroke can occur at any age, but half of all strokes occur in people aged over 70 years. About 80% of all acute strokes are ischaemic, usually resulting from thrombotic or embolic occlusion of a cerebral artery. The remainder are caused either by intracerebral or subarachnoid haemorrhage. METHODS AND OUTCOMES: We conducted a systematic review and aimed to answer the following clinical questions: What are the effects of specialised care in people with acute stroke? What are the effects of medical treatment in people with acute ischaemic stroke? What are the effects of decompressive hemicraniectomy in acute ischaemic stroke? What are the effects of surgical evacuation for intracerebral haematomas? We searched: Medline, Embase, The Cochrane Library, and other important databases up to August 2010 (Clinical Evidence reviews are updated periodically, please check our website for the most up-to-date version of this review). We included harms alerts from relevant organisations such as the US Food and Drug Administration (FDA) and the UK Medicines and Healthcare products Regulatory Agency (MHRA). RESULTS: We found 41 systematic reviews, RCTs, or observational studies that met our inclusion criteria. We performed a GRADE evaluation of the quality of evidence for interventions. CONCLUSIONS: In this systematic review we present information relating to the effectiveness and safety of the following interventions: acute reduction in blood pressure, aspirin, evacuation (early surgical evacuation, or conservative treatment), decompressive hemicraniectomy, neuroprotective agents (calcium channel blockers, citicoline, gamma-aminobutyric acid agonists, glycine antagonists, lubeluzole, magnesium, N-methyl-D-aspartate antagonists), specialised stroke care, systemic anticoagulation (heparinoids, specific thrombin inhibitors, low molecular weight heparin, oral anticoagulants, unfractionated heparin), and thrombolysis.
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Heparina , Accidente Cerebrovascular , Enfermedad Aguda , Anticoagulantes , Arterias Cerebrales , Agonistas del GABA , Humanos , Accidente Cerebrovascular/cirugía , Resultado del TratamientoRESUMEN
INTRODUCTION: Stroke is the third most common cause of death in most resource-rich countries. It is a worldwide problem; about 4.5 million people die from stroke each year. Stroke can occur at any age, but half of all strokes occur in people aged over 70 years. About 80% of all acute strokes are ischaemic, usually resulting from thrombotic or embolic occlusion of a cerebral artery. The remainder are caused either by intracerebral or subarachnoid haemorrhage. METHODS AND OUTCOMES: We conducted a systematic review and aimed to answer the following clinical questions: What are the effects of specialised care in people with acute stroke? What are the effects of medical treatment in people with acute ischaemic stroke? What are the effects of surgical treatment for intracerebral haematomas? We searched: Medline, Embase, The Cochrane Library, and other important databases up to June 2007 (BMJ Clinical Evidence reviews are updated periodically, please check our website for the most up-to-date version of this review). We included harms alerts from relevant organisations such as the US Food and Drug Administration (FDA) and the UK Medicines and Healthcare products Regulatory Agency (MHRA). RESULTS: We found 42 systematic reviews, RCTs, or observational studies that met our inclusion criteria. We performed a GRADE evaluation of the quality of evidence for interventions. CONCLUSIONS: In this systematic review we present information relating to the effectiveness and safety of the following interventions: acute reduction in blood pressure, aspirin, evacuation (early surgical evacuation, or conservative treatment), neuroprotective agents (calcium channel antagonists, citicoline, gamma-aminobutyric acid agonists, glycine antagonists, lubeluzole, magnesium, N-methyl-D-aspartate antagonists, tirilazad), specialised stroke care, systemic anticoagulation (heparinoids, low or specific thrombin inhibitors, molecular weight heparin, oral anticoagulants, unfractionated heparin), and thrombolysis.