RESUMO
OBJECTIVES: To describe incidence, risk factors, and treatment of poststroke epilepsy (PSE) in Germany based on claims data. METHODS: Retrospective analysis of claims data from a German public sickness fund (AOK PLUS). Patients with acute stroke hospitalizations from January 01, 2011 and December 31, 2015 (index hospitalization) were followed for 12-72 months. Outcomes included incidence of PSE (patients with ≥2 seizure claims [during/after index hospitalization], or ≥1 seizure claim after index hospitalization), multivariate Cox-regression analyses of time to seizure claim and death after index stroke hospitalization discharge, and antiepileptic drug (AED) treatment. RESULTS: Among 53 883 patients with stroke (mean follow-up of 829.05 days [median 749]), 6054 (11.24%) had ≥1 seizure claim (mean age 73.95 years, 54.18% female). 2130 (35.18%) patients had a seizure claim during index hospitalization (indicative of acute symptomatic seizures). Estimated incidence of PSE (cases/1000 patient-years) was 94.49 within 1 year. Risk of seizure claim following hospital discharge was higher in patients with hemorrhagic stroke (hazard ratio [HR] =1.13; p <.001) vs those with cerebral infarction. Seizure claim during index hospitalization was a risk factor for seizure claims after hospital discharge (HR =6.97; p <.001) and early death (HR =1.78; p <.001). In the first year of follow-up, AEDs were prescribed in 73.75% of patients with seizure claims. CONCLUSIONS: Incidence of PSE was in line with previous studies. Hemorrhagic stroke and seizure claim during index hospitalization were risk factors for seizure claims after hospital discharge. Most patients with seizure claims received AED treatment.
Assuntos
Epilepsia/epidemiologia , Epilepsia/etiologia , Acidente Vascular Cerebral/complicações , Adulto , Idoso , Anticonvulsivantes/uso terapêutico , Epilepsia/tratamento farmacológico , Feminino , Alemanha/epidemiologia , Humanos , Incidência , Revisão da Utilização de Seguros , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Fatores de RiscoRESUMO
Chemically induced dimerization is an important tool in chemical biology for the analysis of protein function in cells. Here we report the use of the natural product fusicoccin (FC) to induce dimerization of 14-3-3-fused target proteins with proteins tagged to the C terminus (CT) of the H(+)-ATPase PMA2. To prevent nonproductive or detrimental interactions of the 14-3-3 proteins and CT fusions with endogenous cell proteins, their interaction surface was engineered to facilitate FC-induced dimerization exclusively between the introduced protein constructs. Live-cell imaging documented the reversible FC-induced translocation of 14-3-3 and CT to different cell compartments depending on localization sequences fused to their dimerization partner protein. The functionality of this system was demonstrated by the FC-induced importation of the NF-κB-CT into the nucleus. In HeLa cells, FC-mediated dimerization of the NF-κB-CT with a constitutively nuclear-localized 14-3-3 protein led to an NF-κB-specific cellular response by inducing IL-8 secretion.
Assuntos
Glicosídeos/farmacologia , NF-kappa B/química , Multimerização Proteica/efeitos dos fármacos , Transdução de Sinais , Proteínas 14-3-3/química , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Sítios de Ligação , Far-Western Blotting , Núcleo Celular/metabolismo , Glicosídeos/química , Células HEK293 , Células HeLa , Humanos , Interleucina-8/metabolismo , Cinética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Modelos Moleculares , Estrutura Molecular , NF-kappa B/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Fatores de TempoRESUMO
Myeloid leukemia factor 1 (MLF1) is associated with the development of leukemic diseases such as acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). However, information on the physiological function of MLF1 is limited and mostly derived from studies identifying MLF1 interaction partners like CSN3, MLF1IP, MADM, Manp and the 14-3-3 proteins. The 14-3-3-binding site surrounding S34 is one of the only known functional features of the MLF1 sequence, along with one nuclear export sequence (NES) and two nuclear localization sequences (NLS). It was recently shown that the subcellular localization of mouse MLF1 is dependent on 14-3-3 proteins. Based on these findings, we investigated whether the subcellular localization of human MLF1 was also directly 14-3-3-dependent. Live cell imaging with GFP-fused human MLF1 was used to study the effects of mutations and deletions on its subcellular localization. Surprisingly, we found that the subcellular localization of full-length human MLF1 is 14-3-3-independent, and is probably regulated by other as-yet-unknown proteins.
Assuntos
Proteínas 14-3-3/genética , Núcleo Celular/metabolismo , Proteínas/genética , Proteínas 14-3-3/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Células COS , Proteínas de Ciclo Celular , Núcleo Celular/genética , Chlorocebus aethiops , Proteínas de Ligação a DNA , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde , Células HEK293 , Humanos , Imagem Molecular , Mutagênese Sítio-Dirigida , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/metabolismo , Plasmídeos , Proteínas/metabolismo , Transdução de Sinais , TransfecçãoRESUMO
Status epilepticus is a life-threatening emergency, and to date, few studies have reported on its long-term treatment and outcomes. This study aimed to estimate the incidence, the treatment and outcomes, the healthcare resource utilization and the costs of status epilepticus in Germany. Data from 2015 to 2019 were obtained from German claims (AOK PLUS). Patients with ≥1 status epilepticus event and no event in the preceding 12 months (baseline) were included. A subgroup of patients with an epilepsy diagnosis during baseline was also analysed. Of the 2782 status epilepticus patients (mean age = 64.3 years; 52.3% female), 1585 (57.0%) were previously diagnosed with epilepsy. The age- and sex-standardized incidence was 25.5 cases/100 000 persons in 2019. The mortality rate after 12 months was 39.8% overall (19.4% and 28.2% after 30 and 90 days, respectively) and 30.4% in the epilepsy patient subgroup. Factors associated with higher mortality were age, comorbidity status, presence of brain tumours and an acute stroke. An epilepsy-related hospitalization at onset of or 7 days prior to the status epilepticus event as well as prescription of antiseizure medication during baseline was associated with a better survival rate. Overall, 71.6% of patients (85.6% in the epilepsy subgroup) were prescribed with out-patient antiseizure medication and/or rescue medication within 12 months. All patients sustained on average 1.3 status epilepticus-related hospitalizations (20.5% had more than one) during a mean follow-up period of 545.2 days (median 514 days); total direct costs including in-patient and out-patient status epilepticus treatments were 10 826 and 7701 per patient-year overall and for the epilepsy patient subgroup, respectively. The majority of status epilepticus patients received an out-patient treatment in line with epilepsy guidelines, and patients previously diagnosed with epilepsy have a higher likelihood to receive it. The mortality in the affected patient population is high; risk factors were older age, higher comorbidity burden, the presence of brain tumours or an acute stroke.
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One-third of all human cancers harbor somatic RAS mutations. This leads to aberrant activation of downstream signaling pathways involving the RAF kinases. Current ATP-competitive RAF inhibitors are active in cancers with somatic RAF mutations, such as BRAF(V600) mutant melanomas. However, they paradoxically promote the growth of RAS mutant tumors, partly due to the complex interplay between different homo- and heterodimers of A-RAF, B-RAF, and C-RAF. Based on pathway analysis and structure-guided compound identification, we describe the natural product cotylenin-A (CN-A) as stabilizer of the physical interaction of C-RAF with 14-3-3 proteins. CN-A binds to inhibitory 14-3-3 interaction sites of C-RAF, pSer233, and pSer259, but not to the activating interaction site, pSer621. While CN-A alone is inactive in RAS mutant cancer models, combined treatment with CN-A and an anti-EGFR antibody synergistically suppresses tumor growth in vitro and in vivo. This defines a novel pharmacologic strategy for treatment of RAS mutant cancers.
Assuntos
Proteínas 14-3-3/metabolismo , Produtos Biológicos/uso terapêutico , Diterpenos/uso terapêutico , Neoplasias/tratamento farmacológico , Quinases raf/metabolismo , Animais , Produtos Biológicos/farmacologia , Linhagem Celular Tumoral , Diterpenos/farmacologia , Humanos , Camundongos , Camundongos SCID , Modelos Moleculares , Neoplasias/genética , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-raf/metabolismo , Quinases raf/genética , Proteínas ras/genéticaRESUMO
C-RAF kinase is a central component of the Ras-RAF-MEK (mitogen-activated protein kinase/extracellular signal-regulated kinase)-ERK (extracellular signal-regulated kinase) pathway, which has been shown to be activated in 30% of human tumors. 14-3-3 proteins inactivate C-RAF by binding to the two N-terminal phosphorylation-dependent binding sites surrounding S233 and S259. 14-3-3 proteins can bind two target sequences located on one polypeptide chain simultaneously, thereby increasing binding affinity compared to single-site binding and possibly allowing regulated 14-3-3 binding through gatekeeper phosphorylation. To date, it was unclear whether 14-3-3 proteins can bind the two N-terminal phosphorylation-dependent binding sites of C-RAF simultaneously. Fluorescence polarization using phosphorylated peptides demonstrated that S233 is the low-affinity and S259 is the high-affinity binding site, while simultaneous engagement of both sites by 14-3-3ζ enhances affinity compared to single-site binding. Determination of a 1:1 stoichiometry for the di-phosphorylated peptide binding to one 14-3-3ζ dimer with isothermal titration calorimetry was supported by the crystal structure of the 14-3-3ζ/C-RAFpS233,pS259 complex. Cellular localization studies validate the significance of these sites for cytoplasmic retention of C-RAF, suggesting an extended mechanism of RAF regulation by 14-3-3 proteins.
Assuntos
Proteínas 14-3-3/química , Proteínas 14-3-3/metabolismo , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/metabolismo , Sítios de Ligação , Cristalografia por Raios X , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HEK293 , Humanos , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de SinaisRESUMO
Myeloid leukaemia factor 1 (MLF1) binds to 14-3-3 adapter proteins by a sequence surrounding Ser34 with the functional consequences of this interaction largely unknown. We present here the high-resolution crystal structure of this binding motif [MLF1(29-42)pSer34] in complex with 14-3-3ε and analyse the interaction with isothermal titration calorimetry. Fragment-based ligand discovery employing crystals of the binary 14-3-3ε/MLF1(29-42)pSer34 complex was used to identify a molecule that binds to the interface rim of the two proteins, potentially representing the starting point for the development of a small molecule that stabilizes the MLF1/14-3-3 protein-protein interaction. Such a compound might be used as a chemical biology tool to further analyse the 14-3-3/MLF1 interaction without the use of genetic methods. Database Structural data are available in the Protein Data Bank under the accession number(s) 3UAL [14-3-3ε/MLF1(29-42)pSer34 complex] and 3UBW [14-3-3ε/MLF1(29-42)pSer34/3-pyrrolidinol complex] Structured digital abstract ⢠14-3-3 epsilon and MLF1 bind by x-ray crystallography (View interaction) ⢠14-3-3 epsilon and MLF1 bind by isothermal titration calorimetry (View Interaction: 1, 2).
Assuntos
Proteínas 14-3-3/química , Modelos Moleculares , Estrutura Terciária de Proteína , Proteínas/química , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Sítios de Ligação/genética , Calorimetria/métodos , Proteínas de Ciclo Celular , Cristalografia por Raios X , Proteínas de Ligação a DNA , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Serina/química , Serina/genética , Serina/metabolismoRESUMO
The Ras-RAF-mitogen-activated protein kinase (Ras-RAF-MAPK) pathway is overactive in many cancers and in some developmental disorders. In one of those disorders, namely, Noonan syndrome, nine activating C-RAF mutations cluster around Ser(259), a regulatory site for inhibition by 14-3-3 proteins. We show that these mutations impair binding of 14-3-3 proteins to C-RAF and alter its subcellular localization by promoting Ras-mediated plasma membrane recruitment of C-RAF. By presenting biophysical binding data, the 14-3-3/C-RAFpS(259) crystal structure, and cellular analyses, we indicate a mechanistic link between a well-described human developmental disorder and the impairment of a 14-3-3/target protein interaction. As a broader implication of these findings, modulating the C-RAFSer(259)/14-3-3 protein-protein interaction with a stabilizing small molecule may yield a novel potential approach for treatment of diseases resulting from an overactive Ras-RAF-MAPK pathway.