RESUMO
Levels of residual structure in disordered interaction domains determine in vitro binding affinities, but whether they exert similar roles in cells is not known. Here, we show that increasing residual p53 helicity results in stronger Mdm2 binding, altered p53 dynamics, impaired target gene expression and failure to induce cell cycle arrest upon DNA damage. These results establish that residual structure is an important determinant of signaling fidelity in cells.
Assuntos
Proteínas Intrinsicamente Desordenadas/química , Proteínas Proto-Oncogênicas c-mdm2/química , Transdução de Sinais , Proteína Supressora de Tumor p53/química , Ciclo Celular/genética , Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Dano ao DNA , Escherichia coli/genética , Escherichia coli/metabolismo , Raios gama , Regulação da Expressão Gênica , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Moleculares , Mutação , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The level of the p53 transcription factor is negatively regulated by the E3 ubiquitin ligase murine double-minute clone 2 (MDM2). The interaction between p53 and MDM2 is essential for the maintenance of genomic integrity for most eukaryotes. Previous structural studies revealed that MDM2 binds to p53 transactivation domain (p53TAD) from residues 17 to 29. The K24N mutation of p53TAD changes a lysine at position 24 to an asparagine. This mutation occurs naturally in the bovine family and is also found in a rare form of human gestational cancer called choriocarcinoma. In this study, we have investigated how the K24N mutation affects the affinity, structure, and dynamics of p53TAD binding to MDM2. Nuclear magnetic resonance studies of p53TAD show that the K24N mutant is more flexible and has less transient helical secondary structure than the wild type. Isothermal titration calorimetry measurements demonstrate that these changes in structure and dynamics do not significantly change the binding affinity for p53TAD-MDM2. Finally, free-energy perturbation and standard molecular dynamic simulations suggest the negligible affinity change is due to a compensating interaction energy between the K24N mutant and the MDM2 when it is bound. Overall, the data suggest that the K24N-MDM2 complex is able to, at least partly, compensate for an increase in the conformational entropy in unbound K24N with an increase in the bound-state electrostatic interaction energy.
Assuntos
Proteínas Proto-Oncogênicas c-mdm2 , Proteína Supressora de Tumor p53 , Sequência de Aminoácidos , Calorimetria , Humanos , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Mutação/genética , Mutação/fisiologia , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-mdm2/química , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Introduction: This study evaluates trends in drug-related death cases within both Pasco and Pinellas County, Florida, from the calendar years 2011 to 2016. Specifically, it focuses on opioids and the role of fentanyl in overdose-related mortality in rural versus suburban populations. Methods: Two sets of data from each calendar year were obtained from a Medical Examiner's Office. These data were compared by year to assess differences using the nonparametric ANOVA test with the statistical software SAS, University Edition. Binary logistic regression was performed to assess which drugs occurred most frequently in the presence or absence of fentanyl. Results: There was not a significant difference in the month of the year or the day of the week that drug-related fatalities occurred. More drug-related mortalities occurred during daylight hours (e.g., 8:00 AM-4:00 PM) and more fentanyl-related mortalities occurred in Pinellas County compared to Pasco County. Fentanyl and heroin tended to co-occur in mortalities, while ethanol, hydrocodone, morphine, oxycodone, and methadone were negatively associated with fentanyl-related overdose cases. Conclusion: The characteristics of drug-related mortalities identified here may be used to better target interventions against drug abuse and overdose.