ABSTRACT
The CFIm25 subunit of the heterotetrameric cleavage factor Im (CFIm) is a critical factor in the formation of the poly(A) tail at mRNA 3' end, regulating the recruitment of polyadenylation factors, poly(A) site selection, and cleavage/polyadenylation reactions. We previously reported the homologous protein (EhCFIm25) in Entamoeba histolytica, the protozoan causing human amoebiasis, and showed the relevance of conserved Leu135 and Tyr236 residues for RNA binding. We also identified the GUUG sequence as the recognition site of EhCFIm25. To understand the interactions network that allows the EhCFIm25 to maintain its three-dimensional structure and function, here we performed molecular dynamics simulations of wild-type (WT) and mutant proteins, alone or interacting with the GUUG molecule. Our results indicated that in the presence of the GUUG sequence, WT converged more quickly to lower RMSD values in comparison with mutant proteins. However, RMSF values showed that movements of amino acids of WT and EhCFIm25*L135 T were almost identical, interacting or not with the GUUG molecule. Interestingly, EhCFIm25*L135 T, which is the only mutant with a slight RNA binding activity experimentally, presents the same stabilization of bend structures and alpha helices as WT, notably in the C-terminus. Moreover, WT and EhCFIm25*L135 T presented almost the same number of contacts that mainly involve lysine residues interacting with the G4 nucleotide. Overall, our data proposed a clear description of the structural and mechanistic data that govern the RNA binding capacity of EhCFIm25.
Subject(s)
Entamoeba histolytica/chemistry , Leucine/chemistry , Protozoan Proteins/chemistry , RNA, Bacterial/chemistry , RNA, Messenger/chemistry , Tyrosine/chemistry , mRNA Cleavage and Polyadenylation Factors/chemistry , Amino Acid Substitution , Binding Sites , Crystallography, X-Ray , Entamoeba histolytica/genetics , Entamoeba histolytica/metabolism , Leucine/metabolism , Molecular Dynamics Simulation , Mutation , Poly A/chemistry , Poly A/genetics , Poly A/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Multimerization , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Thermodynamics , Tyrosine/metabolism , mRNA Cleavage and Polyadenylation Factors/genetics , mRNA Cleavage and Polyadenylation Factors/metabolismABSTRACT
Introducción y Objetivo. El desbridamiento precoz es la base del tratamiento de las quemaduras. La retirada de la escara durante las primeras 72 horas es la mejor opción para reducir la estancia hospitalaria y los eventos infecciosos. Sin embargo, en muchas ocasiones se compromete la dermis viable necesaria para obtener los mejores resultados estéticos y funcionales, obligando a injertar el lecho. Hay numerosa evidencia acerca de la reducción de las tasas de injerto, la pérdida hemática y el número de intervenciones cuando se utiliza un desbridante enzimático, NexoBrid(R). El objetivo de esta publicación es establecer una guía clínica basada en la opinión de los expertos españoles. Material y Método. Se diseñó un panel de 7 expertos de las principales Unidades de Quemados españolas, con más de 350 pacientes tratados, que discutió las diferentes fases del tratamiento con NexoBrid(R) para obtener una guía clínica de consenso acerca de la indicación, uso y manejo del desbridamiento enzimático. Resultados. Se alcanzó un alto nivel de consenso, con más del 70% de acuerdo en cada una de las fases de tratamiento. Todos los aspectos del tratamiento con NexoBrid(R) fueron discutidos durante la reunión, así como las indicaciones y limitaciones de su uso, incluyendo todas las nuevas evidencias publicadas hasta el momento. También las diversas opciones utilizadas por los diferentes centros de quemados españoles, alcanzando una recomendación global sobre su uso. Conclusiones. Se redactó un documento como guía clínica preliminar sobre el uso de NexoBrid(R) hasta que se creen nuevas guías basadas en evidencia. No existe ningún otro consenso similar publicado hasta el momento (AU)
Background and Objective: Early debridement is considered the keystone of the burn injuries treatment. It is well known that the eschar removal during the first 72 hours is the best option to reduce the hospital stay and the infectious events. However this treatment compromise the preservation of viable dermis, needed to reach the best functional and aesthetic outcome, therefore grafting the wound bed is mandatory. There is increasing evidence that enzymatic debridement with NexoBrid(R) is showing a reduction in the grafting rate, the blood loss and the number of surgical excisions. The objective of this consensus meeting is providing guidelines based in Spanish experts experiences. Methods. A panel of 7 experts from the main Spanish Burn Units were design, with more than 350 patients treated by them, where the different statements were discussed, trying to get a consensus guideline of the indication, use and management of NexoBrid(R). Results. A high level agreement was stated. All the aspects of NexoBrid(R) treatment were discussed showing an agreement of 70%, as well as the indications and limitations of its use. All new evidence published so far this paper was included. Different treatment options used by the Spanish Burn Centers were discussed, showing a global recommendation in their use. Conclusions. A consensus document was created as a preliminary guideline for the NexoBrid(R) use until further guidelines are available. There is no other consensus guideline published so far this document (AU)
