Bacterial co-expression of human Tau protein with protein kinase A and 14-3-3 for studies of 14-3-3-phospho-Tau interactionReportar como inadecuado




Bacterial co-expression of human Tau protein with protein kinase A and 14-3-3 for studies of 14-3-3-phospho-Tau interaction - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.

Abundant regulatory 14-3-3 proteins have an extremely wide interactome and coordinate multiple cellular events via interaction with specifically phosphorylated partner proteins. Notwithstanding the key role of 14-3-3-phosphotarget interactions in many physiological and pathological processes, they are dramatically underexplored. Here, we focused on the 14-3-3 interaction with human Tau protein associated with the development of several neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases. Among many known phosphorylation sites within Tau, protein kinase A PKA phosphorylates several key residues of Tau and induces its tight interaction with 14-3-3 proteins. However, the stoichiometry and mechanism of 14-3-3 interaction with phosphorylated Tau pTau are not clearly elucidated. In this work, we describe a simple bacterial co-expression system aimed to facilitate biochemical and structural studies on the 14-3-3-pTau interaction. We show that dual co-expression of human fetal Tau with PKA in Escherichia coli results in multisite Tau phosphorylation including also naturally occurring sites which were not previously considered in the context of 14-3-3 binding. Tau protein co-expressed with PKA displays tight functional interaction with 14-3-3 isoforms of a different type. Upon triple co-expression with 14-3-3 and PKA, Tau protein could be co-purified with 14-3-3 and demonstrates complex which is similar to that formed in vitro between individual 14-3-3 and pTau obtained from dual co-expression. Although used in this study for the specific case of the previously known 14-3-3-pTau interaction, our co-expression system may be useful to study of other selected 14-3-3-phosphotarget interactions and for validations of 14-3-3 complexes identified by other methods.



Autor: Kristina V. Tugaeva, Philipp O. Tsvetkov, Nikolai N. Sluchanko

Fuente: http://plos.srce.hr/



DESCARGAR PDF




Documentos relacionados