Structure-function analysis of RBPJ-interacting and tubulin-associated (RITA) reveals regions critical for repression of Notch target genes [Protein Structure and Folding]

The Notch pathway is a cell-to-cell signaling mechanism that is essential for tissue development and maintenance, and aberrant Notch signaling has been implicated in various cancers, congenital defects, and cardiovascular diseases. Notch signaling activates the expression of target genes, which are regulated by the transcription factor (CBF1/RBP-J, Su(H), Lag-1). CSL interacts with both transcriptional corepressor and coactivator proteins, functioning as both a repressor and activator, respectively. While Notch activation complexes are relatively well understood at the structural level, less is known about how CSL interacts with corepressors. Recently, a new RBP-J (mammalian CSL ortholog) interacting protein termed RITA has been identified and shown to export RBP-J out of the nucleus, thereby leading to the down-regulation of Notch target gene expression. However, the molecular details of RBP-J/RITA interactions are unclear. Here, using a combination of biochemical/cellular, structural, and biophysical techniques, we demonstrate that endogenous RBP-J and RITA proteins interact in cells, map the binding regions necessary for RBP-J/RITA complex formation, and determine the X-ray structure of the RBP-J/RITA complex bound to DNA. To validate the structure and glean more insights into function, we tested structure-based RBP-J and RITA mutants with biochemical/cellular assays and ITC (isothermal titration calorimetry). While our structural and biophysical studies demonstrate RITA binds RBP-J similar to the RAM (RBP-J associated molecule) domain of Notch, our biochemical and cellular assays suggest RITA interacts with additional regions in RBP-J. Taken together, these results provide molecular insights into the mechanism of RITA-mediated regulation of Notch signaling, contributing to our understanding of how CSL functions as a transcriptional repressor of Notch target genes.

from #AlexandrosSfakianakis via Alexandros G.Sfakianakis on Inoreader http://ift.tt/2qpj00v
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