Abstract
The formation of multiprotein complexes constitutes a key step in determining the function of any translated gene product. Thus, the elucidation of interacting partners for a protein of interest is of fundamental importance to cell biology. Here we describe a simple methodology for the prediction of novel interactors. We have applied this to the developmental transcription factor Brn-3a to predict and verify a novel interaction between Brn-3a and the androgen receptor (AR). We demonstrate that these transcription factors form complexes within the nucleus of ND7 neuroblastoma cells, while in vitro pull-down assays show direct association. As a functional consequence of the Brn-3a-AR interaction, the factors bind cooperatively to multiple elements within the promoter of the voltage-gated sodium channel, Nav1.7, leading to a synergistic increase in its expression. Thus, these data define AR as a direct Brn-3a interactor and verify a simple interacting protein prediction methodology that is likely to be useful for many other proteins.
Highlights
Disrupt protein-protein interactions, the creation of drugs that stabilize a transcriptional complex has been postulated [6], suggesting the possibility that, by modulating protein binding events, genes critical to pathological processes may be switched on or off pharmacologically
Prediction of Brn-3a Interactors—We have developed a method for the prediction of novel interacting proteins of Brn3a, described in detail under “Experimental Procedures.”
To confirm that our observations are likely to hold true for endogenous Nav1.7 gene expression, we examined the effect of Brn-3aL and androgen receptor (AR) overexpression on Nav1.7 mRNA levels by quantitative realtime PCR
Summary
Disrupt protein-protein interactions, the creation of drugs that stabilize a transcriptional complex has been postulated [6], suggesting the possibility that, by modulating protein binding events, genes critical to pathological processes may be switched on or off pharmacologically. 4) Each interaction between Brn-3a-binding proteins and predicted second degree interactors was rated according to experimental evidence supporting it.
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