NPAS4, a typical bHLH-PAS transcriptional factor, and its engagement with 14-3-3 proteins beyond canonical recognition motifs

Abstract

Background: NPAS4 (Neuronal PAS domain containing protein 4) a member of the bHLHPAS (basic helix-loop-helix-Per-ARNT-Sim) is well known as a transcriptional factor serves
as master regulator of neural circuit homeostasis, memory formation, and synaptic plasticity.
On the other side 14-3-3 are scaffolding protein in brain and binds with phosphorylated client
proteins and modulate their localization and activity. Some studies had shown that interaction
of 14-3-3 with some bHLH family members influences on their localization and activity.
However, to date no any data had shown whether NPAS4 interacts with 14-3-3 proteins and
this interaction would be new clue to understand pathways of neurodegeneration development
involving Tau (Microtubule associated protein).
Methods: To address this, our study is aimed to set out to test whether 14-3-3 and NPAS4
actually interact. Using Scansite 4.0 and ELM for in silico prediction, we identified potential
14-3-3 binding sites within NPAS4. Strikingly, these predicted motifs fell inside intrinsically
disordered regions (IDRs). As a first experimental step, we performed co-localization studies
in neuronal cells using confocal microscopy.

Results: Our results revealed that fluorescently tagged NPAS4 and 14-3-3 co-localize in
transfected N2a cells, hinting at a possible interaction. This leads us to propose that 14-3-3
proteins may control NPAS4's subcellular location and activity. To move forward, we plan to
study this interaction in living neuronal cells using pull-down assays. Importantly, we are also
trying to achieve for the first time full length NPAS4 expression in Pichia pastoris primarily
data will be present