Role of 4E-BP2 in neuronal translation

Abstract

Translational control is a powerful means to alter gene expression and regulates synaptic plasticity, learning and memory. 4E-BP2 (Eif4ebp2, Eukaryotic Initiation Factor 4E-Binding Protein 2) is the predominantly expressed 4E-BP in the mammalian brain and represses cap-dependent translation initiation, by binding to eIF4E (Eif4e, eukaryotic Initiation Factor 4E). As a master regulator of protein synthesis in the mammalian brain, 4E-BP2 has been implicated in learning, memory and Autism Spectrum Disorder (ASD). Upon phosphorylation by mTOR (mammalian Target Of Rapamycin, mTOR) which occurs in most tissues, 4E-BP2 cannot bind to eIF4E, failing to repress translation initiation. However, in early postnatal brain development, 4E-BP2 undergoes brain-specific post-translational deamidation on asparagines N99 and N102, which are converted to aspartic acid. Asparagine deamidation is not catalysed by enzymes but can occur spontaneously and is induced by alkaline pH. Deamidated 4E-BP2 was shown to regulate the kinetics of excitatory synaptic transmission in early postnatal brain development, suggesting that it may be important for synaptic function during that crucial developmental period. N99/N102 deamidation decreases the affinity of 4E-BP2 for eIF4E and increases its binding to the mTORC1 protein Raptor. The significance of enhanced Raptor binding to deamidated 4E-BP2 is yet unclear because 4E-BP2 phosphorylation is very low in adult brain. Moreover, the role of deamidated 4E-BP2 and the downstream effects of deamidated 4E-BP2 translational control in the mammalian brain are not known but of cardinal importance given the pervasive role of 4E-BP2 in regulating brain function. In this thesis, we describe a previously unidentified mechanism during early postnatal brain development, whereby the constitutively deamidated form of the cardinal brain translation initiation repressor 4E-BP2 is more susceptible to ubiquitin proteasomal degradation (as compared to unmodified, WT protein) because it binds with higher affinity to a complex, comprising the mTORC1 protein Raptor and the ubiquitin E3 ligase CUL4B. Deamidated 4E-BP2 (2D) stability is regulated by mTORC1 and AMPAR activity but not NMDARs. We also showed that 4E-BP2 deamidation is neuron-specific and occurs in human brain. We explored whether deamidated and WT 4E-BP2 have a similar subcellular distribution in neurons, indicating that there is very low co-localization of them in both soma and dendrites. We studied WT and 2D structures, with Synchrotron radiation circular dichroism (SCRD), Small angle X-ray scattering (SAXS) and Nuclear magnetic resonance (NMR) spectroscopy of full-length recombinant 4E-BP2 (WT or 2D) expressed in E. Coli and purified, and we identified that they share a similar structure, with only minor differences in a few residues. Moreover, using unbiased translatome mapping, we discovered that overexpression of deamidated 4E-BP2 represses the translation of a distinct pool of mRNAs linked to cerebral development, mitochondria and chiefly NF- κB activity. Collectively, these data describe a previously unidentified brain-specific translational control mechanism that could be crucial for postnatal brain development in neurodevelopmental disorders such as ASD.