Role of 4E-BP2 in neuronal translation
View/ Open
Date
25/11/2019Item status
Restricted AccessEmbargo end date
25/11/2020Author
Kouloulia, Stella
Metadata
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.