Maria Vistrup Madsen
Medicinal Chemistry Research
Jagtvej 162, 2100 København Ø
My PhD project is focused at exploring the effect of phosphorylation on the protein-protein interactions (PPIs) of post-synaptic density protein 95 (PSD-95) using genetically encoded phosphoserine.
PSD-95 is one of the most abundant proteins in synapses. It is a key scaffolding protein organizing interactions with receptors and cytosolic proteins in the post-synaptic density (PSD) of excitatory neurons. It comprises three PSD-95/disks large/zonula occludens-1 (PDZ) domains, which are known to be one of the most common regulatory PPI modules. Through its PDZ domains, PSD-95 interacts with glutamatergic receptors, like N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, thereby organizing the PSD and regulating synaptic plasticity and signaling.
Post-translational modification (PTM) of proteins is a key regulatory event in cellular signaling and important for many biological functions. Through mass spectrometry analysis multiple sites of PSD-95 have been found to be post-translationally modified, in particularly by phosphorylation and palmitoylation. The regulation of PSD-95 by PTMs affects the PSD and its plasticity, including long-term potentiation (LTP) and long-term depression (LTD). However, despite the identification of multiple modification sites, it is still largely unknown how PTMs of PSD-95 affect the PPIs of the protein on a molecular level.
This project explores the methodology of unnatural amino acid (UAA) mutagenesis to incorporate genetically encoded phosphorylated serine (pSer) residues at biologically relevant sites in full-length PSD-95. The method utilizes the natural translation machinery of the host cell to synthesize and express proteins carrying pSer. Incorporation of pSer requires a unique aminoacyl synthetase and tRNA pair that is specific for pSer and orthogonal to the natural translation system.
By expressing and studying phosphorylated PSD-95, we expect to contribute to unravelling the importance of phosphorylation on the PPIs of this key scaffolding protein.