The postsynaptic density protein of 95 kDa (PSD-95) is a key scaffolding protein that controls signaling at synapses in the brain through interactions of its PDZ domains with the C-termini of receptors, ion channels, and enzymes. PSD-95 is highly regulated by phosphorylation. To explore the effect of phosphorylation on PSD-95, we used semisynthetic strategies to introduce phosphorylated amino acids at four positions within the PDZ domains and examined the effects on interactions with a large set of binding partners. We observed complex effects on affinity. Most notably, phosphorylation at Y397 induced a significant increase in affinity for stargazin, as confirmed by NMR and single molecule FRET. Additionally, we compared the effects of phosphorylation to phosphomimetic mutations, which revealed that phosphomimetics are ineffective substitutes for tyrosine phosphorylation. Our strategy to generate site-specifically phosphorylated PDZ domains provides a detailed understanding of the role of phosphorylation in the regulation of PSD-95 interactions.

Peptides are of increasing interest as therapeutics in a wide range of diseases, including metabolic diseases such as diabetes and obesity. In the latter, peptide hormones such as peptide YY (PYY) and pancreatic peptide (PP) are important templates for drug design. Characteristic for these peptides is that they contain a C-terminal that is α-amidated, and this amidation is crucial for biological function. A challenge is to generate such peptides by recombinant means and particularly in a production scale. Here, we have examined an intein-mediated approach to generate a PYY derivative in a larger scale. Initially, we experienced challenges with hydrolysis of the intein fusion protein, which was reduced by a T3C mutation in the intein. Subsequently, we further engineered the intein to decrease the absolute size and improve the relative yield of the PYY derivative, which was achieved by substituting 54 residues of the 198 amino acid intein with an eight amino acid linker. The optimized intein construct was used to produce the PYY derivative under high cell density cultivation conditions, generating the peptide thioester precursor in good yields and subsequent amidation provided the target peptide.

SummaryThe mechanism of action of methotrexate (