Isoaspartate-containing galanin in rat hypothalamus

Area of Science:

• Biochemistry
• Neuroscience
• Proteomics

Background:

• Isoaspartate residue is a spontaneous post-translational modification (PTM) linked to protein aggregation.
• This modification is typically considered slow and affects long-lived proteins.

Purpose of the Study:

• Investigate the prevalence and kinetics of isoaspartate formation in the neuropeptide galanin (Gal).
• Determine the impact of isoaspartate modification on Gal aggregation and fibril formation.

Main Methods:

• Liquid chromatography-trapped ion mobility mass spectrometry (LC-TIMS-MS) to quantify isoaspartate levels.
• Protein Isoaspartyl methyltransferase (PIMT) assays.
• In vitro isomerization assays under mildly acidic conditions.
• Transmission electron microscopy (TEM) to analyze fibril morphology.

Main Results:

• 20 ± 2% of mature Gal in Rattus norvegicus hypothalamus contain L-isoaspartate.
• Gal aspartate isomerizes within 48 hours in vitro, faster than previously known.
• L-isoaspartate modified Gal significantly enhanced fibril formation.
• TEM revealed distinct fibril morphologies for modified vs. unmodified Gal.

Conclusions:

• Isoaspartate modification occurs rapidly in short-lived neuropeptides like Galanin.
• This PTM significantly promotes Gal aggregation, potentially explaining in vivo fibril deposits in the brain.
• The findings necessitate a re-evaluation of isoaspartate kinetics and its role in proteinopathies.