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<p>The isoaspartate residue is a spontaneous, time-dependent post-translational modification (PTM) of proteins and peptides, associated with in vivo protein aggregation and changes in molecule lifetime. While this is considered a slow modification impacting long lived proteins, surprisingly, we observed this PTM at high levels within the relatively short-lived neuropeptide galanin (Gal). The combination of liquid chromatography-trapped ion mobility mass spectrometry and protein Isoaspartyl methyltransferase assays demonstrated that 20 ± 2% of the mature Gal contain L-Isoaspartate residue in the hypothalamus of <i>Rattus norvegicus</i>. Aspartate in Gal isomerizes spontaneously under mildly acidic conditions within 48 h in vitro, much faster than previously assumed. Gal with the L-isoaspartate PTM significantly enhanced fibril formation. Transmission electron microscopy revealed differences in morphology of fibrils formed by D17Isoasparte Gal compared to the unmodified peptide. Observed characteristics of D17Isoasparte Gal suggest a potential mechanism for the development of in vivo Gal fibril deposits previously reported in the brain.</p>

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Isoaspartate-containing galanin in rat hypothalamus

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年3月8日

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  • Samuel Okyem,
  • David H. Mast,
  • Elena V. Romanova,
  • Stanislav S. Rubakhin,
  • Jonathan V. Sweedler

摘要

The isoaspartate residue is a spontaneous, time-dependent post-translational modification (PTM) of proteins and peptides, associated with in vivo protein aggregation and changes in molecule lifetime. While this is considered a slow modification impacting long lived proteins, surprisingly, we observed this PTM at high levels within the relatively short-lived neuropeptide galanin (Gal). The combination of liquid chromatography-trapped ion mobility mass spectrometry and protein Isoaspartyl methyltransferase assays demonstrated that 20 ± 2% of the mature Gal contain L-Isoaspartate residue in the hypothalamus of Rattus norvegicus. Aspartate in Gal isomerizes spontaneously under mildly acidic conditions within 48 h in vitro, much faster than previously assumed. Gal with the L-isoaspartate PTM significantly enhanced fibril formation. Transmission electron microscopy revealed differences in morphology of fibrils formed by D17Isoasparte Gal compared to the unmodified peptide. Observed characteristics of D17Isoasparte Gal suggest a potential mechanism for the development of in vivo Gal fibril deposits previously reported in the brain.