TY - JOUR
T1 - Isoaspartate-containing galanin in rat hypothalamus
AU - Okyem, Samuel
AU - Mast, David H.
AU - Romanova, Elena V.
AU - Rubakhin, Stanislav S.
AU - Sweedler, Jonathan V.
N1 - Research reported in this publication was supported by the National Institutes of Health, National Institute of Neurological Disorders and Stroke by Award No. R01NS031609, the National Institute on Aging under award No. 1R01AG078797 and the National Institute on Drug Abuse by Award No. P30DA018310. S.O. was supported through the NSF NRT-UtB (DGE 1735252). The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.
PY - 2025/3/8
Y1 - 2025/3/8
N2 - 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.
AB - 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.
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U2 - 10.1038/s42004-025-01475-5
DO - 10.1038/s42004-025-01475-5
M3 - Article
C2 - 40057589
AN - SCOPUS:86000781638
SN - 2399-3669
VL - 8
JO - Communications Chemistry
JF - Communications Chemistry
IS - 1
M1 - 72
ER -