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Cited by 124Alzheimer's disease research and treatmentsDementia and Cognitive Impairment ResearchAdvanced Proteomics Techniques and Applications

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Dual modification of Alzheimer’s disease PHF-tau protein by lysine methylation and ubiquitylation: a mass spectrometry approach

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Thomas, Stefani N., Funk, Kristen E., Wan, Yunhu, Liao, Zhongping, Davies, Peter, Kuret, Jeff, Yang, Austin J.·2012·Acta Neuropathologica
DOI 10.1007/s00401-011-0893-0PMC PMC3249157
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Abstract

In sporadic Alzheimer's disease (AD), neurofibrillary lesion formation is preceded by extensive post-translational modification of the microtubule associated protein tau. To identify the modification signature associated with tau lesion formation at single amino acid resolution, immunopurified paired helical filaments were isolated from AD brain and subjected to nanoflow liquid chromatography-tandem mass spectrometry analysis. The resulting spectra identified monomethylation of lysine residues as a new tau modification. The methyl-lysine was distributed among seven residues located in the projection and microtubule binding repeat regions of tau protein, with one site, K254, being a substrate for a competing lysine modification, ubiquitylation. To characterize methyl lysine content in intact tissue, hippocampal sections prepared from post mortem late-stage AD cases were subjected to double-label confocal fluorescence microscopy using anti-tau and anti-methyl lysine antibodies. Anti-methyl lysine immunoreactivity colocalized with 78 ± 13% of neurofibrillary tangles in these specimens. Together these data provide the first evidence that tau in neurofibrillary lesions is post-translationally modified by lysine methylation.

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Fig. 1 Summary of modification sites identified by LC–MS/MS on immunopurified PHF-tau. The sequence shown is that of human 2N4R tau (NCBI accession number NP_005901). Bold sequence coverage; Red PHF6 and PHF6* motifs; dashed lines segments encoded by alternatively spliced exons 2, 3, and 10; underline repeat region (as defined in [23]); P Phosphorylated sites, U Ubiquitylated sites, me1 monomethylated sites identified from MS analysis reported herein and in [13]
Fig. 1 Summary of modification sites identified by LC–MS/MS on immunopurified PHF-tau. The sequence shown is that of human 2N4R tau (NCBI accession number NP_005901). Bold sequence coverage; Red PHF6 and PHF6* motifs; dashed lines segments encoded by alternatively spliced exons 2, 3, and 10; underline repeat region (as defined in [23]); P Phosphorylated sites, U Ubiquitylated sites, me1 monomethylated sites identified from MS analysis reported herein and in [13]CC BY-NC · PubMed Central
Fig. 2 Relative abundance of PHF-tau methylation and ubiquitylation. Relative abundance was calculated based on the spectral counts of the modified peptide/(modified peptide + unmodified peptide). Only sites having total spectral counts >3 are shown. Inset quantifies K254 methylation (41%) and ubiquitylation (1%). K254 was the most abundant methylated site identified on PHF-tau (spectral count = 17) and the only site on PHF-tau identified in methylated and ubiquitylated forms
Fig. 2 Relative abundance of PHF-tau methylation and ubiquitylation. Relative abundance was calculated based on the spectral counts of the modified peptide/(modified peptide + unmodified peptide). Only sites having total spectral counts >3 are shown. Inset quantifies K254 methylation (41%) and ubiquitylation (1%). K254 was the most abundant methylated site identified on PHF-tau (spectral count = 17) and the only site on PHF-tau identified in methylated and ubiquitylated formsCC BY-NC · PubMed Central
Fig. 3 MS/MS characterization of K254 modification site. Spectra for peptide aa241–254 derived from Lys-C cleavage of PHF-tau identified in (a) unmodified and (b) methylated (asterisks site of methylation) forms. c Spectrum for tryptic peptide aa243–257 identified in ubiquitylated form (carets site of ubiquitylation). Trypsin did not cleave ubiquitylated aa243–257 at K254 owing to its conjugation to the C-terminal di-Gly fragment of ubiquitin
Fig. 3 MS/MS characterization of K254 modification site. Spectra for peptide aa241–254 derived from Lys-C cleavage of PHF-tau identified in (a) unmodified and (b) methylated (asterisks site of methylation) forms. c Spectrum for tryptic peptide aa243–257 identified in ubiquitylated form (carets site of ubiquitylation). Trypsin did not cleave ubiquitylated aa243–257 at K254 owing to its conjugation to the C-terminal di-Gly fragment of ubiquitinCC BY-NC · PubMed Central
Fig. 4 MS/MS identification of S262 phosphorylated and K267 methylated PHF-tau peptides. Spectra for PHF-tau derived peptide aa258–267 identified in a unmodified, b monomethylated (asterisks site of methylation), c Ser phosphorylated (at sign site of phosphorylation), and d doubly modified (asterisks site of methylation; at sign site of phosphorylation) forms
Fig. 4 MS/MS identification of S262 phosphorylated and K267 methylated PHF-tau peptides. Spectra for PHF-tau derived peptide aa258–267 identified in a unmodified, b monomethylated (asterisks site of methylation), c Ser phosphorylated (at sign site of phosphorylation), and d doubly modified (asterisks site of methylation; at sign site of phosphorylation) formsCC BY-NC · PubMed Central
Fig. 5 Anti-meK antibody specificity. Aliquots of unmodified (−) and in vitro methylated (+) 2N4R tau were separated by SDS-PAGE (8% polyacrylamide) and either a stained with Coomassie Blue (500 ng tau proteins), or b subjected to immunoblot analysis with anti-meK antibody (100 ng tau protein). Molecular mass calibration markers (M) are shown in units of kDa. High-stoichiometry reductive methylation reduced recombinant 2N4R migration on SDS-PAGE. To further test specificity, antibodies AT8 (c, f; green channel) and anti-meK (d, g; red channel) were pre-adsorbed with either unmodified (c–e) or in vitro methylated (f–h) 2N4R tau, and then subjected to double-label confocal immunofluorescence microscopy on sections of AD hippocampus. Lesions labeled with both AT8 and anti-meK are marked by asterisks. AT8 labeling was unaffected by preadsorption with either unmodified or methylated tau, whereas anti-meK immunoreactivity was diminished by pre-adsorption with methylated (f–h; carets), but not unmodified tau (c–e)
Fig. 5 Anti-meK antibody specificity. Aliquots of unmodified (−) and in vitro methylated (+) 2N4R tau were separated by SDS-PAGE (8% polyacrylamide) and either a stained with Coomassie Blue (500 ng tau proteins), or b subjected to immunoblot analysis with anti-meK antibody (100 ng tau protein). Molecular mass calibration markers (M) are shown in units of kDa. High-stoichiometry reductive methylation reduced recombinant 2N4R migration on SDS-PAGE. To further test specificity, antibodies AT8 (c, f; green channel) and anti-meK (d, g; red channel) were pre-adsorbed with either unmodified (c–e) or in vitro methylated (f–h) 2N4R tau, and then subjected to double-label confocal immunofluorescence microscopy on sections of AD hippocampus. Lesions labeled with both AT8 and anti-meK are marked by asterisks. AT8 labeling was unaffected by preadsorption with either unmodified or methylated tau, whereas anti-meK immunoreactivity was diminished by pre-adsorption with methylated (f–h; carets), but not unmodified tau (c–e)CC BY-NC · PubMed Central
Fig. 6 Anti-meK immunoreactivity colocalizes with neurofibrillary lesions in AD hippocampus. Double-label confocal images of hippocampal sections (CA1 region) stained with anti-tau mouse monoclonal antibodies AT8 (a–i; green channel) or Tau5 (j–o; green channel) along with the rabbit anti-meK antibody (red channel). In AD cases, low magnification images (40× objective,1× zoom) show colocalization of anti-meK immunoreactivity with NFTs (d–f; asterisks) and a neuritic plaque (j–l; arrows). High magnification images (100× objective, 3× zoom) show typical morphology of NFTs (g–i and m–o). Image overlays highlight pixel overlap between anti-meK and AT8 (d–i) and Tau5 (j–o) immunoreactivity. Anti-meK immunoreactivity colocalized extensively with NFTs in all fields examined. In contrast, AT8 and anti-meK immunoreactivity did not colocalize in hippocampal sections prepared from cognitively normal cases (a–c)
Fig. 6 Anti-meK immunoreactivity colocalizes with neurofibrillary lesions in AD hippocampus. Double-label confocal images of hippocampal sections (CA1 region) stained with anti-tau mouse monoclonal antibodies AT8 (a–i; green channel) or Tau5 (j–o; green channel) along with the rabbit anti-meK antibody (red channel). In AD cases, low magnification images (40× objective,1× zoom) show colocalization of anti-meK immunoreactivity with NFTs (d–f; asterisks) and a neuritic plaque (j–l; arrows). High magnification images (100× objective, 3× zoom) show typical morphology of NFTs (g–i and m–o). Image overlays highlight pixel overlap between anti-meK and AT8 (d–i) and Tau5 (j–o) immunoreactivity. Anti-meK immunoreactivity colocalized extensively with NFTs in all fields examined. In contrast, AT8 and anti-meK immunoreactivity did not colocalize in hippocampal sections prepared from cognitively normal cases (a–c)CC BY-NC · PubMed Central

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Authors
Thomas, S. N., Funk, K. E., Wan, Y., Liao, Z., Davies, P., Kuret, J., & Yang, A. J.
Affiliations
  • University of Maryland, Baltimore (US)
  • The Ohio State University (US)
  • Albert Einstein College of Medicine (US)
Corresponding author
  • Kuret, Jeff
  • Yang, Austin J.
ORCID
Venue
Acta Neuropathologica
Publisher
Springer Science and Business Media LLC
Published
2011-10-28
Volume
123
Issue
1
Pages
105-117
ISSN
  • 0001-6322 (ISSN-L)
  • 1432-0533
DOI
10.1007/s00401-011-0893-0
PMC
PMC3249157
OpenAlex
W2155022431
Cited by
123
References
67
Citations by year
2026: 7 · 2025: 12 · 2024: 14 · 2023: 8 · 2022: 10 · 2021: 14 · 2020: 7 · 2019: 9 · 2018: 9 · 2017: 14 · 2016: 5 · 2015: 3 · 2014: 7 · 2013: 1 · 2012: 3
FWCI
2.40
Type
journal article
Language
en
Open access
Licence
cc-by-nc
Locations
  • Acta Neuropathologica (publishedVersion, cc-by-nc)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
Funders
  • Ohio State University (P30-NS045758)
  • University of Rochester
  • National Institutes of Health (NS045758)
  • National Institute on Aging (AG 25323, R01AG014452, AG 14452, R01AG025323)
  • National Institute of Mental Health (MH 59786, R01MH059786)
  • National Institute of Neurological Disorders and Stroke (P30NS045758)
Topics
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Alzheimer's disease research and treatments
  • Health Sciences › Medicine › Psychiatry and Mental health › Dementia and Cognitive Impairment Research
  • Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
Keywords
Alzheimer's disease, tau protein, lysine methylation, ubiquitination, post-translational modification, paired helical filaments, neurofibrillary tangles, microtubule-binding repeats, hippocampus
MeSH terms
Aged, Aged, 80 and over, Alzheimer Disease, Amino Acid Sequence, Female, Humans, Lysine, Male, Methylation, Middle Aged, Molecular Sequence Data, Phosphorylation, Protein Processing, Post-Translational, Neurofibrillary Tangles, tau Proteins, Tandem Mass Spectrometry, Ubiquitination

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