alpha-synuclein and epsilon-(gamma-glutamyl)-lysine

The alpha-synuclein immunopositive and chaotrope-insoluble material from human brains with Lewy body pathology was analyzed by mass spectrometry. From the proteinase K-cleavable peripheral fraction of Lewy bodies, which was densely cross-linked by gamma-glutamyl-epsilon-lysine bonds between HspB1 and ubiquitin in a pattern similar to neurofibrillary tangles (Nemes, Z., Devreese, B., Steinert, P. M., Van Beeumen, J., and Fésüs, L. (2004) FASEB J. 18, 1135-1137), 53 proteins were identified. In the core of Lewy bodies only alpha-synuclein was found, and it contained a low amount of intramolecular cross-links between Gln-99 and Lys-58. In vitro cross-linking of alpha-synuclein by transglutaminases 1-3 and 5 produced a heterogeneous population of variably cross-linked alpha-synucleins in solution, which inhibited the aggregation of the protein into amyloid. However, in the presence of phosphatidylserine-rich membranes and micromolar calcium concentrations, the cross-linking by transglutaminases 1, 2, and 5 showed specificity toward the utilization of Gln-99 and Lys-58. As shown by thioflavin T fluorescence monitoring, the formation of this cross-link accelerated the aggregation of native alpha-synuclein. Chemical cross-linking of residues 58-99 triggered amyloid formation, whereas such bonding of residues 99 to 10 was inhibitory. Our findings reveal the pivotal role of membrane attachment and transglutaminase-mediated intermolecular cross-linking for the propagative misfolding and aggregation of alpha-synuclein.

Topics: Aged; Aged, 80 and over; alpha-Synuclein; Amino Acid Sequence; Amyloid; Animals; Brain; Cell Membrane; Cross-Linking Reagents; Dipeptides; Endopeptidase K; Female; Humans; Lewy Bodies; Male; Molecular Sequence Data; Solubility; Transglutaminases; Ubiquitin

In Parkinson's disease (PD), conformational changes in the alpha-synuclein monomer precede the formation of Lewy bodies. We examined postmortem PD and undiseased (control) substantia nigra for evidence of pathological crosslinking of alpha-synuclein by tissue transglutaminase (tTG) using immunohistochemistry, immunoprecipitation, and Western blot. Consistent with previous reports, we found that both tTG and its substrate-characteristic N(epsilon)-(gamma-glutamyl)-lysine crosslink are increased in PD nigral dopamine neurons. Furthermore, both the tTG protein and its substrate crosslink coprecipitated with alpha-synuclein in extracts of PD substantia nigra. Unexpectedly, the isodipeptide crosslink was detected in the alpha-synuclein monomer as well as in higher molecular mass oligomers of alpha-synuclein. Although the intramolecularly crosslinked alpha-synuclein monomer was present in control tissue, it was highly enriched in PD substantia nigra. Conversely, significantly less uncrosslinked alpha-synuclein remained in the postimmunoprecipitate lysate of PD tissue than in control. Crosslinked alpha-synuclein, formed at the expense of the total alpha-synuclein monomer, correlated with disease progression. These results demonstrate that much of the alpha-synuclein monomer in PD nigra is crosslinked by tTG and thus may be functionally impaired. This modification appears to be an early step in PD pathogenesis, preceding the aggregation of alpha-synuclein in Lewy bodies.

Topics: Adult; Aged; Aged, 80 and over; alpha-Synuclein; Biopolymers; Dipeptides; Disease Progression; Dopamine; Female; GTP-Binding Proteins; Humans; Lewy Bodies; Male; Middle Aged; Nerve Tissue Proteins; Neurons; Parkinson Disease; Protein Conformation; Protein Glutamine gamma Glutamyltransferase 2; Solubility; Structure-Activity Relationship; Substantia Nigra; Synucleins; Transglutaminases

The accumulation of misfolded proteins in intracellular inclusions is a generic feature of neurodegenerative disorders. Although heavily ubiquitylated, the aggregated proteins are not degraded by the proteasomes. A possible reason for this phenomenon may be a modification of deposited proteins by transglutaminases forming gamma-glutamyl-epsilon-lysine (GGEL) cross-links between distinct proteins. Here, we show that the frequency of GGEL cross-links is an order of magnitude higher in Alzheimer's brain cortex than in age-matched or younger controls. This difference is due to the accumulation of GGEL cross-links in ubiquitin-immunopositive protein particles present in both Alzheimer's brains and those from aged individuals. The highly cross-linked protein aggregates show immunoreactivity to antibodies against tau and neurofilament proteins, and partially also to alpha-synuclein, indicating that these structures are inherent in Alzheimer's neurofibrillary tangles and Lewy bodies. Using mass sequence analysis, we identified the same six pairs of peptide sequences cross-linked in both senile and Alzheimer's specimens: Gln31 and Gln190 of HSP27 protein are cross-linked with Lys29 and Lys48 of ubiquitin and HSP27 therefore may cross-link two (poly)ubiquitin chains. One lysine residue of parkin and one of alpha-synuclein were also found to be cross-linked. The data suggest that cross-linking of (poly)ubiquitin moieties via HSP27 may have a role in the stabilization of the intraneuronal protein aggregates by interference with the proteasomal elimination of unfolded proteins.

Topics: Adult; Aged; Aged, 80 and over; alpha-Synuclein; Alzheimer Disease; Brain Chemistry; Cerebral Cortex; Chromatography, High Pressure Liquid; Dipeptides; Enzyme-Linked Immunosorbent Assay; Female; Glutamine; Heat-Shock Proteins; Hippocampus; HSP27 Heat-Shock Proteins; Humans; Inclusion Bodies; Lysine; Macromolecular Substances; Male; Molecular Chaperones; Neoplasm Proteins; Nerve Tissue Proteins; Neurofibrillary Tangles; Neurofilament Proteins; Plaque, Amyloid; Proteasome Endopeptidase Complex; Solubility; Synucleins; tau Proteins; Ubiquitin; Ubiquitin-Protein Ligases