Identification of brain substrates of transglutaminase by functional proteomics supports its role in neurodegenerative diseases.

Transglutaminases are calcium-dependent enzymes that catalyze the formation of ε-(γ-glutamyl)lysine isopeptide bonds between specific glutamine and lysine residues. Some transglutaminase isoforms are present in the brain and are thought to participate in the protein aggregation characteristic of neurological diseases such as Huntington, Alzheimer's and Parkinson's disease. We have developed a functional proteomics strategy in which biotinylated amine-donor and amine-acceptor probes were used to identify the transglutaminase substrates present in brain. Bioinformatics analyses revealed that most of the 166 brain substrates identified interacted with huntingtin, the amyloid precursor protein or α-synuclein and that neurological disease was the most significant canonical pathway associated with the substrates. The physiological relevance of the substrates identified by mass spectrometry was confirmed by the fact that three of them (actin, β-tubulin and a neurofilament subunit) were polymerized in neuronal cells when cytosolic calcium concentration was raised. We also showed by in-situ immunolabeling that some of the substrates were part of the protein aggregates found in neurological diseases. These results strongly support the idea that the crosslinking activity of brain transglutaminase participates in the formation of the protein aggregates found in diseases of the central nervous system.