Transglutaminase-catalyzed crosslinking in neurological disease: from experimental evidence to therapeutic inhibition.

Diseases of polyglutamine expansion, Alzheimer's disease and Parkinson's disease are neurodegenerative diseases associated with insoluble protein aggregates and neuronal death. These diseases constitute a group of devastating diseases for which there is currently little treatment. The protein aggregates may be the cause of neuronal death, although there is some controversy as to which form of aggregation (oligomers, polymers or microscopic aggregates) is the most toxic. More than a decade ago, the participation of transglutaminases in the formation of the abnormal protein aggregates was proposed. Transglutaminases are a large family of enzymes that catalyze the formation of N(sigma) (gamma-glutamyl)-lysine isodipeptide crosslinks between proteins. In this review, we summarize the evidence supporting the participation of transglutaminase in diseases of the central nervous system. We also describe newly developed transglutaminase inhibitors and their potential use as therapeutic agents in neurological disease.

The end product of transglutaminase crosslinking: simultaneous quantitation of [Nepsilon-(gamma-glutamyl) lysine] and lysine by HPLC-MS3.

Transglutaminases catalyze the formation of Nepsilon-(gamma-glutamyl) isodipeptide crosslinks between proteins. These enzymes are thought to participate in a number of diseases, including neurological disease and cancer. A method associating liquid chromatography and multiple stage mass spectrometry has been developed for the simultaneous quantitation of [Nepsilon-(gamma-glutamyl) lysine] isodipeptide and lysine on an ion trap mass spectrometer. Highly specific detection has been achieved in MS3 mode. The method includes a derivatization step consisting of butylation of carboxylic groups and acetylation of amide groups, a liquid-liquid extraction, and a 19-min separation on a 100x2.1-mm Beta-basic C18 column with an acetonitrile gradient elution. 13C6-(15)N2 isotopes of the isodipeptide and the lysine serve as internal standards. The assay was linear in the range of 50 pmol/ml to 75 nmol/ml for the isodipeptide and the range of 10 nmol/ml to 3.5 micromol/ml for the lysine, with correlation coefficients greater than 0.99 for both ions. Intra- and inter-day coefficients of variation ranged from 3.5 to 15.9%. The method was successfully applied to human biological samples known to be crosslinked by transglutaminase such as cornified envelopes of epidermis, fibrin, and normal and Huntington disease brain.

Oligomeric and polymeric aggregates formed by proteins containing expanded polyglutamine.

Neurological diseases resulting from proteins containing expanded polyglutamine (polyQ) are characteristically associated with insoluble neuronal inclusions, usually intranuclear, and neuronal death. We describe here oligomeric and polymeric aggregates formed in cells by expanded polyQ. These aggregates are not dissociated by concentrated formic acid, an extremely effective solvent for otherwise insoluble proteins. Perinuclear inclusions formed in cultured cells by expanded polyQ can be completely dissolved in concentrated formic acid, but a soluble protein oligomer containing the expanded polyQ and released by the formic acid is not dissociated to monomer. In Huntington's disease, a formic acid-resistant oligomer is present in cerebral cortex, but not in cerebellum. Cortical nuclei contain a polymeric aggregate of expanded polyQ that is insoluble in formic acid, does not enter polyacrylamide gels, but is retained on filters. This finding shows that the process of polymerization is more advanced in the cerebral cortex than in cultured cells. The resistance of oligomer and polymer to formic acid suggests the participation of covalent bonds in their stabilization.