Letter to the editor

Human genetic diseases due to codon reiteration: Relationship to an evolutionary mechanism

Cysteamine, as a part of CoA plays a crucial role in metabolism by facilitating thioester formation. However, as a free entity, cysteamine is largely absent from metabolism. By contrast, supraphysiological amounts of this simple aminothiol have profound and selective effects of physiology and certain diseases. Most of these effects are explicable in terms of thiol-disulfide exchanges with cystine or specific cystinyl residues, and cysteamine is the drug-of-choice to prevent the accumulation of excess lysosomal cystine in cystinosis. This success and others have prompted studies of cysteamine for the treatment of radiation poisoning, cancer, and various neurological diseases as described in the following article.

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.

Well-defined robust ruthenium(II) complexes 3a–d bearing o-aryloxide-N-heterocyclic carbene ligands with different wingtip substituents (3a (R = Me), 3b (R = Ph), 3c (R = ⁱPr) and 3d (R = Mes)) in the imidazole ring were synthesized in good yields by the reaction of imidazolium proligands with metal precursor [RuHCl(CO)(PPh₃)₃] by transmetallation from the corresponding silver carbene complexes. All the Ru(II)–NHC complexes have been characterized by elemental analyses, spectroscopic methods as well as ESI mass spectrometry. The molecular structure of the complex 3a was identified by means of single-crystal X-ray diffraction analysis, which revealed that the complexes possess a distorted octahedral geometry. In order to explore the catalytic potential of the synthesized complexes, all the four [Ru–NHC] complexes [3a–d] were tested as catalysts for transamidation of carboxamides with amines. Notably, the complex 3a was found to be very efficient and versatile catalyst toward transamidation of a wide range of amides with amines.

Huntington's disease (HD) is a devastating autosomal-dominant neurodegenerative disorder initiated by an abnormally expanded polyglutamine in the huntingtin protein. Determining the contribution of specific factors to the pathogenesis of HD should provide rational targets for therapeutic intervention. One suggested contributor is the type 2 transglutaminase (TG2), a multifunctional calcium dependent enzyme. A role for TG2 in HD has been suggested because a polypeptide-bound glutamine is a rate-limiting factor for a TG2-catalyzed reaction, and TG2 can cross-link mutant huntingtin in vitro. Further, TG2 is up regulated in brain areas affected in HD. The objective of this study was to further examine the contribution of TG2 as a potential modifier of HD pathogenesis and its validity as a therapeutic target in HD. In particular our goal was to determine whether an increase in TG2 level, as documented in human HD brains, modulates the well-characterized phenotype of the R6/2 HD mouse model. To accomplish this objective a genetic cross was performed between R6/2 mice and an established transgenic mouse line that constitutively expresses human TG2 (hTG2) under control of the prion promoter. Constitutive expression of hTG2 did not affect the onset and progression of the behavioral and neuropathological HD phenotype of R6/2 mice. We found no alterations in body weight changes, rotarod performances, grip strength, overall activity, and no significant effect on the neuropathological features of R6/2 mice. Overall the results of this study suggest that an increase in hTG2 expression does not significantly modify the pathology of HD.

Chronological changes of tissue-type transglutaminase (tTG) were observed in the hippocampal CA1 region after transient forebrain ischemia in gerbils. In the sham-operated group, tTG immunoreactivity was weakly detected in blood vessels which were immunostained with platelet endothelial cell adhesion molecule-1 (PECAM-1), and tTG immunoreactivity in blood vessels was highest 5 days after ischemia/reperfusion. In addition, tTG immunoreaction was expressed in microglia which were immunostained with Iba-1 at 4 days post-ischemia, and tTG immunoreactivity in the microglia was also highest at 5 days post-ischemia. In Western blot analysis, tTG protein levels in the CA1 region after ischemia/reperfusion began to increase 3 days after ischemia/reperfusion and peaked 5 days after ischemia/reperfusion. The expression of tTG in PECAM-1-immunoreactive blood vessels may be associated with integrin regulation or transendothelial migration of leukocytes in the ischemic CA1 region. In this study, we also observed the effect of cystamine, a tTG inhibitor, against ischemic damage. Administration of cystamine protected in certain degree neuronal damage from ischemic damage in the CA1 region. These results suggest that tTG may be associated with neuronal death in the hippocampal CA1 region induced by ischemia/reperfusion.

Transglutaminases catalyze the formation of N^ε-(γ-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 [N^ε-(γ-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 100 × 2.1-mm Beta-basic C₁₈ column with an acetonitrile gradient elution. ¹³C₆–¹⁵N₂ 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 μmol/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.