Novel inhibitors against the transglutaminase-catalysed crosslinking of lens proteins.
Post-translational modifications by transglutaminase may contribute to the remodeling of cellular architecture in the development of lens fiber cells, and there is evidence that the enzyme may also play a role in cataract formation. It catalyses hydrolytic deamidations as well as amide exchanges on select glutamine side chains at endo positions in a small subset of proteins of the lens. N epsilon(gamma-glutamyl)lysine crosslinks, the characteristic hallmarks of transglutaminase activity, were identified in polymers isolated from human cataract. Following up on our earlier studies relating to the inhibition of protein crosslinking by the Ca(2+)-activated transglutaminase in the lens, we have now examined the effects of 2-[(2-oxopropyl)thio]-imidazolium derivatives, recently described as active site-directed inhibitors for this family of enzymes. First, we have shown that the compounds at concentrations of 1-2 microM were effective in blocking the transamidating activities of partially purified lens transglutaminase. Then we focused on their efficacy in preventing the formation of the ca. 55 kDa beta crystallin dimers in the whole lens tissue. The production of these dimers, crosslinked by N epsilon(gamma-glutamyl)lysine isopeptide bridges, is an early sign of transglutaminase action in rabbit lens, and it can be readily documented by the SDS-PAGE analysis of proteins remaining in the soluble phase after brief exposure of the homogenate to Ca2+. The new compounds proved to be potent inhibitors of transglutaminase also in this preparation, preventing the crosslinking event at ca. 1 microM concentration. Moreover, even when applied at a 1,000-fold greater concentration (2 mM), they did not interfere with the action of calpain which, similarly to the activation of the transglutaminase system, is triggered by the addition of Ca2+. The high selectivity of the new compounds for differentially blocking only the transglutaminase and not the calpain of the lens, is all the more remarkable because these two enzymes share several mechanistic and structural similarities.
Lorand
L
L
Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL 60611, USA.
Stern
A M
AM
Velasco
P T
PT
D000818:Animals; D002154:Calpain / Q000037:antagonists & inhibitors; D003432:Cross-Linking Reagents / Q000378:metabolism; D003459:Crystallins / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007093:Imidazoles / Q000494:pharmacology*; D007908:Lens, Crystalline / Q000201:enzymology*; D011817:Rabbits; D011503:Transglutaminases / Q000037:antagonists & inhibitors*
D003432:Cross-Linking Reagents; D003459:Crystallins; D004791:Enzyme Inhibitors; D007093:Imidazoles; C088946:1,3,4,5-tetramethyl-2-((2-oxopropyl)thio)imidazolium; C088947:1,3-dimethyl-2-((2-oxopropyl)thio)imidazolium; D011503:Transglutaminases; D002154:Calpain