Transglutaminases (TGases; protein-glutamine: amine gamma-glutamyltransferases) are a diverse family of Ca2+-dependent enzymes produced by distinct genes and possessing diverse structural and biological functions. For example, the enzymes participate in blood clotting, apoptosis, seminal fluid coagulation, extracellular matrix and bone formation, and barrier formation in stratified squamous epithelia. The common reaction catalyzed by each of the eight known active human/rodent TGase isozymes (i.e, Factor XIIIa, TGase 1 through TGase 7) involves attack of a suitable acceptor nucleophile on the gamma-carboxamide group of a glutamine residue in a donor protein/peptide. Four types of TGase-catalyzed nucleophilic reactions at the carboxamide are known: Attack by a polyamine (such as spermidine) results in a mono- or bis-(gamma-glutamyl)polyamine linkage. If the epislon-NH2 group of a protein-bound lysine residue is the attacking nucleophile, an isopeptide Nepsilon-(gamma-glutamyl)lysine cross link is formed. If water is the attacking nucleophile, the net result is deamidation of the glutamine residue to a glutamic acid residue. When the terminal ?-alcohol group of certain long-chain epidermal-specific ceramides is the attacking nucleophile, a glutamate ester linkage results. ? In addition, among the mammalian TGases characterized thus far, TGase 2 and TGase 3 have been shown to bind and hydrolyze GTP to GDP effectively. The binding of GTP to TGase 3 has been extensively studied by biochemical and biophysical assays and by X-ray crystallography. The biochemical analyses showed that TGase 3 interacts with GTP in a coordinated manner. Binding of GTP results in loss of Ca2+ at one site and occupation of this site by a Mg2+ ion. At least three TGase isozymes (i.e., TGases 1, 2, and 3) are expressed in human brain and aberrant TGase activity has been implicated in the pathology associated with several neurodegenerative diseases. Although, most of the work on brain TGases has been carried out with TGase 2, recent evidence suggests that TGases 1, 2 and 3 can catalyze cross linking of polyglutamine domains in huntingtin (the mutated protein in Huntington disease, HD). Thus, TGase inhibitors might be of therapeutic benefit in neurodegenerative diseases, such as HD. A promising candidate for the inhibition of TGase activity in vivo would seem to be cystamine (beta,beta prim-diaminodiethyl disulfide). Cysteamine (beta-mercaptoethylamine), the reduced form of cystamine, has been used successfully for many years to treat children with cystinosis, and a phase I trial of HD patients to test the tolerance to cysteamine has recently been successfully completed. ? Cystamine has been used as an in vitro TGase inhibitor for many years. There are several potential mechanisms whereby cystamine could inhibit TGases. For example, cystamine may act as a competitive inhibitor and/or alternative substrate in the standard reaction in which radiolabeled putrescine (or dansylcadaverine) is covalently incorporated into a Q substrate, such as casein (or methylated or acetylated casein). Cystamine is known to readily participate in thiol disulfide interchange reactions. Therefore, cystamine may also inhibit TGase 2 by forming a cysteine-cysteamine mixed disulfide at the active site. However, if a strong reducing agent is present in the assay mixture, such as beta-mercaptoethanol or dithiothreitol, the cystamine is reduced to cysteamine. In that case, the cysteamine generated by reduction of cystamine becomes an inhibitor of the putrescine/dansylcadaverine binding, by virtue of it being an alternative substrate of TGase 2. This may be important because the thiol redox status in vivo, as a result of the high glutathione/glutathione disulfide ratio, would dictate a cysteamine/cystamine ratio greatly in favor of cysteamine. Several groups have shown that, in cells in culture, cystamine inhibits in situ TGase activity, raises the level of the important antioxidant glutathione, and inhibits caspase activity (presumably as a result of sulfide-disulfide interchange reactions). Thus, based on these studies of cells in culture, cystamine was predicted to have several potentially desirable properties as an agent for the treatment of HD and other neurodegenerative diseases. Indeed, pharmacological doses of cystamine administered in the drinking water or intraperitoneally have been shown robustly to be beneficial in R6/2 and YAC128 transgenic mouse models of HD. However, the levels of brain glutathione were not altered in mice treated with pharmacological doses of cystamine, and cystamine could not be detected (<2 nmol/ mg of protein) in the brains of the treated mice. Moreover, it was recently shown that the beneficial effect of cystamine is additive to the beneficial effect due to loss of TGase activity in R6/2 TGase 2-/- mice. Nevertheless, we considered it would be instructive to determine the interaction of cystamine with a TGase by X-ray crystallography. We chose to use TGase 3 for this study because TGase 3 is the only currently available mammalian TGase isozyme that is readily crystallizable in an activated form. A thorough knowledge of how the relatively simple molecule cystamine binds to and inhibits this TGase should provide an excellent starting point for understanding the mechanism of action of cystamine as a general TGase inhibitor and subsequently for the development of more complex and more specific TGase inhibitors as potential therapeutic agents.? The crystal structure of TGase 3-cystamine complex was determined to 2.5 resolution. We identified a cystamine-binding cavity and showed a reciprocal relationship between Ca2+ and cystamine levels on TGase 3 transamidation activity. Two cystamines bind in the nucleotide-binding pocket, each in a distinct conformation one closed (horseshoe-like) and the other open (hurdle-jump-like). Cystamine binding results in the loss of one of three bound Ca2+ ions, resulting in conformational rearrangements that close a central channel to the active site. We previously showed that the nucleotide-binding pocket in TGase 3 may be exploited to either enhance or inhibit enzyme activity. The present work expands on the previous findings and suggests that knowledge of the mechanism whereby cystamine binds to TGases may provide the basis for the synthesis of rationally designed inhibitors specifically tailored to interact with the GTP-binding site and close off the channel to the active site. Since aberrant TGase activity is associated with a number of diseases, such inhibitors may be therapeutic. Furthermore, these studies have now been extended to include an analysis of the binding of the reduced form of cystamine (i.e. cysteamine) in the nucleotide-binding pocket of activated, recombinant human TGase 3. The crystal structure of this TGase 3 containing bound cysteamine was determined to 2.7 resolution. This study revealed that cystamine and cysteamine occupy the same nucleotide-binding pocket, albeit with different modes of interaction with protein residues. Two moles of cysteamine are bound per mole of crystallized TGase 3. Binding of cysteamine prevents formation of active enzyme by promoting the loss of one of three bound Ca2+ ions, resulting in conformational rearrangements that close a central channel to the active site and in total loss of transamidation activity. The present findings suggest that knowledge of the mechanism by which cystamine and cysteamine bind to TGases may provide the basis for the synthesis of rationally designed inhibitors specifically designed to close off the channel to the active site in which transamidation occurs. Such inhibitors may be useful for the treatment of CAG-repeat expansion diseases, and possibly for other neurodegenerative diseases.
Showing the most recent 10 out of 12 publications
