The studies on transglutaminase-mediated protein modifications in cell stress responses
Date Issued
2008
Date
2008
Author(s)
Lee, Der-Yen
Abstract
Transglutaminases (TGs) have been characterized as a group of enzymes designated for catalyzing protein cross-linking, polyamine incorporation, and deamidation. Meanwhile, an emerging group of proteins has been cataloged as TG protein substrates in numerous studies, and their functions are affected by the post-translational modifications catalyzed by TGs. This proteomics study was performed in a protein sample enriched in TG2 and protein substrates based on the theoretical interactions of enzymes and substrates. The fraction enriched in TG2 obtained by ion exchanger column chromatography was subjected to proteomics identification of potential TG2 protein substrates. Twenty nine putative TG2 protein substrates of interest were documented from the proteomics identification. VCP, ERp72, PDI, 14-3-3, PRDX1, PSC2, and glyoxalase I (GlxI) were verified as novel TG protein substrates by in vitro transamidation assay. Among the putative TG2 protein substrates, GRP78 has been identified as a TG2 protein substrate. Our results from mass spectrometry analysis and in vitro transamidation experiments confirmed previous descriptions. Moreover, TG2 activity was co-purified with GRP78 by affinity column chromatography indicating that the relationship between TG2 and GRP78 may go beyond that of enzyme and substrates. On the other hand, 14-3-3 was identified as a TG protein substrate for the first time. Dimerization of 14-3-3 was found in cells treated with calcium ionophore, possibly caused as TG2-catalyzed cross-linking and this rendered 14-3-3 insoluble. e focused on on glyoxalase I (GlxI) and studied in more detail the effect of TG2 on the substrates. Except for being a TG2 protein substrate, GlxI was found as a polyamine binding protein and the interaction between the enzyme and spermidine or spermine was SDS-resistant. Binding of polyamines alone increased the enzyme activity and protein stability in alkaline solution of GlxI. Moreover, polyamine incorporation and deamidation of GlxI were demonstrated by in vitro transamidation assay and competitive transamidation assay, respectively. TG2 catalyzed polyamine incorporation and deamidation of GlxI, instead of protein cross-linking. With TG2 catalysis, polyamine incorporation or deamidation of GlxI enhanced the enzyme activity. However, polyamine incorporation of GlxI not only increased the enzyme activity but also protein stability. More importantly, the modifications of GlxI by TG2 and up-regulation of Glx enzyme activity were found to increase cells’ resistence to methylglyoxal (MG)-induced cytotoxicity. The TG activity of cultured cells was elevated with MG and suppressed with TG inhibitors treatments. The inhibition of endogenous TG activity by TG inhibitors in cultured cells reduced the cell viability on cells challenged with MG. Furthermore, knockdown of TG2 by RNA interference also reduced the cell resistance to MG treatments. Besides, the endogenous polyamines also modulate the endogenous GlxI activity. Depletion of endogenous polyamine by DENSPM caused more cell death when cells were treated with MG. The results indicated that TG2 is able to regulate downstream protein substrates by posttranslational modifications. In this study, polyamine incorporation and deamidation of GlxI by TG2 catalysis are essential for cells to respond to the increasing MG and remove the toxicity of excessive MG. The results also imply that the drug resistance of cancer cells against MG-induced apoptosis may result from the overexpression of TG2 and GlxI.
Subjects
cell stress response
transglutaminase
protein modification
glyoxalase I
SDGs
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