Multiple Functions of Glutathione Transferases : A Study on Enzymatic Function, Regulatory Role and Distribution in Mouse and Man

To cope with various endogenous toxin and xenobiotics nature has equipped the organisms with a proper protection system. Glutathione transferases (GSTs) are important components of the cellular defense against oxidative stress. These proteins appear to be suited for different tasks. Based on catalytic activity of GSTs with monochlorobimane (MCB), a screening method was developed for identification of active GSTs in bacterial colonies and for characterization of combinatorial GST libraries. Solvent viscosity effects on kcat and kcat/Km on wild-type human GST A1-1 and phenylalanine-220 mutants indicate a physical step being the rate-limiting step in the catalytic mechanism. Three residues that were under evolutionary selection pressure were identified in Mu class GSTs. By changing these residues in human GSTM2-2, a 1000-fold change of catalytic activity towards GSTM1-1 was accomplished. Using peptide phage display, a peptide sequence was found that acts as non-substrate ligand for human GST M2-2. The peptide sequence was shown to be highly similar to the C-terminal region of c-Jun N-terminal kinase (JNK). JNK is a kinase linked to activating protein-1 (AP-1) transcriptional activity, which is part of the regulation of cell proliferation and apoptosis in response to cellular stress. Reporter gene assays in cell lines showed that human GST M2-2 coactivates the transcriptional activity of AP-1. GSTs as part of the cellular defense against oxidative stress could be important in inflammatory processes. The distribution of GSTs in the intestine of both mice and human in abnormal inflammatory state was investigated immunohistochemically. Using an experimental mouse model, it was shown that mouse GST A4-4 is markedly induced while, the expression of Mu and Pi class GSTs is reduced in the colon of conventional and germ-free mice with extensive colitis. Moreover, the expression of mouse GST A4-4 was elevated with time when germ-free mice were exposed to normal bacteria flora. In contrast, Mu and Pi class GSTs showed decreased expression in the colon of germ-free mice associated with commensal flora. The Alpha, Mu and Pi class GST levels in mouse colon were increased when germ-free mice received Lactobacillus strain GG. The distribution of Alpha, Mu and Pi class GST in the intestinal tissues of patients with Crohn’s disease was investigated using immunohistochemistry. All the three classes were consistently expressed in the intestinal epithelium as well as in macrophage-like cells and smooth muscle tissue. The mucus secreting goblet cells, however, did not express Alpha class GST