The primary objectives of this program are to gain insight into physiological functions of glutathione S-transferases (GSTs) by studying their molecular and catalytic properties. By virtue of their catalytic activities GSTs can function to detoxifiy different types of noxious substances including some carcinogenic agents. GSTs can also function as stoichiometric binding proteins for diverse types of ligands. Some strategies to counter drug resistance and for chemoprevention of cancer have been based on manipulations of the GSTs. Our recent kinetic studies on human Mu-class GSTs suggest details of catalytic mechanisms that are not entirely evident from analysis of solid- state crystal structures. That allows us, as proposed, to introduce key point and modular mutations into human Mu class GSTs in order to define specific features of their catalytic mechanisms. Binding of substrate analogs and ligands will be studied in solution by NMR, isothermal titration microcalorimetry and other appropriate methods. Because, natural cellular ligands or substrates for GSTs largely have not been identified, we propose to use immobilized forms of GSTs to isolate from cell extracts low molecular weight ligands that selectively bind to different GSTs. The bound ligands will be fractionated by HPLC and other chromatographic procedures and identified by combined use of mass spectrometry and other analytical methods. We shall also determine whether other cellular proteins form specific complexes with GSTs. Binding of intracellular ligands could influence GST functions and conversely, GSTs could influence functions of bound low molecular weight ligands and of proteins. We have designed a cotranslational system for expression of different binary combinations of the 5 natural human Mu class GST subunits or specific chimeric variants. The system will be used to study homo- and heterodimeric assembly mechanisms during protein synthesis. Results will be compared to those obtained by in vitro reassembly mechanisms and to the observed cellular heterodimeric combinations. The well-characterized structure of these GSTs should provide a paradigm to develop fundamental concepts about cotranslational protein-folding and subunit assembly. Our studies will be performed mainly with a novel GST subclass preferentially expressed in post-meiotic germ cells and certain cortical and hypothalamic neurons. Since the genes for human and rodent forms of this subclass have been cloned and characterized in our laboratory, but specific functions have not been defined, we propose to undertake targeted gene disruption of the mouse mGSTM5 gene from this subclass. Phenotypic characteristics of the offspring will be determined, with regard to reproductive and neuronal functions. Inferred functions will be coordinated with studies of structure and properties of these GSTs. A transgenic model for regulation of expression of the mouse (mGSTM5) and human (hGSTM3) genes will be established to determine a basis for their tissue-specific expression patterns.
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