Gene Regulation, Structure, and Function of Glutathione S-Transferase Systems
Our laboratory uses various glutathione S-transferase (GST) systems to investigate the biological functions of these isozymes and to elucidate mechanisms of regulation in the pathways of their gene expression with emphasis on the post-transcription levels. The GSTs are ubiquitous in nature. They are found in many species ranging from bacteria to man. GSTs are products of two gene superfamilies encoding the cytosolic and the microsomal isozymes, respectively. GSTs are considered to be important detoxification enzymes; they catalyze the conjugation of reduced glutathione (GSH) to a variety of electrophiles (xenobiotic substrates). Some of the GSTs have selenium-independent GSH peroxidase activities and/or ligand binding capability for a variety of organic anions. The multiplicity in the GST genetic reservoir and the inducibility of many GSTs by xenobiotic compounds such as carcinogens, chemotherapeutic agents, barbiturates, plant safeners, etc., make the GST systems part of an organism's defense network against chemical insults.
The Drosophila GST D gene family, mapped at 87B of the polytene chromosome, consists of at least 8 intronless genes and apparent pseudogenes which are separated into two divergently expressed subgroups. The gstD1 gene constitutes its own subgroup and encodes an active enzyme GST D1. It is abundantly expressed throughout development and has activities toward the pesticide DDT and the universal GST substrate, 1-chloro-2,4-dinitrobenzene (CDNB). The other subgroup consists of seven genes and pseudogenes, gstD21 through gstD27. The gstD21 gene encodes a glutathione conjugate binding protein, GSTD21. GST D21 is naturally lacking GST activities, partly because it does not have any catalytically essential serine residue at position 8 or 9.
Barbiturates like phenobarbital or pentobarbital (PB) can sedate the flies just as they do rodents and humans; they also induce GST expression. While gstD1 expression is transcriptionally activated by PB, resulting in a 2-3 fold increase of GST D1 protein and enzyme activity, the 18-20 fold increase in the steady-state level of gstD21 mRNA is most likely due to a change in mRNA stability. The gstD21 mRNA level is considerably lower than gstD1 mRNA under control conditions despite its having a faster transcription rate. Curiously, the increase in gstD21 mRNA under PB treatment did not lead to any net increase of GST D21 protein. It is intriguing that a glutathione conjugate binding protein appears to be tightly regulated in Drosophila. We wish to understand the biological functions of D21 and regulatory mechanisms by which a xenobiotic compound (e.g. PB) changes mRNA stability. We hypothesize that cis-acting element(s) on the D21 mRNA is (are) responsible for its instability. Upon treatment with PB, factors which are capable of binding to D21 mRNA may be induced and/or activated to confer increased stability to gstD21 mRNA. Interaction of such factors or activated complexes with the D21 mRNA may directly or indirectly responsible for the apparent translational regulation and/or enhanced stability.
The current research foci are: (1) to identify and characterize cis-acting element(s) which are responsible for D21 mRNA instability and translational regulation; (2) to identify and characterize trans-acting factors which are responsible for enhanced D21 mRNA stability and translational regulation; (3) to investigate the biochemical and biological function(s) of the GST D21 protein. To achieve the first two goals, we are constructing trangenes which connect various portions of the D21 mRNA sequence to a reporter gene sequence in order to uncover differences in the stability of chimeric RNAs in the presence and absence of PB. We are also making chimeric construct in transgenic flies where the coding region of D21 mRNA is separate from its native 5'- and 3'- UTR. While more transgenic flies are being constructed for in-depth dissection of the D21 mRNA sequences, we are establishing the yeast three-hybrid screening system of Wickens and Field to facilitate the search for any RNA binding proteins which specifically interact with the D21 mRNA sequence. The first two RNAX sequences used for screening are the AT-rich 3'-UTR of D21 mRNA and a region of the coding sequences which are capable of forming two alternative but mutually exclusive inverted repeats. Two cDNA libraries from control and PB-treated flies are specifically constructed for this screening experiment. To complement the molecular genetic approach, we have set up gel shift analysis and in vitro translation assays to biochemically characterize putative D21 mRNA binding proteins. The success of the third goal depends partially on the ability of relief of translational regulation in D21 mRNA. A FLAG-tagged D21 gene has been constructed in E. coli to produce tagged D21 protein. Since D21 does not have enzyme activity, the tagged D21 will be carefully characterized in parallel with tagged D1 to ensure their maintaining the wild type properties. The tagged proteins will be used in biological assays by microinjection and their gene constructs will be engineered for induced expression in transgenic flies. Sequences responsible for translational regulation of D21 expression will be mutated so that in vivo overexpression of D21 and its variants can be achieved in transgenic lines. To complete the analysis, deletions in the D21 gene will be generated from P-element insertions in the 87B region and subjected to molecular analysis.