Dept. of Biochemistry & Organic Chemistry

Françoise Raffalli-Mathieu Group

Project description

Project 1              Mutational analysis of bovine GST A1-1

In spite of its high sequence similarity with human hGST A3-3, bovine GST A1-1 displays low steroid isomerase activity. Our recent cloning and characterization of bovine GST A1-1 has revealed novel residues potentially critical to high steroid isomerase activity. This project deals with exploring the importance of these amino acids for the D5-D4 double-bond isomerase activity of bovine GST A1-1 and of human GST A3-3. Results are expected increase our understanding of the exceptional steroid isomerase activity of hGSTA3-3.

Methods: PCR, cloning, protein expression and purification, enzymology.

Project 2              Characterization of bovine GST A2-2 expressed in ovary.

We have recently cloned and characterized a bovine alpha-class GST selectively expressed in bovine steroidogenic organs (bGST A1-1). The highly similar enzyme GST A2-2 is co-expressed with GST A1-1, but a striking feature of GST A2-2 is the absence of the NT-R-A-I-LC motif common to alpha-class GSTs. Instead, a region lacking homology with GST A1-1 is found in GST A2-2. As GST A1-1, GST A2-2 seems to be tightly connected to steroidogenic processes. Investigation of the enzymatic properties of GST A2-2 is the goal of this project, and should bring important new information concerning the function of this enzyme in steroidogenic tissues.

Methods: PCR, cloning, protein expression and purification, enzymology.

Project 3           Establishment of stable GST A1-1, GST A3-3 knockdown human cell lines.

The function of cellular proteins can be studied by gene knockdown, a technique allowing to specifically silence the expression of a target gene in cultured cells. We are interested in understanding physiological functions of GSTs in human living cells, in particular the relevance of alpha-class GSTs in the biosynthesis of steroid hormones. This project proposes to develop human cell lines where the expression of one or several alpha-class GSTs is stably knocked down. Such cell lines will be used to investigate the function of alpha-class GSTs in steroidogenic processes.

Methods: Cloning, culture of human cell lines, PCR, RT-PCR, transfection, clone selection.

Project 4           Characterization of the promoter region of human hGST A3-3.

Several lines of evidence strongly suggest that human hGST A3-3 is important for the biosynthesis of sex steroid hormones in humans. Steroidogenesis is a highly regulated process, via modulation of the level of expression of key proteins within the steroidogenic pathway. Examination of the promoter of human hGST A3-3 reveals several potential binding sites for transcription factors known to be involved in steroidogenesis. This project deals with the functional analysis of hGST A3-3 promoter region. Results are expected to shed light on the physiological function of hGST A3-3 and on novel mechanisms of regulation of GST enzymes.

Methods: Cloning, culture of human cell lines, transfection, reporter gene analysis

Project 5:              Function of hGST A3-3 splice variants

Several splice variants of hGST A3-3 mRNA have been reported. Certain seem to be expressed at even higher levels than the full-length transcript. Whether these variants are translated into proteins, and what their function(s) might be is unknown. This project proposes to clone hGST A3-3 splice variants, and study their expression and function in human cultured cells.

Methods: cloning and eucaryotic expression of FLAG-tagged constructs, preparation of protein extracts from subcellular fractions, Western-blot.