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Our laboratory works on the structure and mechanism of enzymes that function at membrane surfaces. We utilize a combination of modern biophysical and molecular biology approaches. One project focuses on the structure and function of phosphatidylinositol (PI) specific phospholipase C (PI-PLC). Members of this class of enzymes cleave the lipid PI, producing a membrane-soluble product, diacylglycerol (DAG), and a water-soluble product, inositol phosphate or its phosphorylated derivative PI-4,5-bisphosphate (PIP2). In eukoryotic cells there are three classes of PI-PLCs. Each of these isozymes produce both DAG and PIP2, which then act as second messengers and play an important role in the signal transduction process, initiated by hormones and growth factors binding to receptors at the cell surface. PI-PLCs are also excreted by several pathogenic bacteria, and some of these may be “attack” enzymes that serve as virulence factors.

A specific project involves the PI-PLC produced by Bacillus cereus. We have cloned, sequenced, and crystallized this enzyme. The crystal structure reveals a single domain consisting of a TIM barrel, similar to that first observed for triose phosphate isomerase. The active site has been identified by determining the structure in complex with an inhibitor. Current efforts involve a combination of enzyme kinetics and site-directed mutagenesis to investigate the catalytic mechanism and membrane binding region. NMR and other biophysical techniques are being used to determine the dynamics of the histidines and the hydrogen bonding network at the active site.

The mammalian PI-PLCs are the focus of a related project. New fluorogenic substrate analogues are being synthesized in collaboration with Professor John Keana's group. These compounds will provide sensitive probes of the signal transduction process and translocation in model systems and in cultured cells, utilizing fluorescence spectroscopy.

A third project involves a serine protease activation pathway on human cells. The urokinase plasminogen activator receptor plays a key role in invasive cell behavior in normal development and in tumorigenesis. The receptor is a GPI-anchored protein susceptible to cleavage by bacterial PI-PLC and is both expressed at the cell surface and released into the extracellular environment. The localization and behavior of this receptor in a human glioma cell model system is being studied with a combination of microscopy and biochemical techniques.

We are also interested in advanced methods of imaging. For example, the electron optical analog of fluorescence imaging called photoelectron imaging or photoelectron microscopy, and aberration correction to produce higher quality images in all types of electron and ion microscopes.

Figure: B.cereus phosphatidylinositol-specific phospholipase C with myoinositol bound at the active site.


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