Associate Professor, Biochemistry

B.A. 1991, Bluffton College
Ph.D. 1996, Purdue University
Postdoctoral: 1996-2000, University of Michigan Medical School

Email: jfriese@ilstu.edu
Phone: (309)438-7850
Office: 318 Science Laboratory Building

In my laboratory we use modern molecular biological tools as well as classical biochemical techniques to study the structure and function of enzymes critical for the biosynthesis of phosphatidylcholine, a major phospholipid component of the eukaryotic cell membrane.

Research focuses on the enzyme CTP:phosphocholine cytidylyltransferase (CT), a member of the CDP-choline pathway, which results in the biosynthesis of phosphatidylcholine (PC). PC is the major component of eukaryotic cell membranes and a precursor to vital components of signal transduction pathways such as diacylglycerol and phosphatidic acid. CT is rate-limiting for the CDP-choline pathway and extensively regulated at the cellular level. CT is present as both a soluble and membrane-associated form. In many cells, activation of CT occurs simultaneously with the translocation of the enzyme from a soluble form to membrane-associated form, while in vitro the soluble form of CT is activated by the addition of certain lipids. In addition to regulation via association with membranes, CT from mammals is extensively phosphorylated. The regulation of CT activity is central to a variety of cellular processes, including the cell cycle, cell death, and vesicular traffic. Investigations in my laboratory utilize recombinant forms of CT from rat, yeast, the nematode C. elegans, the fruitfly D. melanogaster and the malaria parasite P. falciparum to explore the mechanism whereby lipids activate the enzyme.

Conservation of amino acid sequence in the putative lipid binding regions of various CTs is very low, making it difficult to predict individual amino acids critical for lipid activation. In vitro CT is activated in the presence of lipid vesicles, providing a test tube model for membrane association. By exploiting the similarities in the lipid response of each form of CT, I hope to establish a common sequence motif necessary for a cytidylyltransferase to be lipid activated. In contrast, by analyzing differences in the lipid response of each CT, I hope to relate the low amino acid sequence conservation to individual preferences for lipid type and lipid concentration.

Previous work has shown that while wild-type rat CT was activated by lipids a truncated form of rat CT (CT236) lacking the C-terminal lipid-binding region was constitutively active; activity was independent of exogenous lipid. It was concluded that the lipid-binding region of CT constitutes an inhibitory domain in the absence of lipid. Recombinant forms of yeast and C. elegans CT have been expressed using a baculovirus expression system and purified to homogeneity. The activity of each enzyme is sensitive to lipids, maximally activated by almost 5-fold (yeast) and 20-fold (C. elegans) less lipid than purified rat CT. The lipid-binding region of C. elegans CT was identified by truncation mutagenesis and truncated mutant enzymes were characterized whose activity was independent of lipid, similar to rat CT236. The lipid-activation region of C. elegans CT was localized to a 21 amino acid stretch between residues 246 and 266. Mutant enzymes lacking these amino acids do not need to associate with lipid to be fully active.

The next step in my research is to investigate the interaction of CT with membranes at a molecular level. Identification of specific amino acids required for lipid activation of CT will employ the polymerase chain reaction (PCR) to alter individual amino acids by overlap extension site-directed mutagenesis. Initial experiments will use C. elegans CT as a model for the cytidylyltransferase family. The region of primary sequence from amino acids 246 to 266 of C. elegans CT will be targeted based on previous data implicating this region in lipid activation. This region of amino acid sequence plots as an amphipathic alpha helix, a secondary structure element that may play a role in membrane association. Once molecular interactions critical for membrane association are identified, analogous amino acids will be mutated in rat and yeast CT to ascertain if each member of the enzyme family utilizes a common mechanism of membrane interaction.

SELECTED PUBLICATIONS

J. R. Stephenson, J. A. Stacey, J. B. Morgenthaler, J. A. Friesen, T. D. Lash, and M. A. Jones,  "Role of aspartate 400, arginine 262, and arginine 401 in the catalytic mechanism of human coproporphyrinogen oxidase." Protein Science January 22, 2007.

S. J. Gitter, C. L. Cooper, J. A. Friesen, T. D. Lash, and M. A. Jones. "Investigation of the Catalytic and Structural Roles of Conserved histidines of Human Coproporphyrinogen Oxidase Using Site-directed Mutagenesis." The Medical Science Monitor 13(1):BR1-10, 2007.

J. B. Morgenthaler, J. R. Stephenson, J. A. Friesen, and M. A. Jones. "A Beneficial Alternative to the Traditional Western Blot," American Journal of Biochemistry and Biotechnology 2(4):146-147, 2006.

J. R. Stephenson, J.B. Morgenthaler, C.L. Cooper, J.A. Stacey, J. Momani, E. Jenkins, J.A. Friesen, and M.A. Jones, "Use of iLAP Plates as a New Rapid Screening Method for the Evaluation of Various Coproporphyrinogen Oxidase Mutants,"e Journal of Young Investigators (www.jyi.org/research; online journal sponsored through the NFS) 14, 2006.

J.R. Stephenson, N.E. Thomas, J.A. Friesen, and M.A. Jones. "Use of Cross-linking to Assess Subunit Interactions of Recombinant Human Coproporphyrinogen Oxidase." American Journal of Biochemistry and Biotechnology 1(2):103-106, 2005.

Li, S.*, Friesen, J. A. Fei, H.*, Ding, X.*, and Borst, D. W. (2004) The lobster mandibular organ produces soluble and membrane-bound forms of 3-hydroxy-3-methylglutaryl-CoA reductase. Biochemical Journal 381, 831-840.

Friesen, J.A., and Rodwell, V.W. (2004) The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductases. Genome Biology 5:248.

Helmink, B. A. and Friesen, J. A. (2004) Characterization of a lipid activated CTP:Phosphocholine Cytidylyltransferase from Drosophila melanogaster. Biochim. Biophys. Acta 1683, 78-88.

Pattridge, K. A., Weber, C. H., Friesen, J. A., Sanker, S., Kent, C., and Ludwig, M. L. (2003) Glycerol-3-phosphate cytidylyltransferase: Structural changes induced by binding of CDP-glycerol and the role of lysine residues in catalysis. J. Biol. Chem. 278, 51863-51871.

Jones, M. A., Shoffner, R.*, and Friesen, J. A. (2003) Use of Computer Modeling of Site-directed Mutagenesis of a Selected Enzyme: A Class Activity for an Introductory Biochemistry Course. Journal of Science Education and Technology, 12, 413-419.