Faculty

Proteolytic Processing and Protein Localization in the Secretory Pathway. We are interested in the biochemistry of proteolytic processing enzymes that function in the eukaryotic secretory pathway and in the mechanisms that govern their cellular compartmentalization. Studies focus on the Kex2/furin family of enzymes required for biosynthesis and regulation of bioactive peptides and proteins from yeast to humans (1). A major focus of our studies is the prototypical yeast processing enzyme, Kex2, but we are also interested in homologous mammalian enzymes, such as human furin, that have essential roles in normal physiology and in disease.

Projects:
1. Calcium-dependent recognition of Arginine. Kex2/furin enzymes recognize sites consisting of clusters of basic amino acid residues and cleave immediately C-terminal to an Arg residue. This is highly selective as Lys substitution lowers kcat/Km by 100-10,000-fold and changes the rate-limiting step of the reaction (2). Our recent structure of the Kex2 catalytic domain (3) reveals a conserved loop in the enzyme and a calcium ion that "organize" Arg recognition. This project will involve determining mechanistic effects of mutagenesis of calcium ligands and residues of the calcium loop and/or effects of substitution of other metals for calcium.

2. Small molecule inhibitors of Kex2 and furin. In pathogenic fungi, Kex2 enzymes are important for cell wall integrity and virulence. In humans, furin and related enzymes are essential factors that activate bacterial toxins (e.g., Anthrax) and viral fusion proteins (e.g. Avian influenza HA). We have developed both protein-based (4) and small-molecule inhibitors of furin and Kex2 (5). This project involves identification and characterization of small molecule inhibitors by high-throughput screening followed by combining these with metal chelate inhibitors to created bi-partite molecules that should show high-affinity, selective and irreversible inhibition.

3. Biochemical reconstitution of vesicular transport events. Localization of Kex2 and furin to late Golgi compartments at steady-state involves continued cycles of vesicular transport of these enzymes between Golgi and post-Golgi, endosomal membranes. We have developed methods to reconstitute these complex reactions in the test tube in order to isolate and anaylze individual vesicular transport events (6). This project will involve (e.g.) determining the role of the dynamin-like GTPase, Vps1, in the reaction by purification of the protein and reconstitution with Vps1-deficient membranes. Several other possible related projects are available.

Awards

Faculty Recognition Award, University of Michigan, 2000
Lucille P. Markey Scholar, 1986-1992
Helen Hay Whitney Postdoctoral Fellow, 1984-1987
Received B.S. degree, magna cum laude, Yale University, 1978

Representative Publications

1. Rockwell, N.C., Krysan, D.J., Komiyama, T. and Fuller, R.S., "Precursor Processing by Kex2/furin Proteases", Chem. Rev., 2002, 102, 4525.

2. Rockwell, N.C. and Fuller, R.S., "Differential Utilization of Enzyme-Substrate Interactions for Acylation but not Deacylation During the Catalytic Cycle of Kex2 Protease", J. Biol. Chem., 2001, 276, 38394.

3. Holyoak, T., Wilson, M.A., Fenn, T.D., Kettner, C.A., Petsko, G.A., Fuller, R.S. and Ringe, D., "The 2.4 Å crystal structure of the processing protease Kex2 in complex with an Ala-Lys-Arg boronic acid inhibitor", Biochem., 2003, 42, 6709.

4. Podsiadlo, P., Komiyama, T., Fuller, R.S. and Blum, O., "Furin inhibition by compounds of copper and zinc", J. Biol. Chem., 279, 36219.

5. Komiyama, T., Swanson, J.A. and Fuller, R.S., "Protection from Anthrax Toxin-Mediated Killing of Microphages by the Combined Effects of Furin Inhibitors and Chloroquine", Antomicrob. Agents Chemother., 2005, in press.

5. Abazeed, M.E., Blanchette, J.M. and Fuller, R.S., "Cell-free Reconstitution of Transport from the TGN to the Late Endosome Requires Factors Involved in Formation and Consumption of Clathrin-coated Vesicles", J. Biol. Chem., 2005, 280, 4442.