Research in my group focuses on the chemistry and biology of protein post-translational modification. Post-translational modifications play an essential role in regulating protein function, with these modifications linked to multiple human diseases such as cancer, cardiovascular disease, and obesity. The enzymes responsible for these chemical transformations face a daunting molecular recognition challenge, needing to efficiently identify and act upon their pool of substrates within a complex mixture of other proteins within the cell. Understanding the strategies employed by these enzymes will help identify novel substrates, while potentially illuminating new targets for therapeutic intervention and inhibitor development. Drawing from chemistry, biochemistry, and molecular biology, my research program investigates enzymes involved in post-translational modification with the long term goal of defining how these enzymes and the modifications they perform control biological activity by altering protein structure, function, and localization. We focus on enzymes involved in protein lipidation pathways, such as prenyltransferases, proteases, and acyltransferases. These enzymes alter protein hydrophobicity through covalent modification, leading to changes in protein localization and activity. Our research examines how these enzymes recognize their substrates and catalyze reactions and the impact of the resulting modification(s) on protein function within the cell.
1. Protease specificity in the prenylation pathway
Protein prenylation is essential for the function of many membrane-associated signaling proteins, with these proteins potentially linked to cancer, cardiovascular disease, and other pressing health conditions. Following the protein prenyltransferase-catalyzed attachment of a prenyl group to the cysteine of the "Ca1a2X" C-terminal sequence of a substrate protein, the three amino acids C-terminal to the prenylcysteine residue are removed by one of two Ca1a2X proteases, Rce1 and Afc1. Proteolysis is thought to increase the membrane affinity of prenylated proteins, potentially serving as a sequence-dependent mechanism for regulating prenylated protein trafficking and function. By probing Ca1a2X protease substrate selectivity both in vitro and in vivo, we aim to define the sequence dependence of Ca1a2X protease activity and identify properties within the "Ca1a2X" sequence recognized by the proteases. This research will lay the foundation of a comprehensive specificity model for these proteases and providing information for the design of protease inhibitors as potential therapeutics.
2. Control of protein localization and downstream pathway activation by protein prenylation
Most prenylated proteins must localize to cellular membranes to function in signal transduction pathways. However, the impact of individual steps of the prenylation pathway on protein localization and function has not been well characterized beyond the use of global enzyme inhibition or gene knockout studies. We aim to correlate protein modification, changes in cellular localization, and modulation of signaling pathways using a variety of techniques including fluorescence microscopy, mass spectrometry, and biochemical assays of downstream kinase activation. The long term goals of this project are to develop a model for sequence-based prediction of prenylated protein localization and to determine the connection between localization of upstream control proteins and activation of cellular signaling pathways. Defining the connection of protein modification, trafficking, and function will aid in deciphering the complex interaction networks involved in membrane-associated biological signaling.
3. Investigation of ghrelin acylation by ghrelin O-acyltransferase
Enzymes in the membrane-bound O-acyltransferase (MBOAT) family modify multiple secreted signaling proteins, such as ghrelin, Hedgehog, and Wnt. Discovery of ghrelin and its stimulating effect on appetite suggests that ghrelin-influenced pathways may provide an avenue for treatment of obesity. Ghrelin undergoes a unique serine O-octanoylation modification that appears essential for binding to its cognate receptor. Ghrelin O-acyltransferase (GOAT), the enzyme that catalyzes this modification, has recently been identified and its biochemical activity confirmed in both mice and humans. Our goal is to characterize the catalytic mechanism and substrate specificity of GOAT, with these studies providing valuable information for development of GOAT inhibitors and identification of other potential GOAT substrates.
Hougland, J. L.; Hicks, K. A.; Hartman, H. L.; Kelly, R. A.; Watt, T. J.; Fierke, C. A. Identification of Novel Peptide Substrates for Protein Farnesyltransferase Reveals Two Substrate Classes with Distinct Sequence Selectivities. J. Mol. Biol. 2010, 395, 176-190.
Hougland, J. L.; Lamphear, C. L.; Scott, S. A.; Gibbs, R. A.; Fierke, C. A. Context-Dependent Substrate Recognition by Protein Farnesyltransferase. Biochemistry 2009, 48, 1691-1701.
Hougland, J. L.; Sengupta, R. N.; Dai, Q.; Deb, S. K.; Piccirilli, J. A. The 2′-Hydroxyl Group of the Guanosine Nucleophile Donates a Functionally Important Hydrogen Bond in the Tetrahymena Ribozyme Reaction. Biochemistry 2008, 47, 7684-7694.
Hougland, J. L.; Kravchuk, A. V.; Herschlag, D.; Piccirilli, J. A. Functional Identification of Catalytic Metal Ion Binding Sites Within RNA. PLoS Biol. 2005, 3, e277.
Hougland, J. L.; Deb, S. K.; Maric, D.; Piccirilli, J. A. An Atomic Mutation Cycle for Exploring RNA's 2′-Hydroxyl Group. J. Am. Chem. Soc. 2004, 42, 13578-9.