Molecular Recognition of Substrates by Protein Farnesyltransferase and Geranylgeranyltransferase-I.

Prenylation is an important post-translational modification that targets proteins to the cellular membrane. Farnesyltransferase (FTase) catalyzes the attachment of the 15-carbon farnesyl moiety from farnesyldiphosphate to a cysteine near the C-terminus of a protein, while geranylgeranyltransferase-I (GGTase-I) catalyzes the analogous attachment of the 20-carbon geranylgeranyl group from geranylgeranyldiphosphate. Substrates of the prenyltransferases are involved in a myriad of signaling pathways and processes within the cell, therefore inhibitors targeting FTase and GGTase-I are being developed as therapeutics for treatment of diseases such as cancer, parasitic infection, and progeria. FTase and GGTase-I were proposed to recognize a Ca1a2X motif, where C is the cysteine where the prenyl group is attached, a1 and a2 are aliphatic amino acids, and X confers specificity between FTase and GGTase-I with X being methionine, serine, glutamine, and alanine for FTase and leucine or phenylalanine for GGTase-I. Recent work indicates that the Ca1a2X paradigm should be expanded; therefore, further studies are needed to define the prenylated proteome, to understand normal cellular processes, and to determine the targets of prenyltransferase inhibitors. In this study, we probed the molecular recognition of GGTase-I by testing a 400 peptide library for activity with GGTase-I. The enzyme modifies two classes of substrates: multiple turnover substrates (MTO) and single turnover-only (STO) which undergo chemistry but not product release. Statistical analysis was used to determine that MTO substrates typically follow the Ca1a2X definition, but the STO sequences are more diverse, further indicating GGTase-I recognizes a broader range of substrates. Additionally, with collaborators at the Hebrew University of Jerusalem, a computational program that predicts FTase substrates was developed, FlexPepBind. This novel method successfully predicted new peptide substrates with FTase and identified a new class of substrates containing a positively charged X residue. Lastly, to examine prenylation in vivo, we created a library of GFP-Ca1a2X fusion proteins and measured protein localization using fluorescence microscopy. The identity of the C-terminal sequence caused the proteins to localize to different cellular compartments presumably due to modification status. Together, these studies provide insight into the in vivo specificity of prenyltransferases and the involvement of prenylation in various cellular processes.Ph.D.Biological ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/91519/1/lamphear_1.pd

Identification of a Novel Class of Farnesylation Targets by Structure-Based Modeling of Binding Specificity

Farnesylation is an important post-translational modification catalyzed by farnesyltransferase (FTase). Until recently it was believed that a C-terminal CaaX motif is required for farnesylation, but recent experiments have revealed larger substrate diversity. In this study, we propose a general structural modeling scheme to account for peptide binding specificity and recapitulate the experimentally derived selectivity profile of FTase in vitro. In addition to highly accurate recovery of known FTase targets, we also identify a range of novel potential targets in the human genome, including a new substrate class with an acidic C-terminal residue (CxxD/E). In vitro experiments verified farnesylation of 26/29 tested peptides, including both novel human targets, as well as peptides predicted to tightly bind FTase. This study extends the putative range of biological farnesylation substrates. Moreover, it suggests that the ability of a peptide to bind FTase is a main determinant for the farnesylation reaction. Finally, simple adaptation of our approach can contribute to more accurate and complete elucidation of peptide-mediated interactions and modifications in the cell