The discovery that Ras proteins are farnesylated and that inhibition of farnesylation prevents cellular transformation has generated an explosion of interest in this post-translational modification;currently, several drug candidates that target farnesyl transferase are in Phase 3 trials. However, despite this success, our current understanding of the enzymology of prenyltransferases remains incomplete. Moreover, many aspects concerning the biological function of protein prenylation remain unclear. In the last three years of this project, we have gained significant insights into the structure and mechanism of prenyltransferases;we have also made progress in understanding functional aspects of this modification.
The specific aims of this renewal application are to: (1) Construct transition state structural models from kinetic isotope effect measurements for the reactions catalyzed by protein farnesyl and geranylgeranyl transferase;(2) Probe for intermediates in the reaction catalyzed by protein farnesyltransferase using caged forms of farnesyldiphosphate in conjunction with time-resolved crystallography;(3) Identify active site residues in the protease, RCE1;(4) Develop proteomic methods for determining the prenylation state of proteins and use them to measure prenylation states and levels in cancer cells treated with inhibitors of protein prenylation;(5) Augment the affinity of peptides that bind to RhoGDI and study the mechanism by which they cause cell death;(6) Study the effect of prenyl group structure on subcellular localization of prenylated peptides and proteins. Completion of the above aims should provide additional insights into the mechanism and function of protein prenylation

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058842-12
Application #
7802058
Study Section
Synthetic and Biological Chemistry B Study Section (SBCB)
Program Officer
Gerratana, Barbara
Project Start
1998-06-01
Project End
2011-04-30
Budget Start
2010-05-01
Budget End
2011-04-30
Support Year
12
Fiscal Year
2010
Total Cost
$285,178
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
555917996
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Palsuledesai, Charuta C; Ochocki, Joshua D; Kuhns, Michelle M et al. (2016) Metabolic Labeling with an Alkyne-modified Isoprenoid Analog Facilitates Imaging and Quantification of the Prenylome in Cells. ACS Chem Biol 11:2820-2828
Diaz-Rodriguez, Veronica; Ganusova, Elena; Rappe, Todd M et al. (2015) Synthesis of Peptides Containing C-Terminal Esters Using Trityl Side-Chain Anchoring: Applications to the Synthesis of C-Terminal Ester Analogs of the Saccharomyces cerevisiae Mating Pheromone a-Factor. J Org Chem 80:11266-74
Palsuledesai, Charuta C; Ochocki, Joshua D; Markowski, Todd W et al. (2014) A combination of metabolic labeling and 2D-DIGE analysis in response to a farnesyltransferase inhibitor facilitates the discovery of new prenylated proteins. Mol Biosyst 10:1094-103
Wollack, James W; Monson, Benjamin J; Dozier, Jonathan K et al. (2014) Site-specific labeling of proteins and peptides with trans-cyclooctene containing handles capable of tetrazine ligation. Chem Biol Drug Des 84:140-7
Wang, Yen-Chih; Dozier, Jonathan K; Beese, Lorena S et al. (2014) Rapid analysis of protein farnesyltransferase substrate specificity using peptide libraries and isoprenoid diphosphate analogues. ACS Chem Biol 9:1726-35
Ochocki, Joshua D; Igbavboa, Urule; Wood, W Gibson et al. (2014) Evaluation of prenylated peptides for use in cellular imaging and biochemical analysis. Methods Mol Biol 1088:213-23
Rashidian, Mohammad; Mahmoodi, Mohammad M; Shah, Rachit et al. (2013) A highly efficient catalyst for oxime ligation and hydrazone-oxime exchange suitable for bioconjugation. Bioconjug Chem 24:333-42
Rashidian, Mohammad; Dozier, Jonathan K; Distefano, Mark D (2013) Enzymatic labeling of proteins: techniques and approaches. Bioconjug Chem 24:1277-94
Rashidian, Mohammad; Kumarapperuma, Sidath C; Gabrielse, Kari et al. (2013) Simultaneous dual protein labeling using a triorthogonal reagent. J Am Chem Soc 135:16388-96
Wang, Hong; Henry, Olivier; Distefano, Mark D et al. (2013) Butyrophilin 3A1 plays an essential role in prenyl pyrophosphate stimulation of human V?2V?2 T cells. J Immunol 191:1029-42

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