Our goal is to define the enzymatic properties of the Ras Converting Enzyme (Rce1p), a protease that is required for CaaX protein biosynthesis. CaaX proteins are lipidated molecules that often function as signaling molecules in important cellular pathways. Ras and RhoB are key examples. Because of the role that CaaX proteins have in cellular transformation (e.g., activated forms of Ras are associated with 30% of all cancers), strategies that regulate the biosynthesis of these proteins are being explored as novel anticancer therapies. Rce1p is a new target in these strategies. Rce1p is an atypical protease, having multiple membrane spans and lacking a canonical protease motif. To date, the mechanism of Rce1p remains undefined. We hypothesize that the Rce1p active site is comprised of a subset of residues that are invariably conserved between Rce1p orthologs. In our preliminary studies, which take advantage of the S. cerevisiae system, we have 1) Identified 4 residues that are critically important for Rce1p function, 2) Discovered a novel compound that is potentially a specific and irreversible inhibitor of Rce1p, and 3) Utilized a dual genetic/ biochemical reporter and deletion analysis to assess the topology and importance of the Rce1p C-terminus. We have developed a coherent set of biochemical, genetic, chemical, and molecular approaches around our findings that will be used for defining the active site and enzymatic properties of Rce1p. Specifically, we will use a novel quantitative genetic assay to detail the importance of charge and position for residues deemed critical for enzymatic activity and to evaluate the substrate specificity of Rce1p mutants. We will use an in vitro assay that monitors cleavage of a quenched fluorescent peptide substrate to evaluate the effect of novel inhibitors and mutations on the kinetic parameters of Rce1p. mutants. Finally, we will use a dual genetic/biochemical topology reporter and deletion approaches to identify the functional domains of Rce1p. In sum, this proposal will clarify the enzymatic properties of Rce1p, a member of an emerging class of multi-span membrane-bound proteases having biomedical importance.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Biochemistry and Biophysics of Membranes Study Section (BBM)
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Ikeda, Richard A
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University of Georgia
Schools of Arts and Sciences
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Mohammed, Idrees; Hampton, Shahienaz E; Ashall, Louise et al. (2016) 8-Hydroxyquinoline-based inhibitors of the Rce1 protease disrupt Ras membrane localization in human cells. Bioorg Med Chem 24:160-78
Alper, Benjamin J; Schmidt, Walter K (2013) Evaluating amyloid Beta (a?) 1-40 degradation by capillary electrophoresis. Methods Mol Biol 984:263-73
Hildebrandt, Emily R; Davis, Dillon M; Deaton, John et al. (2013) Topology of the yeast Ras converting enzyme as inferred from cysteine accessibility studies. Biochemistry 52:6601-14
Kukday, Sayali S; Manandhar, Surya P; Ludley, Marissa C et al. (2012) Cell-permeable, small-molecule activators of the insulin-degrading enzyme. J Biomol Screen 17:1348-61
Noinaj, Nicholas; Song, Eun Suk; Bhasin, Sonia et al. (2012) Anion activation site of insulin-degrading enzyme. J Biol Chem 287:48-57
Kyro, Kelly; Manandhar, Surya P; Mullen, Daniel et al. (2011) Photoaffinity labeling of Ras converting enzyme using peptide substrates that incorporate benzoylphenylalanine (Bpa) residues: improved labeling and structural implications. Bioorg Med Chem 19:7559-69
Dechert, Anne-Marie R; MacNamara, James P; Breevoort, Sarah R et al. (2010) Modulation of the inhibitor properties of dipeptidyl (acyloxy)methyl ketones toward the CaaX proteases. Bioorg Med Chem 18:6230-7
Kyro, Kelly; Manandhar, Surya P; Mullen, Daniel et al. (2010) Photoaffinity labeling of Ras converting enzyme 1 (Rce1p) using a benzophenone-containing peptide substrate. Bioorg Med Chem 18:5675-84
Manandhar, Surya P; Hildebrandt, Emily R; Jacobsen, William H et al. (2010) Chemical inhibition of CaaX protease activity disrupts yeast Ras localization. Yeast 27:327-43
Krishnankutty, Ranjith K; Kukday, Sayali S; Castleberry, Amanda J et al. (2009) Proteolytic processing of certain CaaX motifs can occur in the absence of the Rce1p and Ste24p CaaX proteases. Yeast 26:451-63

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