Browsing by Subject "Zincmetalloprotein Sterile 24 (Ste24)"
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Item Small Molecules And Functionalized Peptides To Study Protein Prenylation(2016-09) Vervacke, JeffreyPost-prenylation processing enzymes Rce1, Ste24, and Icmt function to increase the hydrophobicity of prenylated proteins and assist in targeting them to cellular membranes. Rce1 and Ste24 are CAAX endoproteases that cleave the –AAX residues off the C-termini of prenylated proteins that are then methylated by Icmt. Many prenylated proteins play key roles in the progression of cancer, such as the prenylated G-protein family Ras. Most therapeutic efforts in this area focused on preventing protein prenylation by inhibiting the prenyltransferase enzymes. However, other means of preventing oncogenic signal transduction can be accomplished by disrupting the function of Rce1, Ste24, and Icmt. This thesis focuses on the synthesis and application of small molecules and functionalized peptides to study isoprenoid recognizing enzymes. In order to evaluate Icmt’s topology, mechanism of methylation, and substrate recognition, isoprenoid-containing photoactivatable analogues were developed that crosslink to residues in or near the reactive sites. Initial studies focused on the synthesis and evaluation of benzophenone-containing peptide probes that resemble the natural, prenylated substrate. After the functionalized peptides were determined to be substrates, UV wavelength photolysis was used to cross-link the benzophenone moiety to adjacent Icmt isoprenoid binding site residues. To improve the efficiency of the substrate recognition and enhance labeling of Icmt, a new diazirine isoprenoid analogue was developed. Additionally, the peptide backbone was modified to contain both a biotin moiety and a fluorophore for facile in-gel fluoresces detection of the crosslinked material. These improvements should assist the ability for the cross-linked active site to be determined for Icmt after proteolysis and LC-MS-MS analysis, a task that has yet to be solved. Ste24p has two different proteolysis roles in the maturation of the yeast mating pheromone, a-factor. The first site is the CAAX motif, while the second site is upstream toward the N-terminus, each having a unique amino acid sequences. As a way to observe the reaction kinetics of both sites, peptide analogues that contain a fluorescent donor-quencher pair were developed. By monitoring the increase in fluorescence upon proteolysis, reaction kinetics of both cleavage positions were determined. To explore the pool of cellular proteins that recognize isoprenoid diphosphates, photoaffinity isoprenoid diphosphate analogues were synthesized. Two types of isoprenoid diphosphate derivatives were developed: diphosphate and phosphonophosphate. Each of the derivatives contain a diazirine motif as the photophore. The diphosphate analogue was shown to be an alternative substrate for both yeast and mammalian farnesyltransferase. The photoaffinity phosphonophosphate analogues were proven to be inhibitors of farnesyltransferase, demonstrated the ability to label isoprenoid binding proteins SmgGDS-607 and SmgGDS-558, and should be suitable for identifying unknown binding partners in future cellular lysate labeling experiments. Lastly, effort was put forth to synthesize a small molecule inhibitor for Icmt. The inhibitor incorporates both substrates recognized by Icmt: a prenylcysteine carboxylate, and the methyl donor S-adenosine methionine. In order to make the bisubstrate molecule an inhibitor, the appendage between the two substrates is through an amide bond that replaces the cysteine carboxylate moiety. It is hypothesized that by combining both substrates into one entity, the bisubstrate compound will bind more efficiently with Icmt and enhance inhibitor potency. The bisubstrate inhibitor was shown to inhibit mammalian Icmt, but had little effect on the yeast homologue.