Evaluation of substrate and inhibitor binding to yeast and human isoprenylcysteine carboxyl methyltransferases (Icmts) using biotinylated benzophenone-containing photoaffinity probes

Abstract

Isoprenylcysteine carboxyl methyltransferases (Icmts) are a class of integral membrane protein methyltransferases localized to the endoplasmic reticulum (ER) membrane in eukaryotes. The Icmts from human (hIcmt) and S. cerevisae (Ste14p) catalyze the α-carboxyl methyl esterification step in the post-translational processing of CaaX proteins, including the yeast a-factor mating pheromones and both human and yeast Ras proteins. Herein, we evaluated synthetic analogs of two well-characterized Icmt substrates, N-acetyl-S-farnesyl-L-cysteine (AFC) and the yeast a-factor peptide mating pheromone, that contain photoactive benzophenone moieties in either the lipid or peptide portion of the molecule. The AFC based-compounds were substrates for both hIcmt and Ste14p, whereas the a-factor analogs were only substrates for Ste14p. However, the a-factor analogs were found to be micromolar inhibitors of hIcmt. Together, these data suggest that the Icmt substrate binding site is dependent upon features in both the isoprenyl moiety and upstream amino acid composition and that hIcmt and Ste14p have overlapping, yet distinct, substrate specificities. Photocrosslinking and neutravidin-agarose capture experiments with these analogs revealed that both hIcmt and Ste14p were specifically photolabeled to varying degrees with all of the compounds tested. These data suggest that these analogs will be useful for the future identification of the Icmt substrate binding sites.

Geranylgeranyl diphosphate synthase inhibition induces apoptosis that is dependent upon GGPP depletion, ERK phosphorylation and caspase activation

“…Although membrane association is not always a prerequisite for activity, it is commonly required for activity and its disruption is desirable. 49 , 50 , 51 Ultimately, the localization of many small GTPases was altered with either compound, indicative of engagement with their respective targets. Interestingly, DGBP treatment altered total expression of Rac, RhoA and Rap1, albeit in different ways, with Rac expression decreased and RhoA and Rap1 expression increased.…”

Systematic Proteomic Analysis of Protein Methylation in Prokaryotes and Eukaryotes Revealed Distinct Substrate Specificity

“…In HeLa cells, all these types of methylation were identified. Intriguingly, methylation has been reported for most of these amino acid residues in mammalian cells except glutamic acid and asparagine . Nonetheless, given that glutamic acid and asparagine residues were structurally similar to aspartic acid and glutamine, respectively, and the latter two were found to be methylated in microbes, we reasoned it was likely that the identified methylation of glutamic acid and asparagine were true PTM in mammalian cells.…”

Diazirine-Containing Photoactivatable Isoprenoid: Synthesis and Application in Studies with Isoprenylcysteine Carboxyl Methyltransferase

“… 23 , 28 Farnesylated control peptide 17 and benzophenone-containing peptide 19 were prepared by a similar procedure, whereas 18 was prepared as previously described. 22 …”

“…In vitro assays for methyltransferase activity were performed using crude membranes as previously described. 22 In brief, reactions contained crude membrane preparations, 200 μM AFC, 20 μM S -adenosyl- l -[methyl- 14 C]methionine ([ 14 C]SAM) (50–60 mCi/mmol), and 100 mM Tris-HCl, pH 7.5, in 60 μL. The reactions were incubated at 30 °C for 30 min and terminated by the addition of 50 μL of 1 M NaOH and 1% SDS (v/v).…”