a-Factor Analogues Containing Alkyne- and Azide-Functionalized Isoprenoids Are Efficiently Enzymatically Processed and Retain Wild-Type Bioactivity

Abstract

Protein prenylation is a post-translational modification that involves the addition of one or two isoprenoid groups to the C-terminus of selected proteins using either farnesyl diphosphate or geranylgeranyl diphosphate. Three crucial enzymatic steps are involved in the processing of prenylated proteins to yield the final mature product. The farnesylated dodecapeptide, a-factor, is particularly useful for studies of protein prenylation because it requires the identical three-step process to generate the same C-terminal farnesylated cysteine methyl ester substructure present in larger farnesylated proteins. Recently, several groups have developed isoprenoid analogs bearing azide and alkyne groups that can be used in metabolic labeling experiments. Those compounds have proven useful for profiling prenylated proteins and also show great promise as tools to study how the levels of prenylated proteins vary in different disease models. Herein, we describe the preparation and use of prenylated a-factor analogs, and precursor peptides, to investigate two key questions. First, a-factor analogues containing modified isoprenoids were prepared to evaluate whether the non-natural lipid group interferes with the biological activity of the a-factor. Second, a-factor-derived precursor peptides were synthesized to evaluate whether they can be efficiently processed by the yeast proteases Rce1 and Ste24 as well as the yeast methyltransferase Ste14 to yield mature a-factor analogues. Taken together, the results reported here indicate that metabolic labeling experiments with azide- and alkyne-functionalized isoprenoids can yield prenylated products that are fully processed and biologically functional. Overall, these observations suggest that the isoprenoids studied here that incorporate bio-orthogonal functionality can be used in metabolic labeling experiments without concern that they will induce undesired physiological changes that may complicate data interpretation.

Dual chemical probes enable quantitative system-wide analysis of protein prenylation and prenylation dynamics

“…12 peptides had undergone C-terminal hydrolytic cleavage generating a prenylated C-terminal cysteine, a further 4 peptides were cleaved and methylated, and the remaining 10 featured unprocessed C-termini (Supplementary Data 4). These findings demonstrate that YnF and YnGG labeling is compatible with endogenous post-prenylation processing by Ras-converting enzyme 1 (RCE1) and Isoprenylcysteine carboxyl methyltransferase (ICMT)28. Interestingly, we also found strong evidence for novel C-terminal processing of geranylgeranylated RAB3B, RAB3D, and RAB7A, in which the two terminal amino acids of the canonical XCXC motif have been removed (Supplementary Figure 10).…”

Metabolic labeling with an alkyne probe reveals similarities and differences in the prenylomes of several brain-derived cell lines and primary cells

“…Suppression of the pool of native isoprenoids using statins may allow C15AlkOPP to more effectively compete for labeling of those more difficult to farnesylate with potentially lower native abundances. It is also important to note that the use of this probe does not necessarily impact the function of labeled substrates as previously described 52 . Therefore, this isoprenoid tagging approach may operate in viable cells with minimal perturbations in the physiological function of prenylated proteins.…”

Metabolic Labeling of Prenylated Proteins Using Alkyne‐Modified Isoprenoid Analogues

“…Recently, it has been demonstrated that a-factor, a farnesylated pheromone from yeast retains full activity when the farnesyl group is substituted with several chemically modified isoprenoids used for metabolic labeling. Those results suggest that such probes can be used without concern that they will cause undesired interference in normal cell physiology (Diaz-Rodriguez et al, 2017). …”