Metabolic Labeling of Prenylated Proteins Using Alkyne‐Modified Isoprenoid Analogues

Abstract

Protein prenylation involves the attachment of a farnesyl or geranylgeranyl group onto a cysteine residue located near the C-terminus of a protein, recognized via a specific prenylation motif, and results in the formation of a thioether bond. To identify putative prenylated proteins and investigate changes in their levels of expression, metabolic labeling and subsequent bioorthogonal labeling has become one of the methods of choice. In that strategy, synthetic analogues of biosynthetic precursors for post-translational modification bearing bioorthogonal functionality are added to the growth medium from which they enter cells and become incorporated into proteins. Subsequently, the cells are lysed and proteins bearing the analogues are then covalently modified using selective chemical reagents that react via bioorthogonal processes, allowing a variety of probes for visualization or enrichment to be attached for subsequent analysis. Here, we describe protocols for synthesizing several different isoprenoid analogues and describe how they are metabolically incorporated into mammalian cells, and the incorporation into prenylated proteins visualized via in-gel fluorescence analysis. © 2018 by John Wiley & Sons, Inc.

In vivoprenylomic profiling in the brain of a transgenic mouse model of Alzheimer’s disease reveals increased prenylation of a key set of proteins

“…Consistent with previous findings, 35,38,39 statin treatment augmented isoprenoid analogue incorporation. Metabolic labeling in the brain with C15AlkOPP resulted in the identification of 17 prenylated proteins in the absence of statin.…”

“…In previous work, performed in cell culture, it has been demonstrated that treatment with a statin leads to increased incorporation of C15AlkOPP, as evidenced by a larger number of proteins identified with greater fold changes as well as by an increase in the identification of farnesylated proteins. 35,38,39 When WT mice were treated with C15AlkOPP in conjunction with simvastatin, a similar increase was seen with 37 prenylated proteins found, compared with 17 observed in the absence of statin. This 20protein increase included a significant increase in the number of FTase substrates.…”

“…Therefore, a direct brain injection approach was employed to ensure the delivery of the isoprenoid analogue to the brain. Diphosphate isoprenoid analogue C15AlkOPP manifests superior labeling efficiency compared to alcohol based C15AlkOH in multiple cell lines 38 and therefore, C15AlkOPP was chosen over C15AlkOH in this study. The chemical structures of C15AlkOPP, C15AlkOH, and the endogenous isoprenoids FPP and GGPP are shown in Fig.…”

Splice switching an oncogenic ratio of SmgGDS isoforms as a strategy to diminish malignancy

“…Multiple Small GTPases. To assess whether reducing the 607:558 ratio disrupts prenylation, we tested the incorporation of a synthetic isoprenoid probe, C15AlkOPP (29), after SSO treatment and conducted analysis by in-gel fluorescence and mass spectrometry (Fig. 4A).…”

A Not-So-Ancient Grease History: Click Chemistry and Protein Lipid Modifications

“…409 Furthermore, HeLa cells may not respond to isoprenoid labeling as efficiently as other cell lines do such as COS-7, which displayed excellent labeling with C15-Alk-OPP but not with C15-Alk-OH. 410 This disparity in labeling between cell lines using different forms of the isoprenoid probe can be rationalized with the observation that different cell-types respond with varying degrees of probe labeling with C15-Alk-OH. 411 Profiling of S-prenylated proteome in the macrophage cell line RAW264.7 yielded 23 Ca 1 a 2 X-containing and 12 Rab proteins using C15-Alk-OH.…”