Improved synthesis and application of an alkyne-functionalized isoprenoid analogue to study the prenylomes of motor neurons, astrocytes and their stem cell progenitors

Abstract

Protein prenylation is one example of a broad class of post-translational modifications where proteins are covalently linked to various hydrophobic moieties. To globally identify and monitor levels of all prenylated proteins in a cell simultaneously, our laboratory and others have developed chemical proteomic approaches that rely on the metabolic incorporation of isoprenoid analogues bearing bio-orthogonal functionality followed by enrichment and subsequent quantitative proteomic analysis. Here, several improvements in the synthesis of the alkyne-containing isoprenoid analogue C15AlkOPP are reported to improve synthetic efficiency. Next, metabolic labeling with C15AlkOPP was optimized to obtain useful levels of metabolic incorporation of the probe in several types of primary cells. Those conditions were then used to study the prenylomes of motor neurons (ES-MNs), astrocytes (ES-As), and their embryonic stem cell progenitors (ESCs), which allowed for the identification of 54 prenylated proteins from ESCs, 50 from ES-MNs, and 84 from ES-As, representing all types of prenylation. Bioinformatic analysis revealed specific enriched pathways, including nervous system development, chemokine signaling, Rho GTPase signaling, and adhesion. Hierarchical clustering showed that most enriched pathways in all three cell types are related to GTPase activity and vesicular transport. In contrast, STRING analysis showed significant interactions in two populations that appear to be cell type dependent. The data provided herein demonstrates that robust incorporation of C15AlkOPP can be obtained in ES-MNs and related primary cells purified via magnetic-activated cell sorting allowing the identification and quantification of numerous prenylated proteins. These results suggest that metabolic labeling with C15AlkOPP should be an effective approach for investigating the role of prenylated proteins in primary cells in both normal cells and disease pathologies, including ALS.

Keywords: Click chemistry; Farnesylation; Geranylgeranylation; Metabolic labeling; Prenylation; Proteomics; Synthetic probe.

Figure 1.

Structures of natural prenylation substrates, FPP and GGPP, the alkyne-containing probe C15AlkOPP (1) and the C-terminal structure of a mature prenylated protein.

Figure 2.

Synthetic route for the preparation of C15AlkOPP (1).

Figure 3.

Metabolic labeling using C15AlkOPP in ES-MNs. A) Comparison of prenylome labeling in ES-MNs with previously studied cell lines COS-7 and HeLa. Labeling was performed by preincubation with lovastatin at the concentrations indicated for 6 h followed by treatment with C15AlkOPP for 18 h. B) Different treatment conditions in ES-MNs to improve prenylome labeling. Labeling was performed by preincubation with lovastatin at the indicated concentrations and times (Pre-treatment) followed by treatment with 25 μM C15AlkOPP for the indicated times (probe time).

Figure 4.

C15AlkOPP labeling in motor neurons, astrocytes and their stem cell progenitors. A) Metabolic labeling with C15AlkOPP in ES-MN cells. B) Metabolic labeling with C15AlkOPP in ESC parent cells. C) Metabolic labeling with C15AlkOPP in ES-As cells. In all cases, labeling was performed by preincubation with 2 μM lovastatin for 24 h followed by treatment with 25 μM C15AlkOPP for 72 h. In each gel, lanes 1–3 and 4–6 are from triplicate samples.

Figure 5.

Prenylomic analysis of astrocytes, motor neurons and stem cell parent cells. A) Volcano plots (FDR = 0.01, s0 = 0.5) for the proteins identified after C15AlkOPP treatment and enrichment from ES-MNs, ESC parent cells, and ES-As. B) Comparison of the f identified proteins from the three cell types. C) Comparison of the identified proteins annotated as farnesylated (F), geranylgeranylated (GG), and Rabs. The novel proteins identified containing CaaX box motifs were assigned as farnesylated. Proteins labeled in the volcano plots are proteins noted in the over-representation analysis (ORA) described below. In addition, three novel proteins including Stonin-2, Fam63a and Fam63b, identified in ES-As, are also labeled.

Figure 6.

Comparison of prenylomic data obtained here with that observed in previous studies with COS-7 and E.hy296 cells. A) Comparison of the number of prenylated proteins enriched in ES-As (this work) compared with previous studies in COS-7 and EA.hy926 cells. B) Comparison of the fold change obtained for prenylated protein enrichment in ES-As (this work) and COS-7 (previous work). C) Comparison of the number of prenylated proteins and their prenylation type obtained in ES-MNs (this work) compared with previous results with N2a cells. D) Comparison of the number of prenylated proteins and their prenylation type obtained in ES-As (this work) compared with previous results with primary astrocytes. Data for COS-7, N2A and primary astrocytes is from Suazo et al. and data for EA.hy926 cells is from Storck et al.

Figure 7.

Bioinformatic analysis of ESCs, astrocytes (ES-As) and motor neurons (ES-MN). A) Heatmap of scaled prenylated proteins. B) Cord diagrams for the top 5 enriched pathways, sorted for padj, and resulting from Over Representation Analysis (ORA) performed with gProfiler R package, on the hyper-prenylated proteins for each population as indicated by the color codes bars on the right of the heatmap. Gene color is function of scaled fold change. C) STRING protein connections among selected hyperprenylated proteins.