Chemical Proteomic Profiling of the Interacting Proteins of Isoprenoid Pyrophosphates

Abstract

Isoprenoid pyrophosphates are involved in protein prenylation and assume regulatory roles in cells; however, little is known about the cellular proteins that can interact with isoprenoid pyrophosphates. Here, we devised a chemical proteomic strategy, capitalizing on the use of a desthiobiotin-geranyl pyrophosphate (GPP) acyl phosphate probe for the enrichment and subsequent identification of GPP-binding proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS). By combining stable isotope labeling by amino acids in cell culture (SILAC) and competitive labeling with low vs high concentrations of GPP probe, with ATP vs GPP acyl phosphate probes, or with the GPP probe in the presence of different concentrations of free GPP, we uncovered a number of candidate GPP-binding proteins. We also discovered, for the first time, histone deacetylase 1 (HDAC1) as a GPP-binding protein. Furthermore, we found that the enzymatic activity of HDAC1 could be modulated by isoprenoid pyrophosphates. Together, we developed a novel chemical proteomic method for the proteome-wide discovery of GPP-binding proteins, which sets the stage for a better understanding about the biological functions of isoprenoids.

Figure 1.

Isoprenoid probes for studying protein prenylation (A) and design of the affinity-based probe for the covalent labeling and enrichment of GPP-binding proteins and their component peptides (B).

Figure 2.

SILAC-based competition assay for assessing GPP-binding proteins at the entire proteome scale. (A) The workflow for the competition assay. (B) Quantitative comparisons of the labeling efficiencies with the use of 100 vs 10 μM GPP probe. The data were obtained from 2 forward and 2 reverse labeling experiments; FDPS-a and FDPS-b represent two desthiobiotin-labeled peptides identified from FDPS. (C) Workflow for the competition between GPP and ATP acyl phosphate probes. (D) Quantification results obtained from the GPP/ATP competition assay with 2 forward and 2 reverse SILAC labeling experiments.

Figure 3.

Quantitative analysis for GPP inhibition on probe labeling efficiency. (A) Workflow for the competition experiment. (B) Full-scan ESI-MS of the light- and heavy-labeled tryptic peptide LKEVLEYNAIGGKYNR from FDPS in the presence or absence of free GPP. (C) A heat map showing the quantification results with increasing concentrations of GPP, where the proteins are ranked on the basis of the calculated IC₅₀ values of the labeling efficiencies of the GPP probe.

Figure 4.

Labeling sites for known GPP-binding proteins. (A) Diagrams showing the interaction between the Lys123 and the bound IPP in FDPS (PDB 4H5E); the probe-labeled Lys123 corresponds to Lys57 in the reported crystal structure, and the blue-colored strand represents the labeled peptide identified from LC-MS/MS. (B) A crystal structure of GGPS1 (PDB 2Q80) showing the GGPP-binding pocket and the location of the probe-labeled Lys25. (C, D) Comparison of the detected intensity for the light and heavy peptides for FDPS, GGPS1, and HDAC1 with increasing concentrations of GPP (from left to right: 0, 50, 100, 200, 500, and 1000 μM).

Figure 5.

GPP and GGPP could modulate the enzymatic activity of HDAC1 in vitro. (A) A crystal structure of HDAC1 (PDB 5ICN) showing the interaction of Lys31 with the bound inositol hexaphosphate in the allosteric pocket; H4K16Hx is an inhibitor for HDAC1 and occupies the active site through coordination with Zn²⁺. (B) The chemical structures of GPP and GGPP. (C, D) Western blot results for monitoring the enzymatic activity of HDAC1 in the presence of different concentrations of GPP or GGPP, where calf thymus core histones were used as the substrate.