Guillaume
Charron
,
Lun K.
Tsou
,
William
Maguire
,
Jacob S.
Yount
and
Howard C.
Hang
*
The Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, NY, 10065. E-mail: hhang@mail.rockefeller.edu
First published on 25th November 2010
Protein S-prenylation is a lipid modification that regulates membrane-protein and protein-protein interactions in cell signaling. Though sites of protein S-prenylation can be predicted based upon conserved C-terminal CaaX or CC/CXC motifs, biochemical detection of protein S-prenylation in cells is still challenging. Herein, we report an alkynyl-isoprenol chemical reporter (alk-FOH) as an efficient substrate for prenyltransferases in mammalian cells that enables sensitive detection of S-farnesylated and S-geranylgeranylated proteins using bioorthogonal ligation methods. Fluorescent detection alleviates the need to deplete cellular isoprenoids for biochemical analysis of S-prenylated proteins and enables robust characterization of S-prenylated proteins, such as effectors that are injected into host cells by bacterial pathogens. This alkynyl-prenylation reporter provides a sensitive tool for biochemical analysis and rapid profiling of prenylated proteins in cells.
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Fig. 1 Bioorthogonal reporters of protein prenylation. (A) CaaX protein S-farnesylation and S-geranylgeranylation (top). Rab protein dual S-geranylgeranylation (bottom). (B) Scheme for metabolic labeling with prenylation reporters. (C) Prenylation reporters. (D) Detection tags. |
The significance of S-prenylated proteins has motivated the dissection of their biosynthetic pathways. The precursors for S-prenylation are derived from isoprenoid pyrophosphate substrates. Farnesyltransferase (FTase) catalyzes the transfer of farnesyl diphosphate (FPP) onto proteins while releasing a free diphosphate (Fig. 1A). Geranylgeranyltransferase type 1 and 2 (GGTase-I and GGTase-II) use geranylgeranyl diphosphate (GGPP) as a substrate (Fig. 1A). FTase and GGTase-I recognize a C-terminal CaaX motif where C is the modified cysteine, “a” is an aliphatic amino acid, and X determines the selectivity for either FTase (prefers Cys, Met, Ser, Ala and Gln) or GGTase-I (prefers Leu and Phe), although this rule is not stringent.14 For example, K-Ras (with CVIM or CIIM motifs) becomes geranylgeranylated in vivo upon farnesyltransferase inhibition15 and RhoB (with a CKVL motif) is known to be both farnesylated and geranylgeranylated in vivo.16 Upon prenylation in the cytosol, CaaX proteins are recruited to the endoplasmic reticulum where CaaX prenyl protease 2 (RCE1) cleaves the aaX tri-peptide and isoprenylcysteine carboxylmethyltransferase (ICMT) methylates the newly exposed C-terminal carboxylic acid of the cysteine. GGTase-II (also referred as RabGGTase) exclusively modifies Rab proteins and requires Rab escort proteins (REP) to present the C-terminal CC or CXC double cysteines of Rab proteins, which gets dually geranylgeranylated (Fig. 1A). Rab proteins bearing a C-terminal CXC motif also get carboxymethylated. Based on the conserved CaaX-box and CC/CXC motifs, bioinformatic analyses predict a few hundred S-prenylated proteins in mammalian proteomes.17 The biochemical dissection of protein prenylation pathways sparked significant interest in the development of robust detection methods to better understand the roles of S-prenylated proteins in cell signaling, but still remains challenging.
Prenylated proteins have been traditionally visualized by metabolic labeling with radioactive substrates such as [3H]mevalonic acid, [3H]FPP and [3H]GGPP, which have low sensitivity and require long exposure times for detection (weeks to months). To address these limitations, chemical reporters of protein prenylation have been developed to improve the detection of lipidated proteins.18,19 Fluorescent (NBD-GPP and NBD-FPP)20 and biotinylated (BGPP)21 analogs of isoprenoid diphosphates were successfully incorporated onto prenylated proteins in mammalian cells, but the bulk size of the fluorophore or biotin moieties may perturb membrane targeting of proteins and downstream signaling. Azido- and alkynyl-isoprenoid diphosphates coupled with bioorthogonal ligation22 provide improved methods for visualizing S-prenylated proteins (Fig. 1B).18,19 Azido- and alkynyl-geranyl diphosphates enabled the selective in vitro tagging by FTase of peptides and proteins bearing a CaaX motif,23–25 while azido-geranylgeranyl diphosphate afforded in vitro labeling by GGTase-I and GGTase-II of geranylgeranylated proteins.26 Further improvement was achieved with azido-farnesol (Az-FOH, Fig. 1C)27 and azido-geranylgeraniol28 metabolic incorporation in mammalian cells onto farnesylated and geranylgeranylated proteins, respectively.
Having previously demonstrated that alkyne-functionalized chemical reporters provide more sensitive detection than their azido-counterpart for fatty-acylated proteins,29 acetylated proteins30 as well as newly synthesized proteins,31 we synthesized and evaluated a panel of alkynyl-isoprenols to improve the detection of protein S-prenylation in cells. We demonstrate here that the alkynyl-farnesol (alk-FOH, Fig. 1C) chemical reporter is a readily accessible substrate for FTase and GGTases in cells and allows sensitive fluorescent detection of S-farnesylated and S-geranylgeranylated proteins using copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) methods.
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Fig. 2 Synthesis of prenylation reporters. |
With all three alkynyl-farnesol reporters in hand, we assayed whether these compounds would be metabolically incorporated into mammalian cells and installed onto endogenous target proteins. Jurkat T cells were incubated with alk-FOH, alk-FOH-2, alk-FOH-3 or az-FOH at 50 μM for 4 h, lysed and conjugated to azido-rhodamine29 (in the case of alk-FOHs) or alk-rhodamine29 (for az-FOH) via CuAAC, separated by SDS-PAGE and analyzed by in-gel fluorescence scanning (Fig. 3). The banding pattern of proteins with the alk-FOH probes was similar to az-FOH labeling, suggesting that all three alk-FOHs were incorporated onto farnesylated proteins. Labeling was stronger with alk-FOH and alk-FOH-3 with markedly stronger signal in the 20–25 kDa molecular weight region of small GTPases. We also determined the effect of HMG-CoA reductase inhibitors (lovastatin) on the incorporation efficiency and detection sensitivity in comparison to az-FOH. Robust signal over the background was obtained without HMG-CoA reductase inhibitors using alkynyl-FOHs, whereas only few specific bands could be detected with previously reported az-FOH.28 Optimal concentration for alk-FOH was 50 μM, as judged by labeling of Jurkat cell lysates (Fig. S1‡). Addition of alk-FOH at 100 μM resulted in cellular toxicity that was not observed at lower concentrations (data not shown).
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Fig. 3 Comparative analysis of prenylation reporters. Jurkat cells were treated with either 20 μM lovastatin or with DMSO as a solvent control for 24 h before supplementing the media with prenylation reporters (50 μM, 4 h). Cell lysates labeled with alkynyl-isoprenols (alk-FOH, alk-FOH-2 and alk-FOH-3) or azido-isoprenol (az-FOH) were conjugated via CuAAC to azido-rhodamine (az-rho) or alkynyl-rhodamine (alk-rho), respectively. Lysates (20 μg) were separated by SDS-PAGE and scanned for fluorescence (top panel) or stained with Coomassie blue as a loading control (lower panel). |
To determine whether the alkynyl-farnesol reporters indeed targeted S-prenylated proteins, we evaluated the labeling of known farnesylated protein (H-Ras) and two different classes of geranylgeranylated proteins (RhoA and Rab7). All three reporters successfully visualized Ras farnesylation upon lovastatin treatment (Fig. 4A). Nonetheless, alk-FOH and alk-FOH-3 afforded better detection of Ras farnesylation than alk-FOH-2 and az-FOH, even at endogenous levels (without lovastatin pre-treatment, Fig. 4A). We focused on alk-FOH, as this chemical reporter can be readily synthesized and is stable compared to other alkynyl-farnesol reporters. Alk-FOH was then assayed with a known substrate protein of each prenyltransferase in HeLa cells transiently expressing Ras for FTase, RhoA for GGTase-I and Rab7 for GGTase-II. All three proteins were labeled by alk-FOH (Fig. 4B). Furthermore, selective depletion of the Ras signal was observed when incubating the cells with a FTase inhibitor (FTI-277, 10 μM), and selective diminution of the RhoA signal was observed with a GGTase-I inhibitor (GGTI-2133, 10 μM) while the signal was unaffected in the case of Rab7 (Fig. 4B). This demonstrates that alk-FOH can be incorporated as a substrate for all three prenyltransferases in cells and hence is a useful tool to detect protein S-prenylation. Alk-FOH was also incorporated by a variety of other cell types (fibroblasts: HeLa, 3T3; leukocytes: Jurkat, DC2.4 and Raw264.7) demonstrating the generality of this chemical reporter (Fig. S2‡). Each cell line exhibited a different banding pattern, highlighting the heterogeneity of S-prenylated proteins among unique cell types.
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Fig. 4 Fluorescent visualization of protein S-prenylation on known prenylated proteins. (A) Jurkat cells were treated with either 20 μM lovastatin or with DMSO as a solvent control for 24 h before supplementing the media with prenylation reporters (50 μM, 4 h). Immunopurified Ras labeled with alkynyl-isoprenols (alk-FOH, alk-FOH-2 and alk-FOH-3) was conjugated via CuAAC to azido-rhodamine (az-rho), followed by separation by SDS-PAGE and fluorescence detection (top panels) or immunoblotting as a loading control (lower panel). (B) HeLa cells transiently expressing HA-RasG12V, GFP-RhoA or GFP-Rab7 were treated with either 10 μM FTI-277, GGTI-2133, or with DMSO as a solvent control for 1 h before supplementing the media with prenylation reporter alk-FOH (50 μM, 4 h). Immunopurified proteins labeled with alk-FOH were conjugated via CuAAC to azido-rhodamine (az-rho), followed by separation by SDS-PAGE and fluorescence detection (top panels) or immunoblotting as a loading control (lower panel). |
Having demonstrated the efficient detection of S-prenylated proteins in mammalian cells with alk-FOH, we evaluated S-prenylation of Salmonella type III secretion system effector SifA. The bacterial protein effector SifA is translocated into the host cytoplasm after bacterial infection and localizes to membranes of the Salmonella-containing vacuoles (SCVs).34 SifA induces the formation of tubular membrane extensions of the SCV termed Salmonella-induced filaments (Sifs),34 which are necessary for maintaining the integrity of the SCV and Salmonella virulence.35 The C-terminal motif of SifA (331CLCCFL336) contains an unusual prenylation motif that can be interpreted either as a CaaX motif and/or a Rab protein motif (CCXX) and S. typhimurium lacking this SifA C-terminal hexapeptide exhibited a significant defect in bacterial replication.36 Although the S-prenylation prediction suite (PrePS)14 does not recognize SifA as a substrate for any prenyltransferase, SifA was labeled specifically at cysteine 333 by [3H]mevalonic acid in rabbit reticulocyte lysate in vitro.37 These data suggest that SifA is S-prenylated by the host cell machinery for recruitment to the SCV membrane,36,37 although SifA S-prenylation could not be detected by [3H]mevalonic acid labeling in cells. The precise roles of SifA S-palmitoylation and S-prenylation during Salmonella infection are unclear, as single point mutations of putative lipidation sites do not recapitulate the defect in Salmonella replication observed with the SifA mutant lacking the entire C-terminal hexapeptide.36,37
To provide more insight into SifA S-prenylation in cells, N-terminally HA-tagged SifA (HA-SifA) was transiently expressed in HeLa cells and metabolically labeled with alk-FOH. HA-SifA was then immunopurified, reacted with az-rho via CuAAC and analyzed by in-gel fluorescence scanning. Alk-FOH afforded robust visualization of SifA prenylation in cells for the first time (Fig. 5A). As opposed to previous in vitro radioactive labeling studies,37 we observed residual cellular HA-SifA prenylation (∼40%) in the presence of GGTase-I inhibitors (GGTI-298 or GGTI-2133, 10 μM). Furthermore, inhibition of FTase (with FTI-277, 10 μM) or dual inhibition of FTase and GGTase-I in cells retained residual SifA prenylation (∼20% and ∼50%, respectively) (Fig. 5A), suggesting that SifA is a substrate for more than GGTase-I. Single mutations of SifA C-terminal cysteines to serines (HA-SifAC333S, HA-SifAC334S) each retained about 30% of prenylation compared to wild-type, while dual mutations (HA-SifAC333,334S) or triple cysteine mutations (HA-SifAC331,333,334S) were not prenylated by alk-FOH in cells (Fig. 5B), suggesting that SifA is dually prenylated at cysteines 333 and 334. These results are in contrast with SifA in vitro radioactive labeling studies, where a single mutation (HA-SifAC333S) was sufficient to deplete SifA prenylation.37
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Fig. 5 Alk-FOH analysis of SifA prenylation. (A) HeLa cells transiently expressing HA-SifA were treated with either 10 μM FTI-277, GGTI-298, GGTI-2133 or 10 μM of both FTI-277 and GGTI-2133, or with DMSO as a solvent control, for 1 h before supplementing the media with prenylation reporter alk-FOH (50 μM, 4 h). Immunopurified HA-SifA labeled with alk-FOH was conjugated via CuAAC to azido-rhodamine (az-rho), followed by separation by SDS-PAGE and fluorescence detection (top panels) or immunoblotting as a loading control (lower panel). (B) HeLa cells transiently expressing HA-SifA, HA-SifAC333S, HA-SifAC334S, HA-SifAC333,334S, or HA-SifAC331,333,334S were treated with prenylation reporter alk-FOH (50 μM, 4 h). Immunopurified HA-SifA labeled with alk-FOH was conjugated via CuAAC to azido-rhodamine (az-rho), followed by separation by SDS-PAGE and fluorescence detection (top panels) or immunoblotting as a loading control (lower panel). Fluorescence was quantified by mean fluorescence intensity adjusted for loading and normalized for background (0) and strongest signal (1). |
The differences in SifA prenylation between our results and previous studies may be due to cellular labeling with alk-FOH compared to in vitro labeling with [3H]mevalonic acid. The improved fluorescence detection with alk-FOH in cells may reveal low levels of S-prenylation not visualized by [3H]mevalonic acid in vitro. Prenylation analyses of proteins bearing the unusual double cysteine CCXX C-terminal motif similar to SifA have been described. Geranylgeranyl modification of Rab5A (CCSN) is catalyzed by an enzyme in brain cytosol but not by purified GGTase-I.38 Similarly, Rab5A prenylation with GGTase-II/REP-1 and [3H]GGPP followed by mass spectrometry analysis demonstrated that Rab5A is geranylgeranylated on both cysteines by GGTase-II.39 However, the CCXX motif is not always a marker for GGTase-II substrates. For example, in vitro translational analyses by radioactive labeling in rabbit reticulocyte lysate demonstrated that both Ral-A (CCIL) and Ral-B (CCLL) are geranylgeranylated exclusively at the cysteine in the fourth position from the carboxyl terminus and [3H]GGPP incorporation onto RalA/B could be blocked with GGTase-I inhibitors.40,41 Alternatively, Wrch-1, a Rho family GTPase bearing a CCFV C-terminal motif and a brain-specific isoform of Cdc42 with a CCIF motif were not found to be prenylated, but intead S-fatty acylated.42,43 CCXX can therefore be a motif for prenylation and S-palmitoylation. Nonetheless, our analyses of SifA prenylation with inhibitors of prenyltransferases and cysteine mutants suggest that SifA may be heterogeneously prenylated in host cells and single cysteine mutations do not completely abolish S-prenylation. While our cellular alk-FOH labeling of SifA differ from in vitro [3H]mevalonic acid labeling studies (Fig. 5), the robust and heterogeneous SifA prenylation from our alk-FOH cellular labeling is consistent with residual membrane partitioning of these SifA mutants as well as their activity during Salmonella infection.37
In conclusion, the alkynyl-farnesol, alk-FOH, provides an efficient chemical reporter of S-prenylated proteins in cells. Several improvements over previously reported detection methods were achieved. Alk-FOH is stable, readily accessible through chemical synthesis, and a substrate of all three prenyltransferases in mammalian cells. Importantly, HMG-CoA reductase inhibitors are not required for alk-FOH detection of endogenously prenylated proteins and transfer of hydrophobic proteins onto membranes is circumvented by in-gel fluorescence detection. Our approach enabled the detection of SifA prenylation in cells for the first time, which revealed robust and heterogeneous prenylation of the Salmonella T3SS effector SifA. Overall, the increased sensitivity achieved here should assist in biochemical and functional analysis of protein S-prenylation in cells.
For cell lysates, 50 μg proteins were clicked in 47 μl SDS-buffer (4% SDS, 50 mM triethanolamine pH 7.4, 150 mM NaCl) with 3 μL freshly premixed click reaction cocktail (same as above) for 1 h at room temperature. Proteins were precipitated by adding ice-cold methanol (1 mL), placing at −80 °C overnight, centrifuging at 18000 g for 10 min at 4 °C and discarding the supernatant. The protein pellets were allowed to air-dry, resuspended in 50 μL loading buffer (same as above), heated for 5 min at 95 °C, and 20 μg of protein was loaded on 2 separate SDS-PAGE gels.
Footnotes |
† This article is part of a themed issue of Molecular BioSystems on Post-translational modifications. |
‡ Electronic supplementary information (ESI) available: Fig. S1 and S2; synthesis details. See DOI: 10.1039/c0mb00183j |
This journal is © The Royal Society of Chemistry 2011 |