Metabolic Labeling with an Alkyne-modified Isoprenoid Analog Facilitates Imaging and Quantification of the Prenylome in Cells

Protein prenylation is a post-translational modification that is responsible for membrane association and protein–protein interactions. The oncogenic protein Ras, which is prenylated, has been the subject of intense study in the past 20 years as a therapeutic target. Several studies have shown a correlation between neurodegenerative diseases including Alzheimer’s disease and Parkinson’s disease and protein prenylation. Here, a method for imaging and quantification of the prenylome using microscopy and flow cytometry is described. We show that metabolically incorporating an alkyne isoprenoid into mammalian cells, followed by a Cu(I)-catalyzed alkyne azide cycloaddition reaction to a fluorophore, allows for detection of prenylated proteins in several cell lines and that different cell types vary significantly in their levels of prenylated proteins. The addition of a prenyltransferase inhibitor or the precursors to the native isoprenoid substrates lowers the levels of labeled prenylated proteins. Finally, we demonstrate that there is a significantly higher (22%) level of prenylated proteins in a cellular model of compromised autophagy as compared to normal cells, supporting the hypothesis of a potential involvement of protein prenylation in abrogated autophagy. These results highlight the utility of total prenylome labeling for studies on the role of protein prenylation in various diseases including aging-related disorders.


Imaging Prenylated Proteins
HeLa, A549, MDCK, COS-7, or NIH/3T3 cells were seeded (see above for density) in 35 mm glass bottomed culture dishes and grown to approximately 50% confluence (24 h). The cells were rinsed twice with phosphate buffered saline (PBS), followed by the addition of 10 µM C15Alk in 2 mL complete DMEM media. After 24 h probe incubation, the cells were rinsed twice with PBS, fixed with 4% paraformaldehyde (PFA) in PBS for 10 min at RT, permeabilized with 0.1% Triton X-100 in PBS for 2 min at RT, and subsequently rinsed three times with PBS. The following reagents were added, in order, at the specified concentration (typically 300 uL total volume in PBS): TAMRA-PEG-N 3 or 5-Fam-PEG-N 3 (0.1 mM), tris(2-carboxyethyl)phosphine (TCEP, 1 mM), tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl] amine (TBTA, 0.2 mM), CuSO 4 (1 mM). Following 1 h incubation at RT, the cells were rinsed four times with PBS and Hoescht 34580 nuclear stain was added at a final concentration of 1 µg mL -1 for 10 min. Additionally, for the colocalization experiments, ER Tracker Red (1 µM for 10 min) or Wheat Germ Agglutinin conjugated to Alexa Fluor 594 (5 µg mL -1 for 10 min) were added. The cells were rinsed twice with PBS and imaged using an Olympus FluoView FV1000 IX2 Inverted Confocal Microscope with a 60X objective. All the confocal images in Figure 2 were acquired under the identical microscope settings (including Laser source power, exposure time and PMT gain). Additionally, RGB settings used to prepare colored images in the Figure 2 are identical across each color.
Quantifying the Prenylome Cells were seeded in 6-well culture dishes and grown to approximately 50-60% confluence (24 h). Cell culture media was then supplemented with 25 µM and cells were incubated for 18-24 h.
Media from the cells was then replaced with 1 mL fresh media, followed by addition of 10-50 µM C15Alk (or as indicated). Cells were grown for 24 h in presence of C15Alk, unless otherwise specified. In the inhibitor treatment experiments, 10-100 μM L-744,832, 5 µM GGTI-286 or 1-2 mM 3-PEHPC were added to cells 1 h prior to labeling with 10 µM C15Alk. For competition experiments, 10-50 µM FOH, 10-50 µM GGOH or 50-100 µM mevalonolactone were added immediately following C15Alk addition. After incubation of C15Alk and/or inhibitors for various times (typically 24 h), the cells were rinsed twice with PBS and trypsinized for 5 min. After addition of 4X volume of media to inactivate trypsin, cells were transferred to micro-centrifuge tubes, and pelleted by centrifugation at 100 x g for 5 min. The media was aspirated from the pellet and the cells were fixed with 4% paraformaldehyde in PBS for 10 min at RT, pelleted, and permeabilized with 0.1% Triton X-100 in PBS for 2 min at RT. After permeabilization, cells were pelleted and rinsed twice with 200 µL PBS. Next, the following reagents (at the indicated final concentrations) were mixed immediately prior to use and 100 uL total volume in PBS was added to each cell pellet: 5-Fam-PEG-N 3 (0.5 mM), TCEP (1 mM), TBTA (0.2 mM), CuSO 4 (1 mM).
Following 1 h incubation at RT, the cells were pelleted, and rinsed three times with 200 µL PBS.
Cells were added to a 12 x 75 mm test tube (1.5 mL in PBS) for flow cytometry analysis. A total of 10,000 events (cell counts) for each sample were analyzed using a BD FACSCalibur (BD Biosciences) or a BD Accuri C6 (BD Biosciences).

In-Gel Fluorescence
HeLa cells were seeded in 60 mm or 100 mm culture dishes at 1.9 x 10 4 cells/cm 2 and grown for approximately 24 h (to approximately 50-60% confluence). Cell media were then supplemented with 25 µM lovastatin (when indicated), cells were grown for 18-24 h. Cell media was then replaced with media containing 10 µM C15Alk (unless specified otherwise) and cells were grown for 24 h (unless specified otherwise). Following this, the cells were rinsed twice with icecold PBS and scraped from the plate in 0.6-1.0 mL PBS using a teflon scraper. Cells were pelleted by centrifugation at 100 x g for 5 min. After discarding the supernatant, 200-400 µL of lysis buffer was added to each cell pellet. Lysis buffer consisted of PBS containing 1% SDS, protease inhibitor cocktail (per manufacturers protocols), benzonase (per manufacturers protocols), and 2.4 µM PMSF. Cells were lysed by sonicating with six cycles of 10 seconds on, 20 seconds off on ice. The concentration of protein in the lysate was determined using DC™ protein assay (Bio-Rad). To 100 µg (1 mg mL -1 concentration) of HeLa lysate, CuAAC reagents were added at indicated final concentrations: 25 µM TAMRA-PEG-N 3 , 1 mM TCEP, and 100 µM TBTA. After vortexing, 1.0 mM CuSO 4 (final concentration) was added, and the reaction was allowed to proceed at RT for 1 h. Excess reagents were removed by protein precipitation with a ProteoExtract protein precipitation kit (Calbiochem), following manufacturers protocols. Protein pellets were dissolved in 1x Laemmli SDS-PAGE loading buffer and boiled for 6 min and 30-50 µg of protein samples were loaded onto 12% polyacrylamide gel. Gel was run at 120 V until dye front reached the bottom of the gel. For in-gel TAMRA fluorescence images, gels were scanned on a BioRad FX Molecular Imager. Gels were stained with Coomassie blue staining solution for total protein stain images.

In-Gel Fluorescence Densitometry
Densitometry was performed using ImageJ. In brief, gel images in the TAMRA channel were converted into 8-bit mode and a region between 150 kDa and 20 kDa was selected for all the lanes. Lane profiles were plotted, bands were defined by drawing vertical lines, and the area

Primary Astrocyte Preparation
The brains were removed from C3H/C57BL/6 mice pups at postnatal day 0-2 and placed in 1x Hanks balanced salt solution (HBSS). Cortices and hippocampi were dissected out and meninges removed. The tissues were cut into 1 mm 3 pieces with a sterile disposable scalpel, transferred to a conical tube with 1x trypsin in HBSS, and incubated in a 37 °C water bath for 15 minutes. A stop solution, containing 4% FBS and 0.2 mg mL -1 DNase I in HBSS, was added and the incubation extended for an additional 30 seconds at 37 °C. The tissue mixture was centrifuged at 200 x g for 5 minutes at RT and the supernatant was carefully aspirated from the tube. DMEM containing 10% FBS was added to the cell pellet and the mixture was triturated 20 times with a fire-polished pasteur pipet to dissociate cells. The cell suspension was passed through a 100 μm cell strainer to achieve single-cell suspension. Cells were then plated in T-75 flasks coated with poly-D-lysine, each plate was seeded with approximately 3-5 pups worth of glial cells. Cells were cultured at 37 °C with 5% of CO 2 , in DMEM containing 5% FBS, 16 mM Hepes Buffer, 1x non-essential amino acids, 2 mM glutaMAX, 2.5 µg mL -1 aphotericin-B, and 50 µg mL -1 gentamicin. Media was changed on days 1, 2, 6, 14 and 21 post-plating to remove cellular debris. At this point, mixed glial/astrocyte cultures were trypsinized and approximately 0.5 x 10 6 cells were plated into each well of a poly-D-lysine (0.1 mg mL -1 ) coated 6-well plate.
For purifying astrocytes: 14 days after plating, microglial cells (attached on top of the astrocyte cell monolayer adhered to the culture plate) were harvested by shaking them off the astrocyte monolayer, and media was replaced on astrocyte culture. Seven days later, this process was performed again. At this time, astrocytes were passaged to a new T-75 flask and allowed to grow for 4 days, at which point they were shaken to remove any residual microglia and passaged again. Four days later, the astrocytes were passaged a third time and the total cell number and viability were determined with a hemocytometer by adding 20 µL of cell suspension and 20 µL of Trypan blue. Approximately 0.5 x 10 6 cells were plated into each well of a 6-well plate, which had been previously coated with 0.1 mg mL -1 poly-D-lysine. After 3 days in culture, the astrocytes were subjected to metabolic labeling and quantification of the prenylome as described above. and ATG7 knockdowns. While ATG5 and ATG7 knockdowns resulted in approximately 70% reduction in the corresponding protein levels, we were unable to detect the BECN1 protein using the rabbit-hosted Beclin-1 antibody described above. Hence we were unable to establish the level of knockdown in the C2C12 cells used here. However, it should be noted that BECN1 Silencer ® Select Pre-designed siRNAs have been used by other investigators to successfully knockdown BECN1. 2,3 Kinetic Constant Measurement His-tagged rat FTase (rFTase) was expressed in E. coli and purified similarly to previous methods. 4 The rate of farnesylation of purified peptide by rFTase was determined using the

Western Blot Analysis
Cell lysates were prepared using the following buffer: 50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mg mL -1 aprotinin, 1 mg mL -1 leupeptin, 1 mM Na 3 VO 4 , 1 mM NaF, 20 mM b-glycerophosphate. Lysates were cleared by centrifugation (16,000 x g, 10 min, 4 °C) and proteins (40 µg) were resolved using 15% SDS-polyacrylamide mini-gels, and then transferred to Immobilon-P PVDF membrane. After blocking in a TBST/5% milk solution, immunoblots were incubated overnight at 4 °C using the following primary antibodies: anti-H-ras rabbit polyclonal (1:500) and anti-ERK2 rabbit polyclonal     After 24 h, whole cell lysates were prepared and 40 µg of protein was resolved by 15% SDS-PAGE. The proteins were transferred to a PVDF membrane and incubated with an antibody against H-Ras. Note the faster mobility of processed Ras (P) compared with unprocessed Ras (U). Total ERK2 levels were measured as a loading control because they do not change in response to treatment with lovastatin or alkyne isoprenoids; Bottom: Densitometry analysis of western blot bands in Figure 2 using ImageJ.  1% Triton X-100 in PBS was found to be optimum. In B), cells were permeabilized using Triton X-100 (2 min at RT) and subjected to CuAACs with indicated concentrations of 5-Fam-PEG-N 3 for 1 h. After several additional rinses, the cells were analyzed by flow cytometry and the results are expressed as the percentage of maximum cell count. Data were obtained in triplicate with similar results. 50 µM 5-Fam-PEG-N 3 was found to be sufficient to cause significant labeling over background. Figure S6. Control experiments utilizing a non-reactable farnesyl alkyne analog demonstrate no background labeling. Farnesyl alkyne was incubated with HeLa cells at 50 μM for 24 h followed by fixation, permeabilization, and the CuAAC to 5-Fam-PEG-N 3 . The extent of the prenylated protein labeling with this analog is not over the DMSO background, as this analog is not capable of being incorporated onto proteins as it lacks the ability to be phosphorylated. Further, C15Alk at various concentrations was incubated with 5-Fam-PEG-N 3 in vitro prior to cellular incubation ('C15 pre-click'). This 'pre-clicked' mixture was then added to fixed cells for 1 h, followed by washing and analysis by flow cytometry. The labeling in all cases is less than the DMSO background, indicating that the fluorophore-isoprenoid complex that is formed during the CuAAC isn't being trapped or non-specifically attached to the cells and causing background labeling.   Figure S10. 10-50 µM FOH and GGOH significantly compete away labeling caused by 10 µM C15Alk, particularly in 25 kDa region. HeLa cells were treated for 24 h with 10 µM C15, in the presence or absence of 10 µM C15Alk overnight pre-treatment. 10, 25 or 50 µM farnesol and geranylgeraniol, each, were added simultaneously with C15Alk to compete away cellular labeling of prenylated proteins. Cells were lysed, and lysates were reacted with 25 µM of TAMRA-PEG-N 3 for 1 h and then proteins were separated on a 12% polyacrylamide gel. Densitometry analysis of these images included in Figure 6.     The cells were analyzed via flow cytometry and the results are expressed as the mean fluorescence intensity of 10,000 cells ± standard error of the mean of at least three replicates. A. Cos-7 cells, which have higher cell area (see Figure S12), have less incorporation of C15Alk compared to HeLa cells. B. There is significant incorporation of C15Alk in primary astrocyte cells. Figure S16. In a cellular model of compromised autophagy, a surrogate to study aging disorders, the levels of prenylated proteins are increased over normal control levels. In the defective autophagy cell model, the ATG7 gene controlling autophagy was knocked down with siRNA (denoted as "Aging" L6 cells). Control cells included transfection of a scrambled siRNA. After 24 h transfection period, cells were incubated with C15Alk at 50 μM for 24 h, followed by fixing, permeabilizing, and the CuAAC to 5-Fam-PEG-N 3 and analysis by flow cytometry. After several additional rinses, the cells were analyzed by flow cytometry and the results are expressed as the mean fluorescence intensity of 10,000 cells ± standard error of the mean of at least three replicates. There is a statistically significant (p=0.002) increase in the amount of prenylated proteins in the model of compromised autophagy.