Article pubs.acs.org/jpr
FK506, an Immunosuppressive Drug, Induces Autophagy by Binding to the V‑ATPase Catalytic Subunit A in Neuronal Cells Dongyoung Kim,† Hui-Yun Hwang,† Jin Young Kim,‡ Ju Yeon Lee,‡ Jong Shin Yoo,‡ György Marko-Varga,§ and Ho Jeong Kwon*,†,∥ †
Global Research Laboratory, Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Korea ‡ Biomedical Omics Group, Korea Basic Science Institute, Ochang, Chungbuk 28119, Korea § Clinical Protein Science & Imaging, Biomedical Center, Department of Biomedical Engineering, Lund University, BMC D13, SE-221 84 Lund, Sweden ∥ Department of Internal Medicine, College of Medicine, Yonsei University, Seoul 120-752, Korea S Supporting Information *
ABSTRACT: The drug FK506 (tacrolimus, fujimycin) exerts its immunosuppressive effects by regulating the nuclear factor of the activated T-cell (NFAT) family of transcription factors. However, FK506 also exhibits neuroprotective effects, but its direct target proteins that mediate these effects have not been determined. To identify the target proteins responsible for FK506’s neuroprotective effects, the drug affinity responsive target stability (DARTS) method was performed using labelfree FK506, and LC−MS/MS analysis of the FK506-treated proteome was also performed. Using DARTS and LC−MS/ MS analyses in combination with reference studies, V-ATPase catalytic subunit A (ATP6V1A) was identified as a new target protein of FK506. The biological relevance of ATP6V1A in mediating the neuroprotective effects of FK506 was validated by analyzing FK506 activity with respect to autophagy via acridine orange staining and transcription factor EB (TFEB) translocation assay. These analyses demonstrated that the binding of FK506 with ATP6V1A induces autophagy by activating the translocation of TFEB from the cytosol into the nucleus. Because autophagy has been identified as a mechanism for treating neurodegenerative diseases and because we have demonstrated that FK506 induces autophagy, this study demonstrates that FK506 is a possible new therapy for treating neurodegenerative diseases. KEYWORDS: FK506, neuroprotective activity, DARTS, LC−MS/MS, V-ATPase, autophagy, TFEB translocation
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response.12−15 Furthermore, FK506 has been associated with antineurodegenerative activity, which is a less explored biological aspect of this drug. In recent studies, FK506 has been shown to reduce the negative effects that occur in several neurodegenerative diseases, such as Huntington’s disease, Alzheimer’s disease, and prion diseases.16−20 Neurodegenerative diseases are mainly caused by a loss of neuronal function; it has also been observed that proteins inside the neurons are misfolded and aggregated in patients who experience these diseases.21 The accumulation of these proteins can damage neurons, ultimately leading to neuronal death. Therefore, the removal of misfolded and aggregated proteins is a central goal in treating these diseases. Induction of autophagy, a cellular process that degrades unnecessary cell organelles and proteins via the fusion of an autophagosome to a lysosome, could
INTRODUCTION FK506, also known as tacrolimus or fujimycin, is a macrolide isolated from Streptomyces tsukubaensis.1 FK506 is an immunosuppressive drug used primarily to reduce the risk of organ rejection in patients who have undergone organ transplantation. It is mainly administered to kidney transplant patients but is also given to liver, pancreas, intestine, and heart transplant patients. Thanks to this drug, the use of corticosteroids was decreased significantly.2−5 Like other macrolides, it cannot be produced in vitro, and processes for biosynthesis and purification are needed. Currently, many other bacterial strains that produce FK506 have been generated to maximize production of this drug.6−9 Also, synthetic biological approaches are being used to improve the efficiency of production.10 It is known that FK506 binds to the protein FKBP12, which, in turn, binds to calcineurin and inhibits its activity.11 Calcineurin activates the nuclear factor of the activated T-cell (NFAT) family of transcription factors, which are key regulators of the immune response. The impact of FK506 on calcineurin results in the inhibition of the immune © XXXX American Chemical Society
Special Issue: The Immune System and the Proteome 2016 Received: July 9, 2016
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DOI: 10.1021/acs.jproteome.6b00638 J. Proteome Res. XXXX, XXX, XXX−XXX
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Journal of Proteome Research
to quantify the amounts of proteins present, the protein targets of small molecules can be identified. In this study, we analyzed DARTS samples with LC−MS/MS to identify new protein targets of FK506, specifically those responsible for inducing autophagy and leading to neuroprotective activity.
become one of the main treatment strategies for removing these aggregated proteins.22,23 In the process of autophagy, LC3 and p62 are very pivotal factors. LC3 is a membrane component of autophagosome in its elongation process. LC3 is converted from LC3-I to LC3-II, the latter of which is located on the membrane of the autophagosome; therefore, LC3-II can be used as a means of quantifying the amount of autophagosome in cells.24 On the contrary, p62 recruits misfolded proteins to the autophagosome, localizes mainly to the inside of the autophagosome, and is degraded when a lysosome fuses with that autophagosome, which makes p62 serve as a good marker for the degradation processes that occur during autophagy.25 Small molecules that have effects on autophagy can be classified into two categories: autophagy inducers and the drugs that cause defective autophagy. Autophagy inducers increase both autophagosome formation and lysosomal degradation, but drugs of defective autophagy block the lysosomal degradation, which, in turn, increases the number of autophagosomes in cells. To distinguish these two types of autophagy status, the comparison between quantities of LC3 (LC3-II) and p62 is needed because LC3 represents the number of autophagosomes in cells and p62 represents the degradation status of the cells.26 It has been noted that FK506 shows antineurodegenerative activities that are accomplished through autophagy, but FK506’s protein target for this activity is still not known.17,20 To illuminate the underlying mechanisms of FK506’s impacts on autophagy and antineurodegenerative activity, it is necessary to identify more of its protein targets beyond FKBP12 and calcineurin. A pivotal step in determining the underlying biological mechanisms of a small molecule’s effects is the identification of its protein targets; these may be detected using phenotypic analysis or activity tests. Despite the high demand within the biomedical field for target identification to assist in the treatment of diseases, the targets of many potential drugs are still unknown due to (a) the limitations of existing target identification methods and (b) the complexity of the diseases being treated. The most commonly used methods for identifying the targets of small molecules are pull-down assays and phage display technology, both using an affinity matrix with labeled or tagged small molecules.27−31 Current affinity-based techniques have significant limitations and drawbacks, including the necessity of modifying the small-molecule compound itself.32−34 Modification of a small molecule may have the following disadvantages: (1) the cost of the modification step can be quite high; (2) the modification may disrupt the compound’s native structure; and (3) there are many compounds that are virtually or truly impossible to modify.35−39 To overcome these limitations, new strategies have recently been developed. Some of these new strategies exploit the thermodynamic changes that occur when a small molecule binds to its protein target. These methods employ thermal or proteolytic treatments to study thermodynamic changes in proteins.40 The method that uses proteolytic treatment is called “drug affinity responsive target stability” (DARTS).41−43 In this method, binding between the protein target and the small molecule makes that protein less susceptible to proteolysis than if it had remained an unbound protein; this is because the small-molecule-bound protein undergoes a conformational change that gives it a thermodynamically more stable protein structure. Using liquid chromatography/tandem mass spectrometry (LC−MS/MS)
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MATERIALS AND METHODS Bafilomycin A1, rapamycin, dithiothreitol (DTT), iodoacetamide (IAA), and formic acid were purchased from SigmaAldrich (St. Louis, MO). Dulbecco’s modified Eagle’s medium (DMEM), minimum essential medium (MEM), Hank’s balanced salt solution (HBSS), and fetal bovine serum (FBS) were obtained from Invitrogen (Grand Island, NY). Hoechst 33342, Lipofectamine LTX, and Lipofectamine 2000 were purchased from Invitrogen. Antilight chain 3 (LC3) antibody was purchased from MBL (Nagoya, Japan); antilamin A/C and anti-TFEB antibodies were purchased from Cell Signaling (Beverly, MA), and anti-ATP6V1A, anti-β-tubulin, and anti-βactin antibodies were purchased from Abcam (Cambridge, MA). TFEB-EGFP vector, anti-LAMP1 antibody, and primers for LAMP1 were kindly provided by Dr. Myung-Shik Lee (Yonsei University College of Medicine). Cell Culture
U87-MG cells (human glioblastoma cells) were grown in MEM containing 10% FBS and 1% antibiotics. SH-SY5Y cells (human neuroblastoma cells) were grown in DMEM containing 10% FBS and 1% antibiotics. All cell cultures were maintained at pH 7.4 in a humidified incubator at 37 °C, 5% CO2 in air. Western Blot Analysis
The cell lysates were analyzed by 8, 10, and 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA) via standard electroblotting procedures. The blots were then blocked, immunolabeled, and incubated overnight at 4 °C with primary antibodies. The primary antibodies were blotted with peroxidase-conjugated secondary antirabbit or antimouse antibodies, followed by detection with an enhanced chemiluminescence (ECL) kit (GE Healthcare, Buckinghamshire, U.K.) according to the manufacturer’s instructions. Images were quantified using Image Lab software (Bio-Rad, Hercules, CA). DARTS Method
Cell lysates were obtained from U87-MG cells. Cells were scraped and lysed with 8 M urea lysis buffer. After being centrifuged for 15 min at 10 000g, the supernatant was obtained and protein content was quantified using Bradford solution. Before drug treatment, protein concentration was diluted to 1 mg/mL. Samples were treated with the drugs FK506 and DMSO for 4 h at 4 °C. Samples were then incubated with Pronase or distilled water, as indicated, for 20 min at 25 °C. A small portion of each sample was used for Western blot analysis, and the remainder of each sample was frozen to be used in LC−MS/MS analysis. LC−MS/MS Sample Preparation
For each sample, DTT was added to a final concentration of 10 mM, and the samples were then incubated for 30 min at room temperature, with agitation. Next, IAA was added to each sample to a final concentration of 10 mM, and each sample was then placed in the dark for 30 min at room temperature. Subsequently, protein samples were digested into peptides B
DOI: 10.1021/acs.jproteome.6b00638 J. Proteome Res. XXXX, XXX, XXX−XXX
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Journal of Proteome Research using trypsin; these peptides were incubated with end-over-end rotation at 37 °C for 4 h. The final solutions were dried using a SpeedVac, and the dried peptides were redissolved in an aqueous solution of 0.1% formic acid prior to quantification.
imported ion library; the number of transitions (three) per peptide; a peptide confidence threshold (95%) in percentage that is used to remove the peptides with a confidence below that value; a false discovery rate (FDR < 1%) in percentage, which is used to filter the SWATH extraction results and only export the peptide peak groups displaying a false positive rate below that value; and XIC (Extracted Ion Chromatogram) retention time window (15 min) and mass tolerance window (50 ppm) for RT and m/z tolerance, to pick the transitions. After SWATH peak extraction, the transition ion peak areas, peptide peak areas, and protein peak areas were exported in Excel format for further statistical analysis. The ion peak areas exported by PeakView for each sample were normalized, by total area normalization, using MarkerView 1.2.1. Specifically, the sum total of all ion intensities for each sample was calculated, and the maximum of those totals was determined. The ion intensities of each sample were divided by the ratio of [total ion intensities]/[maximum total ion intensity], ensuring that the normalized ion intensities sum to the same value for all samples. Normalized protein peak areas were automatically performed via PCA (principal component analysis) and then grouped by PC variance in MarkerView; 25 groups were generated. These results were then exported into Excel format according to peptide and protein, which we refer to henceforth as peptide peak area and protein peak area. Finally, the results of proteins showing significant changes in expression level were checked by manual validation, and their peak areas were recalculated based solely on reliable peptide peaks acquired during SWATH extraction.
Proteome Analysis
LC−MS/MS Analysis. IDA (information-dependent acquisition) and SWATH (sequential windowed acquisition of all theoretical fragment-ion spectra) mass spectrum techniques were used to acquire tandem MS data on a Triple TOF 6600 tandem mass spectrometer (AB Sciex, Concord, Ontario, Canada) coupled to an Ekspert nanoLC 400 system (AB Sciex). An autosampler was used to load individual 5 μL aliquots of the peptide solutions into a C18 trap column of inner diameter (i.d.) 180 μm, length 20 mm, and particle size 5 μm (Waters, Milford, MA). Peptides were desalted and concentrated on the trap column for 10 min at a flow rate of 5 μL/ min. Trapped peptides were then back-flushed and separated on a homemade microcapillary C18 column of i.d. 100 μm and length 200 mm (Reprosil Pur 120, C18-AQ, particle size 3 μm) at a flow rate of 300 nL/min. The mobile phases were composed of 100% water (A) and 100% acetonitrile (ACN) (B), each containing 0.1% formic acid. The liquid chromatography (LC) gradient began with 5% mobile phase B and ramped to 10% mobile phase B, for 1 min. 10% mobile phase B was maintained for 4 min. 10% mobile phase B was linearly ramped to 40% for 90 min and then to 95% for 1 min. Then, 95% mobile phase B was maintained for 13 min before descending to 5% B for another 1 min. The column was reequilibrated with 5% B for 10 min before the next run. The voltage of the nanoelectrospray was 2.0 kV. During the chromatographic separation, triple time-of-flight mass spectrometry (TOF-MS) was performed using either an IDA MS or a SWATH MS. In IDA mode, a TOF-MS survey scan was acquired at m/z 400−2000 with 250 ms accumulation time. The 10 most intense precursor ions (2+−5+) in the survey scan were consecutively isolated for product ion scans. Dynamic exclusion was used for 45 s. Product ion spectra were accumulated for 100 ms in the mass range m/z 100−2000, with rolling collision energy. SWATH data were acquired three times for each sample. In SWATH mode, TOF-MS survey scans were acquired (m/z 400−2000, 250 ms); this was followed by 34 product ion scans, with consecutive constant Q1 windows (25 Da) in the mass range m/z 400−1250. Product ion spectra were accumulated for 49 ms in the mass range m/z 100−2000, with rolling collision energy. IDA Data-Based SWATH Library Generation. IDA MS/ MS data were subjected to database searches by ProteinPilot (V5.0, AB Sciex) using the Paragon algorithm. The UniProt human database (August 2014 version) was used for database searches. Search parameters were as follows. Sample type: identification. Cys alkylation: iodoacetamide. Digestion: trypsin. Special factors: none. ID focus: allow biological modifications. The group file from the database search was loaded to PeakView (V2.2 with SWATH Quantitation plug-in) and exported as libraries in CSV format. SWATH Peak Extraction. PeakView V2.2 with SWATH quantitation plug-in (AB Sciex) was used to extract SWATH MS peak areas using the library generated in our study. PeakView uses a set of processing settings to filter the ion library and determine which peptides or transitions should be used for quantification. These settings include: the maximum number of peptides (50) per protein to be included from the
Acridine Orange Staining
SH-SY5Y cells were seeded at a density of 5 × 105 cells/well in six-well plates and incubated overnight. These cells were treated with drugs for the time period indicated, followed by treatment with 2 μg/mL acridine orange (AO). Following incubation, the cells were fixed with 4% paraformaldehyde, washed with phosphate-buffered saline (PBS) three times, and visualized by confocal microscopy. EGFP-TFEB Nuclear Translocation Assay
SH-SY5Y cells were seeded into six-well plates and incubated for 24 h, and EGFP (enhanced green fluorescence protein)TFEB vector was transfected into the cell using Lipofectamine LTX transfection reagent (Invitrogen), according to the manufacturer’s instructions. Then, FK506 treatment was performed, lasting 1−24 h, as indicated. Results were normalized using negative (DMEM medium) and positive (starvation, 3 h, as specified in references) control samples in the same plates. Cells were then washed, fixed, and stained with DAPI. Images were acquired using a Zeiss LSM 700 confocal microscope. Nuclear and Cytosolic Fractionation
SH-SY5Y cells were seeded into cell culture dishes, incubated for 48 h, and treated, as indicated in Figure 4C. Cells were then washed, harvested with trypsin-EDTA, and centrifuged at 500g for 5 min. Subsequently the cells were washed again by resuspending the cell pellet in PBS. Nuclear and cytosolic fractions were extracted using the NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Fisher Scientific, Canoga Park, CA) according to the manufacturer’s protocol. Protein quantification of nuclear and cytosolic extracts was carried out to obtain sample concentrations of 1 mg/mL using C
DOI: 10.1021/acs.jproteome.6b00638 J. Proteome Res. XXXX, XXX, XXX−XXX
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Journal of Proteome Research
Figure 1. FK506 induces autophagosome formation and lysosomal degradation. FK506, rapamycin, and bafilomycin A1 were used to treat SH-SY5Y cells for the indicated times. NT: nontreated. Ra: treated with 10 μM rapamycin. Baf: treated with 10 nM bafilomycin A1. Specific antibodies against LC3 and p62 were used to detect the target proteins. Graphs on the right side represented the quantification results for LC3-II and p62, normalized by comparison to β-actin. The graphs are expressed as the mean ± SD, * p < 0.05 and ** p < 0.01 versus nontreated cells (NT).
Victor 3 Multilabel Plate Readers (PerkinElmer, Waltham, MA).
GTCATTGGAGCACTTGA-3′), respectively. GAPDH mRNA was used as a reference for qRT-PCR.
ATP6V1A RNA Interference and Reverse Transcriptase-Polymerase Chain Reaction Analysis
Statistical Analysis
The results are expressed as the mean standard error (SE) of the raw data. The Student’s t test was used to determine the statistical significance of differences between the control and test groups. A p value of
