Changes in the Proteome after Neuronal Zif268 Overexpression Karsten Baumga¨rtel,†,‡,§ Ry Y. Tweedie-Cullen,†,‡ Jonas Grossmann,| Peter Gehrig,| Magdalena Livingstone-Zatchej,† and Isabelle M. Mansuy*,† Brain Research Institute, Medical Faculty of the University of Zu ¨ rich and Department of Biology, Swiss Federal Institute of Technology Zu ¨ rich (ETHZ), Switzerland, and Functional Genomics Center Zu ¨ rich, University of Zu ¨ rich/Swiss Federal Institute of Technology Zu ¨ rich (ETHZ), Switzerland Received November 18, 2008
Long-lasting forms of brain plasticity are a cellular basis for long-term memory, and their disturbance underlies pathological conditions such as dementia and cognitive impairment. Neuronal plasticity is a complex process that utilizes molecular cascades in the cytoplasm and the nucleus and involves numerous transcription factors, in particular, immediate early genes (IEGs). The signaling cascades that control IEGs are fairly well described, but the downstream transcriptional response is poorly understood, especially its late components. Here, we investigated the response induced by the IEG Zif268 in the adult brain in relation to long-term memory. Using a mouse model with increased neuronal expression of Zif268 that leads to improved memory, we identified an ensemble of proteins regulated by Zif268 expression and differentiated between direct and indirect targets based on the presence of a consensus binding motif in their promoter. We show that Zif268 regulates numerous substrates with diverse biological functions including protein modification and degradation (proteasome-core complex), phosphorylation, cell division, sensory perception, metabolism, and metal ion transport. The results provide a comprehensive and quantitative data set characterizing the Zif268-dependent proteome in the adult mouse brain and offers biologically important new insight into activity-dependent pathways downstream of IEGs. Keywords: Egr-1 • Zif268 • neuronal plasticity • mass spectrometry • iTRAQ • bioinformatics
Introduction Neuronal plasticity is a property of the nervous system that allows nerve cells to dynamically respond to activity. This property underlies cognitive functions and complex behaviors such as learning, memory, and drug addiction.1 Plasticity is mediated by a variety of molecular processes that are distinct in time course, nature, and persistence. In general, transient changes such as post-translational modifications (PTMs) of proteins sustain short-term forms of plasticity, while longlasting changes involving transcriptional regulation underlie persistent forms of plasticity.2 Both forms of plasticity, however, are interdependent and share some common components. For instance, protein PTMs are required for signaling cascades that also control transcription factors (TFs) and chromatin remodelling and thereby regulate gene expression.3 * To whom correspondence should be addressed. E-mail: mansuy@ hifo.uzh.ch, Brain Research Institute, University of Zu ¨ rich/Swiss Federal Institute of Technology Zu ¨ rich, Winterthurerstrasse 190, CH-8057 Zu ¨ rich, Tel. +41 44 635-3360, Fax +41 44 635-3303. † Brain Research Institute, University of Zu ¨ rich/Department of Biology, Swiss Federal Institute of Technology Zu ¨ rich. ‡ These authors contributed equally. § Current Address: Institute for Childhood and Neglected Diseases, Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla 92037, USA. | Functional Genomics Centre Zu ¨ rich, University of Zu ¨ rich/Swiss Federal Institute of Technology Zu ¨ rich.
3298 Journal of Proteome Research 2009, 8, 3298–3316 Published on Web 04/17/2009
Signaling cascades engaged in plasticity and memory have been extensively studied using pharmacological and transgenic approaches,4 and recently also by proteomic methods5-7 (for a review, see ref 8). However, the transcriptional component of neuronal plasticity is only partially understood. It is known to involve the induction of IEGs, as either direct effectors and/ or coordinators of subsequent waves of gene expression9 required for persistent neuronal plasticity. Eliminating or inhibiting specific IEGs thought to mediate this response induces profound deficits in ex vivo models of plasticity and in long-term memory in mice (see, for instance, refs 10-13). The substrates of this late phase of transcription and their functions remain largely unidentified. To better understand how the late transcriptional response contributes to neuronal plasticity and long-term memory, we overexpressed Zif268 (Egr-1, NGFI-A, Tis-8, Krox24, ZENK) inducibly in forebrain neurons by conditional transgenesis in mice.14 Zif268 is an IEG encoding a TF critical for memory processes11,12,15 and drug addiction.16,17 We investigated the molecular basis of this involvement by examining the effect of Zif268 overexpression on protein expression in the amygdala, a brain region involved in emotional memory18,19 and addiction.20,21 Using the technique of quantitative isobaric tags for relative and absolute quantification (iTRAQ) and mass spectrometry (MS), we identified an ensemble of proteins differentially regulated by Zif268 overexpression. Gene ontology 10.1021/pr801000r CCC: $40.75
2009 American Chemical Society
Changes in the Proteome after Neuronal Zif268 Overexpression
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analysis revealed that these proteins compose distinct functional groups. Extensive bioinformatic analyses further differentiated between primary and secondary Zif268 targets and generated lists of putative target genes from the whole mouse genome that were compared to our data set. This provided a comprehensive list of potential Zif268 targets based on published putative Zif268 binding motifs and our own data set. Taken together, this novel and functional data represents a step forward in the elucidation of signaling cascades regulated by Zif268 in the adult mouse brain.
Experimental Procedures Generation of Zif268 Transgenic Mice. Zif268 overexpressing mice were generated as described previously.14 Adult mutant male mice and control littermates (6 months old) were used. Mice were maintained in standard conditions under a reversed light cycle (dark phase, 7 a.m. to 7 p.m.). Doxycyclinesupplemented food (6 mg per 100 g of wet food, West-Ward Pharmaceuticals) was administered to the animals daily for 12 days before mice were sacrificed. All experiments were carried out in accordance with guidelines and regulations of the Cantonal Veterinary Office, Zu ¨ rich. Western Blotting. For nuclear enrichment, isolated cortex was homogenized in lysis buffer (0.32 M sucrose, 10 mM HEPES pH 7.4, 1 mM MgCl2, 0.5 mM CaCl2, 5 mM EDTA, protease inhibitor cocktail (Sigma)), using a Teflon homogenizer (Wheaton). After a 10-min centrifugation at 1000 rpm, the pellet was resuspended in lysis buffer with 2% SDS but without sucrose. After an additional centrifugation, the supernatant was removed, the protein concentration was determined using a Bradford assay, and 50 µg of each sample were resolved by SDSPAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (Biorad). Blocking was performed in 4% milk powder dissolved in 1× TBS-T, and antibodies were diluted in a buffer for near-infrared detection (Rockland). Total Zif268 protein was revealed using an anti-Egr1 antibody (1:1000, Cell Signaling Technology) followed by an IRDye700-conjugated goat anti-rabbit IgG (1:5000, Rockland) and normalized to histone H1 (1:1000, Abcam) followed by IRDye800-conjugated goat anti-mouse IgG (Rockland, final dilution 1:5000). Visualization and quantification were performed using the Odyssey Infrared Imaging system (LI-COR Biosciences). Immunofluorescence. Mice were transcardially perfused with 4% paraformaldehyde in 1× PBS (phosphate buffered saline); their brains were isolated and cryosectioned coronally at 20 µm. After permeabilisation for 30-min in 0.4% Triton X-100 (Sigma) diluted in 1× PBS, sections were washed (3 times for 5-min in 1× PBS) and nonspecific binding was blocked for 1 h using 10% fetal calf serum (GibcoBRL), 0.5% blocking reagent (NEN, Perkin-Elmer), 0.2% Triton X-100 in 1× PBS. The sections were incubated with primary antibody (1:200 rabbit anti-Zif268, Cell Signaling Technology; 1:200 rabbit anti-βgalactosidase, Molecular Probes; 1:200 mouse anti-β-galactosidase, Promega; 1:400 mouse anti-NeuN, Abcam; 1:400 rabbit anti-GFAP, DAKO) diluted into 5% fetal calf serum, 0.05% Triton X-100, 1× PBS, overnight at 4 °C and washed 4 times for 15 min in 1× PBS. Secondary antibody incubation (1:1000 anti-rabbit-FITC, Jackson Immunoresearch; 1:1000 anti-mouseCy3, Jackson Immunoresearch) was conducted for 1 h at room temperature in 5% fetal calf serum, 0.05% Triton X-100 in 1× PBS. After 4 washing steps of 5 min with 1× PBS, sections were mounted on coverslips (Menzel) in MOWIOL mounting medium and stored at 4 °C. Images were recorded on a confocal
Figure 1. Zif268 mutant mice coexpress Zif268 and lacZ in forebrain neurons. (A) Zif268 mutant mice carry two transgenes: a CaMKIIR promoter-rtTA2 transgene and a transgene carrying a bitetO-promoter fused to a β-galactosidase reporter gene and a Zif268 open reading frame. (B) In Zif268 mutant mice, Zif268 protein is increased by 50% in neocortex as analyzed by semiquantitative immunoblotting (normalized to histone H1; mutants: 150 ( 18%, n ) 6, black bar; controls: 100 ( 14%, n ) 6, white bar; ANOVA, main effect of genotype: F1,10 ) 4.98; p < 0.05). Representative bands from the immunoblots are shown in the small inset. (C) Expression of nuclear β-galactosidase coincides with strong Zif268 expression. (D) β-galactosidase expression is neuron-specific and colocalizes with the neuronal nuclear marker NeuN. (E) β-galactosidase expression is absent from glial cells positive for the astrocytic marker glial fabrilliary acidic protein (GFAP).
microscope (Leica) and processed using the Imaris Software (Bitplane AG). Sample Digestion and iTRAQ Labeling. Mice were sacrificed by cervical dislocation, the amygdala was removed and homogenized in 200 µL lysis buffer (50 mM ammonium bicarbonate pH 8, 0.1% SDS) by 10 up and down strokes of a 27 G gauge syringe, sonicated for 2-min and centrifuged at 13 000 g for 10-min to remove insoluble material. Protein fractions (100 µg) were desalted by acetone precipitation then solubilized and cysteine blocked according to the iTRAQ protocol. Samples were digested overnight with trypsin (Promega) at 37 °C (1:13 enzyme:substrate) into peptides. Peptides were differentially Journal of Proteome Research • Vol. 8, No. 7, 2009 3299
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Figure 2. (A) Schematic representation of the workflow of the proteomics experiment. After isolation of the amygdala from Zif268 mutant mice (n ) 6) and littermate controls (n ) 8), the tissue was homogenized, digested with trypsin, and iTRAQ labeled. For each of the four control samples, the amygdala from two control mice were pooled prior to tissue homogenization. The samples were then mixed, fractionated using SCX-HPLC, and spotted after RP-HLPC on three MALDI plates. (B) Order in which samples were labeled and spotted on all three MALDI plates.
labeled with iTRAQ reagents according to manufacturer’s instructions (Applied Biosystems/MDS SCIEX) and as described previously6 and combined as per Figure 2B prior to 2D-LC (twodimensional liquid chromatography) MALDI (matrix assisted laser desorption ionization) MS/MS analysis. SCX-HPLC Fractionation of Peptides. The solution containing peptides was acidified to pH < 3 with 10% TFA, made to 25% acetonitrile, and centrifuged at 16 000 g for 10 min to remove insoluble material. Peptides were loaded onto a 2.1 mm × 200 mm polySULFOETHYL aspartamide A column (PolyLC) on an Agilent HP1100 binary HPLC (high performance liquid chromatography) system and then eluted with an increasing KCl gradient (0 to 150 mM over 35 min, 150 to 300 mM over 10 min, 300 to 500 mM over 10 min, and 500 mM for the following 10 min) in 10 mM KH2PO4, 25% acetonitrile, pH 3. Fractions were pooled based on the UV chromatogram to yield 4 fractions, lyophilized to remove acetonitrile, desalted with 3300
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Baumga¨rtel et al. Sep-Pak reversed-phase (RP) cartridges (Waters), and then lyophilized to dryness. RP-HPLC and MALDI Plate Preparation. Lyophilized samples were resuspended in 5% acetonitrile, 0.1% TFA (solvent A). Peptide separation was performed on an Ultimate chromatography system equipped with a Probot MALDI spotting device and Famos autosampler (Dionex - LC Packings, Hercules, U.S.A.). For each sample, 5 µL was injected and loaded directly onto a 75 µm × 150 mm separation column (Inertsil ODS-3, 5 µm; Dionex - LC Packings). Peptides were eluted with the following gradient: 0-10 min, 0% solvent B (80% acetonitrile, 0.1% TFA); 10-105 min, 0-50% solvent B; 105-115 min, 50-100% solvent B, and 115-124 min, 100% solvent B, with a flow rate of 300 nL/min. The column effluent was directly mixed with MALDI matrix solution (5 mg/mL R-cyano-4hydroxycinnamic acid in 70% acetonitrile, 0.1% TFA, with the addition of neurotensin for internal calibration of the MALDI MS). Fractions were automatically deposited every 10 s onto the MALDI target plate (Applied Biosystems/MDS SCIEX) using a Probot microfraction collector. For each RP-HPLC run, a total of 416 spots were collected from the same pooled SCX fraction. Applied Biosystems 4800 MALDI-MS/MS Analysis. MALDI plates were analyzed on a 4800 Proteomics Analyzer MALDI time-of-flight/time-of-flight (MALDI-TOF/TOF) system (Applied Biosystems/MDS SCIEX). The instrument was equipped with a Nd:YAG laser operating at 200 Hz. Spectra were externally calibrated using peptide standards, and spectra from 416 spots per sample were generated in positive reflector mode by accumulating data from 1000 laser shots. Spectral peaks that met the threshold criteria and were not on the exclusion list were included in the acquisition list for MS/MS spectra. Threshold criteria were set as follows: mass range, 750-4000 Da; minimum signal-to-noise (S/N) ratio, 120; precursors/spot, 8. Peptide collision induced dissociation (CID) was performed at a collision energy of 1 keV and a collision gas pressure of 2 × 10-6 Torr. During MS/MS data acquisition, a method with a stop condition was used. In this method, a minimum of 1000 shots (20 subspectra accumulated from 50 laser shots each) and a maximum of 2000 shots (40 subspectra) were allowed for each spectrum. The accumulation of additional laser shots was halted whenever at least 4 ions with a S/N ratio of at least 100 were present in the accumulated MS/MS spectrum in the region from m/z 200 to the precursor ion mass. Applied Biosystems 4800 MALDI-MS/MS Database Searching and Data Analysis. Database searching of MS/MS spectra was performed using the EBI (European Bioinformatics Institute) mouse protein database (42 656 sequences; 20 120 892 residues, release date: 29/05/2007). Searches using MALDI-MS/ MS spectra were performed for monoisotopic peptides with +1 charge and error limits were set at 25 ppm for precursor masses and 0.2 Da for fragment ions. Peptides modifications were MMTS (C, fixed) and iTRAQ (N-termini and K, fixed). Only strictly tryptic peptides22 with a maximum of 1 missed cleavage were allowed. GPS (Global Proteome Server) Explorer software (Applied Biosystems/MDS SCIEX) was used to process spectra and to submit data for database searching using the search parameters described above. Data were searched against a concatenated forward-reverse database to calculate the false discovery rate.23 Quantification of iTRAQ reporter ion intensity was performed by integrating the area under reporter ion peaks using Applied Biosystems 4800 Explorer software before combining with database search results. Only peptides with both satisfactory database identification and sufficiently intense
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Changes in the Proteome after Neuronal Zif268 Overexpression a
Table 1. List of Proteins Regulated after Zif268 Overexpression from the “A” Comparison protein name
accession number
p-value
% change
S.E.M.
Guanine nucleotide-binding protein G(I)/G(S)/G(O) γ-4 SPARC-like protein 1 BM88 antigen PSD-95/SAP90-binding protein 3 Protein S100B Abi1 protein WD repeat-containing protein 13 Sec31A NADH dehydrogenase [ubiquinone] 1 β-11 Heterogeneous nuclear ribonucleoprotein A0 homologue 40S ribosomal protein S10 NAD-dependent deacetylase sirtuin-2 T-complex protein 1 SU R B 10-formyltetrahydrofolate dehydrogenase Inorganic pyrophosphatase Contactin associated protein 1 Eukaryotic translation initiation factor 4 γ-1 60S ribosomal protein L4 SH3-domain GRB2-like endophilin B2 OCIA domain-containing protein 1 Cytochrome b5 Acetyl-CoA acetyltransferase cAMP-dependent protein kinase type I-β regulatory SU VGF nerve growth factor inducible Cell adhesion molecule 1 Rabphilin-3A Voltage-gated potassium channel subunit β-3 2′,3′-cyclic-nucleotide 3′-phosphodiesterase Probable oxidoreductase KIAA1576 Reticulon-4 Succinyl-CoA ligase β-chain Asparaginyl-tRNA synthetase Membrane-associated phosphatidylinositol transfer protein 1 6-phosphogluconolactonase MKIAA0845 protein 60S ribosomal protein L12 DmX-like protein 2 Acyl carrier protein Shank3 Phosphatase and actin regulator 4 Fatty acid-binding protein, epidermal Cystatin C Centaurin R Cysteine and glycine-rich protein 1 Atp8a1 protein Ubiquitin carboxyl-terminal hydrolase 5 Neuronal-specific septin-3 Plasma membrane calcium-transporting ATPase 2 Calcium/calmodulin-dependent protein kinase type II δ Adenylate kinase 3 R-like 1 Solute carrier family 2, facilitated glucose transporter 3 Homer protein homologue 1 Isocitrate dehydrogenase 1 β-arrestin-1 Ribosomal protein 10 Huntingtin-interacting protein 1-related protein F-box only protein 41 Septin-8 MKIAA0968 protein Phospholipase C, β-1 40S ribosomal protein S26 Protein disulfide-isomerase A3 Protein kinase C γ type Elongation factor Tu Plasminogen activator inhibitor 1 RNA-binding protein Heat shock 70 kDa protein 4 Vacuolar ATP synthase SU H
P50153|GBG4_MOUSE P70663|SPRL1_MOUSE Q9JKC6|CEND_MOUSE A2A7T7|A2A7T7_MOUSE P50114|S100B_MOUSE Q6AXD3|Q6AXD3_MOUSE Q91 V09|WDR13_MOUSE Q3UPL0|SC31A_MOUSE O09111|NDUBB_MOUSE Q9CX86|Q9CX86_MOUSE P63325|RS10_MOUSE Q8 VDQ8|SIRT2_MOUSE P11983|TCPA2_MOUSE Q8R0Y6|FTHFD_MOUSE Q9D819|IPYR_MOUSE Q3U428|Q3U428_MOUSE Q6NZJ6|IF4G1_MOUSE Q9D8E6|RL4_MOUSE A2AWI7|A2AWI7_MOUSE Q9CRD0|OCAD1_MOUSE P56395|CYB5_MOUSE Q8QZT1|THIL_MOUSE P12849|KAP1_MOUSE Q0 VGU4|Q0 VGU4_MOUSE Q8R5M8|CADM1_MOUSE P47708|RP3A_MOUSE P97382|KCAB3_MOUSE P16330|CN37_MOUSE Q80TB8|K1576_MOUSE Q99P72|RTN4_MOUSE Q9Z2I9|SUCB1_MOUSE Q3T9A7|Q3T9A7_MOUSE O35954|PITM1_MOUSE Q9CQ60|6PGL_MOUSE Q80TQ3|Q80TQ3_MOUSE P35979|RL12_MOUSE Q8BPN8|DMXL2_MOUSE Q9CR21|ACPM_MOUSE Q4ACU6|SHAN3_MOUSE Q501J7|PHAR4_MOUSE Q05816|FABPE_MOUSE A2APX3|A2APX3_MOUSE Q8BVR8|Q8BVR8_MOUSE P97315|CSRP1_MOUSE A1L332|A1L332_MOUSE P56399|UBP5_MOUSE Q9Z1S5|SEPT3_MOUSE Q9R0K7|AT2B2_MOUSE Q6PHZ2|KCC2D_MOUSE A2ARF3|A2ARF3_MOUSE P32037|GTR3_MOUSE Q9Z2Y3|HOME1_MOUSE Q3UAV7|Q3UAV7_MOUSE Q8BWG8|ARRB1_MOUSE A2AM96|A2AM96_MOUSE Q9JKY5|HIP1R_MOUSE Q6NS60|FBX41_MOUSE Q8CHH9|SEPT8_MOUSE Q80TN1|Q80TN1_MOUSE Q6PDH1|Q6PDH1_MOUSE P62855|RS26_MOUSE P27773|PDIA3_MOUSE P63318|KPCG_MOUSE Q8BFR5|EFTU_MOUSE Q9CY58|PAIRB_MOUSE Q61316|HSP74_MOUSE Q8BVE3|VATH_MOUSE
