CHAPTER 3. SILAC mouse-based screen to identify differentially expressed proteins in liver of induced Dicer knockout mice

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1 CHAPTER 3 SILAC mouse-based screen to identify differentially expressed proteins in liver of induced Dicer knockout mice 3.1 Introduction As mentioned earlier, few studies have been carried out to investigate proteome alteration associated with induced Dicer knockout. We have reported altered proteome of small intestine upon ablation of Dicer [49]. We utilized inducible Cre-loxP knockout system for deletion of Dicer. For the quantitative profiling of the liver proteome, we spiked SILAC labeled mouse liver. Previously, various in vitro labeling methods including DIGE, 18 O and itraq labeling have been adopted for quantitative proteomic profiling of mouse systems [50-55]. Gelbased quantitative proteomics approaches suffer from low reproducibility and hampered separation due to the limited pi range. Back exchange of 18 O isotope, compression of itraq ratios and probability of introduction of manual errors in labeling are other major limitations of the in vitro quantitation approaches. Label free quantitation has been used as an alternative quantitative proteomic approach. However, it requires highly reproducible LC-MS conditions, which are difficult to achieve especially across multiple sample runs [56]. Therefore, in vivo labeling strategies such as 15 N labeling and SILAC are the preferred approaches for quantitative proteomics [57, 58]. Challenges in achieving complete labeling and complexity of the quantitation data are some of the major limitations of the 15 N labeling. As a result, SILAC has been the method of choice for in vivo labeling. However, until recently, use of SILAC was limited to cell lines. With the development of 13 C 6 -Lysine enriched mice food, mice can be labeled in vivo, extending the application of SILAC to animal systems [49, 58, 22

2 59]. This quantitation approach is free of manual errors in labeling as proteins are completely labeled in vivo. We developed SILAC mice-based quantitative proteomics assay to identify the differentially expressed proteins upon depletion of Dicer in liver. We carried out high resolution mass spectrometry analysis and identified 2,137 proteins. We calculated ratio-of-ratios to identify relative quantitation of proteins. Amongst the 257 proteins upregulated in liver of induced Dicer knockout, we observed enrichment of proteins involved in fatty acid metabolism as the regulated subproteome by Dicer. We further carried out MRM assays to validate the candidate proteins which include peroxisomal bifunctional enzyme, phosphoenolpyruvate carboxykinase 1, Cyp3A13, Cyp3A41 and myristoylated alanine-rich protein kinase C substrate. Our findings highlight crucial roles of Dicer in regulation of proteins involved in lipid transport and metabolism in mouse liver. We propose that the SILAC mice-based proteomic screening coupled with MRM assays provide a robust pipeline for a systematic in vivo quantitative proteomic analysis. 23

3 3.2 Materials and Methods Generation of inducible Dicer knockout mouse We selected Cre-loxP system to generate inducible knockout mice. ROSA26- CreERT2 mice and mice with floxed Dicer exon 21 and 22 were crossed. The progeny was responsive to tamoxifen, resulting in deletion of floxed Dicer exon 21 and 22. ROSA26-CreERT2 mice were used as control. Mice were monitored daily for any obvious pathology. On the day 8 post induction, mice were starved for 3 hr. prior to sacrifice. Livers were harvested and washed with PBS to get rid of blood Generation of SILAC mice As described previously, SILAC mice were generated by feeding the stable isotope-labeled mouse food obtained from Cambridge Isotope Laboratories (Mouse Feed Labeling Kit, catalog number: MLK-LYS-C) [49, 60]. Briefly, twenty-eight days old female mouse (F0) was fed diet containing 13 C 6 -Lysine and mated 14 days later. Heavy diet was maintained until F1 pups were weaned. One F1 female mouse was kept on heavy diet and propagated to F2. Littermate female mice (F0) were fed light diet to generate unlabeled control mice. Labeling efficiency was monitored from blood, liver and lung specimens collected at 10 weeks of age from F0 and 4 weeks of age from the F1 generation Sample preparation and basic RPLC Liver tissues were excised from five uninduced and induced Dicer knockout mice. Tissues were lysed in 9 M urea and protein concentrations were measured using BCA assays. Uninduced and induced Dicer knockout liver lysates were 24

4 pooled and spiked with liver lysate from SILAC mouse at 2:1 (w/w) ratio. The spiked lysates were digested in solution using Lys-C protease. Lys-C was added at 1:50 (w/w) (Lysyl Endopeptidase Mass Spectrometry grade, Wako Chemical USA, Richmond, VA) to the lysates and incubated at 37 C for 4 hr. Additional Lys-C was added to the pre-digested lysate at 1:50 (w/w) ratio and incubated for 12 hr. Basic RPLC (brplc) was carried out as described previously [49]. Briefly, Lys-C digests were reconstituted in brplc solvent A (10 mm triethylammonium bicarbonate, ph 9.5) and were separated on XBridge BEH C18 Column (Waters, UK) with a linear increase in gradient from 5 to 100% of 10 mm TEABC with 90% acetonitrile (ph 9.5) over 30 min. and persisting for 10 minutes. For each condition, 24 fractions were collected and dried before LC- MS/MS analysis LC-MS/MS analysis LC-MS/MS analysis of 48 brplc fractions was carried out using Eksigent nano LC interfaced with the LTQ-Orbitrap XL ETD mass spectrometer (Thermo Scientific, San Jose, CA). The peptides were loaded on a trap column (75 µm 2 cm) packed with C18 material (5 μm Magic C18 AQ) at a flow rate of 5 μl/min of 97% solvent A (3% acetonitrile and 0.1% formic acid) and separated on an analytical column (75 µm 12 cm) packed with the same material using linear gradient of solvent B (0.1% formic acid in 90% acetonitrile) from 10 % to 60% solvent B for 60 min, to 97% solvent B from 74 to 90 min. All the MS spectra were acquired on an Orbitrap analyzer at the resolving power of 60,000 at 400 m/z while the data dependent MS/MS spectra were acquired using an LTQ analyzer. Ten most intense precursor ions from a survey scan within m/z 25

5 range from 350 to 1,800 above 1,000 of intensity were isolated with a 2 Da window and fragmented by CID with 30% normalized collision energy. The precursors were excluded, after fragmentation, for 30 seconds with a 7 ppm window. Maximum ion injection times were set to 500 msec for MS and 200 msec for MS/MS. The automatic gain control targets were set to for MS in the Orbitrap, for MS n in the LTQ Data analysis Mass spectrometry data analysis was carried out using Proteome Discoverer 1.2 suite (Thermo Fisher Scientific, Bremen, Germany). Precursor mass range of 300 to 5,000 Da and signal to noise ratio of 1.5 were used as selection criteria for generation of peak lists. NCBI RefSeq 42 containing mouse proteins with known contaminants (29,109 entries) was used as a reference database. SEQUEST and Mascot algorithms were used to carry out database searches. The parameters used for database searches include trypsin as a protease with allowed one missed cleavage, carbamidomethyl cysteine as a fixed modification and 13 C 6 -Lysine, oxidation of methionine as variable modifications. MS error window of 10 ppm and MS/MS error window of 0.8 Da were allowed. As described earlier, LC-MS/MS data was searched against a reversed database to calculate 1% false discovery rate score cut-off [61]. Ratio-of-ratio was calculated to determine differentially expressed proteins in induced Dicer knockout liver [59] MRM assays MRM assays were designed to detect and validate the protein level changes in induced Dicer knockout mice. Five differentially expressed proteins were selected as candidates for MRM analysis. Skyline v1.2 was used to create a 26

6 transition list of proteotypic peptides from the selected proteins [62]. Preference was given to proteotypic peptides with precursor charge of +2 that did not contain cysteine and methionine. In solution digestion of equal amounts of four uninduced and induced Dicer knockout mice liver samples was carried out and the peptides were acidified and stored at -80 C. All samples were analyzed in triplicate on TSQ Quantum Ultra (Thermo, San Jose, CA) interfaced with Agilent s 1100 series LC. Peptides were enriched on a trap column (5 μm, 75 μm 2 cm.) and separated using analytical column (3 μm, 75 μm 10 cm) with a linear gradient of 90% ACN 0.1% formic acid for 80 min at a constant flow rate of 350 nl/min. Both columns were packed in-house using Magic C18 AQ (Michrom Bioresources). Spray voltage of 2.5 kv was applied and ion transfer tube was maintained at 275 C. The collision energy for each transition was optimized based on the formula C.E. = (m/z) Data was acquired with Q1 and Q3 set at 0.4 and 0.7 unit mass resolutions, respectively Bioinformatics analysis for enriching candidate mirnas Proteins upregulated in the liver of induced Dicer knockout were taken as queries to search for the potential regulating mirnas. From TargetScan (Release 6.2), we downloaded the dataset, Summary Counts to identify seed sites in 3 UTR of mrnas and the representative target mirnas [63]. RefSeq was use as the reference database for mouse mrnas. Gene symbols were used to derive the intersection of the proteins and the predicted regulating mirnas. Only mirnas belonging to the species Mus musculus (mmu-mir) were used for analysis. 27

7 3.3 Results and Discussion Dicer is essential for survival of adult mice To assess the role of Dicer in adult mice and to circumvent embryonic lethality, we adopted an inducible Cre-loxP knockout system. The ubiquitous expression of Cre recombinase (Cre-ERT2) was driven by ROSA26 loci. The Cre-ERT2 mice were crossed to mice with floxed exons 21 and 22 of Dicer gene which encode the RNase III domain. Upon administration of tamoxifen, the Cre recombinase translocates to the nucleus deleting floxed exons 21 and 22. RT- PCR targeting the junctional region of exons 20 and 21 demonstrated that 80% of the mice had disrupted Dicer1. Mice were monitored daily for any obvious abnormalities. We observed that 5/10 mice developed diarrhea. The Dicer ablation proved to be lethal in most of the cases. As described earlier, by day 10 after inducing ablation of Dicer, 80% of the mice died [49]. The major histopathological changes observed by day 8 post induction of Dicer knockout included inflamed small intestine and bone marrow depletion with the reduced number of myeloid lineage cells. Dysregulation of lipid metabolism and proteomic changes associated with ablation of Dicer in small intestine were reported by our group earlier [49]. Liver performs crucial functions including metabolism of lipids, glucose and detoxification. Upon sacrifice of the Dicer knockout mice, we did not observe any gross abnormality of liver. The histological studies of the same specimens did not reveal any evident pathology. 28

8 Figure 3. SILAC mouse based quantitative proteomic workflow to identify differentially expressed proteins in liver of induced Dicer knockout mice. Liver lysates of five uninduced and five induced Dicer knockout mice were spiked at 2:1 ratio with liver lysates of two uninduced SILAC mice. Proteolysis was carried out using Lys-C protease. Digests were separated on brplc to obtain 24 fractions each for uninduced and induced Dicer knockout spiked liver lysates. LC-MS/MS analysis of the brplc fractions was carried out on an Orbitrap-XL ETD mass spectrometer. Database searches and quantitation was carried out using Proteome Discoverer (v 1.2) and ratio of ratio was calculated to identify differentially regulated proteins in the liver of induced Dicer knockout mice. Candidate upregulated proteins in induced Dicer knockout were validated using MRM assays. 29

9 3.3.2 Development of quantitative proteomic screen to identify the target Dicer proteome in mouse liver The aim of designing quantitative proteomic assay was to develop a suitable labeled internal standard that will allow systematic quantitation of proteins. SILAC mice have been previously used to explore proteomic changes associated with aging [59]. We developed SILAC mice-based screen as depicted in Figure 3. In order to minimize the inter-individual variation, we pooled liver lysates from five mice. As described in the methods, the SILAC mice were metabolically labeled by feeding 13 C 6 -Lysine labeled diet. As a consequence, all proteins were labeled in vivo with 13 C 6 -Lysine, making them amenable labeled controls. Uninduced and induced Dicer knockout liver lysates were spiked with liver lysate of SILAC mice at the 2:1 ratio. As generation and maintenance of SILAC mice is expensive, spiking of the SILAC mouse tissues provides as an economical alternative for in vivo quantitative proteomics. Proteolysis was carried out in-solution using Lys-C protease which specifically hydrolyses C-terminal of Lysine to acquire quantitation for the maximum number of peptides with paired 13 C 6 -Lysine containing peptides from SILAC mice. Reversed phase liquid chromatography operating at the macroflow rate in the high ph condition (brplc) was used to obtain 24 fractions per condition. The LC-MS/MS analysis was carried out on nanoflow reversed phase liquid chromatography coupled to LTQ-Orbitrap XL ETD mass spectrometer. MS was acquired at a high resolution (60,000 at 400 m/z) to obtain high accuracy quantitation information. In total, we analyzed 48 LC-MS/MS fractions. At better than 1% FDR, we identified 58,684 peptides corresponding to 2,134 proteins. 30

10 A Peroxisomal bifunctional enzyme (Ehhadh) B LGILDVVVK Phosphoenolpyruvate carboxykinase 1 (Pck1) GLGGVNVEELFGISK Relative Abundance Uninduced Induced knockout Light Heavy Light Relative Abundance m/z Heavy m/z y Relative Abundance Uninduced Light Heavy m/z 100 Relative Abundance Induced knockout Light y Heavy m/z y Relative Abundance b y y y7 y5 y b a4 b b m/z 800 y y Relative Abundance y b a y b7-nh y y b6-nh b m/z b y b b b b Figure 4. Representative MS and MS/MS spectra of upregulated proteins in liver induced Dicer knockout mice. A) MS and MS/MS of LGILDVVVK from upregulated peroxisomal bifunctional enzyme in liver of induced Dicer knockout mice. B) MS and MS/MS of GLGGVNVEELFGISK from upregulated phosphoenolpyruvate carboxykinase 1 in liver of induced Dicer knockout mice Differentially regulated proteins in induced Dicer knockout liver In uninduced mice liver, 1,412 proteins and in induced Dicer knockout mice liver, 1,993 proteins were profiled. We selected 1,217 proteins quantified in common and calculated ratio of SILAC ratios to obtain relative quantitation of proteins. We set an arbitrary threshold of 2-fold to select the differentially regulated proteins in induced Dicer knockout mouse liver. Out of the 319 differentially regulated proteins, 257 proteins were upregulated as listed in appendix I. 31

11 The upregulated proteins include aldehyde oxidase (4.5-fold), myristoylated alanine-rich protein kinase C substrate (Marcks) (4.3-fold), sorbin, SH3 domaincontaining protein 1 (3.4-fold), filamin A (3-fold) and phosphoenolpyruvate carboxykinase 1 (Pck1) (2.7-fold). Representative MS and MS/MS of upregulated proteins, peroxisomal bifunctional enzyme and phosphoenolpyruvate carboxykinase are illustrated in Figure 4. The downregulated proteins include 15-hydroxyprostaglandin dehydrogenase (6- fold), cytochrome P450 2C29 (5.5-fold) and alcohol dehydrogenase 4 (4.5-fold). Our dataset represents enrichment of cytochrome P450 family members which were upregulated in Dicer knockout liver such as cytochrome P450 2d22 (15.4- fold), cytochrome P450 3a41b (9.4-fold), cytochrome P450 4v3 (5.8-fold), cytochrome P450 3a13 (4.3-fold), cytochrome P450 2c70 (2.8-fold) and cytochrome P450 2a12 (2-fold). Cytochrome P450 is a family of heme proteins that function as oxygenases. These perform diverse functions including xenobiotic metabolism, fatty acid metabolism and steroid hormone synthesis. Upregulation of cytochrome P450 family of proteins can indicate elevation in the ROS. Association of the polymorphisms in cytochrome P450 family members and hepatocellular carcinoma has been established in previous studies [64, 65] PPARα targets are upregulated upon depletion of Dicer in liver To identify enriched classes of molecules, we analyzed the differentially regulated proteins using DAVID (v 6.7) [66]. Our dataset reflected enrichment of proteins involved in PPAR signaling. PPARs are steroid hormone nuclear receptors. There are multiple isoforms of PPAR with varying tissue expression. PPARα is the predominant isoform expressed in liver [67]. It is known to target 32

12 genes involved in fatty acid catabolism, gluconeogenesis and ketone body synthesis and liporotein assembly [68, 69]. Figure 5. Microsomal and peroxisomal lipid oxidation pathways Upregulated enzymes involved in ω - oxidation of fatty acids and peroxisomal β - oxidation of fatty acids in liver of induced Dicer knockout mice, most of which have been reported to be PPARα targets. Key enzymes involved in the steps of peroxisomal β-fatty acid oxidation included peroxisomal acyl-coenzyme A oxidase 2 (Acox2), peroxisomal multifunctional enzyme type 2 (Hsd17b4), peroxisomal bifunctional enzyme (Ehhadh) and peroxisomal 3- ketoacyl-coa thiolase A (Acaa1a). PPARα targets involved in lipid transport proteins were also upregulated in the liver of induced Dicer knockout mice. 33

13 One of the key pathways regulated by PPARα is fatty acid catabolism. As depicted in Figure 5, some of the major enzymatic steps involved in peroxisomal β -oxidation include oxidation, hydration, dehydrogenation and thiolytic cleavage of fatty acids. Acox2 acyl-coa oxidase was upregulated (2-fold) in induced Dicer knockout liver which acts upon bile acid intermediates and branched chain fatty acids. The second and third steps of β oxidation involve hydratase and 3-hydroxyacyl-CoaA dehydrogenase, for which peroxisome harbors hydroxysteroid (17-beta) dehydrogenase 4 or peroxisomal bifunctional enzyme type 2 and peroxisomal bifunctional enzyme which show overlapping functions. Hydroxysteroid (17-beta) dehydrogenase 4 was 1.4-fold upregulated while peroxisomal bifunctional enzyme was 3.7-fold upregulated in induced Dicer knockout liver. Peroxisomal bifunctional enzyme is involved in production of medium chain dicarboxylic acids. Acaa1a is a peroxisomal 3- ketoacyl-coa thiolase A which performs the last step of β oxidation. It was 4.1- fold upregulated in induced Dicer knockout liver. The substrates for peroxisomal β oxidation are end products of ω-oxidation of fatty acids which is initiated by microsomal Cyp4 family of cytochromes. Cyp4V3 of mouse is a homolog of human Cyp4V2 which functions as fatty acid ω-hydroxylase. Cyp4V3 was 5.8-fold upregulated in the Dicer knockout liver. Though Cyp4V3 is not reported to be a direct target of PPARα, upregulation of Cyp4V3 along with the enzymes involved in peroxisomal β oxidation, indicate over activation of oxidation of fatty acids. These events might have led to the observed loss of weight in the induced Dicer knockout mice. We also identified several other PPARα targets which were upregulated in. Other upregulated PPARα targets in induced Dicer knockout liver include proteins involved in lipid transport such as ATP-binding cassette sub-family D member 3 (11-fold), 34

14 apolipoprotein A-IV (3.9-fold), perilipin 2 (3.4-fold) and solute carrier family 27 (fatty acid transporter), member 2 (2.3-fold). Representative proteins involved in lipid metabolism or transport which were upregulated in liver of induced Dicer knockout mice are listed in Table1. As described earlier, down-regulation of Dicer is reported to be associated with hepatocellular carcinoma [45, 46]. PPARα pathway is also observed to be associated with hepatocellular carcinoma [70]. In our study, PPARα targets were upregulated upon depletion of Dicer. We also observed over-expression of cytochrome P450 family of proteins which can be attributed to elevation of ROS stress due to over activation of microsomal and peroxisomal fatty acid oxidation. The chronic effect of Dicer depletion on the PPAR signaling and development of hepatocellular carcinoma needs to be further explored. Protein Gene symbol KO/Ctrl 1. Sodium/bile acid cotransporter Slc10a ATP-binding cassette sub-family D member 3 Abcd Cytochrome P450 4V3 Cyp4v Peroxisomal 3-ketoacyl-CoA thiolase A Acaa1a Peroxisomal bifunctional enzyme Ehhadh Perilipin-2 Plin Peroxisomal acyl-coenzyme A oxidase 2 Acox2 2.0 Table 1. A partial list of proteins involved in lipid metabolism List of proteins, gene symbols and fold-change upregulation in liver of induced Dicer knockout mice (KO) compared to uninduced Dicer knockout mice (Ctrl) Validation of candidate targets of Dicer using MRM assays To validate upregulated proteins in liver of induced Dicer knockout mice using a complementary mass spectrometry-based method, we designed MRM assays for peroxisomal bifunctional enzyme (Ehhadh), phosphoenolpyruvate carboxykinase (Pck1), Cyp3a41, Cyp3a13 and myristoylated alanine-rich C- 35

15 kinase substrate (Marcks). We selected 4 uninduced and 4 induced Dicer knockout mice. At least four transitions were monitored for a minimum of one unique peptide for each protein. For normalization of data, housekeeping protein, isocitrate dehydrogenase (Idh2) was selected as an unchanged internal standard from LC-MS/MS data. We monitored LGILDVVVK (z = +2, m/z = 478.3) and GWYQYDKPLGR (z = +2, m/z = 691.8) for validation of Ehhadh, a PPARα target which is involved in peroxisomal β-oxidation of fatty acids. As depicted in Figure 6, LGILDVVVK was 5.2-fold upregulated in liver of induced Dicer knockout mice corroborating our findings. Similarly as listed in Table 2, we also monitored LTPIGYIPK (z = +2, m/z = 501.3), LQEEIDETLPNK (z = +2, m/z 714.8), LQDEIDAALPNK (z = +2, m/z = 663.8), VNGDASPAAAEPGAK (z = +2, m/z: 677.8) corresponding to Pck1, Cyp3a41, Cyp3a13 and Marcks, respectively. Quantitation obtained using MRM-based assays was in agreement with the quantitation obtained from the LC-MS/MS data. We observed that MRM provided as a robust and antibody-independent approach for validation of differentially expressed proteins in our study. Protein 1 Peroxisomal bifunctional enzyme 2 Phosphoenolpyruvate carboxykinase 1 3 Cytochrome P450 3A41 4 Cytochrome P450 3A13 5 Myristoylated alaninerich C-kinase substrate Gene symbol Peptide Induced Dicer knockout / uninduced (MRM) Induced Dicer knockout / uninduced (SILAC spike) Ehhadh LGILDVVVK GWYQYDKPLGR 8.7 Pck1 LTPIGYIPK Cyp3a41 LQEEIDETLPNK b Cyp3a13 LQDEIDAALPNK Marcks VNGDASPAAAEPGA K

16 Table 2. List of validated proteins upregulated in induced Dicer knockout using MRM assays List of proteins, gene symbols, MRM-based quantitation and relative fold-change upregulation data from spiked SILAC mice LC-MS/MS analysis Figure 6. MRM-based validation of upregulated proteins in liver of induced Dicer knockout mice. A) Four transitions were monitored for LGILDVVVK from peroxisomal bifunctional enzyme in uninduced and induced Dicer knockout mice. Box and whiskers plot depict relative abundance in the uninduced and induced liver knockout mice. B) Four transitions were monitored for LQEEIDETLPNK of phosphoenolpyruvate carboxykinase 1 in liver of induced Dicer knockout mice. Box and whiskers plot show relative abundance in the uninduced and induced liver knockout mice Bioinformatics analysis of the enriched upstream mirnas. 37

17 Dicer-dependent mirna generation is the predominant pathway in maturation of mirnas. We hypothesized that upon depletion of Dicer; mirna maturation is severely affected, resulting in dysregulation of downstream mirna targets. Thus, we carried out a bioinformatics analysis using TargetScan algorithm to identify the upstream mirnas of the upregulated proteins in liver of induced Dicer knockout mice. We identified 598 unique mouse mirnas (mmu-mirs) predicted to be regulators of the upregulated proteins in induced Dicer knockout liver. Some of the predicted upstream mirnas targeting multiple upregulated proteins included mmu-mir-124 and mmu-mir-200b. mir-124 has been described as a tumor suppressor gene in recent studies carried out on hepatocellular carcinoma [71, 72]. Vimentin (2-fold upregulated), an EMT marker has been demonstrated previously as direct target of mir-124 [72]. Apart from vimentin, multiple upregulated proteins associated with actin binding and membrane trafficking are predicted targets of mir-124. mir-143 was identified as upstream mirna of multiple proteins involved in lipid metabolism or transport which included peroxisomal bifunctional enzyme, Abcd1 and perilipin 2. Marcks was also predicted as a target of mir-143. mir-143 is known to be involved in regulation of lipid metabolism [73, 74]. It induces adipocyte differentiation with accumulation of triglycerides. We hypothesize that upon downregulation of mir-143, the target genes involved in fatty acid oxidation are upregulated. Further studies are required to investigate functions of these candidate mirnas and their targets in liver. Also, studies need to be carried out to identify the correlation of downregulation of Dicer, mirnas, PPARα downstream targets possibly with carcinogenesis. 38