Protein Dysregulation in Mouse Hippocampus Polytransgenic for Chromosome 21 Structures in the Down Syndrome Critical Region Joo-Ho Shin,†,§ Talin Gulesserian,†,§ Emmanuelle Verger,‡ Jean-Maurice Delabar,‡ and Gert Lubec*,† Department of Pediatrics, Division of Neuroproteomics, Medical University of Vienna, Austria, and Department of Biochemistry, University of Paris 7-Denis Diderot, EA3508; IFR117 Paris, France Received July 26, 2005
Mice polytransgenic for chromosome 21 genes DSCR3, 5, 6, 9, and TTC3 within the Down Syndrome Critical Region-1 represent an animal model for Down Syndrome (DS). In a proteomic approach, we show a series of altered hippocampal protein levels that may be caused by overexpression of at least one of the five chromosome 21 genes and that fit fear- conditioned memory defects and were observed to be dysregulated in human fetal DS. Keywords: polytransgenic mice • chromosome 21 • Down Syndrome critical region • two-dimensional gel electrophoresis • hippocampus
Introduction Down syndrome (DS; trisomy 21) is the most frequent genetic cause of mental retardation with an incidence of 1 in 700 life births.1 Although DS involves dysmorphic features, which collectively constitute its distinctive physical phenotype, endocardial and immunological defects, haematological and endocrinal alterations, behavioral and cognitive deficits, mental retardation, and hypotonia,1 no pathomechanism for the development of the DS phenotype has been described so far. A large series of neurochemical changes have been reported at the nucleic acid and protein level2,3,4 indicating the complexity of the problem. The concept of the gene dosage hypothesis claims that three copies of chromosome 21 (HSA21) lead to overexpression of chromosome 21 gene products and these in turn are responsible for the DS phenotype.1 Moreover, according to another hypothesis, triplication of a so-called Down Syndrome Critical Chromosomal Region-1 (DCR-1) may account for a part of the DS phenotype.5 This 2 Mb spanning region of HSA21 surrounding D21S55 at 21q22.2 harbors several genes that may well be the structures whose alteration could lead to neurological deficits. Smith and co-workers therefore generated several nonmosaic polytransgenic mouse strains (PTGs), 141G6, 285E6, 230E8, and 152F7, by inserting yeast artificial chromosomes (YACs) containing a fragment of the human DCR-1 region into the murine genome.6 These mice with triplicated gene groups were tested for neurological and behavioral deficits. Strain 152F7 presented strong learning impairment and hyperactivity during develop* To whom correspondence should be addressed. CChem, FRSC (UK): Medical University of Vienna, Department of Pediatrics, Wa¨hringer Gu ¨ rtel 18, A-1090 Vienna, Austria. Tel: x43.1.404003215. Fax: x43.1.404003194. E-mail:
[email protected]. † Medical University of Vienna. ‡ University of Paris. § These authors have contributed equally to this paper.
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Journal of Proteome Research 2006, 5, 44-53
Published on Web 12/14/2005
ment and extended brain abnormalities, probably due to overexpressed DYRK1A, a dual-specificity tyrosine (Y) regulated kinase involved in a major signaling cascade in the brain.7 Strain141G6, lines 4 and 28, contain one or three copies of YAC carrying the human genes DSCR3 (91.9% identiy/296 amino acids (a.a) to mouse DSCR3), DSCR5 (91.7% identity/132 a.a to mouse DSCR5), DSCR6 (68.5% identity/143 a.a to mouse DSCR6), DSCR9 (52.9% identity/68 a.a to Retrovirus-related POL polyprotein (P11369)) and tetratricopeptide repeat domain 3 (TTC3, 76% identity/2005 a.a to mouse TTC3), genes with so far unknown or poorly defined function. Previous work showed no cognitive defect by using a Morris water maze paradigm but lower performance in fear-conditioning against sound as acoustic conditional stimulus.8 For strain 141G6 (line 28), our experiments (unpublished results) on rota rod, Morris water maze, multiple T-maze, open field, elevated plus maze and a comprehensive neurological observational battery showed no significant neurological, cognitive or behavioral deficits. However, when these mice were analyzed for brain (hippocampal) protein profiling, significant changes in the expression of proteins involved in diverse biological functions were detectable. None of these changes seems able to produce strong cognitive impairment in our test system.
Experimental Section Animals. Transgenic mice were of an FVB inbred background as described by Smith et al.6,9 All animal handling and experimental procedures were performed at the University of Paris 7 in accordance with the European Communities Council Directives. We used 10 nontransgenic (wild-type, WT) males as controls and we tested the males at 3 months. PCR genotyping was performed with oligonucleotides chosen in DSCR9 sequence (a primate-specific gene). The primer sequences used are indicated in Table 1. Proteomics experiments were performed on 141G6 (line 28) and RNA quantification was 10.1021/pr050235f CCC: $33.50
2006 American Chemical Society
Protein Dysregulation in Polytransgenic Mice Table 1. PCR Primers Used for Genotyping of Mice and Gene Dosage Analysis gene
primer name
primer sequences (5′ to 3′)
For Genotyping DSCR9-F CCCATGTCTGCGATGTAACTGC DSCR9-R CTGAGGTTGCCTCCACATGCTA For Gene Dosage Analysis lzst-like locus lzst-F GCTTCGGGAGCAGGTACCC (MMU 11) lzst-R TGGCGAGCCTCAGATCTTG TTC3 locus TTC3-F GGGTCATTATCGTTATTGTGATGCTCT (MMU16 and TTC3-R TTGTTTTTGTAACTTTACATGCTGCTG HSA21) For Real Time RT-PCR lzst1 (MMU8) lzst1-F CCAGCTGCAGGTGTTGCAGT lzst1-R TCGCTGGCCTTAGTGTTCACC GAPD GAPD-F GGTCGGTGTGAACGGATTTGG GAPD-R TGTTAGTGGGGTCTCGCTCCT human DSCR3 DSCR3-F TCCAGATTATCAACAGCACCA and mouse DSCR3-R AAGCTCTCTCTTTGACGTTCTG DSCR3 PGK1 PGK-F CCCAGAAGTCGAGAATGCCTGT PGK-R GCTCGGAAAGCATCAATTTTGG CaMKII CaMKII-F CCGTGGACTGCCTGAAGAAGTT CaMKII-R CAATGGTGGTGTTGGTGCTCTC
DSCR9
performed on 141G6 mice (lines 28 and 4), both lines carrying the same YAC 141G6. Gene Dosage Analysis. For qPCR gene dosage the reference oligonucleotides (lzst-F and lzst-R) were chosen at the lzst-like locus (MMU 11) (Table 1). The target oligonucleotides (TTC3-F and TTC3-R) were chosen for YAC 141G6 at the TTC3 locus (MMU16 and HSA21). In 141G6 (line 28), TTC3 copy number was found equal to 5 with a ratio 141G6/WT equal to 2.5. In 141G6 (line 4) TTC3 copy number was found equal to 8 with a ratio 141G6/WT equal to 4. Quantitative PCR Expression Analysis. Poly A1 RNA was prepared from hippocampi and thalami of adult mice by using the Fast Track kit (Invitrogen, Cergy Pontoise, France). After a DNAse purification step (Qiagen, Courtaboeuf, France), RNA concentrations were spectrophotometrically assessed. Reverse transcription (RT) was performed using the Retroscript kit with random decamers (Ambion, Huntingdon, UK). From a total volume of 50 µL per cDNA, 8 µL were used to prepare 5 dilutions that were used in the real-time PCR reaction. For gene expression assays the reference oligonucleotides were chosen at crossing point (Cp) levels equivalent to those observed for the target, lzst1 (MMU8) or GAPD (Table 1). The target oligonucleotides for human/mouse DSCR3 (HAS21/MMU16), PGK1 (MMUX), or CaMKII (MMU18) were mentioned (Table 1). Two-Dimensional Gel Electrophoresis (2-DE). Mouse hippocampus tissues were suspended in 1 mL of sample buffer consisting of 40 mM Tris, 7 M urea (Merck, Darmstadt, Germany), 2 M thiourea (Sigma, St. Louis, MO), 4% CHAPS (3[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate) (Sigma), 65 mM DTT (Merck), and 1 mM EDTA, protease inhibitors cocktail (Roche) and 1 mM phenylmethylsulfonyl chloride. The suspension was sonicated for approximately 30 s and centrifuged at 150 000 × g for 60 min. The protein content in the supernatant was determined by the Coomassie blue method.10 2-DE was performed essentially as reported.11 Samples of 0.7 mg protein were applied on immobilized pH 3-10 nonlinear gradient strips (18 cm). Focusing started at 200V and the voltage was gradually increased to 8000 V at 3 V/min (approximately 180 000 Vh total). After the first dimension,
research articles strips were equilibrated for 15 min in the equilibration buffer containing 6 M urea, 20% glycerol, 2% SDS, 2% DTT and then for 15 min in the same equilibration buffer containing 2.5% iodoacetamide instead of DTT. After equilibration, strips were loaded on 9-16% gradient SDS gels for second-dimensional separation. The gels (180 × 200 × 1.5 mm) were run at 40 mA per gel. Immediately after the second dimension run, gels were fixed for 18 h in 50% methanol, containing 10% acetic acid, the gels were then stained with Colloidal Coomassie Blue (Novex, San Diego, CA) for 5 h on a rocking shaker. Molecular masses were determined by running standard protein markers (Bio-Rad Laboratories, Hercules, CA) covering the range 10250 kDa. pI values were used as given by the supplier of the immobilized pH gradient strips (Amersham Bioscience, Uppsala, Sweden). Gels were destained with H2O and scanned with ImageScanner (Amersham bioscience). Images were processed using Photoshop (Adobe) and PowerPoint (Microsoft) software. Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight (MALDI-TOF). Spots were excised with spot picker (PROTEINEER sp, Bruker Daltonics, Germany), placed into 96-well microtiter plates. In-gel digestion and MALDI sample preparation were performed by Digestion kit (Bruker Daltonics, Germany) and an automated procedure (PROTEINEER dp, Bruker Daltonics, Germany). Briefly, spots were excised and washed with 10 mM ammonium bicarbonate and 50% acetonitrile in 10 mM ammonium bicarbonate. After washing, the gel plugs were shrunk by addition of acetonitrile and dried by blowing out the liquid through the pierced well bottom. Dried gel pieces were reswollen with 35 ng/mL trypsin in 4 µL enzyme buffer (consisting of 5 mM octyl-β-D-glucopyranoside (OGP) and 10 mM ammonium bicarbonate) and incubated for 4 h at 30 °C. Peptide extraction was performed with 15 µL/spot of 1% trifluoroacetic acid (TFA is highly corrosive and acute toxic through inhalationa and work was carried out in compliance with. Material Safety Data Sheets, www.msdsonline.com) in 5mM OGP. Extracted peptides were directly applied onto a target (AnchorChip, Bruker Daltonics) loaded with R-cyano-4hydroxycinnamic acid (Bruker Daltonics) matrix (20 mg/mL solution in acetone - 0.1% TFA 97:3 v/v). MALDI-MS(/MS) data were obtained using an Ultraflex TOF/TOF (Bruker Daltonics) equipped with a LIFT-MS/MS facility controlled by the FlexControl 2.0 software package. An accelerating voltage of 25 kV was used for protein mass fingerprinting (PMF). For fragment ion analysis in the tandem TOF/TOF mode, precursors were accelerated by 8 kV and selected in a timed ion gate. Fragment ions generated by laser-induced post-source decomposition of the precursor were further accelerated by 19 kV in the LIFT cell and their masses were analyzed after passing the ion reflector. Measurements were in part performed using postLIFT metastable suppression, which allowed removal of precursor and metastable ion signals produced after extraction out of the second ion source. Masses were annotated and processed with FlexAnalysis 2.0. External calibration of MALDI-TOF mass spectra was performed using singly charged monoisotopic peaks of a mixture of angiotensin I, angiotensin II, substance P, bombesin, and adrenocorticotropic hormones (ACTH 1-17 and 18-39) (Bruker Daltonics). For MALDI-MS/MS, calibrations were performed with fragment ion spectra obtained for the proton adducts of these peptides. Each MS spectrum was produced by accumulating data from 200 consecutive laser shots and spectra were interpreted with the aid of the Mascot Software (Matrix Science Ltd, London, UK). For protein search, Journal of Proteome Research • Vol. 5, No. 1, 2006 45
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a mass tolerance of 25 ppm and 1 missing cleavage site was allowed and oxidation of methionine residues was considered. The probability score calculated by the software was used as criterion for correct identification.12,13 Quantification of Protein Spots. Protein spots were outlined (first automatically and then manually) and quantified using the ImageMaster 2D Elite software (Amersham Pharmacia Biotechnology). The intensity and volume of spots were determined and each spot was normalized in comparison with the total intensity and volume of all spots in the 2-DE gel. The percentage of volume was measured by multiplying 100 to normalized value. Statistical Analysis. Protein abundance is expressed as means ( standard deviation (SD) of percentage of the spot volume in each particular gel after subtraction of the background intensity. Between-group differences were analyzed using Student’s t-test. The level of significance was set at P < 0.05. Database Searches. The protein-protein interaction database DIP14 was employed to detect experimentally proven interaction between altered proteins and hypothetical proteins (HPs) observed herein with all proteins from the database. HPs were identified by MASCOT software directly from the server used for MALDI-identification and by MS-FIT15 searches. Alignments of protein sequences of HP were performed with several BLAST and domain search programs.16
Results Quantitative RT-PCR Assessment of mRNA Levels. To differentiate between transcriptional and post-transcriptional regulation of protein levels we used quantitative RT-PCR experiments. We first assessed the level of one of the genes carried by the YAC transgene (namely, DSCR3) and one gene (DYRK1A) outside of the transgene. This was done by using oligonucleotides amplifying both mouse and human cDNA and comparing WT (with only mouse transcripts) and transgenic mouse (with mouse and human transcripts). The level of DSCR3 gene was increased by 3.5-5.6 times in the hippocampus and thalamus of lines 28 and 4 (strain 141G6), respectively, corresponding to an increase of expression above the increase of the gene copy number. In contrast, the ratio for DYRK1A, present in two copies only, was equal to 1 (Figure 1A). To differentiate between transcriptional and post-transcriptional regulation of the non- transgenic proteins, we have chosen two genes encoding the phosphoglycerate kinase 1 (PGK1) and calcium/calmodulin-dependent protein kinase type II alpha chains (CaMKII) whose protein levels were shown to be decreased in 141G6 mice (line 28): Although mRNA steady state levels for DSCR3 were clearly increased, mRNA levels for PGK1 and CaMKII remained unchanged (Figure 1B). Mouse Hippocampal Proteins on 2-DE Gels. Mouse hippocampal proteins were solubilized in the IEF-compatible reagents urea, thiourea and CHAPS and analyzed by 2-DE gels. 2-DE separation was performed on broad pH range IPG strips and protein spots were visualized following staining with colloidal Coomassie blue. A large series of high abundance (i.e., Coomassie-blue stained) proteins of several categories such as antioxidant, cytoskeleton, chaperone, metabolism, nucleic acidbinding, proteosome, signaling, and HPs were successfully represented in 2-DE gels of control and 141G6 polytransgenic mice (Supplementary figure). Identification of Mouse Hippocampus Proteins. In total, 958 spots were analyzed in gels from 141G6 (line 28) mice, resulting 46
Journal of Proteome Research • Vol. 5, No. 1, 2006
Figure 1. Regions of synteny between human chromosome 21 (HSA21) and mouse chromosomes (MMU16) and gene expression.(A) Hippocampal (hi) and thalamic (th) mRNA ratios between wild-type (WT) and 141G6 mice (L28 and L4) for DYRK1A, an MMU16 gene not present in YAC 141G6, for DSCR3, an MMU16 gene carried by YAC 141G6. The level of DSCR3 gene was increased by 3.5-5.6 times in the hippocampus and thalamus of lines 28 and 4, respectively, corresponding to increase of the gene copy number. In contrast, the ratio for DYRK1A, present in two copies only, was equal to 1. (B) Expression level of CaMKII and PGK1 between WT and 141G6 mice. mRNA levels of CaMKII (MMU18) and PGK1 (MMUX) showing altered expression at protein level were comparable between groups. Each gene localization on chromosome was inserted in diagram. An asterisks (*) indicates P < 0.01.
in the identification of 422 polypeptides, which were the products of 239 different genes. Data for protein identification and assignment are provided including accession numbers in Swissprot, entry names, protein names, peptide matches, scores, theoretical pI and Mr, locus of human homologue and mouse chromosomal localization as well as means ( standard deviation and the p-value from the Student’s t-test are shown (Supplementary table). Some proteins presented with several spots probably representing splice variants or post-translational modifications. Only spots that were confidently identified and separated without overlapping on 2-DE were used for quantification. Unfortunately none of DSCR3, 5, 6, 9, and TTC3 proteins was detectable in this study because their expression
Protein Dysregulation in Polytransgenic Mice
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Figure 2. Representative 2-DE image showing proteins dysregulated in the hippocampus of 141G6 mice. Brain proteins were extracted and separated on an immobilized pH 3-10 nonlinear gradient strip, followed by separation on a 9-16% gradient polyacrylamide gel. The gel was stained with Coomassie blue and spots were analyzed by MALDI-TOF/TOF. Spots identified were annotated by Swiss-Prot accession numbers. White and black arrows are only provided to cope with dark or bright background.
might be too low (low abundance proteins) to visualize them in 2-DE gel with Coomassie staining. Quantitative Variation of Proteins in WT and 141G6 Mice. Expression levels of a series of identified proteins were significantly altered in 141G6 mice. These proteins showing different expression levels can be assigned according to their functional categories (Table 2, Figure 2). Four HPs that were only predicted so far, based upon nucleic acid sequences, were shown to exist and two of them were found to be differentially expressed. They are presented herein with protein names, observed masses and peptide sequences (Table 3). Data mining showed low identities (24-48%) to known structures as shown by BLAST, structural and functional domain searches (Table 2, Supporting Information). All HPs were computed in the protein-protein interation database DIP and no experimentally proven interaction between these structures and other proteins encoded on human chromosome 21 or mouse chromosome 16 was documented.
Discussion Chromosome 21 is the smallest of the human autosomes constituting approximately 1∼1.5% of the haploid genome. The major part of chromosome 21 is the long arm (21q), essential for normal development and function, and harboring
almost all genes of known function, except ribosomal RNA.1 The current human chromosome 21 gene catalog17 (http:// chr21.molgen.mpg.de) contains 238 entries and 168 cognate mouse orthologues were identified. Among 168 genes, the expression of 158 unique genes (referred to as mmu21 genes) was explored by systematic in situ hybridization in neonatal brain (at postnatal day 2). Sixty percent of the mmu21 genes are expressed in discrete or whole brain sections.18 For generation of information on HSA21 gene function, gene targeting, and transgenesis of single genes have been used. Although most valuable, these approaches suffer from low throughput and can be replaced by creation of large deletion and large insert transgenesis. Herein, proteomic analysis was performed with polytrangenic mice containing a fragment of DCR-1. The open reading frame of DSCR3 shows significant homology to Hbeta58, a mouse gene essential for embryogenesis, PEP8, a yeast homologue of Hbeta58 and an expressed sequence tag of Arabidopsis thaliana, suggesting that DSCR3 has some important function conserved during the course of evolution.19 DSCR5 is a member of the GPI-GnT, the complex of enzymes realizing the first step in the glycosylphosphatidylinositol (GPI) anchor mode of protein binding to the plasma membrane.20 This protein consists of two N-terminal hydrophobic regions, one C-terminal hydrophilic region, two putative transmemJournal of Proteome Research • Vol. 5, No. 1, 2006 47
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Table 2. List of Proteins Dysregulated in Hippocampus of Polytransgenic Micea theoretical no.
protein name
LEVELb
MP
Q8BMU7
Electron-transfer flavoprotein alpha-subunit, mitochondrial NADH dehydrogenase (ubiquinone) Fe-S protein 3 (30 kDa) NG, NG-dimethylarginine dimethylaminhydrolase 1
V
17
V
15
V
21
O08917
Flotillin-1
V
24
Q9JJV2
Profilin II
V
9
P05216
Tubulin alpha-6 chain
V
24
Q9ERD7
Tubulin beta-3
v
31
Q9D6F9
Tubulin beta-4 chain
v
36
Q7TMM9 Tubulin, beta 2
vV
33
P20152
Vimentin
V
19
P19226
60 kDa heat shock protein, mitochondrial [Precursor]
V
12
P11499
Heat shock protein HSP 90-beta
V
16
P17742
Peptidyl-prolyl cis-trans isomerase A
V
11
Q8BTZ3
Q9CWS0
48
Journal of Proteome Research • Vol. 5, No. 1, 2006
SCORE
pI
Mr (kDa)
locus of human homolog (acc. no) (mouse locus)
statistics (mean ( SD) wild-type
Antioxidant Proteins 243 8.42 34.95 Chr 15q23-q25 1.011 ( 0.280 (81% identity to P13804) (Chr 9) 126 6.67 30.15 Chr 11p11.11 0.379 ( 0.209 (87% identity to O75489) (Chr 2) 327 5.64 31.25 Chr 1p22 0.218 ( 0.097 (93% identity to O94760) (Chr 3) Cytoskeleton Proteins 133 6.71 47.51 Chr6P21.3 0.070 ( 0.050 (81% identity to 0.131 ( 0.082c O75955) (Chr 17) 62 6.78 14.90 Chr 3q25 1.685 ( 0.293 (99% identity to P35080) (Chr 3) 368 4.96 49.91 Chr12q12-q14 1.608 ( 0.430 (96% identity to 1.899 ( 0.983 Q9BQE3) 1.441 ( 1.126 (Chr 15) 2.153 ( 1.329 0.349 ( 0.190 0.147 ( 0.093 4.417 ( 1.553 4.325 ( 1.409 1.140 ( 0.590 0.717 ( 0.777 2.593 ( 1.739 0.447 ( 0.234 0.278 ( 0.142 0.202 ( 0.107 0.462 ( 0.356 0.269 ( 0.123 365 4.82 50.42 Chr16q24.3 0.078 ( 0.057 (91% identity to 1.014 ( 0.488 Q13509) (Chr 8) 1.054 ( 0.463 312 4.78 49.55 Chr 19p13.3 3.263 ( 0.568 (95% identity to 4.542 ( 3.548 Q969E5) 0.460 ( 0.193 (Chr 17) 2.713 ( 1.927 0.062 ( 0.025 1.969 ( 1.362 351 4.78 49.91 Chr 6p25 0.607 ( 0.481 (95% identity to 1.301 ( 1.156 Q13885) 1.027 ( 0.399 (Chr 13) 0.403 ( 0.194 n.m. 3.284 ( 1.668 0.224 ( 0.047 0.530 ( 0.207 6.557 ( 2.864 5.012 ( 2.401 1.056 ( 0.494 0.346 ( 0.134 n.m. 98 5.06 53.56 Chr 10p12.33 0.217 ( 0.0698 (90% identity to P08670) (Chr 2) Chaperone Proteins 79 5.91 60.96 Chr 2q33.1 0.349 ( 0.190 (97% identity to P10809) (Chr 1) 62 4.97 83.19 Chr 6p12 0.344 ( 0.180 (88% identity to P08238) (Chr 17) 116 7.88 17.84 Chr 7p13-p11.2 0.587 ( 0.237 (96% identity to 1.566 ( 0.529 P62937) (Chr 11) 0.245 ( 0.062 0.592 ( 0.202
141G6 mice
P-value
0.721 ( 0.116
0.008
0.172 ( 0.093
0.027
0.074 ( 0.037
0.001
0.072 ( 0.036 0.054 ( 0.026
0.928 0.024
1.307 ( 0.271
0.014
3.069 ( 1.750 2.086 ( 0.407 1.314 ( 0.355 1.751 ( 0.268 0.166 ( 0.106 0.170 ( 0.060 5.388 ( 1.521 5.388 ( 1.521 1.600 ( 0.343 0.213 ( 0.127 0.846 ( 0.628 0.281 ( 0.036 0.360 ( 0.093 0.208 ( 0.243 0.344 ( 0.278 0.195 ( 0.174 0.225 ( 0.115 1.224 ( 0.202 1.067 ( 0.272 7.171 ( 3.498 1.126 ( 0.524 0.383 ( 0.214 2.115 ( 0.820 0.052 ( 0.028 2.682 ( 1.118 0.195 ( 0.098 1.538 ( 1.156 1.339 ( 0.527 0.377 ( 0.163 n.m. 2.947 ( 0.533 0.653 ( 0.373 0.272 ( 0.128 8.302 ( 2.012 6.020 ( 2.096 1.397 ( 0.424 0.316 ( 0.083 n.m. 0.104 ( 0.041
0.081 0.587 0.738 0.361 0.023 0.559 0.227 0.169 0.070 0.243 0.060 0.112 0.253 0.947 0.455 0.331 0.038 0.259 0.948 0.022 0.064 0.464 0.387 0.477 0.285 0.031 0.666 0.965 0.769 n.m. 0.572 0.010 0.019 0.140 0.370 0.174 0.587 n.m. 0.007
0.166 ( 0.107
0.023
0.148 ( 0.129
0.023
0.645 ( 0.128 0.938 ( 0.423 0.308 ( 0.172 0.345 ( 0.185
0.555 0.020 0.348 0.023
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Protein Dysregulation in Polytransgenic Mice Table 2 (Continued) theoretical no.
protein name
LEVELb MP SCORE
pI
Mr (kDa)
locus of human homolog (acc. no) (mouse locus)
Metabolism Proteins 6.29 56.58 Chr 1p12 (92% identity to O43175) (Chr 3) 211 6.75 46.10 Chr 10q24.1-q25.1 (91% identity to P17174) (Chr 19) 423 9.22 59.75 Chr 18q12-q21 (97% identity to P25705) (Chr 18) 501 5.57 56.55 Chr 8p22-p21 (99% identity to P21281) (Chr 8)
Q8C603
3-phosphoglycerate dehydrogenase
v
22
P05201
Aspartate aminotransferase, cytoplasmic ATP synthase alpha chain, mitochondrial [Precursor]
vV
22
V
32
P50517
Vacuolar ATP synthase subunit B, brain isoform
V
37
Q99LD0
ATPase, H+transporting, V1 subunit E isoform 1
V
18
137
8.44
26.16
P30275
Creatine kinase, ubiquitous mitochondrial [Precursor] Fructose-bisphosphate aldolase A
V
26
211
8.39
47.00
V
20
218
8.40
39.22
P05063
Fructose-bisphosphate aldolase C
vV
25
401
6.79
39.26
Chr17 (97% identity to P09972) (Chr 11)
P17183
Gamma enolase
V
29
499
4.99
47.17
Chr 12p13 (98% identity to P09104) (Chr 6)
P13707
Glycerol-3- phosphate dehydrogenase
v
23
289
6.83
37.44
Q91Z53
Glyoxylate reductase/hydroxypyruvate reductase Guanylate kinase
V
14
182
7.57
35.33
V
11
74
6.14
21.79
Q9DBD7
Isovaleryl coenzyme A dehydrogenase
V
33
336
8.34
46.23
P09411
Phosphoglycerate kinase 1
V
22
382
7.52
44.41
P52480
Pyruvate kinase, isozyme M2
V
27
256
7.42
57.76
Chr12 (93% identity to P21695) (Chr 15) Chr9 (82% identity to Q9UBQ7) (Chr 4) Chr 1q32-q41 (88% identity to Q16774) (Chr 11) Chr 15q14-q15 (87% identity to P26440) (Chr 2) Chr Xq13.3 (97% identity to P00558) (Chr X) Chr 15q22-qter (97% identity to P14618) (Chr 9)
Q8BWM5 Similar to glycerol-3phosphate dehydrogenase (74% identity to Glycerol-3-phosphate dehydrogenase 1, Q8N1B0) Q9DBP5 UMP-CMP kinase
v
17
215
6.46
34.63
V
15
185
5.68
22.16
Q62048
Astrocytic phosphoprotein PEA-15
V
7
P97427
Dihydropyrimidinase related protein-1
v
24
Q03265
P05064
Q64520
292
Chr 22pter-q11.2 (74% identity to P36543) (Chr 6) Chr 15q15 (88% identity to P12532) (Chr 2) Chr 16q22-q24 (92% identity to P04075) (Chr 7)
Chr 3p24.1 (95% identity to Q8N335) (ND)
Chr1 (98% identity to P30085) (Chr 4) Miscellaneous Proteins 92 4.94 15.04 Chr 1q21.1 (99% identity to Q15121) (Chr 1) 287 6.64 62.17 Chr 4p16.1-4p15 (89% identity to Q14194) (Chr 5)
statistics (mean ( SD) wild-type
0.935 ( 0.298 0.801 ( 0.143
141G6 mice
0.941 ( 0.152 0.031 0.843 ( 0.123 0.499
0.209 ( 0.090 0.428 ( 0.164 0.544 ( 0.163 0.920 ( 0.514 1.517 ( 0.414 0.893 ( 0.181 ∑14.153 ( 2.385 ∑11.665 ( 2.620 0.512 ( 0.199 0.260 ( 0.167 0.129 ( 0.048 0.215 ( 0.077 1.018 ( 0.333 ∑2.268 ( 0.815
n.m. n.m. n.m. n.m. ∑15.817 ( 7.653 1.246 ( 0.229 0.223 ( 0.095 0.592 ( 0.252 0.351 ( 0.084 ∑0.220 ( 0.093 0.140 ( 0.050 0.177 ( 0.064 0.270 ( 0.116 0.476 ( 0.318 0.120 ( 0.039
P-value
0.654 ( 9.344 0.114 ( 0.046 0.080 ( 0.028 0.096 ( 0.041 0.602 ( 0.375
0.009 0.688 0.001 0.046
0.323 0.023 0.039 0.002 0.042
∑1.211 ( 0.828 0.027
n.m. n.m. n.m. n.m. ∑8.679 ( 4.668 1.647 ( 1.132 0.294 ( 0.234 0.885 ( 0.252 0.230 ( 0.072 0.110 ( 0.058 0.106 ( 0.039 0.187 ( 0.096 0.255 ( 0.112 0.481 ( 0.339 0.187 ( 0.076
0.030 0.343 0.474 0.026 0.013 0.021 0.264 0.842 0.822 0.983 0.043
0.680 ( 0.332
0.218 ( 0.069 0.004
0.117 ( 0.038
0.083 ( 0.025 0.039
1.421 ( 0.273
1.167 ( 0.179 0.036
0.354 ( 0.103 n.m. 2.209 ( 0.914 0.254 ( 0.110 0.816 ( 0.663 2.749 ( 0.777 0.983 ( 0.128 0.100 ( 0.037 0.451 ( 0.181
0.253 ( 0.092 n.m. 1.162 ( 0.608 0.238 ( 0.072 1.249 ( 0.874 2.915 ( 0.735 0.828 ( 0.119 0.099 ( 0.038 0.791 ( 0.324
0.261 ( 0.087
0.092 ( 0.019 0.0001
0.218 ( 0.097
0.074 ( 0.034 0.0009
0.460 ( 0.146
0.583 ( 0.107 0.006
0.049 n.m. 0.023 0.733 0.254 0.649 0.037 0.949 0.027
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Table 2 (Continued) theoretical no.
O35098
protein name
Dihydropyrimidinase related protein-4
Q9D172
LEVELb
V
MP
v
V
20
Q91ZR9
Heterogeneous nuclear ribonucleoprotein A2/B1
V
12
Q8BGQ8
Heterogeneous nuclear ribonucleoprotein K Laminin receptor 1 (67 kDa, ribosomal protein SA) (92% identity to P38983, RSP4_RAT, 40S ribosomal protein SA
V
20
V
13
Q9R0P9
Ubiquitin carboxylterminal hydrolase isozyme L1
V
18
P11798
Calcium/calmodulindependent protein kinase type II alpha chain EF-hand domaincontaining protein 2 (Synonyms: Swiprosin 1) Voltage-dependent anionselective channel protein 1
V
6
v
7
v
25
Voltage-dependent anionselective channel protein 2
V
21
Q9CYW4 Hypothetical haloacid dehalogenase/epoxide hydrolase family containing protein (35% identity to Q6LBD3, hydrolase of HAD-superfamily) Q8K0V8 Weakly similar to 2-hydroxyhepta-2,4-diene1,7-dioate isomerase (48% identity to Q98DB2, to 2-hydroxyhepta-2,4diene-1,7-dioate isomerase)
v
12
V
21
Q9D8Y0
Q60932
Q60930
pI
Mr (kDa)
Miscellaneous Proteins (continued) 30 343 6.51 61.96 Chr 10q25.2-10q26 (91% identity to O14531) (Chr 7) 16 89 9.00 28.09 Chr 21q22.3 (92% identity to P30042) (Chr 10)
ES1 protein homolog, mitochondrial (92% identity to P30042, ES1_HUMAN, ES1 protein homolog, mitochondrial [Precursor]) Q8BGH2 Protein CGI-51 homolog (93% identity to Q9Y512, SA50_HUMAN, SAM50like protein CGI-51)
Q8BNL2
SCORE
locus of human homolog (acc. no) (mouse locus)
226
6.34
51.86
Chr 22q13.31 (93% identity to Q9Y512) (ND)
statistics (mean ( SD) wild-type
141G6 mice
P-value
0.478 ( 0.226 0.395 ( 0.106 0.333 0.241 ( 0.079 0.161 ( 0.051 0.022 0.229 ( 0.145 0.177 ( 0.044 0.378 0.333 ( 0.152 0.506 ( 0.080 0.013
0.175 ( 0.080
0.095 ( 0.035
0.016
Nucleic Acid-Binding Proteins 141 8.67 35.98 Chr 7p15 0.255 ( 0.115 0.134 ( 0.083 0.039 (99% identity to 0.318 ( 0.229 0.261 ( 0.140 0.535 P22626) (Chr 6) 227 5.69 48.51 Chr 9 (68% identity 0.185 ( 0.079 0.088 ( 0.035 0.005 to P61978) (Chr 13) 113 4.80 32.91 Chr 3p21.3 0.220 ( 0.093 0.110 ( 0.058 0.015 (92, Q6NXQ8) 0.218 ( 0.106 0.102 ( 0.062 0.021 (Chr 9)
Proteosomal Proteins 299 5.14 24.84 Chr 4p14 5 (95% identity to P09936) (Chr 5) Signaling Proteins 88 6.88 54.33 Chr5 (96% identity to Q9UQM7) (Chr 18) 83 5.01 26.79 Chr 1p36 74% identity to Q96C19) (ND) 377 8.55 32.35 Chr 5q31 (98% identity to P21796) (Chr 11) 415
Chr 10q22 (97% identity to Q9BWK8) (Chr 14) Hypothetical Proteins 71 6.31 28.03 Chr9q32 (76% identity to Q9BSH5) (Chr 4)
192
7.44
8.42
31.73
34.68
Chr2p24.3-q11.2 (80% identity to Q96GK7) (ND)
0.902 ( 0.461 0.815 ( 0.396 0.725 0.521 ( 0.168 0.342 ( 0.087 0.032 0.255 ( 0.069
0.081 ( 0.069
0.0002
0.151 ( 0.077
0.393 ( 0.133
0.005
0.590 ( 0.441 0.562 ( 0.339 0.898 1.216 ( 0.533 0.852 ( 0.402 0.169 0.634 ( 0.495 0.245 ( 0.289 0.075 0.434 ( 0.205 1.329 ( 0.835 0.016 0.273 ( 0.148 0.270 ( 0.078 0.962 1.949 ( 0.825 1.595 ( 0.534 0.276 4.812 ( 1.513 3.254 ( 0.741 0.010 1.094 ( 0.410 0.986 ( 0.310 0.534 0.095 ( 0.038
0.149 ( 0.048
0.036
0.311 ( 0.154
0.078 ( 0.038
0.0005
a Mouse hippocampus proteins were separated by 2-DE and identified by MALDI-TOF/TOF, following in-gel digestion with trypsin. The spots representing the identified proteins are indicated in Figure 2 and are designated with their Swiss-Prot accession numbers. The number of matching peptides (MP), probability of assignment (Score), and theoretical molecular weight and pI values are given. Score is -10*log(P), where P is the probability that the observed match is a random event (MASCOT, http://www.matrixscience.com). Spots were quantified using the 2D ImageMaster software following selection of an area of interest. Between-group differences were analyzed with Student’s t-test (p < 0.05). Data are given as means ( SD. b Altered expression level in 141G6 mice was indicated with arrows (vV). c In case of protein representing multispots, expression of spots with bold letter was significantly altered. ND, not determined; NM, not measured.
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Protein Dysregulation in Polytransgenic Mice Table 3. Mass Spectrometric Details of Four Hypothetical Proteins mass spectrometric details protein name
BC013491 protein [Fragment] (Q91WT0)
Hypothetical haloacid dehalogenase/epoxide hydrolase family containing protein (Q9CYW4)
PTD012 homologue (Q91V76)
Weakly similar to 2-hydroxyhepta2,4-diene-1,7-dioate isomerase (Q8K0V8)
theoretical Mr
observed Mr
peptide sequence
1125.50 1362.68 1418.69 1423.72 1666.77 1828.86 1957.95 1967.99 1984.05 1986.05 2002.06 2032.06 2296.17 2298.22 3346.62 985.50 1019.49 1134.64 1160.57 1198.64 1354.75 1403.65 1489.67 1768.89 2300.15 985.50 1019.49 991.48 1152.50 1168.51 1312.58 1328.68 1680.95 2294.13 2332.10 2433.23 2451.26 2463.23 1177.70 1333.81 1557.88 1665.88 1711.87 1909.03 1925.01 2027.04 2054.06 2065.12 2070.05 2077.10 2081.11 2142.13 2174.06 2432.27 2448.29 2498.27 2770.41 2786.46 3713.84
1126.51 1363.69 1419.70 1424.73 1667.78 1829.87 1958.96 1969.00 1985.06 1987.06 2003.06 2033.07 2297.17 2299.23 3347.62 986.51 1020.49 1135.65 1161.58 1199.65 1355.76 1404.66 1490.68 1490.68 1769.89 1769.89 2301.15 992.49 1153.51 1169.51 1313.59 1329.68 1681.95 2295.13 2333.11 2434.24 2452.26 2464.23 1178.71 1334.82 1558.89 1666.88 1712.87 1910.03 1926.01 2028.05 2055.07 2066.13 2071.06 2078.10 2082.11 2143.14 2175.07 2433.27 2449.29 2499.27 2771.42 2787.47 3714.84
NDERQPDPR QRMQQEQQCK DIGLFLSTIERR KTSVVPESILSHK TSVVPESILSHKTNR GALSTSSTSAYTAREEAK EDYPMSKEELLSQVMK + 2 Oxidation (M) MCSESEQLEGRFQVLR MCSESEQLEGRFQVLR + Oxidation (M) TTPLKPPDTMEDSSGAVIK TTPLKPPDTMEDSSGAVIK + Oxidation (M) GRESTAGGVHSQSMGSELSR KLDSSPSLTLSSPQISLVTVPK MLQQDVDKMCSESEQLEGR + Oxidation (M) GRGTGSVSHVTFGDSASAAGPVAMASASASGAPVSSR DVVLHTFR LAVVSNFDR RPVGEEYASK GLTSRQWWK LEDILTGLGLR RLEDILTGLGLR LRRPVGEEYASK AQSHNFPNYGLSR AQSHNFPNYGLSR AHGVVVEDITVEQAFR AHGVVVEDITVEQAFR EHFDFVLTSEAVGCPKPDPR EPFTFPVR WLHFYEMK WLHFYEMK + Oxidation (M) TGELNFVSCMR APLVCLPVFVSK IAEVGGVPYLLPLVNK YHDFGCALLANLFASEGQPGK AHIMPAEFSSCPLNSDEAVNK + Oxidation (M) QTLEEHYGDKPVGMGGTFIVQK ACSEFSFHMPSLEELAEVLQK WLHFYEMKAPLVCLPVFVSK ALATQLPVIPR RALATQLPVIPR SQVTFLAPVTRPDK TFDTFCPLGPALVTK DTIADPHNLKICCR TMVQFLEQGETTLSVAR TMVQFLEQGETTLSVAR + Oxidation (M) LFSALLQVQKRPCQPSR ATDVMAHVAGFTVAHDVSAR TMVQFLEQGETTLSVARR ATDVMAHVAGFTVAHDVSAR + Oxidation (M) FSSSIVGPYDEIILPPESK TMVQFLEQGETTLSVARR + Oxidation (M) GDEVQCEIEELGVIINKVV VICVGLNYADHCQEQNVR HIKATDVMAHVAGFTVAHDVSAR HIKATDVMAHVAGFTVAHDVSAR + Oxidation (M) VICVGLNYADHCQEQNVRVPK TFDTFCPLGPALVTKDTIADPHNLK ATDVMAHVAGFTVAHDVSARDWQMR + Oxidation (M) SQVTFLAPVTRPDKVICVGLNYADHCQEQNVR
brane regions, and is localized in the endoplasmic reticulum.20 In humans, DSCR5 has been detected at the mRNA level in several tissues including fetal brain at a low level21 and it was overexpressed about 2-fold in fetal DS brain at the protein level.22 DSCR6 and DSCR9 with unknown function show no sequence-similarity by BLASTP nor a predicted motif by SMART program. PSORTII predicted a nuclear localization and its expression is detectable using Multiple Tissue cDNA panels in fetal kidney and fetal brain at high levels, suggesting a role in
early embryogenesis.21 However, expression of DSCR6 at the protein level was comparable between control and fetal DS brain.22 Tsukahara et al.23 recently isolated a human TTC3 gene, possessing a motif of the tetratricopeptide repeat (TPR) and examined the expression profile of mouse homologue (mtprd) of TTC3 in mouse embryos. In situ hybridization showed that mtprd is ubiquitously expressed in mouse embryos but predominantly in the central nervous system, including telencephalon, mesencephalon, and metencephalon. Journal of Proteome Research • Vol. 5, No. 1, 2006 51
research articles The main outcome of the study is that mice polytransgenic for five genes from DCR-1 and known to be of comparable cognitive function to control mice in terms of the Morris water maze but with a deficit in fear-conditioning performance8 reveal protein dysregulations in the hippocampus, the major structure for synaptic plasticity i.e., learning and memory formation. Methodologically, proteins were extracted from hippocampus, separated by 2-DE, and confidently identified by the use of MS and MS/MS.24,25 The protein map constructed was highly similar to rodent hippocampal proteomes12,13 identifying our proteomic approach as a reliable and reproducible analytical tool taking into account that some identified proteins could not be quantified due to technical reasons including poor separation. When dysregulated proteins in hippocampus of 141G6 mice were compared to those observed in human fetal or adult DS brain, several similarities could be detected: ES1 protein homologue [mitochondrial precursor],26 electrontransfer flavoprotein, a part of complex I of the respiratory chain,27,28 cytoskeleton elements,29-31 metabolically important structures such as aspartate aminotransferase [cytoplasmic],32 ATP synthase alpha chain,33 phosphoglycerate kinase 1,34,35 fructose biphosphate aldolase C,34 pyruvate kinase M2;32 miscellaneous structures such as dihydropyrimidinase-related protein 4,36 nuclear ribonucleoprotein A2/B1,37 ubiquitincarboxyl-terminal hydrolase isoenzyme L138 were found with altered levels in 141G6 mice and human DS brain. Moreover, signaling proteins such as VDAC1 and VDAC239 and chaperones were dysregulated in both conditions.40 Protein levels of ES1 protein homologue [mitochondrial precursor] showing 92% identity to human ES1 protein homologue (P30042) encoded by HSA21 was upregulated in 141G6 mice hippocampus and human fetal DS brain.26 The protein level of “hypothetical haloacid dehalogenase/ epoxide hydrolase family containing” was increased in 141G6 mice. In context with electron-transfer flavoprotein and Fe-S protein, an ubiquinone-handling enzyme altered in 141G6 mice, this HP probably results in the dysregulation of antioxidant system in terms of increased lipid peroxidation (Table 2). HP “weakly similar to 2-hydroxyhepta-2,4-diene-1,7-dioate isomerase”, with a putative metabolic role, was confidently downregulated in 141G6 mice. This protein also contains a fumarylacetoacetate hydrolase domain as shown by the InterPro database (IPR002529) and its significant decrease may lead to disturbances of tyrosine metabolism in 141G6 mice. Aberrant protein expression of antioxidant, chaperone, cytoskeleton and metabolic pathways, nucleic acid binding and handling systems, and miscellaneous as well as hypothetical proteins may well lead to impairment of most of the cognitive functions. This is in disagreement with findings of own unpublished studies using the Morris water maze and the multiple T-maze. The explanation may be that threshold levels necessary to alter these functions were not surpassed or that these tests are not sensitive enough to detect minor loss of cognitive functions. In fear-conditioning, one of possible learning situations, the number of freezing episodes (NFE) in identical context did not differ between 141G6 and WT mice.8 However, NFE of 141G6 mice in altered context and in altered context plus conditioned stimulus (sound) significantly increased or decreased, respectively.8 The ability to inhibit the generalization of the stimulus reflects the degree of behavioral plasticity. In the altered context version of the fear-conditioning 52
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task, success depends on the ability to inhibit freezing when the context has changed and to adjust behavior to the new conditions. This may be relevant for cognitive impairment of DS patients. And indeed, the level of CaMKII protein was found to be decreased in hippocampus of 141G6 mice. A modification of the CaMK-pathway has been previously shown to lead a downstream alteration of the CREB pathway associated with impairment of fear memory.41 We show here that at least one of the five transgenes is associated with alteration of hippocampal protein levels and might deregulate the production of CaMKII at the posttranscriptional level and that this modification alone or in conjunction with other modifications of proteins from the signaling pathway may well act specifically upon one type of memory involved in fear-conditioning. We here show that mice polytransgenic for five genes of the DCR-1 revealed protein dysregulation comparable to that observed in fetal DS brain. Furthermore, the finding of aberrant protein levels of CaMKII, mandatory for the generation of neuronal information storage and important for mechanisms of cognitive processes, may help to explain fear conditioning abnormalities in this mouse model for DS. Abbreviations: DS, down syndrome; HSA21, human chromosome 21; YAC, yeast artificial chromosome; PTG, transgenic mice containing YAC clone from human chromosome 21q22.2; 2-DE, two-dimensional electrophoresis; MALDI-TOF, matrixassisted laser desorption/ionization-time-of-flight; DSCR, Down Syndrome critical region.
Supporting Information Available: List of proteins identified in hippocampus of polytransgenic mice. This material is available free of charge via the Internet at http:// pubs.acs.org. References (1) Epstein, C. J. In The Metabolic and Molecular Bases of Inherited Disease, 7th ed.; Scriver, C. R., Beaudet, A. L., Sly, W. S., Valle, D., Eds.; McGraw-Hill: New York, 1995; Vol. 1, pp 749-794. (2) Antonarakis, S. E.; Lyle, R.; Dermitzakis, E. T.; Reymond, A.; Deutsch, S. Nat. Rev. Genet. 2004, 5, 725-738. (3) Engidawork, E.; Lubec, G. J. Neurochem. 2003, 84, 895-904. (4) Engidawork, E., Lubec, G. Amino Acids 2001, 21, 331-361. (5) Dufresne-Zacharia, M. C.; Dahmane, N.; Theophile, D.; Orti, R.; Chettouh, Z.; Sinet, P. M.; Delabar, J. M. Genomics 1994, 19, 462469. (6) Smith, D. J.; Zhu, Y.; Zhang, J.; Cheng, J. F.; Rubin, E. M. Genomics 1995, 27, 425-434. (7) Kim, D.; Won, J.; Shin, D. W.; Kang, J.; Kim, Y. J.; Choi, S. Y.; Hwang, M. K.; Jeong, B. W.; Kim, G. S.; Joe, C. O.; Chung, S. H.; Song, W. J. Biochem. Biophys. Res. Commun. 2004, 323, 499504. (8) Chabert, C.; Jamon, M.; Cherfouh, A.; Duquenne, V.; Smith, D. J.; Rubin, E.; Roubertoux, P. L. Behav. Genet. 2004, 34, 559569. (9) Smith, D. J.; Rubin, E. M. Hum. Mol. Genet. 1997, 6, 17291733. (10) Bradford, M. M. Anal. Biochem. 1976, 72, 248-254. (11) Weitzdoerfer, R.; Fountoulakis, M.; Lubec, G. Biochem. Biophys. Res. Commun. 2002, 293, 836-841. (12) Yang, J. W.; Czech, T.; Lubec, G. Electrophoresis 2004, 25, 11691174. (13) Shin, J. H.; London, J.; Le Pecheur, M.; Weitzdoerfer, R.; Hoeger, H.; Lubec, G. Neurochem. Int. 2005, 46, 641-653. (14) Xenarios, I.; Salwinski, L.; Duan, X. J.; Higney, P.; Kim, S. M.; Eisenberg, D. Nucleic Acids Res. 2002, 30, 303-305. (15) Clauser, K. R.; Baker, P.; Burlingame, A. L. Anal. Chem. 1999, 71, 2871-2882.
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Protein Dysregulation in Polytransgenic Mice (16) Altschul, S. F.; Madden, T. L.; Schaffer, A. A.; Zhang, J.; Zhang, Z.; Miller, W.; Lipman, D. J. Nucleic Acids Res. 1997, 25, 33893402. (17) Hattori, M.; Fujiyama, A.; Taylor, T. D.; Watanabe, H.; Yada, T.; Park, H. S.; Toyoda, A.; Ishii, K.; Totoki, Y.; Choi, D. K.; Groner, Y.; Soeda, E.; Ohki, M.; Takagi, T.; Sakaki, Y.; Taudien, S.; Blechschmidt, K.; Polley, A.; Menzel, U.; Delabar, J.; Kumpf, K.; Lehmann, R.; Patterson, D.; Reichwald, K.; Rump, A.; Schillhabel, M.; Schudy, A.; Zimmermann, W.; Rosenthal, A.; Kudoh, J.; Schibuya, K.; Kawasaki, K.; Asakawa, S.; Shintani, A.; Sasaki, T.; Nagamine, K.; Mitsuyama, S.; Antonarakis, S. E.; Minoshima, S.; Shimizu, N.; Nordsiek, G.; Hornischer, K.; Brant, P.; Scharfe, M.; Schon, O.; Desario, A.; Reichelt, J.; Kauer, G.; Blocker, H.; Ramser, J.; Beck, A.; Klages, S.; Hennig, S.; Riesselmann, L.; Dagand, E.; Haaf, T.; Wehrmeyer, S.; Borzym, K.; Gardiner, K.; Nizetic, D.; Francis, F.; Lehrach, H.; Reinhardt, R.; Yaspo, M. L.; Chromosome 21 mapping and sequencing consortium. Nature 2000, 405, 311309. Erratum in: Nature 2000, 407, 110. (18) Gitton, Y.; Dahmane, N.; Baik, S.; Ruiz i Altaba, A.; Neidhardt, L.; Scholze, M.; Herrmann, B. G.; Kahlem, P.; Benkahla, A.; Schrinner, S.; Yildirimman, R.; Herwig, R.; Lehrach, H.; Yaspo, M. L.; HSA21 expression map initiative. Nature 2002, 420, 586-590. (19) Nakamura, A.; Hattori, M.; Sakaki, Y. J. Biochem. (Tokyo) 1997, 122, 872-877. (20) Watanabe, R.; Murakami, Y.; Marmor, M. D.; Inoue, N.; Maeda, Y.; Hino, J.; Kangawa, K.; Julius, M.; Kinoshita, T. EMBO J. 2000, 19, 4402-4011. (21) Shibuya, K.; Kudoh, J.; Minoshima, S.; Kawasaki, K.; Asakawa, S.; Shimizu, N. Biochem. Biophys. Res. Commun. 2000, 271, 693698. (22) Ferrando-Miguel, R.; Cheon, M. S.; Lubec, G. Amino Acids 2004, 26, 255-261. (23) Tsukahara, F.; Urakawa, I.; Hattori, M.; Hirai, M.; Ohba, K.; Yoshioka, T.; Sakaki, Y.; Muraki, T. J. Biochem. (Tokyo) 1998, 123, 1055-1063. (24) Lubec, G.; Krapfenbauer, K.; Fountoulakis, M. Prog. Neurobiol. 2003, 69, 193-211.
(25) Fountoulakis, M. Amino Acids 2001, 21, 363-381. (26) Shin, J. H.; Weitzdoerfer, R.; Fountoulakis, M.; Lubec, G. Neurochem. Int. 2004, 45, 73-79. (27) Kim, S. H.; Fountoulakis, M.; Dierssen, M.; Lubec, G. J. Neural. Trans. Suppl. 2001, 61, 109-116. (28) Schuchmann, S.; Heinemann, U. Free Radic. Biol. Med. 2000, 28, 235-250. (29) Pollak, D.; Cairns, N.; Lubec, G. J. Neural. Trans. Suppl. 2003, 149-158. (30) Oppermann, M.; Cols, N.; Nyman, T.; Helin, J.; Saarinen, J.; Byman, I.; Toran, N., Alaiya, A. A.; Bergman, T.; Kalkkinen, N.; Gonzalez-Duarte, R.; Jornvall, H. Eur. J. Biochem. 2000, 267, 4713-4719. (31) Engidawork, E.; Gulesserian, T.; Fountoulakis, M.; Lubec, G. Neuroscience 2003, 122, 145-154. (32) Bajo, M.; Fruehauf, J.; Kim, S. H.; Fountoulakis, M.; Lubec, G. Proteomics 2002, 2, 1539-1546. (33) Kim, S. H.; Vlkolinsky, R.; Cairns, N.; Lubec, G. Cell Mol. Life Sci. 2000, 57, 1810-1816. (34) Kitzmueller, E.; Greber, S.; Fountoulakis, M.; Lubec, G. J. Neural. Trans. Suppl. 2001, 203-210. (35) Labudova, O.; Kitzmueller, E.; Rink, H.; Cairns, N.; Lubec, G. Clin. Sci (Lond). 1999, 96, 279-285. (36) Weitzdoerfer, R.; Fountoulakis, M.; Lubec, G. J. Neural. Trans. Suppl. 2001, 95-107. (37) Kim, S. H.; Dierssen, M.; Ferreres, J. C.; Fountoulakis, M.; Lubec, G. J. Neural. Trans. Suppl. 2001, 273-280. (38) Engidawork, E.; Juranville, J. F.; Fountoulakis, M.; Dierssen, M.; Lubec, G. J. Neural. Trans. Suppl. 2001, 117-130. (39) Yoo, B. C.; Fountoulakis, M.; Cairns, N.; Lubec, G. Electrophoresis 2001, 22, 172-179. (40) Yoo, B. C.; Vlkolinsky, R.; Engidawork, E.; Cairns, N.; Fountoulakis, M.; Lubec, G. Electrophoresis 2001, 22, 1233-1244. (41) Bourtchuladze, R.; Frenguelli, B.; Blendy, J.; Cioffi, D.; Schutz, G.; Silva, A. J. Cell 1994, 79, 59-68.
PR050235F
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