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Analysis of Presenilin 1 and 2 interacting proteins in mouse cerebral cortex during development
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Contents lists available at ScienceDirect
International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu
Analysis of Presenilin 1 and 2 interacting proteins in mouse cerebral cortex during development
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Ashish Kumar, M.K. Thakur ∗ Biochemistry and Molecular Biology Laboratory, Brain Research Centre, Department of Zoology, Banaras Hindu University, Varanasi 221 005, India
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Article history: Received 21 May 2014 Received in revised form 5 August 2014 Accepted 26 August 2014
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Keywords: Presenilin Notch-1 Neurogenesis Synaptogenesis Brain development
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1. Introduction
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In our previous report, we showed that Presenilin (PS)1 and 2 have differential expression profile from early embryonic stages till adulthood in mouse cerebral cortex, suggesting that both of these proteins are crucial for brain development. Genetic manipulation studies have also shown the involvement of PS1 in brain development, but PS2 remains largely unexplored. In order to understand how PS1 and 2 mediate developmental functions, we have investigated the interaction of PS1 and 2 with proteins of mouse cerebral cortex during development. Co-immunoprecipitation (Co-IP) combined with MALDI-MS/MS analysis revealed 12 interacting partners of PS1 and 11 partners of PS2. The interacting proteins were different for PS1 and 2, and involved in cell division, glycolysis, cell adhesion and protein trafficking. Densitometric analysis of protein bands visualized after SDS-PAGE separation of Co-IP proteins revealed variation in their amount and degree of interaction during different developmental stages of mice. Further, immunoblot based validation of PS1 interacting protein Notch-1 showed maximum interaction at embryonic day (E) 12.5, decline at E18.5, upregulation from postnatal day 0 (P0) to P20 and thereafter reduction at P45 and 20 weeks. In-silico analysis of PS and its interacting proteins indicated conformation based interaction through common type of secondary structures having alpha helical, extended beta strand and random coil, and CK2, PKC phosphorylation and myristoylation motifs. Taken together, our study showed that PS1 and PS2 interact to varying extent with different proteins of mouse cerebral cortex and suggests their interaction based on specific conformation and involvement in diverse functions essential for the brain development. © 2014 Published by Elsevier Ltd. on behalf of ISDN.
Presenilin (PS)1 and 2 are multi-pass transmembrane (TM) proteins of aspartyl protease family. Their primary sequence is evolutionarily conserved in eukaryotes, having homologues in organisms as distant as C. elegans (Levitan and Greenwald, 1995), Drosophila (Boulianne et al., 1997), and lower chordates (MartínezMir et al., 2001), suggesting functional conservation. Mammalian PS1 and 2 share extensive amino acid sequence identity and are synthesized as 50 kDa polypeptides (Hutton and Hardy, 1997). They span the membrane in a serpentine way and consist of nine TM domains. Their main domains are three stretches of hydrophilic domains comprising of N-terminal tail, C-terminal tail and a long loop between TM VI and TM VII (Tomita and Iwatsubo, 2013). Amino terminal and long loop domains of PS1 and PS2 are phosphorylated at serine residues (De Strooper et al., 1997).
∗ Corresponding author. Tel.: +91 542 2313958; fax: +91 542 2368174. E-mail addresses: mkt [email protected], [email protected] (M.K. Thakur).
PS1 and PS2 have been implicated in several brain functions (Zhang et al., 2013) through their interaction with amyloid precursor protein, Notch-1, -catenine, calsenilin, cadherines and others Q2 (Chen and Schubert, 2002). With respect to brain development, PS1 knockout mice showed developmental deformities including cortical dysplasia and died before birth (Wen et al., 2005). Conditional knockouts of PS1 have shown its implication in several processes such as neuronal migration, cortical lamination, neurogenesis, neuronal differentiation (Wines-Samuelson et al., 2005), synaptogenesis (Uchihara et al., 2006), somitogenesis, gliogenesis Q3 (Saura, 2010), and growth (Nakajima et al., 2009). On the other hand, knockout of PS2 survived well without any defect in the brain morphology (Herreman et al., 1999), indicating that the protease might not contribute to brain developmental processes directly and may serve to compensate PS1 functions postnatally (Wang et al., 2003). In our previous report, we showed that PS1 and PS2 expression varied significantly in the cerebral cortex during critical time of development. These findings indicated that PS1 and 2 might have independent involvement during different developmental
http://dx.doi.org/10.1016/j.ijdevneu.2014.08.010 0736-5748/© 2014 Published by Elsevier Ltd. on behalf of ISDN.
Please cite this article in press as: Kumar, A., Thakur, M.K., Analysis of Presenilin 1 and 2 interacting proteins in mouse cerebral cortex during development. Int. J. Dev. Neurosci. (2014), http://dx.doi.org/10.1016/j.ijdevneu.2014.08.010
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stages. In order to understand such involvement, we have analyzed interacting partners of PS1 and 2 during brain development. We have co-immunoprecipitated (Co-IP) PS1 and PS2 specific complexes during different developmental stages, and the resulting proteins were identified by MALDI-MS/MS. Further, the interaction of Notch-1 in Co-IP proteins was validated with PS1 at different developmental stages. In silico analysis for secondary structure prediction of putative interacting proteins was done by bioinformatics GOR-4 and specific motifs were identified by Motif-scan.
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2. Materials and methods
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2.1. Animals
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Swiss albino strain mice were inbred and maintained at 25 ± 2 ◦ C with 12 h light and 12 h dark schedule with ad libitum access to standard mice feed and drinking water in the animal house of the Department of Zoology, Banaras Hindu University, Varanasi, India. They were used according to guidelines of the institutional animal ethical committee, Banaras Hindu University, Varanasi, India. Male mice (n = 54) were used for the present study and the detailed information is provided in the supplementary section. To analyze the interacting proteins of PS during development, mice of embryonic day (E) 12.5, E18.5, postnatal day (P) 0, P20, P45 and 20 weeks (w) were used. The mice were decapitated and embryos were collected by flushing out from uterus for prenatal ages. The telencephalon from E12.5 and the cerebral cortex from other developmental stages were dissected out.
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2.2. Preparation of protein extract
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Cytosolic protein extract was prepared from the mouse cerebral cortex of different developmental ages. Briefly, 10% homogenate was prepared in RIPA buffer [50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% NP-40 and 0.5% sodium deoxycholate] with protease inhibitors (Sigma–Aldrich, USA) at 4 ◦ C (Kim et al., 2012; Jang et al., 2011). The homogenate was centrifuged at 1000 × g for 10 min at 4 ◦ C. The postnuclear supernatant was collected in separate microfuge tube and stored at −70 ◦ C. The amount of protein in the preparation was estimated by Bradford (1976) method.
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For Co-IP, the protein (100 g) was incubated with protein A Sepharose beads at 4 ◦ C for 2 h to avoid nonspecific binding, and then centrifuged at 3000 × g for 10 min. The precleared supernatant was incubated overnight with 2 g of anti-PS1 antibody (Alpha Diagnostics International, USA) or anti-PS2 antibody (Alpha Diagnostics International, USA) in 500 l of IP buffer (50 mM Hepes, pH 7.4, 50 mM NaCl, 0.1% Triton X-100, 10% glycerol, 1 mM PMSF). IgG was used as a negative control. Next day, protein A Sepharose beads were added to the reaction mix at 4 ◦ C for 4 h. Thereafter, the beads containing bound proteins were precipitated by centrifugation, washed three times in Co-IP buffer. Co-immunoprecipitate was denatured by boiling in SDS-sample buffer, resolved on 10% Tris–glycine SDS-PAGE and analyzed by silver staining. To analyze Co-IP bands, the signal intensity of each stained band was calculated as Integrated Density Value (IDV) by spot densitometry tool AlphaEaseFC software (Alpha Innotech Corp., USA).
2.4. Immunoblotting
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Initially, the input level of both Notch-1 full length (Notch-1 FL) and PS1 was determined by their protein expression during development. Briefly, 20 g cytosolic protein from each developmental stages was denatured, resolved by 10% Tris–glycine SDS-PAGE, transferred onto PVDF membrane. The membrane was blocked in 5% (w/v) nonfat milk prepared in 1× PBS for 2 h and incubated overnight with rabbit anti-Notch-1 antibody in 1:1000 dilutions (Developmental Studies Hybridoma Bank, USA). After washing twice in 0.1% PBST, the membrane was incubated with horse-radish peroxidase conjugated goat-anti-rabbit secondary antibody in 1:2000 dilutions (Bangalore Genei, India), washed twice in 0.1% PBST and detected by ECL. Immunoblotting of input PS1 was done as mentioned previously (Kumar and Thakur, 2012). Internal control -actin was detected by reprobing the membrane with antimouse -actin HRP conjugate, 1:10,000 dilutions (Sigma–Aldrich, USA). In order to analyze interaction level of Notch-1 with PS1 protein, equal amount of PS1 Co-IP protein from different developmental stages was subjected to immunblotting of Notch-1 Intracellular domain (Notch-1 NICD). The signal intensities for input Notch-1 FL, input PS1 and Co-IP Notch-1 NICD fragment were normalized against signal intensity of -actin and represented as a histogram with mean of three values calculated as RDV (IDV of Notch-1 FL/-actin, PS1/-actin and Notch-1 NICD/actin).
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The protein bands were excised, minced into 1 mm3 pieces and transferred into a sterile microcentrifuge tube. It was washed three times with 500 l of wash solution (50% acetonitrile, 50 mM ammonium bicarbonate) and incubated at room temperature for 15 min with gentle agitation (vortex mixer on lowest setting). Further, the gel was dehydrated in 100% acetonitrile for 5 min. Acetonitrile was removed and the gel was dried at room temperature for 10–20 min. It was rehydrated in 150 l reduction solution (10 mM DTT, 100 mM ammonium bicarbonate) for 30 min at 56 ◦ C. The reduction solution was discarded and added 100 l alkylation solution (50 mM iodoacetamide, 100 mM ammonium bicarbonate) and incubated for 30 min in the dark at room temperature. The alkylation solution was discarded with a pipette, added 500 l of wash solution and incubated at room temperature for 15 min with gentle agitation. The gel was dehydrated in 100 l 100% acetonitrile for 5 min. Further, acetonitrile was discarded and gel dried at room temperature. The gel was rehydrated with a minimal volume of protease digestion solution (20 g/ml in 50 mM ammonium bicarbonate solution) and digested overnight at 37 ◦ C. Next day, it was centrifuged at 12,000 × g for 30 s. The supernatant was transferred to a sterile centrifuge tube. Further, 25–50 l of extraction solution (60% acetonitrile, 0.1% TFA) was added to gel pieces, agitated gently by vortexing at lowest setting for 10 min and it was centrifuged at 12,000 × g for 30 s. The extracted peptides were pooled and dried to near dryness by centrifugal evaporation. The peptides were resuspended in 5 l of resuspension solution (50% acetonitrile, 0.1% TFA) and agitated gently on a vortex at lowest setting.
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2.6. MALDI-TOF-MS
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The samples were spotted (1 l) on MALDI target plate [MTP 384 ground steel (Bruker Daltonics, Germany)] followed by 0.5 l of alpha-cyano-4-hydroxycinnamic acid matrix [10 mg/mL in 50% acetonitrile, 0.1% TFA (Sigma–Aldrich, USA)]. The peptide spectra was acquired in an AUTOFLEX speed MALDI TOF/TOF instrument (Bruker Daltonics, Germany) having Nd:YAG smart Laser beam of 335 nm wavelength. External calibration was done with peptide calibration standard supplied by Bruker, with masses ranging from 1046 to 3147 Da. The obtained spectra were acquired using Flex Control version 3.3 software in reflectron ion mode with an average of 2000 laser shots at mass detection range between 700 and 4000 m/z. The most three abundant peaks were subjected to further fragmentation using LIFT method. The data were analyzed using Flex Analysis software version 3.3 (Bruker Daltonics, Germany) and searched in MASCOT web server (Matrix Science; http://www.matrixscience.com) using Biotools version 3.2 software (Bruker Daltonics, Germany).
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2.3. Co-IP
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2.5. Sample preparation for matrix assisted laser desorption/ionization (MALDI)-time of flight (ToF) mass spectrometry
2.7. In silico analysis
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Secondary structure of the predicted proteins obtained from MALDIMS analysis were analyzed by GOR4 (http://npsa-pbil.ibcp.fr/cgi-bin/ npsa automat.pl?page=npsa gor4.html, Combet et al., 2000). In order to identify the motifs in the predicted proteins, MOTIF-SCAN (http://myhits. isb-sib.ch/cgi-bin/motif scan) was done.
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2.8. Statistical analysis
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Each experiment was repeated three times (n = 3 × 3 = 9 mice/group). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by post hoc test of Student–Newman–Keuls method through Jindel Scientific SigmaPlot for Windows (standard version 2.0). Values were reported as mean ± SEM and pvalues <0.05 were considered as significant.
3. Results 3.1. Co-IP of PS interacting proteins in the cerebral cortex of mice during development With the optimized conditions for PS1 (Fig. 1A) and PS2 (Fig. 2A), Co-IP analysis showed that interacting proteins varied differentially both for PS1 (Fig. 1B) and PS2 (Fig. 2B) with respect to number, amount and degree of interaction in the cerebral cortex of mice during development. Out of these proteins, 12 partners of PS1 (Fig. 1C) and 11 of PS2 (Fig. 2C) showed significant alterations in their level of interaction in prenatal and postnatal stages as compared to P0. Predicted molecular weight of PS1 interacting partners was in the range of 30–342 kDa while the range was 24–193 kDa for PS2. One way ANOVA analysis showed significant F values for all the proteins (details of F values included in supplementary section).
Please cite this article in press as: Kumar, A., Thakur, M.K., Analysis of Presenilin 1 and 2 interacting proteins in mouse cerebral cortex during development. Int. J. Dev. Neurosci. (2014), http://dx.doi.org/10.1016/j.ijdevneu.2014.08.010
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3.2. MALDI/MS/MS analysis of PS interacting proteins MALDI/MS/MS analysis identified synaptotagmin-4, unnamed protein, aldolase A, creatine kinase B-type, alpha enolase,
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elongation factor 1-␣, tubulin -2A chain, pyruvate kinase isoform M1, GRIN1, Notch-1, clathrin heavy chain 1 and unnamed protein as PS1 interacting partners. Similarly, ICAM5, PRP4 kinase, catenin -1, tyrosine-protein kinase, arrestin  2, tubulin -2A
Fig. 1. Co-immunoprecipitation (Co-IP) analysis of PS1 interacting proteins in the cerebral cortex of mice during development. (A) 10% silver stained SDS-PAGE showing optimization of Co-IP for PS1 interacting proteins [Input protein (Input, Lane-1), Supernatant protein (Supt, Lane-2), final wash of Co-IP (wash, Lane-3), Co-IP (Lane-4), antibody (ab, Lane-5) and Marker (M, Lane-6)], (B) Co-IP proteins of different developmental stages [bold arrows indicate specific bands and N denoted dotted lines representing nonspecific bands]. (C) Interaction level of Co-IP proteins plotted by densitometric analysis (integrated Density Value-IDV) of each band resolved on the gel. Asterisk (*) denotes significant differences (p < 0.05) between the age groups as compared to P0. E12.5 – embryonic day 12.5, E18.5 – embryonic day 18.5, P0 – neonatal, P20 – postnatal day 20, P45 – postnatal day 45, 20w – postnatal 20 weeks.
Please cite this article in press as: Kumar, A., Thakur, M.K., Analysis of Presenilin 1 and 2 interacting proteins in mouse cerebral cortex during development. Int. J. Dev. Neurosci. (2014), http://dx.doi.org/10.1016/j.ijdevneu.2014.08.010
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Fig. 2. Co-IP analysis of PS2 interacting proteins in the cerebral cortex of mice during development. (A) 10% silver stained SDS-PAGE showing optimization of Co-IP for PS2 interacting proteins [Input protein (Input, Lane-1), Supernatant protein (Supt, Lane-2), final wash of Co-IP (wash, Lane-3), Co-IP (Lane-4), antibody (ab, Lane-5) and Marker (M, Lane-6)], (B) Co-IP proteins of different developmental stages [bold arrows indicate specific bands and N denoted dotted lines representing non-specific bands]. (C) Interaction level of Co-IP proteins plotted by densitometric analysis (integrated Density Value-IDV) of each band resolved on the gel. Asterisk (*) denotes significant differences (p < 0.05) between the age groups as compared to P0. E12.5 – embryonic day 12.5, E18.5 – embryonic day 18.5, P0 – neonatal, P20 – postnatal day 20, P45 – postnatal day 45, 20w – postnatal 20 weeks.
Please cite this article in press as: Kumar, A., Thakur, M.K., Analysis of Presenilin 1 and 2 interacting proteins in mouse cerebral cortex during development. Int. J. Dev. Neurosci. (2014), http://dx.doi.org/10.1016/j.ijdevneu.2014.08.010
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Table 1 MALDI-MS/MS analysis of imunoprecipitated PS interacting proteins. GI number
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Protein
Function
Unnamed protein Clathrin heavy chain 1 Notch-1
Not known Major protein of the polyhedral coat of coated pits and vesicles. Receptor for membrane-bound ligands to regulate cell-fate determination. Cerebral cortex development. Involved in glutamate neurotransmission and calcium channel activity Glycolytic enzyme, multiple nonglycolytic functions with possible diverse implications Major constituent of microtubules. Promotes the GTP-dependent binding of amino- acyl-tRNA to the A-site of ribosomes during protein biosynthesis. Multifunctional enzyme that, as well as its role in glycolysis, plays a part in various processes such as growth control, hypoxia tolerance and allergic responses Play a central role in energy transduction in tissues with large, fluctuating energy demands. Role in glycolysis and gluconeo-genesis. May also function as scaffolding protein Not known Mediate calcium-dependent regulation of membrane trafficking.
gi|5901688
4
80
GRIN1
gi|359807367
5
58
Pyruvate kinase isoform M1
gi|4507729 gi|407261700
6 7
52 48
Tubulin -2A chain Elongation factor 1-␣
gi|70794816
8
46
Alpha enolase
gi|10946574
9
42
Creatine kinase B (brain)-type
gi|7548322
10
39
Aldolase A
gi|74192167 gi|60218904
11 12
34 30
Unnamed protein Synaptotagmin-4
PS2 interacting proteins 1 gi|148670188 gi|20330556 2
193 117
3 4
83 75
Catenin -1 Tyrosine-protein kinase
5 6 7 8 9 10 11
65 52 45 34 32 28 24
Arrestin  2 Tubulin -2A chain Integrin ␣-6 mKIAA0303 protein Forkhead box protein K2 Coronin-1A Unnamed protein
gi|123234448 gi|4507729 gi|148695149 gi|37359892 gi|238018071 gi|6753492 gi|309267211
192
Mw (kDa) 342 193 110
gi|26337435 gi|31560493
191
Band no.
PS1 interacting proteins gi|26329341 1 gi|51491845 2 gi|224967064 3
ICAM5 PRP4 kinase
chain, integrin ␣-6, mKIAA0303 protein, forkhead box protein K2, coronin-1A, unnamed protein were identified as interacting partners of PS2 (Table 1).
Cell adhesion molecule Serine/arginine-rich protein-specific kinase regarded as a CDK-like kinase Cell adhesion molecule Tyrosine kinases catalyze the phosphorylation of tyrosine residues in proteins Regulate G-protein coupled receptor signaling Major constituent of microtubules. Involved in cell signaling and the regulation of cell cycle. Function not known, predicted in neuronal migration Cellular transcription factor ILF-1 involved in embryonic development Involved in calcium ion transport Not known.
and its interacting proteins showed high number of CK2, PKC phosphorylation and myristoylation motifs (Table 3).
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3.3. Analysis of Notch-1 interaction with PS1 in the cerebral cortex of mice during development To analyze the data, values from neonatal (P0) mice were considered as 100%. Western blot analysis of input Notch-1 FL showed significant alteration at E18.5, P45 and 20w as compared to P0 (E12.5 – 97%, E18.5 – 173%, P20 – 112%, P45 – 200% and 20w – 309%) (Fig. 3). One way ANOVA of PS1 showed significant F value [F(5, 12) = 56.2, p < 0.05]. Analysis PS1 input protein showed significant alteration (E12.5 – 135%, E18.5 – 75%, P20 – 130%, P45 – 156%, 20w – 56%) (Fig. 3). One way ANOVA of PS1 showed significant F value [F(5, 12) = 48.4, p < 0.05]. Notch-1 interaction with PS1, Co-IP followed by western blot analysis showed significant alteration in Notch-1 level in prenatal (E12.5 – 144% and E18.5 – 50%) and postnatal stages (P20 – 115%, P45 – 77% and 20w – 80%) (Fig. 3). One way ANOVA showed significant F value [F(5, 12) = 65.895, p < 0.05].
3.4. In silico analysis of PS interacting proteins Secondary structure prediction analysis showed that PS and its interacting proteins have common type of secondary structure, i.e. alpha helical (Hh), extended beta strand (Ee) and random coil (Cc) but other secondary structures, i.e. 310 helix (Gg), Pi helix (Ii), Beta bridge (Bb), Beta turn (Tt), Bend region (Ss), Ambigous states (?) were not present (Table 2). Motif scan analysis of PS
We have investigated the interaction pattern of PS1 and PS2 with proteins of mouse cerebral cortex at prenatal, neonatal and postnatal stages, covering the wide range of brain developmental processes including neurogenesis, neuronal processing and differentiation. In present study, we have identified 12 interacting partners of PS1 involved in diverse functions. Notch-1 and GRIN1 are involved in brain development, pyruvate kinase isoform M1, alpha enolase, creatin kinase B-type and aldolase A in energy metabolism, clathrin heavy chain 1 is the major protein of the polyhedral coat of coated pits and vesicles involved in protein trafficking, synaptotagmin-4 mediate calcium-dependent regulation of membrane trafficking, elongation factor 1-␣ promotes the GTPdependent binding of aminoacyl-tRNA to the A-site of ribosomes during protein biosynthesis. PS1 interacting partner Notch-1 protein is involved in early neurodevelopmental processes including neurogenesis, somitogenesis and vasculogenesis. In this report, we observed that Notch-1 interaction pattern is similar to the expression profile of PS1 during development up to P20 and thereafter show decreased interaction. Previous study using Co-IP also showed interaction of Notch-1 with PS1 in HEK293 and Drosophila cells (Ray et al., 1999). PS1 interacting proteins pyruvate kinase isoform M1, alpha enolase, creatin kinase B-type and aldolase A are involved in energy metabolism. Our study is partially supported by previous report that showed decrease in the brain oxidative markers including pyruvate kinase isoform M1 and enolase in aged
Please cite this article in press as: Kumar, A., Thakur, M.K., Analysis of Presenilin 1 and 2 interacting proteins in mouse cerebral cortex during development. Int. J. Dev. Neurosci. (2014), http://dx.doi.org/10.1016/j.ijdevneu.2014.08.010
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Hh
Gg
Ii
Bb
Ee
Tt
Ss
Cc
(?)
Others
PS1 and its interacting proteins Presenilin 1, Sequence: 467 Unnamed protein, Sequence: 2473 Clathrin heavy chain 1, Sequence: 1675 Notch 1, Sequence: 2531 GRIN1, Sequence: 827 Pyruvate kinase isoform M1, Sequence: 531 Tubuline  2A chain, Sequence: 445 Elongation factor 1-␣, Sequence: 415 Alpha enolase, Sequence: 434 Creatine kinase B-type, Sequence: 381 Aldolase A, Sequence: 364 Unnamed protein, Sequence: 218 Synaptotagmin 4, Sequence: 425
179 is 38.33% 1729 is 69.92% 801 is 47.82% 320 is 12.64% 130 is 15.72% 233 is 43.88% 148 is 33.26% 117 is 28.19% 194 is 44.70% 114 is 29.92% 126 is 34.62% 85 is 38.99% 126 is 29.65%
0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00%
0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00%
0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00%
98 is 20.99% 121 is 4.89% 222 is 13.25% 472 is 18.65% 134 is 16.20% 100 is 18.83% 81 is 18.20% 112 is 26.99% 53 is 12.21% 63 is 16.54% 102 is 28.02% 34 is 15.60% 71 is 16.71%
0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00%
0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00%
190 is 40.69% 623 is 25.19% 652 is 38.93% 1739 is 68.71% 563 is 68.08% 198 is 37.29% 216 is 48.54% 186 is 44.82% 187 is 43.09% 204 is 53.54% 136 is 37.36% 99 is 45.41% 228 is 53.65%
0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00%
0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00%
PS2 and its interacting proteins Presenilin 2, Sequence: 448 ICAM5, Sequence: 917 PRP4 kinase, Sequence: 1007 Catenin -1, Sequence: Tyrosine-protein kinase, Sequence: 937 Arrestin  2, Sequence: 406 Tubulin -2A chain, Sequence: 445 Integrin ␣ 6, Sequence:1073 mKIAA0303 protein, Sequence: 950 Forkhead box protein K2, Sequence: 651 Coronin 1A, Sequence: 461 Unnamed protein, Sequence: 325
125 is 27.90% 189 is 20.61% 445 is 44.19% 399 is 51.09% 167 is 17.82% 107 is 26.35% 148 is 33.26% 253 is 23.58% 169 is 17.79% 133 is 20.43% 80 is 17.35% 134 is 41.23%
0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00%
0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00%
0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00%
108 is 24.11% 210 is 22.90% 99 is 9.83% 80 is 10.24% 204 is 21.77% 78 is 19.21% 81 is 18.20% 223 is 20.78% 95 is 10.00% 110 is 16.90% 119 is 25.81% 48 is 14.77%
0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00%
0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00%
215 is 47.99% 518 is 56.49% 463 is 45.98% 302 is 38.67% 566 is 60.41% 221 is 54.43% 216 is 48.54% 597 is 55.64% 686 is 72.21% 408 is 62.67% 262 is 56.83% 143 is 44.00%
0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00%
0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00%
* (Hh)- Alpha helix, (Gg)- 310 helix, (Ii)- Pi helix, (Bb)- Beta bridge, (Ee)- Extended strand, (Tt)- Beta turn, (Ss)- Bend region, (Cc)- Random coil, (?)- Ambigous states
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Protein
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Please cite this article in press as: Kumar, A., Thakur, M.K., Analysis of Presenilin 1 and 2 interacting proteins in mouse cerebral cortex during development. Int. J. Dev. Neurosci. (2014), http://dx.doi.org/10.1016/j.ijdevneu.2014.08.010
Table 2 Secondary structure analysis of PS interacting proteins.
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PS1 and its interacting proteins Protein
AMIDATION
GLYCOSYL
ATP GTP A
cAMP PHOSPHO SITE
CK2 PHOSPHO SITE
LEUCINE ZIPPER
MYRISTYL
PKC PHOSPHO SITE
PRENYLATION
TYR PHOSPHO SITE
3 17 9 9 3 1 3 3 – 2 –
– 1 – – – – – 1 – – – – –
– 2 2 2 1 – 2 – – – – – –
7 64 14 14 17 5 7 6 3 4 3 4 8
– 10 – – – – – – – – – – –
4 12 9 9 20 8 9 6 5 8 5 2 4
4 60 10 15 35 10 5 8 4 9 4 3 10
– 1 – – – – – – – – – – –
– 1 – – 3 1 – – – – – – –
2
Protein
AMIDATION
GLYCOSYL
ATP GTP A
cAMP PHOSPHO SITE
CK2 PHOSPHO SITE
LEUCINE ZIPPER
MYRISTYL
PKC PHOSPHO SITE
TYR PHOSPHO SITE
RGD
PS2 ICAM5 PRP4 kinase Catenin -1 Tyrosine-protein kinase Arrestin  2 Tubulin -2A chain Integrin ␣ 6 mKIAA0303 protein Forkhead box protein K2 Coronin 1A Unnamed protein
– – 1 5 1 1 – – 2 – – –
1 13 6 – 7 1 3 8 2 2 – 3
– – – – – – – – – – – –
– 1 13 2 2 2 2 3 3 – 2 –
8 12 17 9 11 2 7 21 21 4 8 4
– – – – – – – – – – 1 –
2 25 3 15 9 2 9 15 7 9 8 2
5 19 27 11 9 10 5 11 27 8 10 3
– – – – – – – – – – – –
– – – – – – – – – – – –
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Presenilin 1 – – Unnamed protein – Clathrin heavy chain 1 Notch-1 – GRIN1 – Pyruvate kinase M1 – – Tubuline -2A chain Elongation factor 1-␣ – – Alpha enolase – Creatine kinase B-type 1 Aldolase A Unnamed protein – Synaptotagmin 4 – PS2 and its interacting proteins
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Table 3 Motif scan analysis of PS interacting proteins.
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Fig. 3. Analysis of Notch-1 interaction with PS1 in the cerebral cortex of mice during development. Western blotting showing Co-IP of Notch-1 Intracellular domain (NICD) fragment (110 kDa), Input Notch-1 (280 kDa) and input PS1 (32 kDa) in the cerebral cortex of mice during development. Histogram represents Relative Density Value, RDV (IDV of Co-IP Notch-1 NICD/-actin, Input Notch-1/-actin and PS1/-actin) from three independent experiments (±) SEM. Asterisk (*) denotes significant differences (p < 0.05) between the age groups as compared to P0. E12.5 – embryonic day 12.5, E18.5 – embryonic day 18.5, P0 – neonatal, P20 – postnatal day 20, P45 – postnatal day 45, 20w – postnatal 20 weeks.
senescence accelerated mice administered with antisense of PS1 (Fiorini et al., 2013). Development dependent analysis of these proteins showed increased pattern of interaction in postnatal mice up 248 to P45 and thereafter decrease in 20w. It is interesting to note that 249 energy metabolism increases postnatally up to young and further 250 decreases in adult (Goyal et al., 2014). 251 PS1 interacting partner clathrin heavy chain 1 is the major pro252 tein of the polyhedral coat of coated pits and vesicles involved in 253 protein trafficking. Previous study showed that PS1 was enriched in 254 the somatodendritic clathrin-coated vesicle membranes targeted 255 to neuronal vesicles where they may regulate vesicular function 256 (Efthimiopoulos et al., 1998). PS1 is also reported to play an essen257 tial role in modulating trafficking and metabolism of a selected 258 set of membrane and secretory proteins in neurons (Naruse et al., 259 1998). 260 We have also identified 11 interacting partners of PS2 involved 261 in various functions. The interacting proteins ICAM5 and catenin 262 1 are cell adhesion molecules, coronin-1A is a crucial component of 263 the cytoskeleton, tubulin -2A chain is major constituent of micro264 tubules, integrin 1 ␣ is cell cycle regulatory protein, PRP4 kinase and 265 tyrosin kinase are signaling molecules involved in cellular morpho266 genesis, arrestin  2 regulate G-protein coupled receptor signaling, 267 forkhead box protein K2 is a cellular transcription factor involved 268 in embryonic development. 269 The interacting proteins ICAM5 and catenin -1 are cell adhe270 sion molecules. Cell adhesion is a very important process in normal 271 development and cellular functions. In vitro and in vivo studies 272 have shown that ICAM-5 binds to PS2 (Yang, 2012). PS also play 273 important role in maturation and transport of cell cycle regulatory 274 protein integrin 1 (Zou et al., 2008). PS2 interacting proteins PRP4 275 kinase and tyrosin kinase are signaling molecules involved in cellu276 lar morphogenesis. Previous reports have shown that tyrosin kinase 277 is processed by PS2-dependent regulated intramembrane proteo278 lysis in epithelial cells (Foveau et al., 2009). PS2 interacting protein 279 -arrastin 2 is involved in regulation of G-protein coupled receptor 280 signaling (Ma and Pei, 2007). Previous study showed that arrestin 281 Q4 2  expression upregulated in Alzheimers disease (Thathiah et al., 282 2013). 283 In present study, male mice were used. In earlier report, Piscopo Q5 284 et al. (2013) showed altered expression of PS1 and PS2 at postnatal 285 stages of rat cerebral cortex development. The alteration in expres286 sion pattern was similar in male and female for PS1 as well as PS2 287 during development but the extent of alteration was variable. In 288 246 247
light of this information, we presume that male and female might not differ in presence or absence of any new interacting partner, but their degree of binding may vary during development. Interaction between proteins is dependent on the proteins structure or functional motifs. In present study, in-silico analysis of PS and its interacting proteins showed common type of secondary structure having alpha helical, extended beta strand and random coil. Motif scan analysis showed that PS and its interacting proteins have CK2, PKC phosphorylation and myristoylation motifs. Such similarity in secondary structure and motifs suggests their conformation based interaction in the mouse cerebral cortex is development-dependent.
5. Conclusion In conclusion, we showed that PS1 and PS2 interact with different proteins of mouse cerebral cortex to varying extent and suggest their conformation based interaction and involvement in various functions essential for the brain development. Further studies are warranted to confirm the specific roles of PS interacting proteins in brain development.
Disclosure statement The authors do not have any actual or potential conflicts of interest including any financial, personal or academic.
Acknowledgement We acknowledge DSHB for proving Notch-1 antibody. The work was supported by grants from the Department of Science and Tech- Q6 nology (No. SR/SO/HS-54/2008) and Department of Biotechnology (BT/PR3996/MED/97/57/2011), Government of India, to M.K.T.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.ijdevneu.2014.08.010.
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Contents lists available at ScienceDirect
International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu
Analysis of Presenilin 1 and 2 interacting proteins in mouse cerebral cortex during development
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Ashish Kumar, M.K. Thakur ∗ Biochemistry and Molecular Biology Laboratory, Brain Research Centre, Department of Zoology, Banaras Hindu University, Varanasi 221 005, India
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a r t i c l e
i n f o
a b s t r a c t
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Article history: Received 21 May 2014 Received in revised form 5 August 2014 Accepted 26 August 2014
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Keywords: Presenilin Notch-1 Neurogenesis Synaptogenesis Brain development
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1. Introduction
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In our previous report, we showed that Presenilin (PS)1 and 2 have differential expression profile from early embryonic stages till adulthood in mouse cerebral cortex, suggesting that both of these proteins are crucial for brain development. Genetic manipulation studies have also shown the involvement of PS1 in brain development, but PS2 remains largely unexplored. In order to understand how PS1 and 2 mediate developmental functions, we have investigated the interaction of PS1 and 2 with proteins of mouse cerebral cortex during development. Co-immunoprecipitation (Co-IP) combined with MALDI-MS/MS analysis revealed 12 interacting partners of PS1 and 11 partners of PS2. The interacting proteins were different for PS1 and 2, and involved in cell division, glycolysis, cell adhesion and protein trafficking. Densitometric analysis of protein bands visualized after SDS-PAGE separation of Co-IP proteins revealed variation in their amount and degree of interaction during different developmental stages of mice. Further, immunoblot based validation of PS1 interacting protein Notch-1 showed maximum interaction at embryonic day (E) 12.5, decline at E18.5, upregulation from postnatal day 0 (P0) to P20 and thereafter reduction at P45 and 20 weeks. In-silico analysis of PS and its interacting proteins indicated conformation based interaction through common type of secondary structures having alpha helical, extended beta strand and random coil, and CK2, PKC phosphorylation and myristoylation motifs. Taken together, our study showed that PS1 and PS2 interact to varying extent with different proteins of mouse cerebral cortex and suggests their interaction based on specific conformation and involvement in diverse functions essential for the brain development. © 2014 Published by Elsevier Ltd. on behalf of ISDN.
Presenilin (PS)1 and 2 are multi-pass transmembrane (TM) proteins of aspartyl protease family. Their primary sequence is evolutionarily conserved in eukaryotes, having homologues in organisms as distant as C. elegans (Levitan and Greenwald, 1995), Drosophila (Boulianne et al., 1997), and lower chordates (MartínezMir et al., 2001), suggesting functional conservation. Mammalian PS1 and 2 share extensive amino acid sequence identity and are synthesized as 50 kDa polypeptides (Hutton and Hardy, 1997). They span the membrane in a serpentine way and consist of nine TM domains. Their main domains are three stretches of hydrophilic domains comprising of N-terminal tail, C-terminal tail and a long loop between TM VI and TM VII (Tomita and Iwatsubo, 2013). Amino terminal and long loop domains of PS1 and PS2 are phosphorylated at serine residues (De Strooper et al., 1997).
∗ Corresponding author. Tel.: +91 542 2313958; fax: +91 542 2368174. E-mail addresses: mkt [email protected], [email protected] (M.K. Thakur).
PS1 and PS2 have been implicated in several brain functions (Zhang et al., 2013) through their interaction with amyloid precursor protein, Notch-1, -catenine, calsenilin, cadherines and others Q2 (Chen and Schubert, 2002). With respect to brain development, PS1 knockout mice showed developmental deformities including cortical dysplasia and died before birth (Wen et al., 2005). Conditional knockouts of PS1 have shown its implication in several processes such as neuronal migration, cortical lamination, neurogenesis, neuronal differentiation (Wines-Samuelson et al., 2005), synaptogenesis (Uchihara et al., 2006), somitogenesis, gliogenesis Q3 (Saura, 2010), and growth (Nakajima et al., 2009). On the other hand, knockout of PS2 survived well without any defect in the brain morphology (Herreman et al., 1999), indicating that the protease might not contribute to brain developmental processes directly and may serve to compensate PS1 functions postnatally (Wang et al., 2003). In our previous report, we showed that PS1 and PS2 expression varied significantly in the cerebral cortex during critical time of development. These findings indicated that PS1 and 2 might have independent involvement during different developmental
http://dx.doi.org/10.1016/j.ijdevneu.2014.08.010 0736-5748/© 2014 Published by Elsevier Ltd. on behalf of ISDN.
Please cite this article in press as: Kumar, A., Thakur, M.K., Analysis of Presenilin 1 and 2 interacting proteins in mouse cerebral cortex during development. Int. J. Dev. Neurosci. (2014), http://dx.doi.org/10.1016/j.ijdevneu.2014.08.010
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stages. In order to understand such involvement, we have analyzed interacting partners of PS1 and 2 during brain development. We have co-immunoprecipitated (Co-IP) PS1 and PS2 specific complexes during different developmental stages, and the resulting proteins were identified by MALDI-MS/MS. Further, the interaction of Notch-1 in Co-IP proteins was validated with PS1 at different developmental stages. In silico analysis for secondary structure prediction of putative interacting proteins was done by bioinformatics GOR-4 and specific motifs were identified by Motif-scan.
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2. Materials and methods
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2.1. Animals
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Swiss albino strain mice were inbred and maintained at 25 ± 2 ◦ C with 12 h light and 12 h dark schedule with ad libitum access to standard mice feed and drinking water in the animal house of the Department of Zoology, Banaras Hindu University, Varanasi, India. They were used according to guidelines of the institutional animal ethical committee, Banaras Hindu University, Varanasi, India. Male mice (n = 54) were used for the present study and the detailed information is provided in the supplementary section. To analyze the interacting proteins of PS during development, mice of embryonic day (E) 12.5, E18.5, postnatal day (P) 0, P20, P45 and 20 weeks (w) were used. The mice were decapitated and embryos were collected by flushing out from uterus for prenatal ages. The telencephalon from E12.5 and the cerebral cortex from other developmental stages were dissected out.
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2.2. Preparation of protein extract
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Cytosolic protein extract was prepared from the mouse cerebral cortex of different developmental ages. Briefly, 10% homogenate was prepared in RIPA buffer [50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% NP-40 and 0.5% sodium deoxycholate] with protease inhibitors (Sigma–Aldrich, USA) at 4 ◦ C (Kim et al., 2012; Jang et al., 2011). The homogenate was centrifuged at 1000 × g for 10 min at 4 ◦ C. The postnuclear supernatant was collected in separate microfuge tube and stored at −70 ◦ C. The amount of protein in the preparation was estimated by Bradford (1976) method.
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For Co-IP, the protein (100 g) was incubated with protein A Sepharose beads at 4 ◦ C for 2 h to avoid nonspecific binding, and then centrifuged at 3000 × g for 10 min. The precleared supernatant was incubated overnight with 2 g of anti-PS1 antibody (Alpha Diagnostics International, USA) or anti-PS2 antibody (Alpha Diagnostics International, USA) in 500 l of IP buffer (50 mM Hepes, pH 7.4, 50 mM NaCl, 0.1% Triton X-100, 10% glycerol, 1 mM PMSF). IgG was used as a negative control. Next day, protein A Sepharose beads were added to the reaction mix at 4 ◦ C for 4 h. Thereafter, the beads containing bound proteins were precipitated by centrifugation, washed three times in Co-IP buffer. Co-immunoprecipitate was denatured by boiling in SDS-sample buffer, resolved on 10% Tris–glycine SDS-PAGE and analyzed by silver staining. To analyze Co-IP bands, the signal intensity of each stained band was calculated as Integrated Density Value (IDV) by spot densitometry tool AlphaEaseFC software (Alpha Innotech Corp., USA).
2.4. Immunoblotting
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Initially, the input level of both Notch-1 full length (Notch-1 FL) and PS1 was determined by their protein expression during development. Briefly, 20 g cytosolic protein from each developmental stages was denatured, resolved by 10% Tris–glycine SDS-PAGE, transferred onto PVDF membrane. The membrane was blocked in 5% (w/v) nonfat milk prepared in 1× PBS for 2 h and incubated overnight with rabbit anti-Notch-1 antibody in 1:1000 dilutions (Developmental Studies Hybridoma Bank, USA). After washing twice in 0.1% PBST, the membrane was incubated with horse-radish peroxidase conjugated goat-anti-rabbit secondary antibody in 1:2000 dilutions (Bangalore Genei, India), washed twice in 0.1% PBST and detected by ECL. Immunoblotting of input PS1 was done as mentioned previously (Kumar and Thakur, 2012). Internal control -actin was detected by reprobing the membrane with antimouse -actin HRP conjugate, 1:10,000 dilutions (Sigma–Aldrich, USA). In order to analyze interaction level of Notch-1 with PS1 protein, equal amount of PS1 Co-IP protein from different developmental stages was subjected to immunblotting of Notch-1 Intracellular domain (Notch-1 NICD). The signal intensities for input Notch-1 FL, input PS1 and Co-IP Notch-1 NICD fragment were normalized against signal intensity of -actin and represented as a histogram with mean of three values calculated as RDV (IDV of Notch-1 FL/-actin, PS1/-actin and Notch-1 NICD/actin).
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The protein bands were excised, minced into 1 mm3 pieces and transferred into a sterile microcentrifuge tube. It was washed three times with 500 l of wash solution (50% acetonitrile, 50 mM ammonium bicarbonate) and incubated at room temperature for 15 min with gentle agitation (vortex mixer on lowest setting). Further, the gel was dehydrated in 100% acetonitrile for 5 min. Acetonitrile was removed and the gel was dried at room temperature for 10–20 min. It was rehydrated in 150 l reduction solution (10 mM DTT, 100 mM ammonium bicarbonate) for 30 min at 56 ◦ C. The reduction solution was discarded and added 100 l alkylation solution (50 mM iodoacetamide, 100 mM ammonium bicarbonate) and incubated for 30 min in the dark at room temperature. The alkylation solution was discarded with a pipette, added 500 l of wash solution and incubated at room temperature for 15 min with gentle agitation. The gel was dehydrated in 100 l 100% acetonitrile for 5 min. Further, acetonitrile was discarded and gel dried at room temperature. The gel was rehydrated with a minimal volume of protease digestion solution (20 g/ml in 50 mM ammonium bicarbonate solution) and digested overnight at 37 ◦ C. Next day, it was centrifuged at 12,000 × g for 30 s. The supernatant was transferred to a sterile centrifuge tube. Further, 25–50 l of extraction solution (60% acetonitrile, 0.1% TFA) was added to gel pieces, agitated gently by vortexing at lowest setting for 10 min and it was centrifuged at 12,000 × g for 30 s. The extracted peptides were pooled and dried to near dryness by centrifugal evaporation. The peptides were resuspended in 5 l of resuspension solution (50% acetonitrile, 0.1% TFA) and agitated gently on a vortex at lowest setting.
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2.6. MALDI-TOF-MS
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The samples were spotted (1 l) on MALDI target plate [MTP 384 ground steel (Bruker Daltonics, Germany)] followed by 0.5 l of alpha-cyano-4-hydroxycinnamic acid matrix [10 mg/mL in 50% acetonitrile, 0.1% TFA (Sigma–Aldrich, USA)]. The peptide spectra was acquired in an AUTOFLEX speed MALDI TOF/TOF instrument (Bruker Daltonics, Germany) having Nd:YAG smart Laser beam of 335 nm wavelength. External calibration was done with peptide calibration standard supplied by Bruker, with masses ranging from 1046 to 3147 Da. The obtained spectra were acquired using Flex Control version 3.3 software in reflectron ion mode with an average of 2000 laser shots at mass detection range between 700 and 4000 m/z. The most three abundant peaks were subjected to further fragmentation using LIFT method. The data were analyzed using Flex Analysis software version 3.3 (Bruker Daltonics, Germany) and searched in MASCOT web server (Matrix Science; http://www.matrixscience.com) using Biotools version 3.2 software (Bruker Daltonics, Germany).
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2.7. In silico analysis
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Secondary structure of the predicted proteins obtained from MALDIMS analysis were analyzed by GOR4 (http://npsa-pbil.ibcp.fr/cgi-bin/ npsa automat.pl?page=npsa gor4.html, Combet et al., 2000). In order to identify the motifs in the predicted proteins, MOTIF-SCAN (http://myhits. isb-sib.ch/cgi-bin/motif scan) was done.
159
2.8. Statistical analysis
164
Each experiment was repeated three times (n = 3 × 3 = 9 mice/group). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by post hoc test of Student–Newman–Keuls method through Jindel Scientific SigmaPlot for Windows (standard version 2.0). Values were reported as mean ± SEM and pvalues <0.05 were considered as significant.
3. Results 3.1. Co-IP of PS interacting proteins in the cerebral cortex of mice during development With the optimized conditions for PS1 (Fig. 1A) and PS2 (Fig. 2A), Co-IP analysis showed that interacting proteins varied differentially both for PS1 (Fig. 1B) and PS2 (Fig. 2B) with respect to number, amount and degree of interaction in the cerebral cortex of mice during development. Out of these proteins, 12 partners of PS1 (Fig. 1C) and 11 of PS2 (Fig. 2C) showed significant alterations in their level of interaction in prenatal and postnatal stages as compared to P0. Predicted molecular weight of PS1 interacting partners was in the range of 30–342 kDa while the range was 24–193 kDa for PS2. One way ANOVA analysis showed significant F values for all the proteins (details of F values included in supplementary section).
Please cite this article in press as: Kumar, A., Thakur, M.K., Analysis of Presenilin 1 and 2 interacting proteins in mouse cerebral cortex during development. Int. J. Dev. Neurosci. (2014), http://dx.doi.org/10.1016/j.ijdevneu.2014.08.010
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165 166 167 168 169
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3.2. MALDI/MS/MS analysis of PS interacting proteins MALDI/MS/MS analysis identified synaptotagmin-4, unnamed protein, aldolase A, creatine kinase B-type, alpha enolase,
3
elongation factor 1-␣, tubulin -2A chain, pyruvate kinase isoform M1, GRIN1, Notch-1, clathrin heavy chain 1 and unnamed protein as PS1 interacting partners. Similarly, ICAM5, PRP4 kinase, catenin -1, tyrosine-protein kinase, arrestin  2, tubulin -2A
Fig. 1. Co-immunoprecipitation (Co-IP) analysis of PS1 interacting proteins in the cerebral cortex of mice during development. (A) 10% silver stained SDS-PAGE showing optimization of Co-IP for PS1 interacting proteins [Input protein (Input, Lane-1), Supernatant protein (Supt, Lane-2), final wash of Co-IP (wash, Lane-3), Co-IP (Lane-4), antibody (ab, Lane-5) and Marker (M, Lane-6)], (B) Co-IP proteins of different developmental stages [bold arrows indicate specific bands and N denoted dotted lines representing nonspecific bands]. (C) Interaction level of Co-IP proteins plotted by densitometric analysis (integrated Density Value-IDV) of each band resolved on the gel. Asterisk (*) denotes significant differences (p < 0.05) between the age groups as compared to P0. E12.5 – embryonic day 12.5, E18.5 – embryonic day 18.5, P0 – neonatal, P20 – postnatal day 20, P45 – postnatal day 45, 20w – postnatal 20 weeks.
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Fig. 2. Co-IP analysis of PS2 interacting proteins in the cerebral cortex of mice during development. (A) 10% silver stained SDS-PAGE showing optimization of Co-IP for PS2 interacting proteins [Input protein (Input, Lane-1), Supernatant protein (Supt, Lane-2), final wash of Co-IP (wash, Lane-3), Co-IP (Lane-4), antibody (ab, Lane-5) and Marker (M, Lane-6)], (B) Co-IP proteins of different developmental stages [bold arrows indicate specific bands and N denoted dotted lines representing non-specific bands]. (C) Interaction level of Co-IP proteins plotted by densitometric analysis (integrated Density Value-IDV) of each band resolved on the gel. Asterisk (*) denotes significant differences (p < 0.05) between the age groups as compared to P0. E12.5 – embryonic day 12.5, E18.5 – embryonic day 18.5, P0 – neonatal, P20 – postnatal day 20, P45 – postnatal day 45, 20w – postnatal 20 weeks.
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Table 1 MALDI-MS/MS analysis of imunoprecipitated PS interacting proteins. GI number
193
Protein
Function
Unnamed protein Clathrin heavy chain 1 Notch-1
Not known Major protein of the polyhedral coat of coated pits and vesicles. Receptor for membrane-bound ligands to regulate cell-fate determination. Cerebral cortex development. Involved in glutamate neurotransmission and calcium channel activity Glycolytic enzyme, multiple nonglycolytic functions with possible diverse implications Major constituent of microtubules. Promotes the GTP-dependent binding of amino- acyl-tRNA to the A-site of ribosomes during protein biosynthesis. Multifunctional enzyme that, as well as its role in glycolysis, plays a part in various processes such as growth control, hypoxia tolerance and allergic responses Play a central role in energy transduction in tissues with large, fluctuating energy demands. Role in glycolysis and gluconeo-genesis. May also function as scaffolding protein Not known Mediate calcium-dependent regulation of membrane trafficking.
gi|5901688
4
80
GRIN1
gi|359807367
5
58
Pyruvate kinase isoform M1
gi|4507729 gi|407261700
6 7
52 48
Tubulin -2A chain Elongation factor 1-␣
gi|70794816
8
46
Alpha enolase
gi|10946574
9
42
Creatine kinase B (brain)-type
gi|7548322
10
39
Aldolase A
gi|74192167 gi|60218904
11 12
34 30
Unnamed protein Synaptotagmin-4
PS2 interacting proteins 1 gi|148670188 gi|20330556 2
193 117
3 4
83 75
Catenin -1 Tyrosine-protein kinase
5 6 7 8 9 10 11
65 52 45 34 32 28 24
Arrestin  2 Tubulin -2A chain Integrin ␣-6 mKIAA0303 protein Forkhead box protein K2 Coronin-1A Unnamed protein
gi|123234448 gi|4507729 gi|148695149 gi|37359892 gi|238018071 gi|6753492 gi|309267211
192
Mw (kDa) 342 193 110
gi|26337435 gi|31560493
191
Band no.
PS1 interacting proteins gi|26329341 1 gi|51491845 2 gi|224967064 3
ICAM5 PRP4 kinase
chain, integrin ␣-6, mKIAA0303 protein, forkhead box protein K2, coronin-1A, unnamed protein were identified as interacting partners of PS2 (Table 1).
Cell adhesion molecule Serine/arginine-rich protein-specific kinase regarded as a CDK-like kinase Cell adhesion molecule Tyrosine kinases catalyze the phosphorylation of tyrosine residues in proteins Regulate G-protein coupled receptor signaling Major constituent of microtubules. Involved in cell signaling and the regulation of cell cycle. Function not known, predicted in neuronal migration Cellular transcription factor ILF-1 involved in embryonic development Involved in calcium ion transport Not known.
and its interacting proteins showed high number of CK2, PKC phosphorylation and myristoylation motifs (Table 3).
4. Discussion 194 195
196 197 198 199 200 201 202 203 204 205 206 207 208 209
210
211 212 213 214 215 216
3.3. Analysis of Notch-1 interaction with PS1 in the cerebral cortex of mice during development To analyze the data, values from neonatal (P0) mice were considered as 100%. Western blot analysis of input Notch-1 FL showed significant alteration at E18.5, P45 and 20w as compared to P0 (E12.5 – 97%, E18.5 – 173%, P20 – 112%, P45 – 200% and 20w – 309%) (Fig. 3). One way ANOVA of PS1 showed significant F value [F(5, 12) = 56.2, p < 0.05]. Analysis PS1 input protein showed significant alteration (E12.5 – 135%, E18.5 – 75%, P20 – 130%, P45 – 156%, 20w – 56%) (Fig. 3). One way ANOVA of PS1 showed significant F value [F(5, 12) = 48.4, p < 0.05]. Notch-1 interaction with PS1, Co-IP followed by western blot analysis showed significant alteration in Notch-1 level in prenatal (E12.5 – 144% and E18.5 – 50%) and postnatal stages (P20 – 115%, P45 – 77% and 20w – 80%) (Fig. 3). One way ANOVA showed significant F value [F(5, 12) = 65.895, p < 0.05].
3.4. In silico analysis of PS interacting proteins Secondary structure prediction analysis showed that PS and its interacting proteins have common type of secondary structure, i.e. alpha helical (Hh), extended beta strand (Ee) and random coil (Cc) but other secondary structures, i.e. 310 helix (Gg), Pi helix (Ii), Beta bridge (Bb), Beta turn (Tt), Bend region (Ss), Ambigous states (?) were not present (Table 2). Motif scan analysis of PS
We have investigated the interaction pattern of PS1 and PS2 with proteins of mouse cerebral cortex at prenatal, neonatal and postnatal stages, covering the wide range of brain developmental processes including neurogenesis, neuronal processing and differentiation. In present study, we have identified 12 interacting partners of PS1 involved in diverse functions. Notch-1 and GRIN1 are involved in brain development, pyruvate kinase isoform M1, alpha enolase, creatin kinase B-type and aldolase A in energy metabolism, clathrin heavy chain 1 is the major protein of the polyhedral coat of coated pits and vesicles involved in protein trafficking, synaptotagmin-4 mediate calcium-dependent regulation of membrane trafficking, elongation factor 1-␣ promotes the GTPdependent binding of aminoacyl-tRNA to the A-site of ribosomes during protein biosynthesis. PS1 interacting partner Notch-1 protein is involved in early neurodevelopmental processes including neurogenesis, somitogenesis and vasculogenesis. In this report, we observed that Notch-1 interaction pattern is similar to the expression profile of PS1 during development up to P20 and thereafter show decreased interaction. Previous study using Co-IP also showed interaction of Notch-1 with PS1 in HEK293 and Drosophila cells (Ray et al., 1999). PS1 interacting proteins pyruvate kinase isoform M1, alpha enolase, creatin kinase B-type and aldolase A are involved in energy metabolism. Our study is partially supported by previous report that showed decrease in the brain oxidative markers including pyruvate kinase isoform M1 and enolase in aged
Please cite this article in press as: Kumar, A., Thakur, M.K., Analysis of Presenilin 1 and 2 interacting proteins in mouse cerebral cortex during development. Int. J. Dev. Neurosci. (2014), http://dx.doi.org/10.1016/j.ijdevneu.2014.08.010
217 218
219
220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245
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6
Hh
Gg
Ii
Bb
Ee
Tt
Ss
Cc
(?)
Others
PS1 and its interacting proteins Presenilin 1, Sequence: 467 Unnamed protein, Sequence: 2473 Clathrin heavy chain 1, Sequence: 1675 Notch 1, Sequence: 2531 GRIN1, Sequence: 827 Pyruvate kinase isoform M1, Sequence: 531 Tubuline  2A chain, Sequence: 445 Elongation factor 1-␣, Sequence: 415 Alpha enolase, Sequence: 434 Creatine kinase B-type, Sequence: 381 Aldolase A, Sequence: 364 Unnamed protein, Sequence: 218 Synaptotagmin 4, Sequence: 425
179 is 38.33% 1729 is 69.92% 801 is 47.82% 320 is 12.64% 130 is 15.72% 233 is 43.88% 148 is 33.26% 117 is 28.19% 194 is 44.70% 114 is 29.92% 126 is 34.62% 85 is 38.99% 126 is 29.65%
0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00%
0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00%
0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00%
98 is 20.99% 121 is 4.89% 222 is 13.25% 472 is 18.65% 134 is 16.20% 100 is 18.83% 81 is 18.20% 112 is 26.99% 53 is 12.21% 63 is 16.54% 102 is 28.02% 34 is 15.60% 71 is 16.71%
0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00%
0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00%
190 is 40.69% 623 is 25.19% 652 is 38.93% 1739 is 68.71% 563 is 68.08% 198 is 37.29% 216 is 48.54% 186 is 44.82% 187 is 43.09% 204 is 53.54% 136 is 37.36% 99 is 45.41% 228 is 53.65%
0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00%
0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00%
PS2 and its interacting proteins Presenilin 2, Sequence: 448 ICAM5, Sequence: 917 PRP4 kinase, Sequence: 1007 Catenin -1, Sequence: Tyrosine-protein kinase, Sequence: 937 Arrestin  2, Sequence: 406 Tubulin -2A chain, Sequence: 445 Integrin ␣ 6, Sequence:1073 mKIAA0303 protein, Sequence: 950 Forkhead box protein K2, Sequence: 651 Coronin 1A, Sequence: 461 Unnamed protein, Sequence: 325
125 is 27.90% 189 is 20.61% 445 is 44.19% 399 is 51.09% 167 is 17.82% 107 is 26.35% 148 is 33.26% 253 is 23.58% 169 is 17.79% 133 is 20.43% 80 is 17.35% 134 is 41.23%
0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00%
0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00%
0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00%
108 is 24.11% 210 is 22.90% 99 is 9.83% 80 is 10.24% 204 is 21.77% 78 is 19.21% 81 is 18.20% 223 is 20.78% 95 is 10.00% 110 is 16.90% 119 is 25.81% 48 is 14.77%
0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00%
0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00%
215 is 47.99% 518 is 56.49% 463 is 45.98% 302 is 38.67% 566 is 60.41% 221 is 54.43% 216 is 48.54% 597 is 55.64% 686 is 72.21% 408 is 62.67% 262 is 56.83% 143 is 44.00%
0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00%
0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00% 0 is 0.00%
* (Hh)- Alpha helix, (Gg)- 310 helix, (Ii)- Pi helix, (Bb)- Beta bridge, (Ee)- Extended strand, (Tt)- Beta turn, (Ss)- Bend region, (Cc)- Random coil, (?)- Ambigous states
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Table 2 Secondary structure analysis of PS interacting proteins.
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PS1 and its interacting proteins Protein
AMIDATION
GLYCOSYL
ATP GTP A
cAMP PHOSPHO SITE
CK2 PHOSPHO SITE
LEUCINE ZIPPER
MYRISTYL
PKC PHOSPHO SITE
PRENYLATION
TYR PHOSPHO SITE
3 17 9 9 3 1 3 3 – 2 –
– 1 – – – – – 1 – – – – –
– 2 2 2 1 – 2 – – – – – –
7 64 14 14 17 5 7 6 3 4 3 4 8
– 10 – – – – – – – – – – –
4 12 9 9 20 8 9 6 5 8 5 2 4
4 60 10 15 35 10 5 8 4 9 4 3 10
– 1 – – – – – – – – – – –
– 1 – – 3 1 – – – – – – –
2
Protein
AMIDATION
GLYCOSYL
ATP GTP A
cAMP PHOSPHO SITE
CK2 PHOSPHO SITE
LEUCINE ZIPPER
MYRISTYL
PKC PHOSPHO SITE
TYR PHOSPHO SITE
RGD
PS2 ICAM5 PRP4 kinase Catenin -1 Tyrosine-protein kinase Arrestin  2 Tubulin -2A chain Integrin ␣ 6 mKIAA0303 protein Forkhead box protein K2 Coronin 1A Unnamed protein
– – 1 5 1 1 – – 2 – – –
1 13 6 – 7 1 3 8 2 2 – 3
– – – – – – – – – – – –
– 1 13 2 2 2 2 3 3 – 2 –
8 12 17 9 11 2 7 21 21 4 8 4
– – – – – – – – – – 1 –
2 25 3 15 9 2 9 15 7 9 8 2
5 19 27 11 9 10 5 11 27 8 10 3
– – – – – – – – – – – –
– – – – – – – – – – – –
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Presenilin 1 – – Unnamed protein – Clathrin heavy chain 1 Notch-1 – GRIN1 – Pyruvate kinase M1 – – Tubuline -2A chain Elongation factor 1-␣ – – Alpha enolase – Creatine kinase B-type 1 Aldolase A Unnamed protein – Synaptotagmin 4 – PS2 and its interacting proteins
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Table 3 Motif scan analysis of PS interacting proteins.
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Fig. 3. Analysis of Notch-1 interaction with PS1 in the cerebral cortex of mice during development. Western blotting showing Co-IP of Notch-1 Intracellular domain (NICD) fragment (110 kDa), Input Notch-1 (280 kDa) and input PS1 (32 kDa) in the cerebral cortex of mice during development. Histogram represents Relative Density Value, RDV (IDV of Co-IP Notch-1 NICD/-actin, Input Notch-1/-actin and PS1/-actin) from three independent experiments (±) SEM. Asterisk (*) denotes significant differences (p < 0.05) between the age groups as compared to P0. E12.5 – embryonic day 12.5, E18.5 – embryonic day 18.5, P0 – neonatal, P20 – postnatal day 20, P45 – postnatal day 45, 20w – postnatal 20 weeks.
senescence accelerated mice administered with antisense of PS1 (Fiorini et al., 2013). Development dependent analysis of these proteins showed increased pattern of interaction in postnatal mice up 248 to P45 and thereafter decrease in 20w. It is interesting to note that 249 energy metabolism increases postnatally up to young and further 250 decreases in adult (Goyal et al., 2014). 251 PS1 interacting partner clathrin heavy chain 1 is the major pro252 tein of the polyhedral coat of coated pits and vesicles involved in 253 protein trafficking. Previous study showed that PS1 was enriched in 254 the somatodendritic clathrin-coated vesicle membranes targeted 255 to neuronal vesicles where they may regulate vesicular function 256 (Efthimiopoulos et al., 1998). PS1 is also reported to play an essen257 tial role in modulating trafficking and metabolism of a selected 258 set of membrane and secretory proteins in neurons (Naruse et al., 259 1998). 260 We have also identified 11 interacting partners of PS2 involved 261 in various functions. The interacting proteins ICAM5 and catenin 262 1 are cell adhesion molecules, coronin-1A is a crucial component of 263 the cytoskeleton, tubulin -2A chain is major constituent of micro264 tubules, integrin 1 ␣ is cell cycle regulatory protein, PRP4 kinase and 265 tyrosin kinase are signaling molecules involved in cellular morpho266 genesis, arrestin  2 regulate G-protein coupled receptor signaling, 267 forkhead box protein K2 is a cellular transcription factor involved 268 in embryonic development. 269 The interacting proteins ICAM5 and catenin -1 are cell adhe270 sion molecules. Cell adhesion is a very important process in normal 271 development and cellular functions. In vitro and in vivo studies 272 have shown that ICAM-5 binds to PS2 (Yang, 2012). PS also play 273 important role in maturation and transport of cell cycle regulatory 274 protein integrin 1 (Zou et al., 2008). PS2 interacting proteins PRP4 275 kinase and tyrosin kinase are signaling molecules involved in cellu276 lar morphogenesis. Previous reports have shown that tyrosin kinase 277 is processed by PS2-dependent regulated intramembrane proteo278 lysis in epithelial cells (Foveau et al., 2009). PS2 interacting protein 279 -arrastin 2 is involved in regulation of G-protein coupled receptor 280 signaling (Ma and Pei, 2007). Previous study showed that arrestin 281 Q4 2  expression upregulated in Alzheimers disease (Thathiah et al., 282 2013). 283 In present study, male mice were used. In earlier report, Piscopo Q5 284 et al. (2013) showed altered expression of PS1 and PS2 at postnatal 285 stages of rat cerebral cortex development. The alteration in expres286 sion pattern was similar in male and female for PS1 as well as PS2 287 during development but the extent of alteration was variable. In 288 246 247
light of this information, we presume that male and female might not differ in presence or absence of any new interacting partner, but their degree of binding may vary during development. Interaction between proteins is dependent on the proteins structure or functional motifs. In present study, in-silico analysis of PS and its interacting proteins showed common type of secondary structure having alpha helical, extended beta strand and random coil. Motif scan analysis showed that PS and its interacting proteins have CK2, PKC phosphorylation and myristoylation motifs. Such similarity in secondary structure and motifs suggests their conformation based interaction in the mouse cerebral cortex is development-dependent.
5. Conclusion In conclusion, we showed that PS1 and PS2 interact with different proteins of mouse cerebral cortex to varying extent and suggest their conformation based interaction and involvement in various functions essential for the brain development. Further studies are warranted to confirm the specific roles of PS interacting proteins in brain development.
Disclosure statement The authors do not have any actual or potential conflicts of interest including any financial, personal or academic.
Acknowledgement We acknowledge DSHB for proving Notch-1 antibody. The work was supported by grants from the Department of Science and Tech- Q6 nology (No. SR/SO/HS-54/2008) and Department of Biotechnology (BT/PR3996/MED/97/57/2011), Government of India, to M.K.T.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.ijdevneu.2014.08.010.
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301
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Please cite this article in press as: Kumar, A., Thakur, M.K., Analysis of Presenilin 1 and 2 interacting proteins in mouse cerebral cortex during development. Int. J. Dev. Neurosci. (2014), http://dx.doi.org/10.1016/j.ijdevneu.2014.08.010
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