A Central Nervous System-Dependent Intron-Embedded Gene Encodes a Novel Murine Fyn Binding Protein

The interplay between the nervous and immune systems is gradually being unraveled. We previously reported in the mouse the novel soluble immune system factor ISRAA, whose activation in the spleen is central nervous system-dependent. We also showed that ISRAA plays a role in modulating anti-infection immunity. Herein, we report the genomic description of the israa locus, along with some insights into the structure-function relationship of the protein. Our findings revealed that israa is nested within intron 6 of the mouse zmiz1 gene. Protein sequence analysis revealed a typical SH2 binding motif (Y102TEV), with Fyn being the most likely binding partner. Docking simulation showed a favorable conformation for the ISRAA-Fyn complex, with a specific binding mode for the binding of the YTEV motif to the SH2 domain. Experimental studies showed that in vitro, recombinant ISRAA is phosphorylated by Fyn at tyrosine 102. Cell transfection and pull-down experiments revealed Fyn as a binding partner of ISRAA in the EL4 mouse T-cell line. Indeed, we demonstrated that ISRAA downregulates T-cell activation and the phosphorylation of an activation tyrosine (Y416) of Src-family kinases in mouse splenocytes. Our observations highlight ISRAA as a novel Fyn binding protein that is likely to be involved in a signaling pathway driven by the nervous system.


Introduction
The nervous and immune systems have long been considered to be separate compartments that fulfill different functions, but recent studies showed the existence of a mutual interaction between the two systems in both physiological and pathological conditions. Indeed, cytokines released by immune cells are able to signal to the brain the occurrence of infections [1,2] by directly accessing the CNS [3] or by stimulating afferent neurons [4]. In response to environmental changes, the brain can modulate immune system functions and maintain homeostasis through a network of neurotransmitters and specific receptors expressed on immune cells [5]. The modulation of immune functions by the CNS occurs in different ways: the autonomic nervous system, the catecholaminergic pathway, or neuropeptide and hormone release [6]. In the spleen, brain immunoregulatory actions can be mediated by the autonomic nervous system through sympathetic and vagus nerve innervations. Vagus nerves excitation induces neuroephinepherine (NE) release from splenic nerves. NE binds to adrenergic receptors on splenic T cells, stimulates acetylcholine production [7] and acts as an immune suppressor. This phenomenon is known as the inflammatory reflex, in which cytokine production is reduced following the binding of acetylcholine to α7 nAChR receptors on macrophages in the spleen red-pulp and marginal zone [8,9]. These observations pointed toward spleen innervations as a key step in the regulation of immune system activity. As reported by our group in 2008 [10], we used a differential display approach to demonstrate that a novel 2.1 Kb mouse gene (israa), which encodes a 125 amino acid protein (ISRAA), is overexpressed in splenocytes less than one minute after Trypanosoma brucei parasite inoculation in mice. This rapid induction of israa expression in the spleen occurred seconds after the subcutaneous inoculation of the parasite. This induction is significantly inhibited by surgical denervation of the spleen. ISRAA was shown to be secreted by splenocytes and to modulate the immune response in a dosedependent manner [10]. These observations suggested that israa could be involved in the nervous system's immune-regulatory function. In an attempt to further characterize israa, we performed in silico structure to function analysis, followed by experimental functional characterization of the protein. The gene (GenBank: EU552928) was mapped to chromosome 14. To investigate the function(s) of this gene, we first analyzed israa's gene structure and genomic environment in the mouse genome. Then, we used a computational approach to gain insights into ISRAA's structure-function(s) relationship. Protein motif analysis revealed the presence of a typical linear SH2 binding motif exposed on ISRAA's surface. Experimental work provided evidence that ISRAA is likely to be a novel binding protein of Fyn kinase and is likely involved in the regulation of early T-cell activation and signaling.

Genomic Analysis
Ensembl Blast was used to map the location of israa in the mouse genome. The NCBI Blast program was used to perform a similarity search and to investigate expression data. Complete and annotated mRNA sequences of the zmiz-1 gene in mouse (REFSEQ accession NM_183208.4)) were downloaded in GenBank format [11] from the NCBI database. The Artemis [12] software available from the Wellcome Trust Sanger Institute website was used to visualize and annotate sequences. Regulatory elements were analyzed via the TIS Miner servers.

Protein Sequence Analysis
Signal peptide, trans membrane topology and protein localization predictions were performed with the SignalP 4.1 server [13], TMHMM server v2.0, SecretomeP [14] and the Phobius server [15] using default parameters for eukaryotic systems. The prediction of putative mitochondrial and ER targeting sequences was performed using the Predotar server, and peroxisomal targeting signal prediction was performed using PTS1 predictor [16]. Leucine-rich nuclear export signal prediction was performed using the NetNES 1.1 server [17]. Putative serine/tyrosine and threonine phosphorylation were predicted using the NetPhos 2.0 server [18], and kinase specificity was predicted using the NetPhosK1.0 server [19]. The conserved domain database (CDD) [20] and SMART [21] servers were used to identify and annotate putative conserved domains. Short protein sequence motifs were analyzed using the Scansite 3.0 server [22] at high stringency.

Protein Modeling
ISRAA was submitted to the full-chain structure prediction server ROBETTA [23], and four models were generated. The stereochemical quality and accuracy of the predicted models were evaluated with PROCHECK [24] via Ramachandran plot analysis [25]. The best model was selected based on the overall G-factor and the number of residues in the core, allowed, generously allowed and disallowed regions. The selected model was further analyzed using Verify 3D [26]. ProSA [27] was used to display Z-scores. Models were visualized manually using Pymol [28].

Protein-Protein Docking
Protein models of ISRAA and the crystal structure of the murine Fyn SH2-SH3 domains (PDB id: 3UF4) were refined with the GROMOS 96 implementation of Swiss-PDB Viewer [29]. Protein-protein docking between the Fyn SH2-SH3 domains as a receptor and the ISRAA model as a ligand was performed using the Hex 8.0.0 software [30] in unbound mode. Docking was performed 10 times, and the solutions were generated and clustered. Out of 1,280 clusters, the best conformation was selected manually based on energy and previously described binding modes for SH2 domains. The following parameters were used in the Docking controls of Hex 8.0.0: Correlation type-Shape only; FFT mode-3D; Grid dimension-0.6; Solutions-2,000; Receptor range-180; Step size-7.5; Ligand range-180; Step size-7.5; Twist range-360; Step size-5.5; Distance range-40; Scan step-0.8; Substeps-0; Steric scan-18; and Final search-25. In contrast, the parameters used in clustering controls to obtain the best results are as follows: Max Clusters-500; Sort solutions by-Cluster; Display clusters-Best; Cluster window-200; RMS threshold-3.0; and Bumps threshold-3. Complexes were visualized with Pymol [26].

Recombinant Protein Production in Escherichia coli and Purification
The recombinant ISRAA protein (rISRAA) and a mutated form (Y102A) were expressed as Nter 6-His-tagged proteins in a Bl21 (DE3 pLysS) E. coli strain. Briefly, coding sequences were synthesized by GeneCust (GeneCust, Dudelange, Luxemburg) and cloned into the pET-28a + system (Novagen, Madison, WI, USA). Competent cells were transformed with recombinant plasmids using the CaCl 2 method [31]. The selected clones were grown overnight in LB broth at 37°C. Subcultures were grown for 4 h at 37°C then induced for 16 h with 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) at 25°C. Purification of the fusion protein was performed via affinity chromatography using imidazole gradient elution over a Ni-NTA resin according to the manufacturer's recommendations (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA)). Purity was demonstrated via 15% polyacrylamide gel electrophoresis (PAGE) and silver staining. The selected fractions were desalted via gel filtration chromatography over PD-10 columns according to the manufacturer's recommendations (GE Healthcare, Buckinghamshire, UK). Endotoxins were removed using the Pierce High-Capacity Endotoxin Removal Spin Column (Thermo Fisher Scientific, MA, USA), according to the manufacturer's recommendations. Protein concentrations were determined using the Bradford method. Protein purity was assessed using 12% SDS-PAGE and silver staining.

Tyrosine Kinase Activity Assay
Fyn Tyrosine kinase (TK) activity was determined using the Universal Tyrosine Kinase Activity Kit (Takara Bio, Shiga, Japan) according to the manufacturer's recommendations. Briefly, recombinant active mouse Fyn (Life Technologies, Carlsbad, CA, USA) was incubated at various concentrations (0.5 to 5 μg/ml) for 20 min at room temperature on a coated universal substrate tyrosine-containing peptide. The reactions were started by the addition of 10 mM ATP and incubated for 30 min at 37°C. The samples were removed, and the wells were blocked for 30 min at 37°C. An anti-phospho-tyrosine antibody (PY-20) coupled to horseradish peroxidase was added, and the plates were incubated for 30 min at 37°C. The antibody was discarded, and the substrate, 3,3'-,5,5'-Tetramethylbenzidin (TMBZ), was added to the wells for 15 min at room temperature. Solution coloration was stopped and measured at 450 nm using a Spectra-Max microplate reader (Molecular Devices, Sunnyvale, CA, USA). One unit (U) of enzymatic activity is defined as the amount needed to incorporate 1 pmol of phosphate into the substrate per min. To investigate if rISRAA is a potential substrate of Fyn tyrosine kinase activity, we used a universal kinase activity kit (R&D Systems, Minneapolis, MN, USA). The principle of the assay is to measure the inorganic phosphate that is released from ADP via substrate phosphorylation by the kinase. Different concentrations of mouse Fyn (0.5 and 1.5 μg/ml) were incubated in the presence of 200 μM ATP and 1 μM concentrations of rISRAA and the rIS-RAA-Y102A mutant for 10 min at room temperature. ADP was used as a positive control, and inorganic phosphate was used for standard curve determination. The reaction was stopped by malachite green. The plates were incubated for 20 min at room temperature. The optical density was measured at 620 nm using a SpectraMax microplate reader (Molecular Devices, Sunnyvale, CA, USA), and the reaction mixtures were analyzed for phospho-tyrosine content via western blotting. For all experiments, each condition was tested in triplicate to ensure the accuracy of the results, and standard deviations were used for error representation.

Cell Culture and Transient Transfection
The EL4 (Mus musculus lymphoma, ATCC id: TIB 39) mouse T-cell line and Expi293F cells (catalog id: A14527, Thermo Fisher Scientific, Waltham, MA, USA) were transfected with the p-SELECT-zeo-rISRAA construct expressing N-terminal 6His-tagged rISRAA or with p-SELECT-zeo-HGh expressing human growth hormone (HGh) as a positive control for expression. p-SELECT-zeo was used for mock transfection. Transfection was performed with the Expifectamine kit (Gibco, Gaithersburg, MD, USA) according to the manufacturer's protocol. Briefly, the cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Gibco, Gaithersburg, MD, USA), 2 mM glutamine, 0.1 mg/ml of streptomycin and 100 U/ml of penicillin at 37°C with 5% CO 2 . Growth was performed in Expi293F expression medium (Thermo Fisher Scientific, MA, USA) until the cells reached a density of 2.5x10 6 cells/ml. Viability was confirmed to be 95% using the Trypan Blue dye exclusion method. DNA (30 μg) was mixed with 80 μl of the Expifectamine reagent in an appropriate volume of OptiMEM media and added to the cells. Zeocin was subsequently added for selection at concentrations ranging from 100 to 200 μg/ml. Forty-eight hours post-transfection, the cells were harvested and assayed for recombinant protein expression via western blotting.

Pull-Down Experiment
Expi293F cells transfected with p-SELECT-zeo-rISRAA were harvested after 48 hours and lysed. Pull-down assays were performed using the Pierce His Protein Interaction Pull-Down Kit (Thermo Fisher Scientific, MA, USA) according to manufacturer's recommendations. Briefly, equilibrated cobalt resin beads were incubated with Expi293F cell lysates for 30 min on ice. Untransfected cells were used as a negative control. The beads were washed 5 times and stored. A bead fraction was boiled in SDS buffer and then analyzed for rISRAA immobilization via western blotting using a polyclonal rabbit anti-His-Tag antibody at a final dilution of 1:1000 (Cell Signaling Technology, Danvers, MA, USA) and an anti-rISRAA monoclonal antibody used at a final dilution of 1:100 (GenScript, Piscataway Township, NJ, USA). EL4 cell lysates were prepared and incubated with rISRAA immobilized beads for 2 hours with gentle rotation at 4°C. After washing, the protein complexes were eluted from the beads with 300 mM imidazole. The eluted fractions were analyzed for Fyn protein detection via western blotting using a rabbit polyclonal anti-Fyn antibody (Abcam, Cambridge, UK) used at a final concentration of 1:1000.

Preparation of Splenocytes
This work was approved by the Ethics and Research Committee of the Arabian Gulf University and was carried out in strict accordance with the recommendations in the committee's guide for the use of laboratory animals. All animal euthanasia was performed via barbiturate overdose (100 mg/Kg) through intravenous injection, and all efforts were made to minimize animal suffering. Eight-week-old female Balb/c mice were used to prepare splenocytes. The spleens were dissected, and splenocytes were prepared by forcing the spleens through a stainless steel mesh. The freed cells were washed once in PBS. Hemolysis of erythrocytes in the cell pellets was performed by adding 2 ml of cold water for 30 s, followed by the addition of 1 ml of Phosphate-buffered saline (PBS buffer). The cells were washed twice in RPMI medium supplemented with L-glutamine, penicillin and streptomycin (Sigma-Aldrich, MO, USA) and then re-suspended in the same medium supplemented with 10% Fetal Calf Serum (FCS) (Life Technologies, CA, USA) to obtain a concentration of 2x10 6 cells/ml.

Anti-CD3 Cell Activation Assay
The 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) Cell Proliferation Assay (ATCC, Manassas, VA, USA) was used to measure cellular activation by anti-CD3. The plates were coated overnight at 4°C with anti-mouse-CD3 (BD Biosciences, San Jose, CA, USA) at a concentration of 5 μg/ml in PBS. The control wells were coated with PBS alone. The cell suspension was harvested by centrifugation and re-suspended at 2x10 6 cells/ml in complete RPMI medium supplemented with 10% FCS (Sigma-Aldrich, St. Louis, MO, USA). One hundred microliters of the cell suspension were plated in each well. Control wells with culture medium alone provided the blanks for absorbance readings. An equal volume of medium containing rISRAA at a final concentration of 1 μM was added to the test wells. Medium alone was added to the control wells. All conditions were tested in triplicate. The plates were incubated at 37°C in 5% CO 2 for 24 hours. Later, 10 μl of 5 mg/ml MTT reagent was added to each well, including the controls, and incubated for 4 hours at 37°C. The plates were centrifuged, and the medium was removed from the wells. Then, 100 μl of DMSO were added to all of the wells. The plates were covered with aluminum foil, agitated on an orbital shaker for 15 min and incubated for 4 h at 37°C to completely dissolve the Formazan crystals. Finally, the absorbance was recorded at 570 nm, with 620 nm as a reference wavelength using a SpectraMax microplate reader (Molecular Devices, Sunnyvale, CA, USA), and the stimulation index was calculated as follows:

SFK Phosphorylation
The splenocyte suspension was harvested by centrifugation and re-suspended at 2x10 6 cells/ml in complete RPMI medium supplemented with 10% FCS, and 400 μl of the cell suspension were plated in 12-well plates. An equal volume of medium containing recombinant rISRAA was added to the test wells at a final concentration of 1 μM, and cells alone served as a negative control. After different stimulation times (

Statistical Analysis
The two-tailed t-test for independent samples was used to calculate the level of significance ( Ã p<0.05, ÃÃ p<0.01, and ÃÃÃ p<0.001) for all experiments. All calculations were performed using SPSS statistics software version 19 (www.ibm.com).

Israa Is an Intron-Embedded Gene
Ensembl Blast analysis showed that israa is a 2,093 bp gene embedded within intron 6 of the zmiz1 gene on mouse chromosome 14 ( Fig 1A). zmiz1 encodes a 1,072 amino acid protein: the zinc finger, MIZ-type containing 1 protein (NP_899031.2), which is also known as zimp10 and is located in region 14qa3 of mouse chromosome 14. The zmiz1 gene is composed of 24 exons and is transcribed into a 207,563 bp pre-mRNA. israa is located approximately 22 kb downstream of zmiz1 exon 6 and 30 kb upstream of exon 7. NCBI blast analysis showed that the israa coding DNA sequence (CDS) (378 bp) is present with 100% identity in two mouse cDNA libraries of ESTs (dbEST ids: 2406142 and 8663286). An exhaustive Blast search revealed no orthologs for the israa gene, and an analysis of israa's transcript showed that it consists of two exons. The junction between the exons is located at base 1,162, and the DNA segment corresponds to a fragment 79 bp in length. An analysis of this junction region revealed the presence of a conventional splicing acceptor sequence but no conventional donor site. israa encodes a 125 residue protein (GenBank: ACB55610.1). Both the ATG initiation and STOP codons are located in exon 1 (Fig 1B). Prediction of the Kozak consensus region using the TIS Miner server showed a significant score (0.786) for the sequence AgaATGG at the translational start site of the israa CDS, which indicates that the israa mRNA can be translated in mammalian cells. In addition, another nested gene (4931406H21RIK) is located 12,886 bp upstream of israa (Gen-Bank: NR_033492.1), with an open reading frame (ORF) of 3,858 bp ( Fig 1A). The RIK gene is constituted by a unique exon and encodes a putative protein of 145 residues in length (GenBank: AAI47213). No ortholog for this gene has been found in the human genome, and no function has been reported for this gene. Expression data obtained via in situ RNA analysis showed tissue-specific gene transcription in the nervous system in mouse embryos [32]. The 4931406H21RIK gene product showed no significant similarity with other proteins or with known conserved protein motifs and functional domains.

ISRAA Protein Analysis and Modeling
ISRAA is formed by 125 amino acids and has a predicted molecular weight of 13.5 KDa, with a pHi of 5.77. Using the NetPhos 2.0 server, we identified several residues that are potential phosphorylation sites: serine residues 22, 28, 32, and 118 and tyrosine residues 61 and 102 ( Fig  1C). Kinase specificity analysis revealed Src-family kinases (SFKs) as the putative specific kinases for Tyr 61 and 102, with a score of 0.5 (Fig 1C). A potential palmitoylation site is located at cysteine 13, and an O-ß-GlcNAc attachment site is located at threonine 57. In addition, ISRAA is predicted to be secreted in an unconventional manner (secretome 2.0 score of 0.931). No potential protein trafficking motifs were identified using different prediction programs, except for a potential leucine-rich nuclear export signal at positions 87-97 (L A I S L L T L P S Q L), with a score slightly superior to the threshold defined by the NetNES server. This signal could be involved in protein export from the nucleus to the cytoplasm. No protein similar to ISRAA was found using multiple blast searches. A comprehensive conserved domain search showed no common conserved functional domains in the protein. Meanwhile, the short protein sequence motif search revealed the presence of a linear "SH2 binding motif" located at tyrosine residue 102 (YTEV), with the lowest score of 0.304 corresponding to Fyn as the best binding candidate (Fig 1C). This motif allows the docking of ISRA into the Fyn-SH2 domain via phospho-tyrosine (p-Tyr) recognition. The ISRAA sequence showed no significant similarity with Protein Data Bank entries. To further characterize the protein structure, we built a de novo model using the ROBETTA full-chain structure prediction server [24]. The best model (Fig 2A) was selected based on the overall G-factor and the number of residues in the core, allowed, generously allowed and disallowed regions. The model showed an overall G-factor of -0.11 and a Z-factor of -3.21. Verify3D analysis showed an overall score of 0.28, indicating a satisfactory model. The folding of the protein is shown in Fig 2A. The model shows favorable surface exposure of the Y 102 residue and partial surface exposure of the Y 61 residue (Fig 2B).

Protein-Protein Docking Simulation
Protein-protein docking between the refined model of the mouse FYN SH2-SH3 domain structure (PDB id: 3UF4) (receptor) and the ISRAA model (ligand) was performed using the Hex 8.0.0 software in unbound mode, as described in the Methods section. Docking was performed 10 times, and the best solutions were selected based on previously described Fyn-SH2 interaction modes with phosphopeptides [33]; R 156 and/or R 176 were shown to bind p-Tyr residue ligands, and a hydrophobic pocket of the SH2 domain provides specificity for a hydrophobic residue in the C-terminal position of the pTyr-containing peptide (Fig 3A). Selected solutions satisfying these conditions were shown to be clustered in a single conformation (Fig 3B). The best solution was chosen based on free energy and showed a total energy of -30 KJ/mol, indicating satisfactory stability. Interaction analysis showed a conventional binding mode for the ISRAA Y 102 -containing peptide and the Fyn SH2 domain (Fig 3C and 3D), which will be discussed later. and 102 are colored by elements. Y 102 , which is part of the predicted SH2 binding motif, is exposed on the protein surface, whereas Y 61 is shown to be partially exposed.

ISRAA Is a Substrate of Fyn
To investigate the in vitro interaction of the Fyn protein with ISRAA, we transfected EL4 cells, a mouse T-cell line, which failed to over-express the rISRAA protein to a level allowing purification for subsequent use in interaction studies (data not shown). We then over-expressed rIS-RAA fused to an N-terminal His-tag in Expi293F cells (Fig 4). The recombinant protein was shown to be pulled down by cobalt beads and specifically detected with both an anti-His antibody and a monoclonal anti-ISRAA antibody generated by GenScript (GenScript, NJ, USA) ( Fig 5). The rISRAA protein was then immobilized on beads and used as bait for EL4 cell lysates. Western blotting analysis of protein complexes revealed Fyn as prey, showing a specific interaction with the rISRAA protein (Fig 6).
To investigate the nature of the ISRAA-Fyn interaction, we performed a tyrosine kinase assay of the recombinant Fyn protein to determine its specific activity. The protein showed a significant specific tyrosine kinase activity of 7.5x10 3 U/mg. We then measured the total Fyn activity where Y 102 is located 3.5 A from R 156 (C). An unconventional binding mode is observed for L 108 (pTyr+6), which is located in a binding pocket between the βD-sheet and the BC loop (D) that is different from the classic pocket. In the entire figure, the Fyn-SH2 domain is represented in gray, and the ISRAA peptide (Y 102 TEVSTL) is represented in green.  Novel Fyn Binding Protein Encoded by an Intron-Embedded Gene in Mouse in the presence of rISRAA and rISRAA-Y102A as substrates. Fig 7A shows the concentrationdependent enzymatic activity of Fyn (0.03 to 0.05 nmol/min) in the presence of 1 μM rISRAA, while no significant activity was shown when the rISRAA-Y102A mutant was used as a substrate. This experiment shows that rISRAA is a substrate for Fyn, primarily through its Y 102 residue. The mutation of that residue to alanine abolishes the kinase activity of Fyn on rISRAA. Western blotting analysis of the reaction mixture using an anti-p-tyrosine (PY-20) antibody before the addition of malachite green (Fig 7B) confirmed the p-Tyr content of recombinant rIS-RAA. In contrast, no significant tyrosine phosphorylation was detected in the rISRAA-Y102A mutant.

ISRAA Downregulates Early T-cell Activation and SFK Activation
The MTT cell activation assay was used to measure T-cell activation by anti-CD3 in the presence and absence of 1 μM rISRAA. After 24 h of incubating splenocytes with 5 μg/ml of anti-CD3, the stimulation index was calculated. The positive control (no rISRAA) showed a stimulation index of 2 compared to non-stimulated cells (Fig 8A). Splenocytes incubated with 1 μM rISRAA downregulated T-cell activation (SI < 1) (Fig 8A). Splenocytes treated with 1 μM rIS-RAA at various times (1, 5, 10, 15 and 30 minutes) and probed using an anti SFK p-Y416 antibody showed a time-dependent dephosphorylation of the SFK members Fyn and Src, as shown by specific protein molecular weights (59 KDa), in comparison to untreated cells (Fig 8B). An analysis of the total actin, Src and Fyn proteins in the cell lysates showed the presence of equivalent amounts of proteins.

Discussion
Israa has been identified in mice and described as a novel immune system modulator whose transcription is activated in vivo in a manner dependent on intact splenic innervations. Interestingly, we found that the gene that encodes ISRAA is an intron-embedded gene in intron 6 of the mouse zmiz1 gene on chromosome 14. This intron also contains another gene, 4931406H21RIK, whose function is also unknown. The CDS of israa matched two EST libraries with 100% identity, showing that this ORF is transcribed into mRNA in the same sense as the zmiz1 transcript. The observation that israa contains a Kozak consensus region argues in favor of its translation. These features, along with the original observation of Bakhiet et al. [10], confirm that israa is an active mouse gene. However, the mechanism of israa transcription and mRNA maturation must be investigated further because no typical splicing features were found in the gene. More intriguing is the fact that we found no orthologs of israa in lower Novel Fyn Binding Protein Encoded by an Intron-Embedded Gene in Mouse vertebrates or primates. Meanwhile, the analysis of the ISRAA protein revealed an extra-cytoplasmic predicted location, although no common signal peptide was identified. The presence of a putative palmitoylation site at Cys13 in the N-terminal part of the protein could indicate potential lipid-raft localization of the protein, therefore strengthening the possibility that ISRAA is released effectively [34]. Indeed, short sequence motif analysis showed the presence of a short linear motif (Y 102 TEV) located at Tyr102 that could bind to SH2 domains, with the best score matching the Fyn SH2 domain, which is prone to phosphorylation by an SFK. In addition, a de novo model of ISRAA showed that the Y 102 TEV motif is well exposed on the protein surface, which facilitates its involvement in protein-protein interactions. Fyn is a member of the SFKs, which are involved in essential signaling pathways, such as cell growth, survival, migration, motility and adhesion [35]. Fyn is localized primarily to the cytoplasmic leaflet of the plasma membrane [35]. Two different isoforms of Fyn are expressed in mice; isoform 1 or B is expressed primarily in the brain, and isoform 2 or T is strongly expressed in T-cells [36]. Fyn plays a central role in immune system signaling and early T-cell activation signaling by phosphorylating T-cell receptor ITAMs in the CD3 and z chains. This phosphorylation allows the recruitment of ZAP-70, which activates T-cell adaptor proteins, such as SLP-76 and LAT, leading to secondary messenger generation and the T-cell activation cascade [37]. The protein shares a common multi-domain architecture with other SFKs, including an SH 1 kinase domain, SH2 and SH3 binding domains, and a C-terminal negative regulatory domain [33]. The SH2 domain is important for directing specific protein-protein interactions that are necessary for substrate specificity, activity regulation, and protein recruitment. Hence, identifying the proteins that bind to this specific domain could help to elucidate the mechanism by which Fyn-related pathways are regulated. Docking simulation of ISRAA within Fyn's SH2 domain showed a favorable binding mode in which the Y 102 residue is located, as expected, in the pTyr-binding cavity between the central anti-parallel βC-sheet and the α-helix αA. When phosphorylated, the phosphate group is 3.5 A from R 156 , which allows H-bond formation. The C-terminal residues of the YTEV motif were expected to be located in the hydrophobic binding pocket near the EF loop; however, the analysis showed a different conformation in which Leu108 is located in a binding pocket between the βD-sheet and the BC loop. In fact, previous studies showed that the binding mode of the p-Tyr-containing motif to the SH2 domain depends strongly on its conformation; a 2010 study showed different binding modes of the same p-Tyr peptides to the Src-SH2 domain depending on whether the peptides adopt an extended or helical conformation [38]. Indeed, a major structural characterization of SH2 binding modes was carried out using short linear peptides and failed to provide a complete picture. Hence, further investigations of the binding mode of ISRAA to the Fyn-SH2 domain using mutagenesis and structural characterization could reveal new insights into p-Tyr recognition and binding regulation.
Cell transfection, pull-down experiments and tyrosine kinase assays provided evidence that cellular Fyn binds to the rISRAA protein and that Fyn is able to phosphorylate rISRAA in vitro. Indeed, ISRAA's Y 102 residue appears to play a major role in the ISRAA-Fyn interaction and ISRAA phosphorylation. This finding could indicate a complex mode of binding of ISRAA to Fyn, which is known as "processive phosphorylation" [39]. In this process, the kinase domain of a kinase phosphorylates a substrate at the tyrosine site, allowing it to bind to the SH2 domain, leading to further phosphorylation of other tyrosine residues in the substrate or another protein bound to the substrate. In the case of ISRAA, we propose that upon phosphorylation by Fyn, the Y 102 TEV motif binds to the SH2 domain of Fyn, which exposes another tyrosine (e.g. Y 61 ) for phosphorylation by the kinase domain of Fyn and leads to the recruitment of other substrates or effectors to the complex. In this case, ISRAA could constitute a new adaptor protein that binds to Fyn and may be involved in the Fyn-dependent signaling pathway. In previous studies, similar approaches succeeded in identifying novel signaling proteins that are involved in Src-family signaling [40].
ISRAA was previously described as a secreted factor that promotes the reactivation and proliferation of Trypanosome-induced immunosuppressed splenocytes following rISRAA treatment (10); however, that study was performed using a mixed population of immune cells without targeting a specific cell population. Hence, that study could not indicate a specific role for the ISRAA protein in a specific immune cell population. In an attempt to understand the role of ISRAA in the early T-cell activation process due to its binding with Fyn, we chose to study the effect of rISRAA on T-cell activation induced by anti-CD3. We have shown that CD3-induced activation is down regulated by soluble rISRAA treatment and that this effect is due in part to the dephosphorylation of the activation tyrosine of the SFK members Fyn and Src (p-Src family Tyr-416) in the early minutes after cell treatment with the rISRAA protein.
Interestingly, in contrast to the findings reported in 2008 [10], this study shows that ISRAA appears to have a negative effect on early T-cell activation by triggering the dephosphorylation of SFKs at Y416.
Taken together, the evidence presented in this report strongly suggests that israa is the first described intron-embedded gene to encode a Fyn binding protein that could be involved in the control of immune homeostasis by the nervous system. Although no israa ortholog was found in the human genome, a comparative genomics study revealed several ORFs within the intron 6 region of the human zmiz1 homolog that could represent candidates with a similar function. A comprehensive study of the ISRAA signaling pathway and the identification of partner proteins and the mechanisms by which israa is regulated could provide an improved understanding of the control of immune function by the nervous system.