In Silico Screening for Palmitoyl Substrates Reveals a Role for DHHC1/3/10 (zDHHC1/3/11)-mediated Neurochondrin Palmitoylation in Its Targeting to Rab5-positive Endosomes*

Background: The identification of palmitoyl substrate-enzyme pairs is important for elucidating physiological roles of protein palmitoylation. Results: In silico screening revealed neurochondrin palmitoylation. DHHC1/3/10 were identified as neurochondrin palmitoylating enzymes and were essential for targeting of neurochondrin to Rab5-positive endosomes. Conclusion: Neurochondrin and the DHHC1/10 and DHHC3/7 subfamilies represent novel substrate-enzyme pairs. Significance: In silico palmitoyl screening is useful for clarifying functions of palmitoylation. Protein palmitoylation, a common post-translational lipid modification, plays an important role in protein trafficking and functions. Recently developed palmitoyl-proteomic methods identified many novel substrates. However, the whole picture of palmitoyl substrates has not been clarified. Here, we performed global in silico screening using the CSS-Palm 2.0 program, free software for prediction of palmitoylation sites, and selected 17 candidates as novel palmitoyl substrates. Of the 17 candidates, 10 proteins, including 6 synaptic proteins (Syd-1, transmembrane AMPA receptor regulatory protein (TARP) γ-2, TARP γ-8, cornichon-2, Ca2+/calmodulin-dependent protein kinase IIα, and neurochondrin (Ncdn)/norbin), one focal adhesion protein (zyxin), two ion channels (TRPM8 and TRPC1), and one G-protein-coupled receptor (orexin 2 receptor), were palmitoylated. Using the DHHC palmitoylating enzyme library, we found that all tested substrates were palmitoylated by the Golgi-localized DHHC3/7 subfamily. Ncdn, a regulator for neurite outgrowth and synaptic plasticity, was robustly palmitoylated by the DHHC1/10 (zDHHC1/11; z1/11) subfamily, whose substrate has not yet been reported. As predicted by CSS-Palm 2.0, Cys-3 and Cys-4 are the palmitoylation sites for Ncdn. Ncdn was specifically localized in somato-dendritic regions, not in the axon of rat cultured neurons. Stimulated emission depletion microscopy revealed that Ncdn was localized to Rab5-positive early endosomes in a palmitoylation-dependent manner, where DHHC1/10 (z1/11) were also distributed. Knockdown of DHHC1, -3, or -10 (z11) resulted in the loss of Ncdn from Rab5-positive endosomes. Thus, through in silico screening, we demonstrate that Ncdn and the DHHC1/10 (z1/11) and DHHC3/7 subfamilies are novel palmitoyl substrate-enzyme pairs and that Ncdn palmitoylation plays an essential role in its specific endosomal targeting.

Protein palmitoylation, a post-translational modification of proteins with lipid palmitate, increases the hydrophobicity of proteins, promotes their association with intracellular and plasma membranes, and regulates protein trafficking and functions (1)(2)(3)(4)(5). Unlike other lipid modifications, such as myristoylation or prenylation, palmitoylation is a reversible reaction that may be regulated by extracellular signals. The reversible nature of palmitoylation allows proteins to shuttle between intracellular compartments, relocalize in physiological contexts, and participate in diverse aspects of cellular signaling (1)(2)(3)(4)(5).
The dynamic palmitoylation level is finely controlled by palmitoyl acyltransferases/palmitoylating enzymes and palmitoyl protein thioesterases/depalmitoylating enzymes. Palmitoylating enzymes were originally identified by forward genetic screening in yeast and contains the conserved Cys-rich DHHC (Asp-His-His-Cys) domain and four or six transmembrane domains (6 -8). A family of the mammalian DHHC proteins was identified (9), and it can be categorized into several subfamilies based on the homology of catalytic DHHC domains (9). The discovery of the mammalian DHHC protein family and the establishment of the simple screening system using the DHHC palmitoylating enzyme library have facilitated identification of palmitoyl substrate-enzyme pairs (4,5). Importantly, a subfamily of DHHC proteins often shares its substrates. However, the identified number of the substrate-enzyme pairs is still limited. Thus, the enzymatic activities of some DHHC proteins, such as the DHHC1/10 (zDHHC1/11; z1/11) subfamily, remain unknown because none of their physiological substrates have been found (4,5).
Recently, several groups have developed proteomic methods by which palmitoylated proteins are purified from cultured cells or tissues and identified by mass spectrometry. These purification methods of palmitoylated proteins include the acylbiotinyl exchange (ABE) 2 method (10 -13) and click chemistry (14 -16). Although these powerful proteomic analyses have identified many new palmitoyl proteins (11, 12, 14 -16), the method using mass spectrometry has some limitations due to the biochemical properties of proteins. First, some proteins are expressed at too low levels to be identified by mass spectrometry. Second, some membrane proteins, such as G-protein-coupled receptors or ion channels, are barely extracted from cells by detergent and also not efficiently ionized for mass spectrometry (17,18). Third, some proteins that are not palmitoylated but are just co-purified with palmitoylated proteins might be contaminated (i.e. false-positive results). Last, identifying palmitoylation sites by mass spectrometry is not easily accessible to every laboratory (19). Our method used in this study, in silico prediction of palmitoylation sites of proteins, is able to complementally overcome those problems and identify novel palmitoyl substrates.
Here, we identified neurochondrin (Ncdn)/norbin as a novel palmitoyl substrate. Ncdn was originally identified as a gene whose expression is up-regulated when long term potentiation is chemically induced in rat hippocampus (20). Ncdn is a 75-kDa neuronal cytoplasmic protein with no obvious domain structures (21). Overexpression of Ncdn in Neuro2a cells promotes neurite outgrowth (20). KO of Ncdn in mice leads to early embryonic lethality (22). Nervous system-specific conditional KO of Ncdn in mice results in epileptic seizure (23). Forebrain-specific Ncdn KO attenuates metabotropic glutamate receptor 5 (mGluR5)-dependent stable change in synaptic transmission in the hippocampus and results in a behavioral phenotype associated with a rodent model of schizophrenia (24). This study also showed that Ncdn regulates the surface expression of mGluR5 (24). Taken together, these findings suggest that Ncdn is a promising central nervous system regulator and functions as a scaffolding protein or an adaptor protein.
However, its precise subcellular localization in neurons and regulatory mechanism remains unclear.
In this study we systematically screened the mouse whole protein database for palmitoyl substrates through computational prediction and experimentally verified at least 10 novel palmitoyl substrates including Ncdn. We found that the DHHC1/10 (z1/11) and DHHC3/7 subfamilies quantitatively palmitoylated Ncdn at Cys-3 and Cys-4. We also found that Ncdn palmitoylation by DHHC1/3/10 (z1/3/11) plays an essential role in targeting Ncdn to Rab5-positive early endosomes in dendrites.

EXPERIMENTAL PROCEDURES
Global in Silico Prediction of Palmitoyl Proteins-For the global prediction of palmitoyl proteins with their sites, we used the software, CSS-Palm 2.0 (Palmitoylation Site Prediction Using a Clustering and Scoring Strategy) (25). This software algorithm is based on experimentally verified palmitoylation sites: 263 palmitoylation sites from 109 distinct palmitoyl proteins (25). The mouse protein sequence data consisting of 62,695 redundant sequences was downloaded from the UniProt database (26). Sequence files were saved as separate text files in FASTA format programmed by Perl and used as the input of the locally installed CSS-Palm 2.0 (without setting a threshold). To run CSS-Palm 2.0 automatically for all sequence files, the applications used were Windows XP Professional SP3 32 bit, UWSC Version 4.6c, and Java Access Bridge for Microsoft Windows Operating System Version 2.0.1. The output CSV file included results with positions of predicted palmitoyl cysteines, surrounding sequences, and CSS-Palm scores for each protein. All the datasets from CSS-Palm 2.0 included 59,157 files of proteins that have cysteine residues. Protein files with only score 0 cysteines were then removed. The resultant 59,136 proteins (with score Ͼ0) were listed in descending order of scores in the CSV file format (supplemental Table S1), which included ϳ19,000 proteins with a score greater than the cut-off score of 1.8. Redundant sequences and sequences with cysteines in the signal sequence were then removed.
To find palmitoyl candidate proteins that are expressed predominantly in the brain, the listed protein sequences were further analyzed by a gene expression database, BodyMap-Xs (27). The entire dataset for mouse genes (38,632 gene data) was automatically downloaded from BodyMap-Xs, and data files were saved as CSV files. Information about accession numbers (Unigene no.) included in each output data file from BodyMap-Xs was collated with the UniProt annotation in the initial output data files from CSS-Palm 2.0. Then the CSS-Palm-predicted candidates included in datasets from the BodyMap-Xs analysis were further extracted. The brain enrichment score was calculated by dividing the score for brain by the sum of scores for all tissues. Because the present expression database is not sufficient for all the proteins, this narrowing-down process may omit 27% of CSS-Palm 2.0 data that is not covered in the Body-Map-Xs database. Proteins with brain enrichment scores Ն0.75 were listed, and proteins containing candidate cysteines only in the extracellular region were manually excluded. The resultant list consisted of 573 promising candidates (supplemental Table  S2). In addition, we manually inspected the omitted candidate proteins (with Ն1.8 CSS-Palm scores, but without Body-Map-Xs data), and selected homer 1C, Syd-1, paxillin, zyxin, and Par3 as additional palmitoyl candidates that are enriched in specific membrane domains such as pre-and postsynapses and cell adhesion sites. Instead of cornichon-3 (CSS-Palm score 1.852), cornichon-2 (1.730) was selected because the potential palmitoyl cysteine is conserved in both isoforms and cornichon-2 is predominantly expressed in the hippocampus and has been extensively studied as an AMPA receptor modulator (28).
Cell Culture-Hippocampal and cortical neuron cultures were prepared from rat embryonic day 18 -20 embryos or postnatal day 1 pups. All animal experiments described herein were reviewed and approved by the ethics committee in our institutes and were performed according to the institutional guidelines concerning the care and handling of experimental animals. Neurons were seeded in neurobasal medium (Invitrogen) supplemented with B-27 supplement (Invitrogen) or B-27 plus (MACS) and 2 mM Glutamax (Invitrogen).
Metabolic Palmitate Labeling and Subcellular Fractionation-Both experiments were performed as previously described (9,29).
ABE Method-The ABE method was performed as previously described (10 -12, 29). Briefly, hippocampal neurons were solubilized and incubated with N-ethylmaleimide to mask free cysteines. Free N-ethylmaleimide was removed by chloroform-methanol precipitation. Precipitated proteins were incubated with hydroxylamine (NH 2 OH) to cleave a thioester bond or Tris/Cl as a control followed by incubation with biotin-HPDP (N-[6-biotinamido]hexyl]-3Ј-(2Ј-pyrdyldithio)propionamide) to label new free cysteines. Biotinylated proteins were then purified with NeutrAvidin-agarose (Thermo Fisher Scientific) and analyzed by Western blotting with indicated antibodies. To detect Ncdn palmitoylation, the improved ABE protocol (13) was used to reduce background signals. Here, to extract more proteins, lysis buffer included 4% SDS instead of 2% SDS. Also, to cleave disulfide bonds before alkylation with N-ethylmaleimide, we used tris(2-carboxyethyl)phosphine, a potent reducing agent that does not cleave the thioester bond between palmitate and cysteine residue.
Tandem Affinity Purification-To pull down associated proteins with DHHC palmitoylating enzymes from the brain membrane fraction, HEK293 cells transfected with His 6 -FLAG-DHHC2, 3 or 10 (z11) were scraped with immunoprecipitation buffer (20 mM Tris/Cl at pH 7.5, 1 mM EDTA, 100 mM NaCl, 2.0% Triton X-100, 50 g/ml PMSF) and homogenized followed by centrifugation at 100,000 ϫ g at 4°C for 30 min. P2 membrane fraction was prepared by homogenization of rat whole brain in homogenizing buffer (20 mM Tris/Cl at pH 7.5, 2 mM EDTA, 0.32 M sucrose, 100 g/ml PMSF) and centrifuga-tion at 20,000 ϫ g at 4°C for 1 h. The resultant pellet was suspended and mixed with the HEK293 cell supernatant containing tagged DHHC proteins prepared above. After incubation for 1 h at 4°C, the homogenate was centrifuged at 100,000 ϫ g at 4°C for 1 h. The supernatant was incubated with FLAG-M2 beads (Sigma) for 2 h at 4°C. After washing the beads, His 6 -FLAG-DHHC was eluted with FLAG peptide (0.25 mg/ml) for 1 h at 4°C. The FLAG eluate was incubated with nickel-nitrilotriacetic acid-agarose for 1 h at 4°C. After washing the beads, His 6 -FLAG-DHHC was eluted with the buffer containing 250 mM imidazole.
Transfection and Immunofluorescence Analysis-Cortical or hippocampal neurons (5 ϫ 10 4 cells) on 12-mm coverslips were transfected by Lipofectamine 2000 (Invitrogen). For knockdown experiments, 6 -7 days after transfection, neurons were fixed with 4% paraformaldehyde at room temperature for 10 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with PBS containing 10 mg/ml BSA for 10 min. Neurons were then labeled with rabbit polyclonal anti-Ncdn (N-rNcdn) and Cy3-conjugated donkey anti-rabbit IgG. Neurons transfected with miR-Ncdn expression vector were visualized by co-cistronic expression of GFP. For Fig. 5, D and E, hippocampal neurons were fixed with methanol at Ϫ30°C for 10 min. For confocal imaging, fluorescent images were obtained using an LSM5 Exciter system (Carl Zeiss) with a Plan-Apochromat 63ϫ NA 1.40 oil immersion objective lens. For two-color stimulated emission depletion (STED) imaging, neurons were immunostained by rabbit anti-Ncdn and chicken anti-GFP or anti-V5 antibodies followed by ATTO425 (Rockland) and Alexa488 (Invitrogen)-conjugated secondary antibodies. STED images were obtained by a Leica TCS STED CW with a 100ϫ NA 1.40 oil immersion objective lens. Obtained images were further deconvoluted with the built-in deconvolution algorithms of the Leica LAS-AF software. Also, the same region was acquired by the confocal mode of a Leica TCS SP5 II and directly compared with that of STED imaging. For statistical analysis, one-way analysis of variance with Tukey's HSD or Dunnett post hoc test was performed. Data are presented as the mean Ϯ S.E.

Global in Silico Screening for Novel Palmitoyl Substrates-To
identify novel palmitoyl substrates, we took advantage of computational prediction using CSS-Palm 2.0 algorithm, which was recently developed for palmitoylation site prediction (25) and is freely available. A pilot study using protein sequences of known palmitoyl and non-palmitoyl proteins showed that CSS-Palm 2.0 predicted the reported palmitoyl cysteines of representative palmitoyl substrates with high scores (e.g. PSD-95, 5.096; GAP-43, 16.844; H-Ras, 4.024) but gave low scores toward nonpalmitoyl proteins (␤-actin, 1.296; ␤-catenin, 0.687). We next applied the comprehensive protein sequences to CSS-Palm 2.0, predicting many novel palmitoyl substrates.
As a comprehensive protein database, we used the UniProt database and selected Mus musculus as an organism (26). To automatically retrieve about 60,000 sequences with redundancies from the UniProt database and apply them to the CSS-Palm 2.0 prediction program, we developed the automatic program (see "Experimental Procedures). Proteins that have cysteines with CSS-Palm score Ͼ0 were listed (supplemental Table S1). Because most known palmitoylated substrates showed more than a score 1.8, we set 1.8 as the cut-off value for the CSS-Palm 2.0 score. Brain enrichment score (see "Experimental Procedures") was used as another criterion for selection (cut-off point is Ն0.75), and 573 proteins were retrieved as candidates for brain-enriched palmitoyl substrates (supplemental Table  S2). Of the 573 proteins, 84.6% are novel candidates for palmitoyl proteins (Fig. 1A). The palmitoyl candidates included the substantial number of putative transmembrane proteins (35.7%) (Fig. 1B). Many of the candidate proteins are known to function in signal transduction, for example, as receptors, channels, transporters, GTPases, and kinases (supplemental Table S2). We selected 12 proteins as candidates for novel palmitoyl substrates based on their brain enrichment (Fig. 1C, Table 1, and supplemental Table S2). Given that palmitoyl proteins accumulate at specialized membrane compartments, such as pre-and postsynaptic membranes, focal adhesions, and tight junctions, we selected five proteins whose CSS-Palm 2.0 scores are Ն1.8 but whose brain enrichment scores are Ͻ0.75 or not calculated (Table 1).
At the postsynapse, many proteins are subjected to palmitoylation (1,4,12). Examples include AMPA-type glutamate receptor (AMPAR, subunits GluA1, -2, -3, and -4) (31), FIGURE 1. In silico screening for neuronal palmitoyl candidates. A-C, based on our criteria (Ն1.8 CSS-Palm score and Ն0.75 brain enrichment score), we chose 573 proteins as brain-enriched palmitoyl candidates (supplemental Table S2). Note that secreted proteins and proteins with target cysteines only in extracellular regions were excluded. Of the candidate proteins, 84. 6% have not yet been reported and are considered as promising new palmitoyl substrates (A). 35.7% of neuronal substrate candidates are membrane proteins (B). TMD, transmembrane domain. Our 12 selected candidates (red) and PSD-95 (blue), a representative neuronal palmitoyl protein, are marked on the scattergram, consisting of CSS-Palm scores (Ն1.8) on the y axis and brain-enrichment scores (Ն0.75) on the x axis (C).

Examination of Novel Palmitoyl Substrates by Experimental
Approaches-To examine whether those 17 substrate candidates are actually palmitoylated in cells, cDNAs of individual candidate and DHHC3 palmitoylating enzyme were co-transfected into HEK293 cells, and palmitoyl modified proteins were metabolically labeled with [ 3 H]palmitate. Based on our previous studies, DHHC3 functions as a general palmitoylating enzyme (i.e. all palmitoyl substrates we have tested are palmitoylated by DHHC3) (4). Among postsynaptic candidate proteins, TARP ␥-2, TARP ␥-8, CNIH2, CaMKII␣, and Ncdn were robustly palmitoylated ( Fig. 2A and Table 1). Neither kalirin7 nor homer 1C were palmitoylated. The presynaptic candidate Syd-1 was palmitoylated, whereas Rab3A ( Fig. 2A) and liprin-␣2 (data not shown) were not palmitoylated. Among cell adhesion-related proteins, zyxin incorporated [ 3 H]palmitate, whereas paxillin and Par3 were not palmitoylated. We noted that the expression level of paxillin-GFP was relatively reduced in the presence of DHHC3. Even when the amounts of paxillin-GFP loaded were adjusted between samples, we did not see clear incorporation of [ 3 H]palmitate to paxillin-GFP in the presence of DHHC3 (data not shown). Other candidate TRPM8, TRPC1 and orexin 2 receptor, but not KIF5C, were efficiently palmitoylated by DHHC3. Thus, of the 17 candidates, 10 were verified as palmitoyl substrates (Table 1).
This assay, however, might possibly include false-positive substrates, as both DHHC3 enzyme and a substrate protein are overexpressed in cells. To test whether endogenous proteins are palmitoylated in neurons, we purified palmitoylated proteins in primary hippocampal neurons by the ABE method and analyzed them by Western blotting. Because of the availability of the specific antibodies, only TARP ␥-8, CaMKII␣, Ncdn, and

candidates in this study
Highest CSS-Palm score, Position, and Sequence indicate the highest score in the protein given by CSS-Palm 2.0, the position of the corresponding cysteine (underlined in sequence), and the sequence around the cysteine with amino acid positions, respectively. N (bold), N terminus; C (bold), C terminus. ND means no data retrieved from Body-Map database. "Metabolic" indicates the result of metabolic labeling assay with [  Syd-1 were tested. All four endogenous proteins were palmitoylated in the primary hippocampal neurons (Fig. 2B and Table 1).
Ncdn Palmitoylation Occurs at Cysteines 3 and 4-Ncdn protein contains 25 cysteine residues, 2 near the amino terminus at positions 3 and 4, and other cysteines scattered throughout the protein. CSS-Palm 2.0 strongly predicted cysteines 3 and 4 as Ncdn palmitoylation sites (Scores are 13.016 and 12.562 at Cys-3 and Cys-4, respectively; Fig. 4A). In contrast, the scores of the other cysteines were less than the 1.8 cut-off value. These scores of Cys-3 and Cys-4 of Ncdn are much higher than those of the other novel substrates, TARP ␥-8 (2.461), CaMKII␣ (3.765), and Syd-1 (3.078) and even a well known substrate, PSD-95 (5.096) (Fig. 1C and Table 1). In fact, the CSS-Palm score of Ncdn (13.016) was the seventh highest among proteins with high brain enrichment score (Ն0.75) (supplemental Table  S2). To determine whether Cys-3, Cys-4, or both in Ncdn serves as the palmitoylation sites, those cysteine residues were mutated to serines (Ncdn C3S, Ncdn C4S, and Ncdn C3S,C4S). HEK293 cells were co-transfected with GFP-fused wild-type (WT) or mutant Ncdn together with DHHCs, and palmitoyl proteins were metabolically labeled with [ 3 H]palmitate. Fluorography showed that a mutation on either Cys-3 or Cys-4 and dual mutations completely eliminated palmitoylation of Ncdn  Fig. 2A. Arrows indicate the positions of palmitoylated substrates. Palmitoylation of Ncdn was enhanced by DHHC1, -3, -7, and -10 (z11), whereas TARP ␥-8, CaMKII␣, and Syd-1 were palmitoylated by the DHHC3/7 subfamily. HA-GST was used for mock indicated as -. B, palmitoylation of Ncdn was increased by the DHHC1/10 (z1/11) and DHHC3/7 subfamilies. The band intensity of palmitoylated Ncdn was quantified and normalized to total Ncdn. The -fold increase in relative band intensity (in the presence of DHHC proteins) to the basal intensity (in the absence of DHHC proteins) is indicated below each lane. The average values of three independent experiments are represented. DHHC1 and -10 (z11) belong to the same subfamily in green, and DHHC3 and -7 belong to the other subfamily in blue. C, expression of 23 HA-DHHC proteins was confirmed by Western blotting (WB) with anti-HA antibody (a representative result). (Fig. 4B). These results indicate that the Ncdn palmitoylation sites are Cys-3 and Cys-4 and that DHHC1, -3, -7, and -10 (z11) mediate Ncdn palmitoylation at specific cysteine residues.
Ncdn Is Localized Specifically in Neuronal Dendrites-Because palmitoylation regulates protein localization, we generated an antibody to Ncdn (N-rNcdn) to examine its subcellular localization in neurons. The antibody to Ncdn specifically recognized a single ϳ75-kDa band in rat cultured cortical neurons (Fig. 5A). We first examined the developmental change of Ncdn expression in cultured cortical neurons. At 3 DIV, when an axon just differentiates from the immature neurite, Ncdn was not detected (Fig. 5A). Ncdn expression began to be detected at 7 DIV and was the highest at 14 DIV, when dendrites differentiate from the other immature neurites. Ncdn expression then slightly declined but remained higher than that at 7 DIV. Notably, this expression pattern of Ncdn was very similar to that of mGluR5.

DISCUSSION
In Silico Prediction of Palmitoyl Proteins-The recent development of purification methods for palmitoylated proteins (the ABE method or click chemistry) has greatly contributed to the identification of novel palmitoyl proteins (11)(12)(13)(14)(15)(16). However, these methods have several potential problems that could lead to false-negative or false-positive results. The biochemical properties of proteins, such as detergent insolubility and posttranslational modifications may hinder the purification and mass spectrometry detection, leading to false-negative results. Also, non-palmitoylated proteins might be co-purified with palmitoyl substrates, leading to false-positive results. In fact, non-palmitoylated, secreted protein LGI1, which indirectly associates with palmitoylated PSD-95 through ADAM22, was listed as a candidate palmitoyl protein (12). In this study we searched for novel palmitoyl substrates through global in silico screening by the CSS-Palm 2.0 prediction program, which requires only protein sequences and ignores any biochemical properties of target proteins. By automatically applying all mouse protein sequences to CSS-Palm 2.0, we classified the 59,136 obtained protein sequences according to the CSS-Palm scores (supplemental Table S1). In fact, this list included many transmembrane proteins such as G-protein-coupled receptors, which are difficult to be solubilized for purification. Of the 17 candidates we selected, 10 proteins were experimentally verified as novel palmitoyl substrates that were not captured by the proteomic methods. This indicates that the in silico approach is useful for the discovery of novel palmitoyl substrates and complementally functions with the experimental methods. However, of the 17 candidates, palmitoylation of seven proteins were not detected by metabolic labeling assay (i.e. false-positive results). In terms of accuracy, this prediction program may have room for improvement. CSS-Palm 2.0 was developed by referring 263 palmitoylation sites from 109 proteins, which had been identified experimentally (25). Very recently, CSS-Palm has been updated to Version 3.0 based on 439 palmitoylation sites from 194 proteins. We repeated the in silico analysis with the 17 candidate substrates by CSS-Palm 3.0 (supplemental Table S3). However, the scores predicted by CSS-Palm 3.0 did not always reflect our experimental results. For example, the score of palmitoyl TARP ␥-2 was 1.9 by CSS-Palm 2.0, whereas the score was 0.7 by CSS-Palm 3.0. Further improvements need to be made for more precise prediction. For example, the increased number of references of known substrates and their palmitoylation sites and the clarification of DHHC subfamily-specific consensus sequences will make the software a much more powerful tool.
Physiological Role of Ncdn Palmitoylation-Previous studies showed that overexpression of Ncdn promotes neurite outgrowth in Neuro2a cells (20,48). The first 100 amino acids of Ncdn, including palmitoylation sites, were thought to contain the activity site that induces the differentiation (48). In polarized neurons, which develop two differential processes, an axon and dendrites, we found that Ncdn protein is highly expressed during dendrite development (Fig. 5A) and localized specifically in dendrites (Fig. 5, D and E). These results imply that Ncdn is involved in dendritogenesis. In fact, we found that overexpression of NcdnWT in hippocampal neurons significantly increased the total length of dendrites compared with control neurons (data not shown). However, Ncdn palmitoylation had no effects on dendrite outgrowth because there was no significant difference in effect of overexpression of NcdnWT versus CS mutant. We also examined whether Ncdn palmitoylation affects dendrite morphology using the molecular replacement strategy. However, we could see no significant effects of knockdown or molecular replacement of Ncdn on dendrite morphology (dendrite length and branching) (data not shown). Further studies on physiological roles of Ncdn and its palmitoylation in neurons will be required.
Ncdn palmitoylation may participate in the regulation of synaptic plasticity. Ncdn was originally identified as an inducible gene during the induction of long-term potentiation (20). Very recently, it was reported that Ncdn interacts with mGluR5 and increases its cell surface expression (24). Forebrain-specific Ncdn KO mice show the reduction of dihydroxyphenylglycineinduced long term potentiation and impairment of the induction of long term potentiation in the Schaffer collateral to CA1 synapses (24). Given that Rab5 is required for internalization of AMPAR during long term depression (52), Ncdn might regulate internalization of AMPAR, directly acting on early endosome dynamics or indirectly modulating a signal through mGluR5. Because conditional Ncdn KO mice showed apparent pathological phenotypes (epileptic seizures and schizophreniarelevant behaviors) (23,24), genetic experiments using knock-in mice in which Ncdn palmitoylation-deficient mutant is expressed should reveal the roles of Ncdn palmitoylation in these pathophysiological phenotypes.
In this study we attempted the global identification of palmitoyl substrates by using an in silico computational approach and identified several unexpected substrates. Among them, we focused on Ncdn palmitoylation and found that the DHHC1/10 (z1/11) and DHHC3/7 subfamilies enhance Ncdn palmitoylation and regulate its localization to early endosomes in hippocampal neurons. This finding demonstrates for the first time that the DHHC1/10 (z1/11) subfamily proteins are functional palmitoylating enzymes in mammalian cells. In the final stage of our manuscript preparation, another group reported that human DHHC1 and -10 (z11) show the palmitoylating activity in the yeast system (53). Thus, this in silico approach can complement experimental approaches to clarify protein functions regulated by post-translational modifications.