@misc{oai:ir.soken.ac.jp:00002712,
 author = {奥, 慎一郎 and オク, シンイチロウ and OKU, Shinichiro},
 month = {2016-02-17, 2016-02-17},
 note = {  Posttranslational modifications, including phosphorylation, ubiquitination, glycosylationand lipidation, provide proteins with additional regulatory systems beyond genomic information.Protein palmitoylation is the most common reversible lipid modification and plays significant rolesin protein trafficking and function. Palmitoylation modifies numerous classes of proteins includingsignaling proteins, synaptic scaffolding proteins, and various transmembrane proteins. Identificationof palmitoyl substrates is important for elucidating physiological roles of protein palmitoylation.Recently, a large family of 23 DHHC proteins has emerged as mammalian palmitoylating enzymes,and several enzyme-substrate pairs have begun to be reported. Also, the recent development of thepalmitoyl-proteomic methods has identified a lot of novel substrates. However, the conventionalproteomic analysis depends on the biochemical property of the target proteins and thereby may omitimportant substrates to understand whole “palmitome.”
   Here, to systematically identify novel palmitoyl substrates, the author performed in silicowhole-genome screening using CSS-Palm 2.0 program, which is a free software for palmitoylationsite prediction. The author lined up about 60,000 mouse protein sequences from UniProt databaseaccording to the CSS-Palm scores, and selected 17 candidates as novel palmitoyl substrates, whichaccumulate at the specific membrane domains like synaptic membranes, and cell-to-cell andcell-to-substratum junctions. Then, the author experimentally verified their palmitoylation. Theauthor investigated by the metabolic labeling method whether these candidate proteins are actuallypalmitoylated by a general palmitoylating enzyme, DHHC3, in HEK293 cells and found thatDHHC3 palmitoylated 10 candidates. These proteins included: a presynaptic protein, Syd-1;postsynaptic proteins, transmembrane AMPA receptor regulatory proteins (TARP) γ2, TARP γ8,cornichon-2, CaMKIIα and neurochondrin (Ncdn); a focal adhesion protein, zyxin; transientreceptor potential (TRP) channels, M8 and C1; and G-protein coupled receptor, orexin receptortype2. Of these novel substrates, the author selected four proteins, Syd-1, Ncdn, TARP γ8 andCaMKIIα, and confirmed that all tested endogenous proteins were palmitoylated in hippocampalneurons by the acyl-biotinyl exchange (ABE) method. By the subsequent screening of 23 DHHCproteins, the author found that Ncdn was robustly palmitoylated by the DHHC1/10 subfamily,whose palmitoylating activity has not yet been reported, and the DHHC3/7 subfamily. Therefore,the author focused on this novel substrate-enzyme pair, Ncdn and DHHC1/10. Ncdn is aneuron-specific cytosolic protein and regulates neurite-outgrowth and mGluR-related synapticplasticity. Also, genetic evidence using the knockout mouse indicates that Ncdn associates withepileptic seizure and schizophrenia.

As predicted by CSS-Palm 2.0, Ncdn was palmitoylated at the amino-terminal cysteine residues (atpositions 3 and 4), which were shared with both DHHC1/10 and DHHC3/7 subfamilies. DHHC3palmitoylated many proteins including Ncdn as well as previously reported palmitoyl substrates,GluA2 and Gαq, whereas DHHC10 palmitoylated only Ncdn. DHHC1/10 subfamily, but notDHHC3/7 subfamily, was co-purified with Ncdn from the brain lysate indicating the physicalinteraction between DHHC1/10 subfamily members and Ncdn. Overexpression of DHHC1/10subfamily specifically relocalized Ncdn near peri-nuclear structures in a palmitoylation-dependentmanner in COS7 cells. Thus, DHHC3/7 subfamily and DHHC1/10 subfamily may differentlyrecognize Ncdn as a palmitoyl substrate.

   The author found that Ncdn expression was increased during dendrite development in culturedneurons and that Ncdn protein was biochemically fractionated into both cytosolic and membranefractions of the brain tissue. Ncdn was specifically localized at somato-dendritic regions inhippocampal neurons and partly co-localized with HA-tagged DHHC1/10 subfamily in dendriticshafts and dendritic spines. Palmitoylation-deficient mutant of Ncdn delocalized from dendriticmembrane structures in hippocampal neurons. Furthermore, knockdown of DHHC1, but neitherDHHC3 nor DHHC10, significantly reduced dendritic localization of Ncdn, indicating that DHHC1is a physiological palmitoylating enzyme to regulate the specific Ncdn localization in neurons.Future functional studies on DHHC1-mediated Ncdn palmitoylation will clarify how this novelsubstrate-enzyme pair is involved in neuronal differentiation and functions such as dendritedevelopment.

&nbsp Finally, the author systematically screened mouse whole-genome for palmitoyl-substrates throughcomputational prediction (in silico proteomics), and found that Ncdn is the first substrate for theDHHC1/10 subfamily, and that DHHC1 plays a critical role in proper localization of Ncdn todendrites in neurons. Thus, this study indicates that in silico approach is useful for the discovery ofnovel palmitoyl substrates and complementally functions with previous experimental methods.Increased information of palmitoyl substrate-enzyme pairs will be the solid foundation forunderstanding molecular mechanisms and regulatory roles of protein palmitoylation in the dynamiccellular functions, and will importantly reveal consensus sequences for palmitoylation based on theDHHC subfamily-specific rules for substrate recognition., application/pdf, 総研大甲第1463号},
 title = {Identification of palmitoyl substrate-enzyme pairs in neurons through in silico proteomics},
 year = {}
}