title: 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.
abstract: 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, Ca(2+)/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.
authors.affiliation: Division of Membrane Physiology, Department of Cell Physiology, National Institute for Physiological Sciences, National Institutes of Natural Sciences, Okazaki, Aichi 444-8787, Japan.
authors.forename: Shinichiro
authors.initials: S
authors.lastname: Oku
authors.forename: Naoki
authors.initials: N
authors.lastname: Takahashi
authors.forename: Yuko
authors.initials: Y
authors.lastname: Fukata
authors.forename: Masaki
authors.initials: M
authors.lastname: Fukata
mesh_terms: D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D011992:Endosomes / Q000235:genetics / Q000378:metabolism*; D022001:Focal Adhesions / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D054878:Lipoylation / Q000502:physiology*; D051379:Mice; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D016501:Neurites / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D040901:Proteomics / Q000379:methods*; D051381:Rats; D012984:Software; D013379:Substrate Specificity; D013569:Synapses / Q000235:genetics / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D009419:Nerve Tissue Proteins; C119189:neurochondrin; D000217:Acyltransferases; C574550:ZDHHC3 protein, human; C486179:ZDHHC1 protein, human; D020696:rab5 GTP-Binding Proteins; D003545:Cysteine
keywords: DHHC Protein; Endosomes; Enzymes; Neurobiology; Neurochondrin; Neuroscience; Post Translational Modification; Protein Palmitoylation; Rab5