title: Neuronal palmitoyl acyl transferases exhibit distinct substrate specificity.
abstract: Palmitoylation, a post-translational modification of cysteine residues with the lipid palmitate, has recently emerged as an important mechanism for regulating protein trafficking and function. With the identification of 23 DHHC mammalian palmitoyl acyl transferases (PATs), a key question was the nature of substrate-enzyme specificity for these PATs. Using the acyl-biotin exchange palmitoylation assay, we compared the substrate specificity of four neuronal PATs, namely DHHC-3, DHHC-8, HIP14L (DHHC-13), and HIP14 (DHHC-17). Exogenous expression of enzymes and substrates in COS cells reveals that HIP14L and HIP14 modulate huntingtin palmitoylation, DHHC-8 modulates paralemmin-1 palmitoylation, and DHHC-3 shows the least substrate specificity. These in vitro data were validated by lentiviral siRNA-mediated knockdown of endogenous HIP14 and DHHC-3 in cultured rat cortical neurons. PATs require the presence of palmitoylated cysteines in order to interact with their substrates. To understand the elements that influence enzyme/substrate specificity further, we fused the HIP14 ankryin repeat domain to the N terminus of DHHC-3, which is not a PAT for huntingtin. This modification enabled DHHC-3 to behave similarly to HIP14 by modulating palmitoylation and trafficking of huntingtin. Taken together, this study indicates that individual PATs have specific substrate preference, determined by regulatory domains outside the DHHC domain of the enzymes.
authors.affiliation: Centre for Molecular Medicine and Therapeutics, University of British Columbia, Vancouver, BC V5Z 4H4, Canada.
authors.forename: Kun
authors.initials: K
authors.lastname: Huang
authors.forename: Shaun
authors.initials: S
authors.lastname: Sanders
authors.forename: Roshni
authors.initials: R
authors.lastname: Singaraja
authors.forename: Paul
authors.initials: P
authors.lastname: Orban
authors.forename: Tony
authors.initials: T
authors.lastname: Cijsouw
authors.forename: Pamela
authors.initials: P
authors.lastname: Arstikaitis
authors.forename: Anat
authors.initials: A
authors.lastname: Yanai
authors.forename: Michael R
authors.initials: MR
authors.lastname: Hayden
authors.forename: Alaa
authors.initials: A
authors.lastname: El-Husseini
mesh_terms: D000217:Acyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D006056:Golgi Apparatus / Q000201:enzymology; D000071058:Huntingtin Protein; D066298:In Vitro Techniques; D054878:Lipoylation / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism; D008958:Models, Molecular; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000201:enzymology*; D009687:Nuclear Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: C499524:Htt protein, rat; D000071058:Huntingtin Protein; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; C523858:Palm protein, rat; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D000217:Acyltransferases