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Neuronal palmitoyl acyl transferases exhibit distinct substrate specificity

Kun Huang^*, Shaun Sanders^*,1, Roshni Singaraja^*,1, Paul Orban^*, Tony Cijsouw, Pamela Arstikaitis, Anat Yanai^*, Michael R. Hayden^*,,2 and Alaa El-Husseini^,3

^* Centre for Molecular Medicine and Therapeutics and

Department of Psychiatry, University of British Columbia, Vancouver, British Columbia, Canada;

Department of Functional Genomics, Center for Neurogenomics and Cognitive Research, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands; and

Children and Family Research Institute, British Columbia Children’s Hospital, Vancouver, British Columbia, Canada

² Correspondence: Centre for Molecular Medicine and Therapeutics, University of British Columbia, Vancouver, BC V5Z 4H4, Canada. E-mail: mrh{at}cmmt.ubc.ca

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.—Huang, K., Sanders, S., Singaraja, R., Orban, P., Cijsouw, T., Arstikaitis, P., Yanai, A., Hayden, M. R., El-Husseini, A. Neuronal palmitoyl acyl transferases exhibit distinct substrate specificity.

Key Words: acyl-biotin exchange • DHHC proteins • HIP14 • HIP14L • huntingtin • SNAP25

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