Abstract
Post-translational modification by the lipid palmitate is crucial for the correct targeting and function of many proteins. Here we show that huntingtin (htt) is normally palmitoylated at cysteine 214, which is essential for its trafficking and function. The palmitoylation and distribution of htt are regulated by the palmitoyl transferase huntingtin interacting protein 14 (HIP14). Expansion of the polyglutamine tract of htt, which causes Huntington disease, results in reduced interaction between mutant htt and HIP14 and consequently in a marked reduction in palmitoylation. Mutation of the palmitoylation site of htt, making it palmitoylation resistant, accelerates inclusion formation and increases neuronal toxicity. Downregulation of HIP14 in mouse neurons expressing wild-type and mutant htt increases inclusion formation, whereas overexpression of HIP14 substantially reduces inclusions. These results suggest that the expansion of the polyglutamine tract in htt results in decreased palmitoylation, which contributes to the formation of inclusion bodies and enhanced neuronal toxicity.
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Acknowledgements
We thank O. Sadiq and E. Eyu for technical assistance. M.R.H. is supported by grants from the Canadian Institutes for Health Research, the Huntington Disease Society of America, the Jack and Doris Brown Foundation and the Huntington Society of Canada. A.E.H. is supported by grants from the Canadian Institutes for Health Research, the EJLB foundation and Neuroscience Canada. R.C.D. and W.N.G. are supported by funding from the National Institute of Neurological Diseases and Stroke, the National Institute of Drug Abuse and the Alzheimer's Association. D.C.R. is funded by a Wellcome Trust Senior Fellowship in Clinical Science, an M.R.C. programme grant and E.U. Framework VI (EUROSCA). A.Y. is supported by funding from the Michael Smith Foundation for Health Research. KH is supported by University Graduate Fellowship and HighQ. M.R.H. and A.E.H. are supported by funding from the HighQ foundation and are investigators of the Fundamental Innovation in Neurodegenerative Diseases (FIND) Research Infrastructure Unit, funded by the Michael Smith Foundation for Health Research. M.R.H. holds a Canada Research Chair in Human Genetics and is a University Killam Professor.
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A.Y. performed all DNA manipulations to generate truncated and full-length mutant htt and HIP14 proteins, and performed most of the [3H]palmitoylation assays and data analyses. K.H. conducted all the Btn-BMCC palmitoylation assays for full-length htt, scored cells for inclusions in htt-transfected COS cells and neurons, designed and characterized HIP14 siRNA, performed all experiments (and corresponding data analysis) in which the alteration of htt trafficking was investigated by knocking down HIP14, conducted the virus production and infection experiments, and scored for TUNEL assay. R.K. performed endogenous htt [3H]palmitoylation assay, scored inclusions in neurons transfected with full-length htt, and performed fragment-htt toxicity assay and the corresponding data analysis. R.R.S. and L.G. performed the htt and HIP14 coimmunoprecipitation experiment. P.C.O. generated HIP14 siRNA lentiviral constructs and supervised the virus production experiment. P.A. conducted the time-lapse experiments. A.M. assisted in the characterization of full-length htt palmitoylation. C.M.C. and L.A.R. were involved in developing the NMDA-induced excitotoxicity TUNEL assay. R.K. and K.H. conducted the TUNEL assays. W.N.G. and R.C.D. developed and supervised the Btn-BMCC palmitoylation assays. B.R. and D.C.R. performed toxicity assays in COS cells. M.R.H., A.E.H., A.Y. and K.H. wrote the manuscript. M.R.H. and A.E.H. supervised the project.
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Supplementary Fig. 2
Altered distribution of palmitoylation-resistant htt in HEK-293 cells. (PDF 1609 kb)
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Yanai, A., Huang, K., Kang, R. et al. Palmitoylation of huntingtin by HIP14is essential for its trafficking and function. Nat Neurosci 9, 824–831 (2006). https://doi.org/10.1038/nn1702
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DOI: https://doi.org/10.1038/nn1702
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