Identification of Binding Sites in Huntingtin for the Huntingtin Interacting Proteins HIP14 and HIP14L

Shaun S Sanders,Katherine K N Mui,Michael R Hayden,Liza M Sutton,Hiroyoshi Ariga

doi:10.1371/journal.pone.0090669

Shaun S Sanders, Katherine K N Mui + Show 3 more

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https://doi.org/10.1371/journal.pone.0090669

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Abstract

Huntington disease is an adult onset neurodegenerative disease characterized by motor, cognitive, and psychiatric dysfunction, caused by a CAG expansion in the HTT gene. Huntingtin Interacting Protein 14 (HIP14) and Huntingtin Interacting Protein 14-like (HIP14L) are palmitoyl acyltransferases (PATs), enzymes that mediate the post-translational addition of long chain fatty acids to proteins in a process called palmitoylation. HIP14 and HIP14L interact with and palmitoylate HTT and are unique among PATs as they are the only two that have an ankyrin repeat domain, which mediates the interaction between HIP14 and HTT. These enzymes show reduced interaction with and palmitoylation of mutant HTT, leading to increased mutant HTT inclusion formation and toxicity. The interaction between HIP14 and HTT goes beyond that of only an enzyme–substrate interaction as HTT is essential for the full enzymatic activity of HIP14. It is important to further understand and characterize the interactions of HTT with HIP14 and HIP14L to guide future efforts to target and enhance this interaction and increase enzyme activity to remediate palmitoylation of HTT and their substrates, as well as to understand the relationship between the three proteins. HIP14 and HIP14L have been previously shown to interact with HTT amino acids 1–548. Here the interaction of HIP14 and HIP14L with N- and C-terminal HTT 1–548 deletion mutations was assessed. We show that HTT amino acids 1–548 were sufficient for full interaction of HTT with HIP14 and HIP14L, but partial interaction was also possible with HTT 1–427 and HTT 224–548. To further characterize the binding domain we assessed the interaction of HIP14-GFP and HIP14L-GFP with 15Q HTT 1-548Δ257-315. Both enzymes showed reduced but not abolished interaction with 15Q HTT 1-548Δ257-315. This suggests that two potential binding domains exist, one around residues 224 and the other around 427, for the PAT enzymes HIP14 and HIP14L.

Highlights

Huntington disease (HD) is an autosomal dominant neurodegenerative disease characterized by motor, cognitive, and psychiatric dysfunction with onset in mid-life and death following, on average, 20 years later [1,2]
The same interaction pattern was observed with a Huntingtin Interacting Protein 14 (HIP14)-FLAG tagged construct and HTT 1–548 did not interact with GFP alone
HIP14 and Huntingtin Interacting Protein 14-like (HIP14L) are HTT interacting and palmitoylating proteins and their interaction and palmitoylation of HTT are decreased in the presence of mutant HTT (mHTT) [5,6,13]

Summary

Introduction

Huntington disease (HD) is an autosomal dominant neurodegenerative disease characterized by motor, cognitive, and psychiatric dysfunction with onset in mid-life and death following, on average, 20 years later [1,2]. One approach that has been taken to determine the functions of the HTT protein and to understand the pathogenesis of HD is to identify and characterize HTT interacting proteins and to determine how these interactions are altered in the presence of the HD mutation. Huntingtin Interacting Protein 14 (HIP14; ZDHHC17; NP_056151) was first identified as a HTT interactor in a yeast 2-hybrid screen. HIP14 was further shown to interact with HTT in mammalian systems and to interact less with mutant HTT (mHTT) [4,5].

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