High-Throughput Screening for Ligands of the HEPN Domain of Sacsin.

Xinlu Li,Zheng Dai,Zheping Hu,Guennadi Kozlov,Bernard Brais,Marie Ménade,Peter S Mcpherson,Kalle Gehring,Oleg Y Dmitriev

doi:10.1371/journal.pone.0137298

Abstract

Sacsin is a large protein implicated in the neurodevelopmental and neurodegenerative disease autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS), which features the loss of Purkinje neurons in the cerebellum. Although the domain architecture of sacsin suggests that it is a neuronal chaperone assisting in protein quality control, the precise function of sacsin remains elusive. Using fluorescence polarization (FP) assays, we confirmed that the HEPN domain of sacsin binds to nucleotides with low micromolar affinities. FP competition assays with a variety of nucleotides and nucleotide analogs revealed that the binding is primarily mediated by the phosphate groups of nucleotides. A high-throughput screen subsequently identified novel small molecule ligands of HEPN, providing new chemical probes for cell culture studies and drug development. Together, the results are consistent with the HEPN domain contributing to the functional activity of sacsin by binding to nucleotides or other multiply charged anionic compounds in neurons.

Highlights

Mutations in sacsin, a large 4579 amino acid protein, are causative of the neurodevelopmental and neurodegenerative disease autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS)
We developed an fluorescence polarization (FP) assay with the higher eukaryotes and prokaryotes nucleotide-binding domain (HEPN) domain of sacsin and fluorescein-labeled ATP to investigate the ligand-binding properties of sacsin
The FP competition assay performed with nucleotides and nucleotide analogs revealed that HEPN preferentially binds to nucleotides with higher number of accessible phosphate groups

Summary

Introduction

A large 4579 amino acid protein, are causative of the neurodevelopmental and neurodegenerative disease autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS). The disease-associated gene (SACS) was discovered by Engert et al in 2000, which allowed for molecular identification of the disease in a large number of patients [2]. ARSACS is currently the second most common recessive ataxia in the world, with over 170 different SACS mutations identified in patients from more than 20 countries [3]. The domain architecture of sacsin suggests that it is a chaperone contributing to protein quality control. At its N-terminus, Sacsin contains a ubiquitin-like (UbL) domain that was shown to interact with the 26S proteasome and mediate protein degradation [4]. The bulk of the protein, over 80% of the amino acids, consists of three large repeating regions named sacsin

Methods

Results

Conclusion