title: High-Throughput Screening for Ligands of the HEPN Domain of Sacsin.
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.
authors.affiliation: Department of Biochemistry, McGill University, Montreal, Quebec, Canada; Groupe de recherche axé sur la structure des protéines, Montreal, Quebec, Canada.
authors.forename: Xinlu
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Biochemistry, McGill University, Montreal, Quebec, Canada; Groupe de recherche axé sur la structure des protéines, Montreal, Quebec, Canada.
authors.forename: Marie
authors.initials: M
authors.lastname: Ménade
authors.affiliation: Department of Biochemistry, McGill University, Montreal, Quebec, Canada; Groupe de recherche axé sur la structure des protéines, Montreal, Quebec, Canada.
authors.forename: Guennadi
authors.initials: G
authors.lastname: Kozlov
authors.affiliation: Department of Biochemistry, McGill University, Montreal, Quebec, Canada; Groupe de recherche axé sur la structure des protéines, Montreal, Quebec, Canada.
authors.forename: Zheping
authors.initials: Z
authors.lastname: Hu
authors.affiliation: Department of Biochemistry, McGill University, Montreal, Quebec, Canada; Groupe de recherche axé sur la structure des protéines, Montreal, Quebec, Canada.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Dai
authors.affiliation: Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada.
authors.forename: Peter S
authors.initials: PS
authors.lastname: McPherson
authors.affiliation: Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada.
authors.forename: Bernard
authors.initials: B
authors.lastname: Brais
authors.affiliation: Department of Biochemistry, McGill University, Montreal, Quebec, Canada; Groupe de recherche axé sur la structure des protéines, Montreal, Quebec, Canada.
authors.forename: Kalle
authors.initials: K
authors.lastname: Gehring
mesh_terms: D004492:Edetic Acid / Q000737:chemistry; D016231:Fluorescence Polarization Immunoassay; D006360:Heat-Shock Proteins / Q000737:chemistry*; D057166:High-Throughput Screening Assays; D006801:Humans; D008024:Ligands; D008274:Magnesium / Q000737:chemistry; D009711:Nucleotides / Q000737:chemistry; D017434:Protein Structure, Tertiary
chemical_list: D006360:Heat-Shock Proteins; D008024:Ligands; D009711:Nucleotides; C404403:SACS protein, human; D004492:Edetic Acid; D008274:Magnesium