title: In vitrocharacterization of sixSTUB1variants in spinocerebellar ataxia 16 reveals altered structural properties for the encoded CHIP proteins.
abstract: Spinocerebellar ataxia, autosomal recessive 16 (SCAR16) is caused by biallelic mutations in the STIP1 homology and U-box containing protein 1 (STUB1) gene encoding the ubiquitin E3 ligase and dimeric co-chaperone C-terminus of Hsc70-interacting protein (CHIP). It has been proposed that the disease mechanism is related to CHIP's impaired E3 ubiquitin ligase properties and/or interaction with its chaperones. However, there is limited knowledge on how these mutations affect the stability, folding, and protein structure of CHIP itself. To gain further insight, six previously reported pathogenicSTUB1variants (E28K, N65S, K145Q, M211I, S236T, and T246M) were expressed as recombinant proteins and studied using limited proteolysis, size-exclusion chromatography (SEC), and circular dichroism (CD). Our results reveal that N65S shows increased CHIP dimerization, higher levels of α-helical content, and decreased degradation rate compared with wild-type (WT) CHIP. By contrast, T246M demonstrates a strong tendency for aggregation, a more flexible protein structure, decreased levels of α-helical structures, and increased degradation rate compared with WT CHIP. E28K, K145Q, M211I, and S236T also show defects on structural properties compared with WT CHIP, although less profound than what observed for N65S and T246M. In conclusion, our results illustrate that someSTUB1mutations known to cause recessive SCAR16 have a profound impact on the protein structure, stability, and ability of CHIP to dimerizein vitro.These results add to the growing understanding on the mechanisms behind the disorder.
authors.affiliation: Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, Bergen, Norway.|Department of Clinical Science, University of Bergen, Bergen, Norway.
authors.forename: Yasaman
authors.initials: Y
authors.lastname: Pakdaman
authors.affiliation: Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, Bergen, Norway.
authors.forename: Monica
authors.initials: M
authors.lastname: Sanchez-Guixé
authors.affiliation: K.G. Jebsen Centre for Neuropsychiatric Disorders, Department of Biomedicine, University of Bergen, Bergen, Norway.
authors.forename: Rune
authors.initials: R
authors.lastname: Kleppe
authors.affiliation: Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, Bergen, Norway.
authors.forename: Sigrid
authors.initials: S
authors.lastname: Erdal
authors.affiliation: Department of Biomedicine, University of Bergen, Bergen, Norway.
authors.forename: Helene J
authors.initials: HJ
authors.lastname: Bustad
authors.affiliation: Department of Biomedical Laboratory Sciences and Chemical Engineering, Western Norway University of Applied Sciences, Bergen, Norway.
authors.forename: Lise
authors.initials: L
authors.lastname: Bjørkhaug
authors.affiliation: Department of Neurology, Haukeland University Hospital, Bergen, Norway.|Department of Clinical Medicine, University of Bergen, Bergen, Norway.
authors.forename: Kristoffer
authors.initials: K
authors.lastname: Haugarvoll
authors.affiliation: Department of Neurology, Haukeland University Hospital, Bergen, Norway.|Department of Clinical Medicine, University of Bergen, Bergen, Norway.
authors.forename: Charalampos
authors.initials: C
authors.lastname: Tzoulis
authors.affiliation: Department of Medical Genetics, Oslo University Hospital, Oslo, Norway.
authors.forename: Ketil
authors.initials: K
authors.lastname: Heimdal
authors.affiliation: Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, Bergen, Norway.|K.G. Jebsen Centre for Neuropsychiatric Disorders, Department of Clinical Science, University of Bergen, Bergen, Norway.
authors.forename: Per M
authors.initials: PM
authors.lastname: Knappskog
authors.affiliation: Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, Bergen, Norway.|K.G. Jebsen Centre for Neuropsychiatric Disorders, Department of Clinical Science, University of Bergen, Bergen, Norway.
authors.forename: Stefan
authors.initials: S
authors.lastname: Johansson
authors.affiliation: Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, Bergen, Norway ingvild.aukrust@helse-bergen.no.|Department of Clinical Science, University of Bergen, Bergen, Norway.
authors.forename: Ingvild
authors.initials: I
authors.lastname: Aukrust
mesh_terms: D006801:Humans; D009154:Mutation; D066329:Protein Aggregates; D011487:Protein Conformation; D055503:Protein Multimerization; D055550:Protein Stability; D058767:Protein Unfolding; D059748:Proteolysis; D020754:Spinocerebellar Ataxias / Q000235:genetics* / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D066329:Protein Aggregates; C467728:STUB1 protein, human; D044767:Ubiquitin-Protein Ligases
keywords: CHIP; co-chaperone; protein aggregation; protein misfolding; ubiquitin ligases