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. 2021 May 30;22(11):5870.
doi: 10.3390/ijms22115870.

Genetic Dominant Variants in STUB1, Segregating in Families with SCA48, Display In Vitro Functional Impairments Indistinctive from Recessive Variants Associated with SCAR16

Yasaman Pakdaman  1   2 , Siren Berland  1 , Helene J Bustad  3 , Sigrid Erdal  1 , Bryony A Thompson  4   5 , Paul A James  6   7 , Kjersti N Power  8 , Ståle Ellingsen  9 , Martin Krooni  10   11 , Line I Berge  10   12 , Adrienne Sexton  6   7 , Laurence A Bindoff  13   14 , Per M Knappskog  1   2 , Stefan Johansson  1   2 , Ingvild Aukrust  1   2
Affiliations

Genetic Dominant Variants in STUB1, Segregating in Families with SCA48, Display In Vitro Functional Impairments Indistinctive from Recessive Variants Associated with SCAR16

Yasaman Pakdaman et al. Int J Mol Sci. .

Abstract

Variants in STUB1 cause both autosomal recessive (SCAR16) and dominant (SCA48) spinocerebellar ataxia. Reports from 18 STUB1 variants causing SCA48 show that the clinical picture includes later-onset ataxia with a cerebellar cognitive affective syndrome and varying clinical overlap with SCAR16. However, little is known about the molecular properties of dominant STUB1 variants. Here, we describe three SCA48 families with novel, dominantly inherited STUB1 variants (p.Arg51_Ile53delinsProAla, p.Lys143_Trp147del, and p.Gly249Val). All the patients developed symptoms from 30 years of age or later, all had cerebellar atrophy, and 4 had cognitive/psychiatric phenotypes. Investigation of the structural and functional consequences of the recombinant C-terminus of HSC70-interacting protein (CHIP) variants was performed in vitro using ubiquitin ligase activity assay, circular dichroism assay and native polyacrylamide gel electrophoresis. These studies revealed that dominantly and recessively inherited STUB1 variants showed similar biochemical defects, including impaired ubiquitin ligase activity and altered oligomerization properties of the CHIP. Our findings expand the molecular understanding of SCA48 but also mean that assumptions concerning unaffected carriers of recessive STUB1 variants in SCAR16 families must be re-evaluated. More investigations are needed to verify the disease status of SCAR16 heterozygotes and elucidate the molecular relationship between SCA48 and SCAR16 diseases.

Keywords: CHIP; E3 ubiquitin ligase; SCA48; SCAR16; STUB1; spinocerebellar ataxia.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure A1
Figure A1
Secondary structure content of wild-type and mutant MBP–CHIP proteins. (A) Far-UV spectra were recorded in the range of 0–260 nm. Final graph was prepared using GraphPad Prism after smoothing. (B) Estimation of the secondary structure content in mutant MBP–CHIP proteins compared to the wild-type, using BeSTSel.
Figure 1
Figure 1
Pedigrees of three families with SCA48. Clinically affected individuals are indicated by filled symbols. Arrows denote the probands. Genetic statuses of examined individuals are provided below the symbols, where “mut” represents the mutant allele and “wt” represents the wild-type allele.
Figure 2
Figure 2
Ubiquitination activity of wild-type and mutant MBP–CHIP proteins. In vitro ubiquitination activity test was performed on MBP–CHIP recombinant proteins using Hsc70 (A) and MBP–CHIP (B) as the substrates. A separate wild-type reaction without ubiquitin was used as the negative control (CNTL) for each assay.
Figure 3
Figure 3
Thermal unfolding profiles of wild-type and mutant MBP–CHIP proteins monitored by circular dichroism spectroscopy. Thermal unfolding curves were obtained for MBP–CHIP proteins by following changes in molar ellipticity at 222 nm wavelength as a function of temperature. Wild-type MBP–CHIP shows three distinct transitions at approximately 42.8 °C, 54.6 °C, and 65.1 °C, as indicated by arrows, whereas all three variants show loss in both ellipticity and transitions.
Figure 4
Figure 4
Oligomerization states of wild-type and mutant MBP–CHIP proteins. Native structures of 5 µg MBP–CHIP proteins were studied by native–PAGE on a 10% gel. Protein bands were visualized by Coomassie blue staining and analyzed by Fiji (ImageJ) software [15].
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