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. 2014 Sep 26:9:146.
doi: 10.1186/s13023-014-0146-0.

STUB1 mutations in autosomal recessive ataxias - evidence for mutation-specific clinical heterogeneity

Ketil Heimdal, Monica Sanchez-Guixé, Ingvild Aukrust, Jens Bollerslev, Ove Bruland, Greg Eigner Jablonski, Anne Kjersti Erichsen, Einar Gude, Jeanette A Koht, Sigrid Erdal, Torunn Fiskerstrand, Bjørn Ivar Haukanes, Helge Boman, Lise Bjørkhaug, Chantal M E Tallaksen, Per M Knappskog, Stefan Johansson

STUB1 mutations in autosomal recessive ataxias - evidence for mutation-specific clinical heterogeneity

Ketil Heimdal et al. Orphanet J Rare Dis. .

Abstract

Background: A subset of hereditary cerebellar ataxias is inherited as autosomal recessive traits (ARCAs). Classification of recessive ataxias due to phenotypic differences in the cerebellum and cerebellar structures is constantly evolving due to new identified disease genes. Recently, reports have linked mutations in genes involved in ubiquitination (RNF216, OTUD4, STUB1) to ARCA with hypogonadism.

Methods and results: With a combination of homozygozity mapping and exome sequencing, we identified three mutations in STUB1 in two families with ARCA and cognitive impairment; a homozygous missense variant (c.194A > G, p.Asn65Ser) that segregated in three affected siblings, and a missense change (c.82G > A, p.Glu28Lys) which was inherited in trans with a nonsense mutation (c.430A > T, p.Lys144Ter) in another patient. STUB1 encodes CHIP (C-terminus of Heat shock protein 70 - Interacting Protein), a dual function protein with a role in ubiquitination as a co-chaperone with heat shock proteins, and as an E3 ligase. We show that the p.Asn65Ser substitution impairs CHIP's ability to ubiquitinate HSC70 in vitro, despite being able to self-ubiquitinate. These results are consistent with previous studies highlighting this as a critical residue for the interaction between CHIP and its co-chaperones. Furthermore, we show that the levels of CHIP are strongly reduced in vivo in patients' fibroblasts compared to controls.

Conclusions: These results suggest that STUB1 mutations might cause disease by impacting not only the E3 ligase function, but also its protein interaction properties and protein amount. Whether the clinical heterogeneity seen in STUB1 ARCA can be related to the location of the mutations remains to be understood, but interestingly, all siblings with the p.Asn65Ser substitution showed a marked appearance of accelerated aging not previously described in STUB1 related ARCA, none display hormonal aberrations/clinical hypogonadism while some affected family members had diabetes, alopecia, uveitis and ulcerative colitis, further refining the spectrum of STUB1 related disease.

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Figures

Figure 1
Figure 1
The dual role of CHIP as both a co-chaperone and an E3 ligase targeting misfolded proteins to proteasome degradation. CHIP binds to HSC70 by its TPR domain and bridges HSC70 to the misfolded protein. An E2 enzyme binds to the U-box domain and CHIP catalyses the ubiquitination reaction by attaching ubiquitin to the HSC70-client protein, targeting it to the proteasome. HSC70 and CHIP are also ubiquitinated, however this is not a signal for proteasomal degradation, but might play a role in their self-regulation.
Figure 2
Figure 2
Cerebral MRI (1.5 Tesla). (A) Cerebral MRI (T1 serie, midline sagittal) of the proband in Family 1 at the time of investigation. Severe atrophy of the whole cerebellum and the anterior part of the corpus callosum. (B) Same examination, but T2 axial scan at the level of the superior cerebellar peduncle. There is an atrophy of both cerebellar hemispheres with widened sulci, and vermis atrophy. The fourth ventricle is moderately dilated. There are a few diffuse hyperintensity signals in the brainstem. The cerebral hemispheres look normal.
Figure 3
Figure 3
Functional domains of the CHIP protein and illustration of amino acid substitutions/deletions found in patients. Presentation of the CHIP E3-Ligase with its three functional domains: Tetratricopeptide repeat (TPR), coiled-coil (CC) and U-box. Patient 1 (P1[II-1]) is homozygous for a point mutation resulting in CHIP-N65S located in the TPR domain. Patient 2 (P2) is compound heterozygous for two point mutations; one resulting in CHIP-E28K in the TPR domain and another causing the deletion mutant CHIP-K144*, a truncated protein lacking most of the CC domain and the entire U-box domain. The mutation resulting in the CHIP-T246M mutant is located in the U-box domain and has previously been described [5] and indicated here in Patient 3 (P3).
Figure 4
Figure 4
Differential levels of CHIP protein in fibroblasts from patients. Fibroblasts from Patient 1 (P1[II-1]) and Patient 2 (P2) show lower steady-state levels of CHIP protein compared with normal fibroblasts (WT), as analyzed by SDS-PAGE and immunoblotting using CHIP-specific antibody. In addition, the band corresponding to CHIP-N65S mutant in P1 reveals slightly faster migration rate on SDS-PAGE compared to the CHIP-WT band, probably due to protein conformational changes induced by the mutation. In fibroblasts from the father of Patient 1 (P1[I-1]), a heterozygous carrier of the N65S mutant allele, a double band can be observed (CHIP-WT and CHIP-N65S). In P2 a weak band most likely corresponding to CHIP-E28K is detected, while the band corresponding to the lower molecular weight CHIP-K144* form of ~16 kDa is not observed on this blot. Actin-specific protein bands are shown to compare the relative amounts of total protein loaded per lane.
Figure 5
Figure 5
CHIP-N65S causes a migration shift when analyzed by SDS-PAGE. (A) CHIP-WT and CHIP-N65S were translated in a TNT coupled transcription/translation system in the presence of [35S]Met, as described in Material & Methods. Samples were analyzed by SDS-PAGE and autoradiography. A double band can be observed for CHIP-WT, in which the lower band migrates at the same rate as CHIP-N65S. CTL; empty vector as negative control. (B) CHIP expression of transfected HEK293 cells with CHIP-WT or CHIP-N65S, tagged with V5 and His, and detected by SDS-PAGE and immunoblotting using anti-CHIP. Endogenous CHIP is observed at 35 kDa. The shift is observed only for transfected CHIP. A secondary isoform of CHIP appears at 32 kDa, lacking the first 72 amino acids, only observed in vitro [9]. CTL; empty vector as negative control. (C) Recombinant WT and CHIP variants expressed and purified from E. coli as MBP-fusion proteins and cleaved by TEV protease as described in Materials & Methods. Samples were analyzed by SDS-PAGE and coomassie staining. The migration shift is only observed for CHIP-N65S. CHIP-K144* appears at a lower molecular weight (truncated version of 16 kDa).
Figure 6
Figure 6
Different E3 ubiquitin ligase activity is observed for various CHIP mutants. In vitro ubiquitination was assessed using CHIP-WT and CHIP-mutant forms as E3 ligases and HSC70 recombinant protein as substrate for ubiquitination. Samples were analyzed by SDS-PAGE followed by immunoblotting using HSC70- and CHIP-specific antibodies. A reaction with WT-CHIP and without ubiquitin was used as a negative control (CTL). Both the levels of ubiquitination of HSC70 and auto-ubiquitination of CHIP itself was investigated using MBP-CHIP fusion protein (A) and tag-free (cleaved) CHIP (B). The lower molecular weight CHIP-K144* deletion mutant were detected as a MBP fusion protein, but not as a tag-free mutant, presumably due to reduced protein stability after removal of the MBP. The asterisks indicate CHIP forms mostly observed for CHIP-T246M and possibly representing protein dimers.
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