Most mutations that cause spinocerebellar ataxia autosomal recessive type 16 (SCAR16) destabilize the protein quality-control E3 ligase CHIP

Abstract

The accumulation of misfolded proteins promotes protein aggregation and neuronal death in many neurodegenerative diseases. To counteract misfolded protein accumulation, neurons have pathways that recognize and refold or degrade aggregation-prone proteins. One U-box-containing E3 ligase, C terminus of Hsc70-interacting protein (CHIP), plays a key role in this process, targeting misfolded proteins for proteasomal degradation. CHIP plays a protective role in mouse models of neurodegenerative disease, and in humans, mutations in CHIP cause spinocerebellar ataxia autosomal recessive type 16 (SCAR16), a fatal neurodegenerative disease characterized by truncal and limb ataxia that results in gait instability. Here, we systematically analyzed CHIP mutations that cause SCAR16 and found that most SCAR16 mutations destabilize CHIP. This destabilization caused mutation-specific defects in CHIP activity, including increased formation of soluble oligomers, decreased interactions with chaperones, diminished substrate ubiquitination, and reduced steady-state levels in cells. Consistent with decreased CHIP stability promoting its dysfunction in SCAR16, most mutant proteins recovered activity when the assays were performed below the mutants' melting temperature. Together, our results have uncovered the molecular basis of genetic defects in CHIP function that cause SCAR16. Our insights suggest that compounds that improve the thermostability of genetic CHIP variants may be beneficial for treating patients with SCAR16.

Keywords: molecular chaperone; neurodegenerative disease; protein misfolding; proteostasis; ubiquitin ligase.

Figure 1.

Mutations in CHIP that cause SCAR16 decrease CHIP's capacity to ubiquitinate substrate. A, schematic of the domains of CHIP illustrating positions of SCAR16 mutants. B, illustration of SCAR16 mutations (red) imposed on the crystal structure of CHIP (Protein Data Bank code 2c2l). The domains of CHIP are color-coded to match A. C, Western blot of Hsp70 ubiquitination by wildtype CHIP and SCAR16 mutants. In vitro ubiquitination assays were performed with wildtype or SCAR16 mutants for 1 h at 37 °C using UbcH5c as the E2. D, Coomassie stain of CHIP variants to standardize CHIP loading for in vitro ubiquitination assays. Ub, ubiquitin.

Figure 2.

Some SCAR16 mutants have defects in chaperone binding. A, crystal structure of the TPR domain of CHIP bound to a C-terminal peptide that corresponds to the C terminus of Hsp90. Asn-65 interacts with the Hsp90 peptide and is indicated in red. B, binding curves for FP assays of wildtype and SCAR16 mutants at 37 °C. Increasing amounts of wildtype or SCAR16 mutants were incubated with 20 nm rhodamine-labeled peptide corresponding to the C terminus of Hsc70. Samples were incubated for 30 min at 37 °C prior to data collection. Error bars represent S.D. mP, millipolarization units.

Figure 3.

Most SCAR16 mutants retain the ability to form free polyubiquitin chains. A, fluorogram of TR-FRET ubiquitination assays performed with wildtype or SCAR16 mutants. Ubiquitination reactions were carried out for 1 h at 37 °C prior to SDS-PAGE and imaging. B, quantification of TR-FRET ubiquitination assays. TR-FRET ubiquitination assays were performed for 1 h at 37 °C prior to being read on a plate reader. Error bars represent S.D., ** indicates p < 0.001, and **** indicates p < 0.0001 (n ≥ 3).

Figure 4.

The majority of SCAR16 mutants form soluble oligomers. A–C, 10 mm wildtype or SCAR16 CHIP mutants was allowed to equilibrate on ice for 1 h prior to being spun down at 20,000 relative centrifugal force for 30 min. Samples were then injected on a gel filtration column, and protein was observed by absorbance at 280 nm. Mutations in CHIP's TPR (A), linker (B), and U-box (C) domains are aligned in columns.

Figure 5.

The levels of most SCAR16 mutants are significantly decreased in HEK293 cells. A, Western blot of HEK293 cells expressing wildtype or SCAR16 mutants. Immunoblotting was performed with anti-myc or anti-tubulin antibodies as indicated. B, quantification of CHIP levels normalized to tubulin from experiments in A. Error bars indicate S.D., ** indicates p < 0.005, *** indicates p < 0.001, and **** indicates p < 0.0001 (n ≥ 3).

Figure 6.

Some SCAR16 mutants recover chaperone binding at 25 °C. A, binding curves for FP assays of wildtype and SCAR16 mutants at 25 °C. Increasing amounts of wildtype or SCAR16 mutants were incubated with 20 nm rhodamine-labeled peptide corresponding to the C terminus of Hsc70. Samples were incubated for 30 min at 25 °C prior to data collection. B–E, the L123V (B), N130I (C), M240T (D), and T246M (E) SCAR16 mutants all partially recovered their ability to bind chaperones at 25 °C compared with 37 °C. Error bars indicate S.D. mP, millipolarization units.

Figure 7.

Most SCAR16 mutants readily ubiquitinate substrate at 25 °C. Shown is a Western blot of Hsp70 ubiquitination by wildtype CHIP and SCAR16 mutants. In vitro ubiquitination assays were performed with wildtype or SCAR16 mutants for 1 h at 25 °C using UbcH5c as the E2. Ub, ubiquitin.