A conserved eEF2 coding variant in SCA26 leads to loss of translational fidelity and increased susceptibility to proteostatic insult

Abstract

The autosomal dominant spinocerebellar ataxias (SCAs) are a genetically heterogeneous group of disorders exhibiting cerebellar atrophy and Purkinje cell degeneration whose subtypes arise from 31 distinct genetic loci. Our group previously published the locus for SCA26 on chromosome 19p13.3. In this study, we performed targeted deep sequencing of the critical interval in order to identify candidate causative variants in individuals from the SCA26 family. We identified a single variant that co-segregates with the disease phenotype that produces a single amino acid substitution in eukaryotic elongation factor 2. This substitution, P596H, sits in a domain critical for maintaining reading frame during translation. The yeast equivalent, P580H EF2, demonstrated impaired translocation, detected as an increased rate of -1 programmed ribosomal frameshift read-through in a dual-luciferase assay for observing translational recoding. This substitution also results in a greater susceptibility to proteostatic disruption, as evidenced by a more robust activation of a reporter gene driven by unfolded protein response activation upon challenge with dithiothreitol or heat shock in our yeast model system. Our results present a compelling candidate mutation and mechanism for the pathogenesis of SCA26 and further support the role of proteostatic disruption in neurodegenerative diseases.

Figure 1.

Cerebellar sections from an SCA26 patient show significant loss of Purkinje cells. (Left) Normal human cerebellar cortex with Purkinje cells at the boundary between the molecular and granular layers. (Right) SCA26 cerebellar cortex with loss of Purkinje cells and thinning of the molecular layer. Hematoxylin and eosin. Scale bar = 50 µm.

Figure 2.

C-to-A transversion at Chr19:3929097 (GI 83656775 1870) co-segregates with disease in SCA26. The C-to-A transversion produces a P596H substitution in the eEF2 protein.

Figure 3.

Proline 596 (H. sapiens) is conserved throughout many eukaryotes. Alignment of the EF2 protein sequence demonstrates that P596 is highly conserved throughout eukaryotes.

Figure 4.

WT and P596H eEF2 co-localize with rER. WT and P596H eEF2 were expressed via transient transfection in SH-SY5Y cells. (Left) WT eEF2 with a C-terminal 3 × HA epitope tag co-localizes strongly with rER in SH-SY5Y cells upon staining for HA and calnexin (rER), 156×. (Middle) P596H eEF2 with a C-terminal 3 × FLAG tag co-localizes strongly with rER in SH-SY5Y cells upon staining for FLAG and calnexin (rER), 170×. (Right) WT eEF2 with a C-terminal 3 × HA epitope tag and P596H eEF2 with a C-terminal 3 × FLAG tag co-localize strongly together in SH-SY5Y cells upon staining for HA and FLAG, 220×

Figure 5.

P580H EF2 shows a significant increase in the rate of frameshifting. The increase in −1 frameshifting is similar to that found in the positive control EF2 mutant, H699Q. The double mutant, P580H–H699Q, exhibits an even greater rate of frameshifting than either single mutation individually. *P < 5 × 10⁻⁴.

Figure 6.

P580H EF2-bearing yeast exhibit more robust unfolded protein response (UPR) induction in response to DTT and heat shock challenge. Yeast were transformed with pJC104, a plasmid containing four tandem repeats of the HAC1 binding element, that normally drives chaperone expression in response to UPR activation, driving β-galactosidase expression. Yeast were challenged either with 10 mm DTT or heat shock at 42°C for 15 min, and β -galactosidase activity was measured using the Promega Beta-glo assay. *P < 0.01.

Figure 7.

Yeast P580 lies in domain IV adjacent to the diphthamide-bearing moiety, H699. Diagram of the crystal structure of EF2 in complex with the yeast 40S ribosomal subunit (PDB: 1S1H), illustrating the proximity of P580 to both H699 and AA1492 and AA1493, all crucial elements in maintaining the fidelity of the translocation process. In a conformational shift of EF2 during translocation, the diphthamide moiety (essentially a 4-carbon chain extending from the imidazole ring of H699) swing down (arrow), breaking crucial hydrogen bonds between AA1492 and AA1493 and the minor groove of the codon-anticodon pair, stabilizing the codon–anticodon pair as it moves from the A- to the P-site within the ribosome, and allowing AA1492 and AA1493 to be available for the next codon–anticodon. P580 is only 10 Å from H699, highly suggesting that the bulky histidine side chain introduced in a P580H substitution would contribute steric hindrance and interfere with this delicate process. Figure courtesy of Rene Jørgensen, Statens Serum Institute, Copenhagen, Denmark.