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An alpha II spectrin mutant peptide with unstable scaffold structure and increased sensitivity to calpain cleavage
- Source :
- Biochemical and Biophysical Research Communications. 581:68-73
- Publication Year :
- 2021
- Publisher :
- Elsevier BV, 2021.
-
Abstract
- A spontaneous missense mutation in the alpha II spectrin (αII) gene, replacing a highly conserved arginine 1098 with the glutamine (R1098Q), causes progressive neurodegeneration in heterozygous mutant mice. The molecular mechanism underlying this phenotype is unknown but the accumulation of 150kD αII breakdown products in brains of homozygous mutant embryos suggests an imbalance in the substrate level control of αII cleavage by calpains. This is further supported by in silico simulation predicting unmasked calpain target site and increased spectrin scaffold bending and flexibility of R1098Q mutant peptide. Here, using spectroscopic and in situ enzymatic techniques, we aimed at obtaining direct experimental support for the impact of R1098Q mutation on the αII stability and its propensity for calpain-mediated degradation. Thermal circular dichroism analyses performed on recombinant wildtype and R1098Q mutant αII peptides, composed of spectrin repeat 9–10 revealed that although both had very similar secondary structure contents, thermal stability curve profiles varied and the observed midpoint of the unfolding transition (Tm) was 5.5 °C lower for the R1098Q peptide. Yet, the dynamic light scattering profiles of both peptides closely overlapped, implying the same thermal propensity to aggregate. Calpain digestion of plate-bound αII peptides with and without added calmodulin revealed an enhancement of the R1098Q peptide digestion rate relative to WT control. In summary, these results support the unstable scaffold structure of the R1098Q peptide as contributing to its enhanced intrinsic sensitivity to calpain and suggest physiologic relevance of a proper calpain/spectrin balance in preventing neurodegeneration.
- Subjects :
- Calmodulin
Glutamine
Mutant
Mutation, Missense
Biophysics
Gene Expression
Enzyme-Linked Immunosorbent Assay
Peptide
Arginine
Biochemistry
Humans
Spectrin
Molecular Biology
Protein secondary structure
chemistry.chemical_classification
biology
Calpain
Protein Stability
Chemistry
Wild type
Spectrin repeat
Cell Biology
Recombinant Proteins
Solutions
Amino Acid Substitution
Proteolysis
biology.protein
Peptides
Subjects
Details
- ISSN :
- 0006291X
- Volume :
- 581
- Database :
- OpenAIRE
- Journal :
- Biochemical and Biophysical Research Communications
- Accession number :
- edsair.doi.dedup.....f0a9f424c5537f1d76055b86ce023038