Functional Rescue of Cataract-Causing αA-G98R-Crystallin by Targeted Compensatory Suppressor Mutations in Human αA-Crystallin
| Autor: | Sundararajan Mahalingam, K. Krishna Sharma, Ashutosh S. Phadte, Puttur Santhoshkumar |
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| Rok vydání: | 2019 |
| Předmět: |
Cell Survival
Mutant alpha-Crystallin A Chain Biochemistry Article Cell Line law.invention 03 medical and health sciences Suppression Genetic law Crystallin Humans Insulin Gene Protein Unfolding Alcohol dehydrogenase 0303 health sciences Base Sequence biology Protein Stability Chemistry 030302 biochemistry & molecular biology Alcohol Dehydrogenase Hydrogen Peroxide Molecular biology Recombinant Proteins Targeted Mutation Chaperone (protein) Mutation Mutagenesis Site-Directed biology.protein Recombinant DNA Unfolded protein response Protein Multimerization |
| Zdroj: | Biochemistry |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/acs.biochem.9b00374 |
| Popis: | The G98R mutation in αA-crystallin is associated with the onset of pre-senile cataract and is characterized biochemically by an increased oligomeric mass, altered chaperone function, and loss of structural stability over time. Thus far, it is not known whether the inherent instability caused by gain of charge mutation could be rescued by a compensatory loss of charge mutation elsewhere on the protein. To answer this question, we investigated whether αA-G98R-mediated instability could be rescued through suppressor mutations by introducing site-specific ‘compensatory’ mutations in αA-G98R crystallin – αA-R21Q/G98R, αA-G98R/R116C, and αA-R157Q/G98R. The recombinant proteins were expressed, purified, characterized, and evaluated by circular dichroism (CD), intrinsic fluorescence and bis-ANS binding studies. Chaperone-like activities of recombinant proteins were assessed using alcohol dehydrogenase (ADH) and insulin as unfolding substrates. Far-UV CD studies revealed an increased α-helical content in αA-G98R in comparison to αA-WT, αA-R21Q, R157Q, and the double-mutants, αA-R21Q/G98R, and αA-R157Q/G98R. Compared to αA-WT, αA-R21Q, and αA-G98R, the double mutants showed an increased intrinsic tryptophan fluorescence, whereas the highest hydrophobicity (bis-ANS binding) was shown by αA-G98R. Introduction of a second mutation in αA-G98R reduced its bis ANS-binding activity. Both αA-R21Q/G98R and αA-R157Q/G98R showed greater chaperone-like activity against ADH aggregation than αA-G98R. However, among the three G98R mutants, only αA-R21Q/G98R protected ARPE-19 cells from H(2)O(2)-induced cytotoxicity. These results suggest that the lost chaperone-like activity of αA-G98R-crystallin can be rescued by another targeted mutation and that substitution of αA-R21Q-crystallin at the N-terminal region can rescue a deleterious mutation in the conserved α-crystallin domain of the protein. |
| Databáze: | OpenAIRE |
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