Dataset Information

Functional Rescue of Cataract-Causing ?A-G98R-Crystallin by Targeted Compensatory Suppressor Mutations in Human ?A-Crystallin.

ABSTRACT: The G98R mutation in ?A-crystallin is associated with the onset of presenile 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 ?A-G98R-mediated instability could be rescued through suppressor mutations by introducing site-specific "compensatory" mutations in ?A-G98R-crystallin, ?A-R21Q/G98R, ?A-G98R/R116C, and ?A-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 ?-helical content in ?A-G98R in comparison to ?A-WT, ?A-R21Q, R157Q, and the double mutants, ?A-R21Q/G98R, and ?A-R157Q/G98R. Compared to ?A-WT, ?A-R21Q, and ?A-G98R, the double mutants showed an increased intrinsic tryptophan fluorescence, whereas the highest hydrophobicity (bis-ANS-binding) was shown by ?A-G98R. Introduction of a second mutation in ?A-G98R reduced its bis-ANS-binding activity. Both ?A-R21Q/G98R and ?A-R157Q/G98R showed greater chaperone-like activity against ADH aggregation than ?A-G98R. However, among the three G98R mutants, only ?A-R21Q/G98R protected ARPE-19 cells from H2O2-induced cytotoxicity. These results suggest that the lost chaperone-like activity of ?A-G98R-crystallin can be rescued by another targeted mutation and that substitution of ?A-R21Q-crystallin at the N-terminal region can rescue a deleterious mutation in the conserved ?-crystallin domain of the protein.

SUBMITTER: Phadte AS

PROVIDER: S-EPMC7266266 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Functional Rescue of Cataract-Causing αA-G98R-Crystallin by Targeted Compensatory Suppressor Mutations in Human αA-Crystallin.

Phadte Ashutosh S AS Mahalingam Sundararajan S Santhoshkumar Puttur P Sharma Krishna K KK

Biochemistry 20190920 40

The G98R mutation in αA-crystallin is associated with the onset of presenile 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 ...[more]

PMID: 31523965

Similar Datasets

Project description:BackgroundA substitution mutation in human ?A-crystallin (?AG98R) is associated with autosomal dominant cataract. The recombinant mutant ?AG98R protein exhibits altered structure, substrate-dependent chaperone activity, impaired oligomer stability and aggregation on prolonged incubation at 37 °C. Our previous studies have shown that ?A-crystallin-derived mini-chaperone (DFVIFLDVKHFSPEDLTVK) functions like a molecular chaperone by suppressing the aggregation of denaturing proteins. The present study was undertaken to determine the effect of ?A-crystallin-derived mini-chaperone on the stability and chaperone activity of ?AG98R-crystallin.Methodology/principal findingsRecombinant ?AG98R was incubated in presence and absence of mini-chaperone and analyzed by chromatographic and spectrometric methods. Transmission electron microscope was used to examine the effect of mini-chaperone on the aggregation propensity of mutant protein. Mini-chaperone containing photoactive benzoylphenylalanine was used to confirm the interaction of mini-chaperone with ?AG98R. The rescuing of chaperone activity in mutant?-crystallin (?AG98R) by mini-chaperone was confirmed by chaperone assays. We found that the addition of the mini-chaperone during incubation of ?AG98R protected the mutant crystallin from forming larger aggregates that precipitate with time. The mini-chaperone-stabilized ?AG98R displayed chaperone activity comparable to that of wild-type ?A-crystallin. The complexes formed between mini-?A-?AG98R complex and ADH were more stable than the complexes formed between ?AG98R and ADH. Western-blotting and mass spectrometry confirmed the binding of mini-chaperone to mutant crystallin.Conclusion/significanceThese results demonstrate that mini-chaperone stabilizes the mutant ?A-crystallin and modulates the chaperone activity of ?AG98R. These findings aid in our understanding of how to design peptide chaperones that can be used to stabilize mutant ?A-crystallins and preserve the chaperone function.

Project description:PURPOSE: To identify the underlying genetic defect in a north Indian family with seven members in three-generations affected with bilateral congenital cataract. METHODS: Detailed family history and clinical data were recorded. Linkage analysis using fluorescently labeled microsatellite markers for the already known candidate gene loci was performed in combination with mutation screening by bidirectional sequencing. RESULTS: Affected individuals had bilateral congenital cataract. Cataract was of opalescent type with the central nuclear region denser than the periphery. Linkage was excluded for the known cataract candidate gene loci at 1p34-36, 1q21-25 (gap junction protein, alpha 8 [GJA8]), 2q33-36 (crystallin, gamma A [CRYGA], crystallin, gamma B [CRYGB], crystallin, gamma C [CRYGC], crystallin, gamma D [CRYGD], crystallin, beta A2 [CRYBA2]), 3q21-22 (beaded filament structural protein 2, phakinin [BFSP2]), 12q12-14 (aquaporin 0 [AQP0]), 13q11-13 (gap junction protein, alpha 3 [GJA3]), 15q21-22, 16q22-23 (v-maf musculoaponeurotic fibrosarcoma oncogene homolog [MAF], heat shock transcription factor 4 [HSF4]), 17q11-12 (crystallin, beta A1 [CRYBA1]), 17q24, 21q22.3 (crystallin, alpha A [CRYAA]), and 22q11.2 (crystallin, beta B1 [CRYBB1], crystallin, beta B2 [CRYBB2], crystallin, beta B3 [CRYBB3], crystallin, beta A4 [CRYBA4]). Crystallin, alpha B (CRYAB) at chromosome 11q23-24 was excluded by sequence analysis. However, sequencing the candidate gene, crystallin, gamma S (CRYGS), at chromosome 3q26.3-qter showed a heterozygous c.176G-->A change that resulted in the replacement of a structurally highly conserved valine by methionine at codon 42 (p.V42M). This sequence change was not observed in unaffected family members or in the 100 ethnically matched controls. CONCLUSIONS: We report a novel missense mutation, p.V42M, in CRYGS associated with bilateral congenital cataract in a family of Indian origin. This is the third report of a mutation in this exceptional member of the beta-/gamma-crystallin superfamily and further substantiates the genetic and clinical heterogeneity of autosomal dominant cataract.

Dataset Information

Functional Rescue of Cataract-Causing ?A-G98R-Crystallin by Targeted Compensatory Suppressor Mutations in Human ?A-Crystallin.

Publications

Functional Rescue of Cataract-Causing αA-G98R-Crystallin by Targeted Compensatory Suppressor Mutations in Human αA-Crystallin.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets