Dataset Information

Expression of Cataract-linked ?-Crystallin Variants in Zebrafish Reveals a Proteostasis Network That Senses Protein Stability.

ABSTRACT: The refractivity and transparency of the ocular lens is dependent on the stability and solubility of the crystallins in the fiber cells. A number of mutations of lens crystallins have been associated with dominant cataracts in humans and mice. Of particular interest were ?B- and ?D-crystallin mutants linked to dominant cataracts in mouse models. Although thermodynamically destabilized and aggregation-prone, these mutants were found to have weak affinity to the resident chaperone ?-crystallin in vitro To better understand the mechanism of the cataract phenotype, we transgenically expressed different ?D-crystallin mutants in the zebrafish lens and observed a range of lens defects that arise primarily from the aggregation of the mutant proteins. Unlike mouse models, a strong correlation was observed between the severity and penetrance of the phenotype and the level of destabilization of the mutant. We interpret this result to reflect the presence of a proteostasis network that can "sense" protein stability. In the more destabilized mutants, the capacity of this network is overwhelmed, leading to the observed increase in phenotypic penetrance. Overexpression of ?A-crystallin had no significant effects on the penetrance of lens defects, suggesting that its chaperone capacity is not limiting. Although consistent with the prevailing hypothesis that a chaperone network is required for lens transparency, our results suggest that ?A-crystallin may not be efficient to inhibit aggregation of lens ?-crystallin. Furthermore, our work implicates additional inputs/factors in this underlying proteostasis network and demonstrates the utility of zebrafish as a platform to delineate mechanisms of cataract.

SUBMITTER: Wu SY

PROVIDER: S-EPMC5207241 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Expression of Cataract-linked γ-Crystallin Variants in Zebrafish Reveals a Proteostasis Network That Senses Protein Stability.

Wu Shu-Yu SY Zou Ping P Fuller Alexandra W AW Mishra Sanjay S Wang Zhen Z Schey Kevin L KL Mchaourab Hassane S HS

The Journal of biological chemistry 20161021 49

The refractivity and transparency of the ocular lens is dependent on the stability and solubility of the crystallins in the fiber cells. A number of mutations of lens crystallins have been associated with dominant cataracts in humans and mice. Of particular interest were γB- and γD-crystallin mutants linked to dominant cataracts in mouse models. Although thermodynamically destabilized and aggregation-prone, these mutants were found to have weak affinity to the resident chaperone α-crystallin in ...[more]

PMID: 27770023

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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

Expression of Cataract-linked ?-Crystallin Variants in Zebrafish Reveals a Proteostasis Network That Senses Protein Stability.

Publications

Expression of Cataract-linked γ-Crystallin Variants in Zebrafish Reveals a Proteostasis Network That Senses Protein Stability.

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