Congenital Cataract-Causing Mutation G129C in γC-Crystallin Promotes the Accumulation of Two Distinct Unfolding Intermediates That Form Highly Toxic Aggregates

Xi, Yi-Bo; Chen, Xiangjun; Zhao, Weijie; Yan, Yong-Bin

doi:10.1016/j.jmb.2015.07.001

Cited by 31 publications

(19 citation statements)

References 68 publications

(110 reference statements)

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“…The aggregates formed by S228P-GFP were p62-positive, while those by GFP-S228P were p62-negative. Most crystallin mutants formed p62-positive aggresomes in human cells (Zhao et al, 2015 ; Xi et al, 2015 ). We further confirmed that GFP-S228P formed p62-negative aggregates by fusing S228P by a Flag tag at the N-terminus (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The recombinant plasmids were exogenously expressed in HEK 293T cells by transient transfection using lipofectamine 3000. The intracellular distribution of the exogenously expressed proteins was studied using the same procedure as those reported previously (Zhao et al, 2015 ; Xi et al, 2015 ). In brief, the 293T cells were cultured in the DMEM medium with the addition of 10% FBS at 37°C in 5% CO 2 incubator.…”

Section: Methodsmentioning

confidence: 99%

“…The cell viability was determined by Cell Counting Kit-8 (CCK-8) using the same procedure as those reported previously (Xi et al, 2015 ). In brief, the HeLa cells were transiently transfected by the empty vector pcDNA 3.1 N-Flag and the recombinant plasmids containing the WT CRYBB1 and S228P genes.…”

Section: Methodsmentioning

confidence: 99%

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Cataract-causing mutation S228P promotes βB1-crystallin aggregation and degradation by separating two interacting loops in C-terminal domain

Liu

et al. 2016

Protein Cell

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β/γ-Crystallins are predominant structural proteins in the cytoplasm of lens fiber cells and share a similar fold composing of four Greek-key motifs divided into two domains. Numerous cataract-causing mutations have been identified in various β/γ-crystallins, but the mechanisms underlying cataract caused by most mutations remains uncharacterized. The S228P mutation in βB1-crystallin has been linked to autosomal dominant congenital nuclear cataract. Here we found that the S228P mutant was prone to aggregate and degrade in both of the human and E. coli cells. The intracellular S228P aggregates could be redissolved by lanosterol. The S228P mutation modified the refolding pathway of βB1-crystallin by affecting the formation of the dimeric intermediate but not the monomeric intermediate. Compared with native βB1-crystallin, the refolded S228P protein had less packed structures, unquenched Trp fluorophores and increased hydrophobic exposure. The refolded S228P protein was prone to aggregate at the physiological temperature and decreased the protective effect of βB1-crystallin on βA3-crystallin. Molecular dynamic simulation studies indicated that the mutation decreased the subunit binding energy and modified the distribution of surface electrostatic potentials. More importantly, the mutation separated two interacting loops in the C-terminal domain, which shielded the hydrophobic core from solvent in native βB1-crystallin. These two interacting loops are highly conserved in both of the N- and C-terminal domains of all β/γ-crystallins. We propose that these two interacting loops play an important role in the folding and structural stability of β/γ-crystallin domains by protecting the hydrophobic core from solvent access.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-016-0284-3) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Cataract-causing mutation S228P promotes βB1-crystallin aggregation and degradation by separating two interacting loops in C-terminal domain

Liu

et al. 2016

Protein Cell

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“…At that point the aggregates grow in size until they become large enough to scatter light, with resulting opacity. There will certainly be exceptions to this general process, e.g., amyloid-like fibrils appear to be involved in some cataracts (Meehan et al, 2004; Sandilands et al, 2002; Xi et al, 2015), although these mechanisms are not mutually exclusive and can overlap (Xi et al, 2015). However, based on the present state of knowledge regarding inherited cataracts, this appears to be a reasonable working hypothesis at the present time.…”

Section: Age Related Cataractmentioning

confidence: 99%

Mutations and mechanisms in congenital and age-related cataracts

Shiels

Hejtmancik

2017

Experimental Eye Research

201

138

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The crystalline lens plays an important role in the refractive vision of vertebrates by facilitating variable fine focusing of light onto the retina. Loss of lens transparency, or cataract, is a frequently acquired cause of visual impairment in adults and may also present during childhood. Genetic studies have identified mutations in over 30 causative genes for congenital or other early-onset forms of cataract as well as several gene variants associated with age-related cataract. However, the pathogenic mechanisms resulting from genetic determinants of cataract are only just beginning to be understood. Here, we briefly summarize current concepts pointing to differences in the molecular mechanisms underlying congenital and age-related forms of cataract.

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“…Protein aggregation is the single most important factor in cataract formation [ 27 ]. Protein aggregation problems have been observed extensively in many types of crystallin proteins, such as αA (R116C) [ 7 ], αB (R120G) [ 28 ], βB2 (V187E) [ 29 ], γC (G129C) [ 30 ], and γD (W43R) [ 31 ], which are known to cause congenital cataracts. Furthermore, Zhao L [ 32 ] reported that when the protein aggregation caused by mutant crystallin proteins was decreased by lanosterol treatment, the lens clarity in animal models was increased and the cataract severity was reduced.…”

Section: Discussionmentioning

confidence: 99%

A novel mutation in the CRYAA gene associated with congenital cataract and microphthalmia in a Chinese family

Song

Xiao

2018

BMC Med Genet

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BackgroundCongenital cataract is the leading cause of blindness in children worldwide. Approximately half of all congenital cataracts have a genetic basis. Protein aggregation is the single most important factor in cataract formation.MethodsA four-generation Chinese family diagnosed with autosomal dominant congenital cataracts and microphthalmia was recruited at the Shengjing Hospital of China Medical University. Genomic DNA was extracted from the peripheral blood of the participants. All coding exons and flanking regions of seven candidate genes (CRYAA, CRYBA4, CRYBB2, CRYGC, GJA8, MAF, and PITX3) were amplified and sequenced. Restriction fragment length polymorphism (RFLP) assays were performed to confirm the candidate causative variant, c.35G > T in the CRYAA gene. We constructed pcDNA3.1(+)-CRYAA expression plasmids containing either the wild-type or the R12L mutant alleles and respectively transfected them into HEK293T cells and into HeLa cells. Western blotting was performed to determine protein expression levels and protein solubility. Immunofluorescence was performed to determine protein sub-cellular localization.ResultsA heterozygous variant c.35G > T was identified in exon 1 of CRYAA, which resulted in a substitution of arginine to leucine at codon 12 (p.R12L). The nucleotide substitution c.35G > T was co-segregated with the disease phenotype in the family. The mutant R12L-CRYAA in HEK293T cells showed a significant increase in the expression level of the CRYAA protein compared with the wild-type cells. Moreover, a large amount of the mutant protein aggregated in the precipitate where the wild-type protein was not detected. Immunofluorescence studies showed that the overexpressed mutant CRYAA in HeLa cells formed large cytoplasmic aggregates and aggresomes.ConclusionsIn summary, we described a case of human congenital cataract and microphthalmia caused by a novel mutation in the CRYAA gene, which substituted an arginine at position 12 in the N-terminal region of αA-crystallin. The molecular mechanisms that underlie the pathogenesis of human congenital cataract may be characterized by the prominent effects of the p.R12L mutation on αA-crystallin aggregation and solubility. Our study also expands the spectrum of known CRYAA mutations.

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Congenital Cataract-Causing Mutation G129C in γC-Crystallin Promotes the Accumulation of Two Distinct Unfolding Intermediates That Form Highly Toxic Aggregates

Cited by 31 publications

References 68 publications

Cataract-causing mutation S228P promotes βB1-crystallin aggregation and degradation by separating two interacting loops in C-terminal domain

Cataract-causing mutation S228P promotes βB1-crystallin aggregation and degradation by separating two interacting loops in C-terminal domain

Mutations and mechanisms in congenital and age-related cataracts

A novel mutation in the CRYAA gene associated with congenital cataract and microphthalmia in a Chinese family

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