Gamma crystallins of the human eye lens

Vendra, Venkata Pulla Rao; Khan, Ismail F.; Chandani, Sushil; Anbukkarasi, Muniyandi; Balasubramanian, D.

doi:10.1016/j.bbagen.2015.06.007

Cited by 72 publications

(56 citation statements)

References 74 publications

(78 reference statements)

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“…Cataract is lens opacity that results when these misfolded crystallins assemble into lightscattering aggregates, making the lens turbid. Cataract is a disease of aging, affecting 17% of all people aged 40 and over (46,47), though many congenital or early-onset mutations are also known (48)(49)(50). Due to the high cost of surgery and lack of a therapeutic treatment, cataract remains the leading cause of blindness in the world (47,51).…”

Section: Introductionmentioning

confidence: 99%

Conformational catalysis of cataract-associated aggregation in a human eye lens crystallin occurs via interface stealing

Serebryany

et al. 2019

Preprint

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Human γD-crystallin (HγD) is an abundant and highly stable two-domain protein in the core region of the eye lens. Destabilizing mutations and post-translational modifications in this protein are linked to onset of aggregation that causes cataract disease (lens turbidity). Wild-type HγD greatly accelerates aggregation of the cataract-related W42Q variant, without itself aggregating. The mechanism of this "inverse prion" catalysis of aggregation remained unknown. Here we provide evidence that an early unfolding intermediate with an opened domain interface enables transient dimerization of the C-terminal domains of wildtype and mutant, or mutant and mutant, HγD molecules, which deprives the mutant's Nterminal domain of intramolecular stabilization by the native domain interface and thus accelerates its misfolding to a distinct, aggregation-prone intermediate. A detailed kinetic model predicts universal power-law scaling relationships for lag time and rate of the resulting aggregation, which are in excellent agreement with the data. The mechanism reported here, which we term interface stealing, can be generalized to explain how common domain-domain interactions can have surprising consequences, such as conformational catalysis of unfolding, in multidomain proteins. SignificanceMost known proteins in nature consist of multiple domains. Interactions between domains may lead to unexpected folding and misfolding phenomena. This study of human γD-crystallin, a two-domain protein in the eye lens, revealed one such surprise: conformational catalysis of misfolding via intermolecular domain interface "stealing." An intermolecular interface between the more stable domains outcompetes the native intramolecular domain interface. Loss of the native interface in turn promotes misfolding and subsequent aggregation, especially in cataractrelated γD-crystallin variants. This phenomenon is likely a contributing factor in the development of cataract disease, the leading worldwide cause of blindness. However, interface stealing likely occurs in many proteins composed of two or more interacting domains.

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Section: Introductionmentioning

confidence: 99%

Conformational catalysis of cataract-associated aggregation in a human eye lens crystallin occurs via interface stealing

Serebryany

et al. 2019

Preprint

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“…31,32 Examples for human γD-crystallin include the G61C variant 33 and W42Q variant, which has increased aggregation propensity in the presence of the wild-type protein. 34 In γS-crystallin, the G18V, 35 G57W, 36 and D26G variants are associated with hereditary cataract.…”

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confidence: 99%

Multiple Aggregation Pathways in Human γS-Crystallin and Its Aggregation-Prone G18V Variant

Roskamp

Montelongo

Anorma

et al. 2017

Invest. Ophthalmol. Vis. Sci.

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PurposeCataract results from the formation of light-scattering precipitates due to point mutations or accumulated damage in the structural crystallins of the eye lens. Although excised cataracts are predominantly amorphous, in vitro studies show that crystallins are capable of adopting a variety of morphologies depending on the preparation method. Here we characterize thermal, pH-dependent, and UV-irradiated aggregates from wild-type human γS-crystallin (γS-WT) and its aggregation-prone variant, γS-G18V.MethodsAggregates of γS-WT and γS-G18V were prepared under acidic, neutral, and basic pH conditions and held at 25°C or 37°C for 48 hours. UV-induced aggregates were produced by irradiation with a 355-nm laser. Aggregation and fibril formation were monitored via turbidity and thioflavin T (ThT) assays. Aggregates were characterized using intrinsic aromatic fluorescence, powder x-ray diffraction, and mass spectrometry.ResultsγS-crystallin aggregates displayed different characteristics depending on the preparation method. γS-G18V produced a larger amount of detectable aggregates than did γS-WT and at less-extreme conditions. Aggregates formed under basic and acidic conditions yielded elevated ThT fluorescence; however, aggregates formed at low pH did not produce strongly turbid solutions. UV-induced aggregates produced highly turbid solutions but displayed only moderate ThT fluorescence. X-ray diffraction confirms amyloid character in low-pH samples and UV-irradiated samples, although the relative amounts vary.ConclusionsγS-G18V demonstrates increased aggregation propensity compared to γS-WT when treated with heat, acid, or UV light. The resulting aggregates differ in their ThT fluorescence and turbidity, suggesting that at least two different aggregation pathways are accessible to both proteins under the conditions tested.

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“…The TM26-004 clone showed the integration of the DT vector in the gamma-crystallin E (Cryge) gene located at Chromosome 1 harboring the gamma-crystallin gene cluster region (Figure 4c), while the TM 28-026 clone showed the integration of the DT176 vector in the Perilipin-1 gene. Gamma crystallins are predominant proteins in the eye lens (Vendra, Khan, Chandani, Muniyandi, & Balasubramanian, 2016;Wistow, 2012), and genetic mutations in CRYG have been shown to induce cataracts in patients (Klopp, Loster, & Graw, 2001;Nag et al, 2007). We generated chimeric mice using the TM26-004-driven FLPo-treated clone, and bred mice to produce F1 mice showing EGFP-labeled lens (Figure 4e).…”

Section: The Dt176 Vector Deleted a Gene In The Differentiated Cellmentioning

confidence: 99%

Diphtheria toxin‐mediated transposon‐driven poly (A)‐trapping efficiently disrupts transcriptionally silent genes in embryonic stem cells

Bai

Kondo

Mayasari

et al. 2020

Genesis

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Summary Random gene trapping is the application of insertional mutagenesis techniques that are conventionally used to inactivate protein‐coding genes in mouse embryonic stem (ES) cells. Transcriptionally silent genes are not effectively targeted by conventional random gene trapping techniques, thus we herein developed an unbiased poly (A) trap (UPATrap) method using a Tol2 transposon, which preferentially integrated into active genes rather than silent genes in ES cells. To achieve efficient trapping at transcriptionally silent genes using random insertional mutagenesis in ES cells, we generated a new diphtheria toxin (DT)‐mediated trapping vector, DTrap that removed cells, through the expression of DT that was induced by the promoter activity of the trapped genes, and selected trapped clones using the neomycin‐resistance gene of the vector. We found that a double‐DT, the dDT vector, dominantly induced the disruption of silent genes, but not active genes, and showed more stable integration in ES cells than the UPATrap vector. The dDT vector disrupted differentiated cell lineage genes, which were silent in ES cells, and labeled trapped clone cells by the expression of EGFP upon differentiation. Thus, the dDT vector provides a systematic approach to disrupt silent genes and examine the cellular functions of trapped genes in the differentiation of target cells and development.

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Gamma crystallins of the human eye lens

Cited by 72 publications

References 74 publications

Conformational catalysis of cataract-associated aggregation in a human eye lens crystallin occurs via interface stealing

Conformational catalysis of cataract-associated aggregation in a human eye lens crystallin occurs via interface stealing

Multiple Aggregation Pathways in Human γS-Crystallin and Its Aggregation-Prone G18V Variant

Diphtheria toxin‐mediated transposon‐driven poly (A)‐trapping efficiently disrupts transcriptionally silent genes in embryonic stem cells

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