Expression of Cataract-linked �-Crystallin Variants inZebrafish Reveals a Proteostasis Network That Senses ProteinStability*□S

Received for publication, July 20, 2016, and in revised form, October 21, 2016 Published, JBC Papers in Press, October 21, 2016, DOI 10.1074/jbc.M116.749606

Shu-Yu Wu‡, Ping Zou‡, Alexandra W. Fuller‡, Sanjay Mishra‡, Zhen Wang§¶, Kevin L. Schey§¶,and Hassane S. Mchaourab‡1

From the Departments of ‡Molecular Physiology and Biophysics and §Biochemistry and the ¶Mass Spectrometry Research Center,Vanderbilt University School of Medicine, Nashville, Tennessee 37232

Edited by Paul Fraser

The refractivity and transparency of the ocular lens is depen-dent on the stability and solubility of the crystallins in the fibercells. A number of mutations of lens crystallins have been asso-ciated with dominant cataracts in humans and mice. Of partic-ular interest were �B- and �D-crystallin mutants linked to dom-inant cataracts in mouse models. Although thermodynamicallydestabilized and aggregation-prone, these mutants were foundto have weak affinity to the resident chaperone �-crystallin invitro. To better understand the mechanism of the cataract phe-notype, we transgenically expressed different �D-crystallinmutants in the zebrafish lens and observed a range of lensdefects that arise primarily from the aggregation of the mutantproteins. Unlike mouse models, a strong correlation was ob-served between the severity and penetrance of the phenotypeand the level of destabilization of the mutant. We interpret thisresult to reflect the presence of a proteostasis network that can“sense” protein stability. In the more destabilized mutants, thecapacity of this network is overwhelmed, leading to the observedincrease in phenotypic penetrance. Overexpression of �A-crys-tallin had no significant effects on the penetrance of lens defects,suggesting that its chaperone capacity is not limiting. Althoughconsistent with the prevailing hypothesis that a chaperone net-work is required for lens transparency, our results suggest that�A-crystallin may not be efficient to inhibit aggregation oflens �-crystallin. Furthermore, our work implicates additionalinputs/factors in this underlying proteostasis network and dem-onstrates the utility of zebrafish as a platform to delineate mech-anisms of cataract.

Lens crystallins are water-soluble proteins that play a centralrole in conferring the optical properties of the ocular lens. Lensdevelopment entails terminal differentiation of epithelial cells

into fiber cells that have negligible protein synthesis and turn-over activities (1, 2). Mammalian crystallins consist of threeclasses of proteins, �-, �-, and �-crystallins, which togetherconstitute 90% of the total weight of lens fiber cells (3). Theglass-like, short range order packing of the crystallins in fibercells is critical to the transparency and refractivity of the lens (4,5). Protein-protein interactions between crystallins are consis-tent with a uniform protein distribution at dimensions compa-rable with the wavelength of visible light (6). From this perspec-tive, the vertebrate lens is a thermodynamic wonder of proteinevolution. The unusual stability of the crystallins along withtheir carefully tuned interactions was evolved to avert, for along period of the human lifespan, the unavoidable thermody-namic fates of aggregation and precipitation expected for highlyconcentrated protein solutions.

The �-crystallins are molecular chaperones that belong tothe small heat shock proteins superfamily (7–11), They canbind thermodynamically destabilized proteins and sequesteraggregation-prone proteins, whereas �- and �-crystallins areconsidered structural proteins with undefined function in thedevelopment of the lens. In his seminal work, Horwitz (12, 13)hypothesized that once lens proteins cross an energetic thresh-old of destabilization, they become substrates for �-crystallin,which sequesters them away inhibiting their aggregation,thereby delaying light scattering. Much effort has been dedi-cated to identifying age-related modifications of the crystallinsand understanding the consequent effects on their stability andpropensity to aggregate (14 –19). The unfolding of destabilizedlens proteins following the exhaustion of �-crystallin bindingcapacity has been hypothesized to lead to the onset of age-related cataract, although changes in the surface properties ofmolecules leading to loss of solubility is another importantmechanism of cataracts (20).

Horwitz’s hypothesis stimulated an expansive but successfuleffort to understand the mechanism of �-crystallin chaperoneactivity in vitro. �-Crystallins bind thermodynamically destabi-lized proteins as well as sequester aggregation-prone proteinsin vitro (10, 12, 13, 21–25). Binding can involve conformationalchanges to expose otherwise unavailable binding sites. The oli-gomer plasticity of �-crystallin is critical to the binding capacityand affinity (26 –29).

Despite the successful outline of its function, a divideemerged between the mechanistic principles of �-crystallin

* This work was supported by National Institutes of Health Grants R01EY12018 (to H. S. M.), R01 EY13462 (to K. L. S.), and P30 EY008126 (toH. S. M. and K. L. S.) and by the Vanderbilt University Proteomics Facility inthe Mass Spectrometry Research Center and Vanderbilt University MedicalCenter Cell Imaging Shared Resource. The authors declare that they haveno conflicts of interest with the contents of this article. The content is solelythe responsibility of the authors and does not necessarily represent theofficial views of the National Institutes of Health.

□S This article contains supplemental Figs. S1–S4.1 To whom correspondence should be addressed. E-mail: hassane.

mchaourab@vanderbilt.edu.

crossmarkTHE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 291, NO. 49, pp. 25387–25397, December 2, 2016

© 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

DECEMBER 2, 2016 • VOLUME 291 • NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 25387

by guest on June 24, 2020http://w

ww

.jbc.org/D

ownloaded from

chaperone activity established in vitro and the role of this activ-ity in lens aging and cataract. Although reliance on model sub-strates is justifiable in the context of mechanistic studies, itsconsequence is to overlook the potential specificity of �-crys-tallin interactions with �- and �-crystallins. A particularly dis-concerting example of this divide is the failure of current mod-els to explain the weak chaperone-based interactions between�-crystallin and destabilized mutants of �-crystallin, whichcause dominant cataract in mouse models. For example,�B-crystallin mutant I4F and �D-crystallin mutant V76D wereidentified in mouse lines with dominant cataracts (30, 31). Indetailed analyses, Mishra et al. (32) and Moreau and King (33)demonstrated that these �-crystallin mutants have reducedthermodynamic stability. Moreover, they undergo a slow pro-cess of aggregation that is relevant in long-lived cells such aslens fiber cells. However, neither �-crystallin subunits can bindthese mutants with high affinity or preempts their aggregationin vitro. Together, the two studies reveal a class of mutationsthat can escape detection by the �-crystallins, a finding seem-ingly consistent with their ability to induce cataracts in mice(30, 31).

In this paper, we explore the in vivo mechanisms by whichthe two thermodynamically destabilized �-crystallin mutantscan lead to lens defects (30, 31). To better define the role of�-crystallin in lens development and dissect its chaperoneactivity in vivo, we have initiated studies using zebrafish as amodel system. The zebrafish lens affords many advantagesand simplifications relative to mouse models that include thecost and speed of generation of transgenic and knock-outlines and the size of embryo clutches, a factor that is criticalfor the studies reported here. In a previous study, we dem-onstrated that �A-crystallin plays a critical role in the devel-opment of the embryonic zebrafish lens (34). Partial rescueby the transgenic expression of rat �A-crystallin reveals aconserved function for this protein in lens development.Here we challenge the proteostasis network of the zebrafishlens with targeted and controlled expression of the �D-crys-tallin mutants. Not unexpectedly, we find that embryosexpressing destabilized and aggregation-prone mutantsshow lens defects. In contrast to mouse models, we observeda range of penetrance and severity determined by the under-

lying stability of the mutants. The mechanism of lens defectsappears to be primarily linked to the tendency of the mutantsto aggregate, whereas other typical cellular dysfunctions arenot prominently detected. We interpret these findings asrevealing the presence of a chaperone network (35) capableof “sensing” the thermodynamic stability and aggregationpropensity of proteins. Consistent with in vitro studies ondestabilized �D-crystallin mutants (32, 33), as well as onother modified crystallin proteins (36, 37) demonstratingthat �A-crystallin does not interact strongly with these ag-gregation-prone mutant proteins, the buffering capacity of�A-crystallin in the lens is not limiting, suggesting the con-tribution of other chaperones to lens proteostasis.

Results

Transgenic Expression of Human �D-Crystallin VariantsInduce Embryonic Lens Defects in Zebrafish—To assess the invivo interactions between cataract-linked �D-crystallin vari-ants and lens chaperones in the context of the native environ-ment of the lens fiber cells, we generated transgenic zebrafishlines that express three destabilizing human �D-crystallin(Hsa.CRYGD) variants I4F, V76D, and I4F/V76D (32). Usingtransgenesis protocols described previously (34), the �D-crys-tallin mutants were expressed specifically in the lens under thecontrol of the zebrafish cryaa promoter. We opted for this pro-moter to keep the overall level of the mutants low comparedwith native �-crystallins because aggregation is stronglyconcentration-dependent. In addition, all transgenic linesexpressed myl7 promoter-driven Cerulean fluorescent proteinto provide a convenient selection marker for transgenic animals(Fig. 1A, panel b).

Expression of the �D-crystallin variants led to apparentabnormalities in the embryonic lens starting at 3 dpf2 (Fig. 1B),whereas the overall morphology of the embryos were com-pletely unaffected (Fig. 1A, panel a). The nature of the lensdefects was similar to those previously described for �A-crys-tallin knock-out lines (34). The phenotypic features appeared as

2 The abbreviations used are: dpf, days post-fertilization; WSF, water-solublefraction; WIF, water-insoluble fraction; DIC, differential interferencecontrast.

FIGURE 1. Transgenic expression of human CRYGD mutants induce lens defects in 4-dpf zebrafish embryos. A, embryos of lens-specific Hsa.CRYGDtransgenes displayed Cerulean marker in the heart. Panel a, an overlay image of DIC and Cerulean fluorescence. Panel b, a white arrow marks the Cerulean markerin the heart. B, comparison of percentage of embryos showing lens defects between WT and three Hsa.CRYGD transgenes: I4F, V76D, and I4/F/V76D.

Mechanisms of Cataract-linked �-Crystallin Mutations

25388 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 291 • NUMBER 49 • DECEMBER 2, 2016

by guest on June 24, 2020http://w

ww

.jbc.org/D

ownloaded from

either round, shiny crystal-like droplets spread across the lensthat were classified as “minor” defects (Fig. 2A, panel e) or largeirregular protuberances located in the center of the lens classi-fied as “major” defects (Fig. 2A, panel f, arrows; also shown insupplemental Fig. S1). The latter class of lens defects led toincreased light reflectance compared with the WT siblings in allthree transgenic lines expressing �D-crystallin variants (Fig.2A, panels a– c, and B; V76D not shown). Transverse and sag-ittal sections of �D I4F/V76D transgene (Fig. 2C, panels b andd) retinae at 4 dpf stained with toluidine blue showed normalretinal laminar architecture when compared with the wild type(Fig. 2C, panels a and c) and further confirmed that theobserved defects were localized in the lens, showing multipledarkly stained speckles or inclusion bodies (Fig. 2C, panel b andd, arrowheads) that resembled cytosolic protein aggregates(31).

We screened a large number of embryos for each mutantscoring them based on the severity of the phenotype. A strongcorrelation emerged between the penetrance of lens defectsand the reduction in the thermodynamic stability of �D-crys-

tallin mutant proteins (Fig. 1B). In the embryos carrying�D-crystallin I4F/V76D transgene, which is the most unstableamong the three mutants, the majority of embryos (�80%)showed abnormalities in lens morphology, and more than halfof these were categorized as major lens defects (Fig. 2A, panel f).In the lines expressing the less destabilizing �D-crystallinmutants, I4F and V76D, we observed lower percentages ofembryos exhibiting lens defects (I4F, �30%; V76D, �62%), butnonetheless more frequent and severe when compared withWT embryos.

The difference of severity and penetrance between the�D-crystallin transgenic lines could be due to differentialexpression level of each line given the random nature of thegenomic insertion events, collectively termed position effects(38 – 40). That is, the higher the level of expression of �D-crys-tallin transgenes, the more penetrant lens phenotype will beobserved. To exclude position effects as the origin of the vari-able phenotype, we performed quantitative RT-PCR to com-pare the expression level between �D-crystallin V76D and I4Ftransgenes, as well as between �D-crystallin V76D embryos

FIGURE 2. The lens defects and reflectance in zebrafish embryos expressing human CRYGD mutants. A, compared with WT siblings (non-transgeniccarriers), embryos carrying Tg(cryaa:Hsa.CRYGD_I4F) and Tg(cryaa:Hsa.CRYGD_I4F/V76D) at 4 dpf exhibited various degrees of lens defects (panels d–f, arrows),as well as changes in reflectance (panels a– c). B, reflectance analysis of WT lens and Hsa.CRYGD_I4F and Hsa.CRYGD_I4F/V76D lens at 4 dpf suggested that thelens of embryos expressing �D-crystallin variants scattered more lights than WT lens (t test; p � 0.001). C, histological sections of the lens in Tg(cryaa:Hsa.CRYGD_I4F/V76D) embryos stained by toluidine blue showed distinct dark inclusion bodies (panels b and d) compared with WT siblings (panels a and c).Panels a and b, transverse section; panels c and d, sagittal section.

Mechanisms of Cataract-linked �-Crystallin Mutations

DECEMBER 2, 2016 • VOLUME 291 • NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 25389

by guest on June 24, 2020http://w

ww

.jbc.org/D

ownloaded from

showing major and minor defects (supplemental Fig. S2). Eventhough the V76D transgenic line displayed lens phenotypes in ahigher percentage of embryos relative to the I4F transgene (Fig.1B), there was actually a slightly lower expression for the V76Dtransgene (supplemental Fig. S2A). Moreover, no significantdifference was detected in the transgene expression betweenembryos exhibiting major and minor lens defects for the samemutant (supplemental Fig. S2B). Thus, we conclude that thelevel of transgenic expression of �D-crystallin mutants does notdictate the severity or penetrance of the lens phenotype; rather,the correlation with the stability of the mutants demonstratesthat the variability is a function of the mutation sites/types. Weinterpret this finding to manifest the presence of an intrinsicmechanism(s) in the lens that can buffer these aggregation-prone mutants. Titration of the chaperone capacity of this net-work accounts for the observed dependence on the stability ofthe mutants.

Destabilized �D-Crystallin Proteins Form Aggregates in theLens—Previous studies inferred that congenital mutations of�-crystallins identified in humans and mouse tend to formaggregates (31, 41– 43), which then result in opacification andcataract formation. However, this conclusion is drawn based onsparse biochemical evidence rather than on in vivo data.

To directly test the hypothesis that the observed lens pheno-types arise from the propensity of the destabilized �D-crystal-lins to form protein aggregates, we tagged the �D-crystallinmutants with red fluorescent protein to visualize their associa-tion state (see “Experimental Procedures” for details). TheGal4/UAS targeted gene expression system (44) was utilized togenerate lens fiber mosaics by injecting embryos derived from alens-specific transgenic driver line, Tg[cryaa:Gal4VP16] (45),with UAS responder constructs expressing fluorescently taggedhuman �D-crystallin (Hsa.CRYGD-mCherry) wild type (ascontrol) and three variants, I4F, V76D, and I4F/V76D double

FIGURE 3. Destabilized �D-crystallin proteins forms aggregation in the lens. A, panel a, schematic of the experiment utilizing the Gal4/UAS targeted geneexpression system. Double transgenic line, Tg[cryaa:Gal4]; Tg[UAS:GFP], males were outcrossed to AB females. Fertilized zygotes were injected with tol2 mRNAand UAS responder Tol2 constructs expressing fluorescently tagged human �D-crystallin (Hsa.CRYGD-mCherry), including wild type and three variants: I4F,V76D, and I4F/V76D double mutant. Panel b, embryos possessing lens fibers positive for mCherry expression were selected and imaged at 4 dpf. Panel b�,examples of green lens (from UAS:GFP) and mosaic expression of mCherry-tagged �D-crystallin. B, mosaic expression of mCherry-tagged human �D-crystal-lins, wild type (panels a– c) and I4F/V76D double mutant (panels d–f). Apparent mCherry punctuates/aggregates were observed in the lens expressingHsa.CRYGD_I4F/V76D-mCherry (white arrow in panels e and f), but not in those expressing wild-type �D-crystallin construct (mostly shown a diffused andelongated pattern, which is consistent with the shape of lens fiber cells). DIC images (panels a and d), fluorescent images (red channel; panels b and e),and merged images (panels c and f) are shown. C, the frequency of embryos showing mCherry punctuates in the lens (wild type and three variants: I4F, V76D,and I4F/V76D double mutant).

Mechanisms of Cataract-linked �-Crystallin Mutations

25390 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 291 • NUMBER 49 • DECEMBER 2, 2016

by guest on June 24, 2020http://w

ww

.jbc.org/D

ownloaded from

mutant (Fig. 3A). When the fluorescence of tagged �D-crystal-lins was measured at 4 dpf, we frequently observed distinct redpunctates in lenses expressing �D-crystallin I4F/V76D mutant(Fig. 3B, panels d–f), whereas the fluorescence was diffuse withrare punctates in the lens expressing wild-type �D-crystallin(Fig. 3B, panels a– c). Importantly, the highest frequency of thefluorescent punctate formation was observed in the lenses ofembryos expressing the most destabilized �D-crystallin variant(I4F/V76D double mutant; Fig. 3C) compared with �D-crystal-lin I4F or V76D. This correlation between the occurrence offluorescent punctates in the lens and the free energy of unfold-ing of �D-crystallin mutants suggests that reduction of�D-crystallin stability does enhance the likelihood of aggregateformation in native cellular environments, i.e. in lens fiber cells.

Supporting this conclusion, the fluorescent punctates werefound to co-localize with the lens defects in parallel DIC andfluorescence microscopy (Fig. 3B, panel f, arrow). Thus, the lensphenotypes associated with expression of the �-crystallinmutants are primarily because of formation of protein aggre-gates consistent with the histological analysis as describedabove (Fig. 2C).

Quantitative Proteomics Reveals Formation of Water-insolu-ble Aggregates in Adult Zebrafish Lens—In addition to theembryonic lens defects, we set out to examine the long termconsequence of the expression of destabilized human �D-crys-tallin variants in adult transgenic fishes. For this purpose, weutilized the iTRAQ quantitative proteomics strategy (46, 47) toanalyze the relative changes of global protein abundance inboth water-soluble (WSF) and -insoluble (WIF) fractions forthe lenses of 3- and 15-month-old adult fish. The detailed anal-yses will be described elsewhere. Directly relevant to this studyis the finding that in the 15-month-old adult lens samples, therewas a significant increase of human �D-crystallin peptides(enzymatically digested for iTRAQ label) in the WIF observedin the fish lens expressing the mutant �D-crystallin I4F/V76Dcompared with I4F (Fig. 4 and supplemental Fig. S3). Togetherwith the fluorescence detection of aggregation described above,these results suggest that aggregates of the more destabilized�D-crystallin mutants do not remain soluble and preferentiallyreside in the insoluble fraction, thus providing a molecular basisfor the increase in light scattering by the lens.

Exogenous Expression of rat ��-Crystallin Showed No Pro-tective Effects on Lens Defects Induced by �D-crystallinI4F/V76D—In vitro biochemical studies showed that cataract-linked �D-crystallin mutants could evade quality control by�-crystallins (32, 33), consistent with their ability to induce cat-aracts in mouse models (30, 31). We set to probe the interac-tions between �D-crystallin and �-crystallins in vivo using ourtransgenic zebrafish model. The goal was to test whether the�-crystallin binding capacity is limiting, being titrated out bythe mutant �-crystallins. By crossing the fish line carrying�D-crystallin I4F/V76D transgene with a transgenic lineexpressing rat ��-crystallin in the lens under the control of thesame promoter (34) and subsequently using PCR genotyping toseparate embryos with single and double transgenes, we foundno suppression for the penetrance of lens defects in doubletransgenic embryos (�D-crystallin I4F/V76D; �� WT) com-

pared with embryos with �D-crystallin I4F/V76D transgenealone (Fig. 5A).

Together with the fluorescence data, these genetic resultssupport the observation that these destabilized �D-crystallinscause lens abnormalities in zebrafish and, we presume, cata-racts in mouse models because of their propensity to form pro-tein aggregates in the lens and thus scatter light (see above). Thelack of an effect of �A-crystallin overexpression is consistentwith lack of significant interactions with �D-crystallin mutantsas previously reported in vitro (32). Thus, the pool of available�A-crystallin binding sites is not limiting, and its titration bythe mutant is not at the origin of aggregation and formation oflens defects.

Reduced Dosage of �-Crystallin Exacerbated the Lens DefectsInduced by �D-crystallin I4F Expression—To further assess therole of �-crystallin in modulating the phenotype, we expressedthe �-crystallin mutants in zebrafish strains with reduced chap-erone capacity. We hypothesized that expression of destabi-lized �D-crystallin variants in embryos with an inheritablycompromised chaperone network would accentuate the sever-ity and penetrance of the lens defects. The �D-crystallin I4Ftransgene that elicited abnormalities in the zebrafish lens withmoderate penetrance (�30%; Fig. 1B) provides an ideal plat-form to study genetic synergism or antagonism for modifyinggenes/factors. In light of our previous finding showing that defi-ciency in ��-crystallin resulted in compromised lens develop-ment in zebrafish (34), we tested our hypothesis by expressing�D-crystallin I4F transgene in the cryaa heterozygous back-ground (cryaa�/�).

FIGURE 4. Human CRYGD mutants show different solubility in adult lens.In the 15-month-old adult lens analyzed by iTRAQ quantitative proteomics,there was a significantly increased amount of human �D-crystallin peptidesdetected in the WIF in the fish lens expressing the �D-crystallin I4F/V76Dcompared with I4F, based on the ratio between �WIF and �WSF. The �WIFand �WSF from each sample (�D-crystallin I4F/V76D and �D-crystallin I4F)were calculated as fold changes of the expression level relative to the lensexpressing �D-crystallin WT.

Mechanisms of Cataract-linked �-Crystallin Mutations

DECEMBER 2, 2016 • VOLUME 291 • NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 25391

by guest on June 24, 2020http://w

ww

.jbc.org/D

ownloaded from

Specifically, crossing the �D-crystallin I4F transgenic linewith the cryaa homozygous mutant (cryaa�/�) and the wild-type fish, we observed a significant increase (�2-fold) in thenumber of embryos exhibiting major lens defects induced by�D-crystallin I4F at 4 dpf when one copy of endogenous cryaagene was deleted (Fig. 5B; comparing I4F; cryaa�/� with IF4).In addition, when compared with the non-transgenic siblings(cryaa�/�), there was also a marked increase in the percentageof embryos showing major lens defects at 4 dpf in the cryaa�/�

embryos that carry the �D-crystallin I4F transgene (Fig. 5B;comparing [I4F; cryaa�/�] with [cryaa�/�] only). These resultsindicated that compromising the level of �A-crystallin exacer-bated the lens defects induced by �D-crystallin I4F expressionin zebrafish lens. This observed additive effect does not neces-sarily imply a direct physical interaction between the two crys-tallin proteins. Rather it may reflect that a single copy loss ofendogenous cryaa gene essentially creates a “sensitized” back-ground, allowing two weak perturbations to synergistically con-verge on the downstream chaperone network. Furthermore, itimplicates a requirement for maintaining a proper “gene dos-age” of �A-crystallin to keep the underlying chaperone net-works operating properly.

Expression of �D-Crystallin Variants Do Not Induce Apopto-sis or Disrupt the Denucleation Process in the Lens—Cell deathhas been implicated in cataract phenotypes, including radia-tion-induced cataracts and diabetic retinopathy-associated cat-aracts (48 –51) and age-related cataract formation (52). Stain-ing with TUNEL (Fig. 6, G and H) and acridine orange(supplemental Fig. S4) to detect apoptotic cell death, we foundno evidence of elevated apoptosis in the embryos carrying�D-crystallin I4F/V76D transgene, as well as I4F and V76Dvariants (not shown), when compared with WT. Therefore, celldeath by apoptosis did not seem to play a major role in the lensdefects observed in the �D-crystallin transgenes (Fig. 2, A andC). Similar phenotypes (i.e. the lack of cell death) were alsoobserved in loss of function analyses by morpholino knock-downs of crybgx, cryaa, and crybb1c in zebrafish (53).

Failure of lens fiber cell denucleation has been associatedwith various congenital cataracts (54 –57). Utilizing nuclearstaining (DAPI; Fig. 6, D and E) and a transgenic line expressingnuclear localized GFP (Fig. 6, A and B), we found no evidenceof any noticeable impairment or delay in the lens fiber celldenucleation process, whereas we clearly observed an incom-plete denucleation in cloche mutants (Fig. 6, C and F), consis-tent with published results (58). Thus, the lens defects inducedby mutant �D-crystallin expression were unlikely a result of thedisruption of the lens fiber cell denucleation process.

Discussion

The finding that expression of destabilized mutant �D-crys-tallins induces defects in the embryonic zebrafish lens is con-sistent with previous studies in mouse models, which associ-ated these mutants with cataract formation (30, 31). However,the penetrance and severity of the phenotype was heterogene-ous in zebrafish, revealing a critical difference between the twomodel systems. The phenotypic variability was not only depen-dent on the degree of destabilization but was also observedwithin the embryo population expressing the same mutant.Hence, there are embryos expressing the V76D mutant thathave undisturbed lenses, whereas others show severe lensdefects.

Given the dependence of the phenotype on the thermody-namic stability of the mutants but not on their level of expres-sion, we propose that the severity and penetrance reflects thepresence of a chaperone network that functions to bind,sequester, and possibly refold the destabilized, aggregation-prone mutants. The buffering capacity of this network effec-tively imparts a capability to “sense” the stability of theexpressed mutant proteins, which is manifested in the remark-able dependence on the free energy of unfolding. Thus, at thelow level of unfolded and aggregation-prone protein popula-tion expected for the I4F mutant, the chaperone network ismore efficient at sequestering aggregation-prone proteins thanfor the double mutant I4F/V76D where the free energy of

FIGURE 5. Percentages of the lens defects induced by human CRYGD_I4F expression at 4-dpf embryos were not suppressed by exogenous expressionof �A-crystallin but were exacerbated by reducing dosage of endogenous �A-crystallin. A, adult Tg(cryaa:Hsa.CRYGD_I4F/V76D) and Tg(cryaa:Rno.Cryaa)transgenic zebrafish lines were crossed. The lens defects of resulting embryos (I4F/V76D) were examined and classified into three severity classes as previouslymentioned. The proportion of I4F/V76D embryos showing lens defects showed no significant changes with or without the presence of Rno.Cryaa transgene.B, a significantly higher proportion of Tg(cryaa:Hsa.CRYGD_I4F) embryos showed major lens defects when losing one copy of cryaa (I4F; cryaa�/�; �39%),compared with those in wild-type background (I4F; �14%), as well as to the non-transgenic cryaa�/� or cryaba�/� embryos.

Mechanisms of Cataract-linked �-Crystallin Mutations

25392 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 291 • NUMBER 49 • DECEMBER 2, 2016

by guest on June 24, 2020http://w

ww

.jbc.org/D

ownloaded from

unfolding is significantly reduced, and partially and/or globallyunfolded states are highly populated even at equilibrium. Thismechanistic model is supported by the detection of aggregatesof �D-crystallin mutants that localize morphologically withlens defects. Our results not only confirm previous studies inmouse models that these mutants are associated with cataracts(30, 31) but also uncover an important role for a chaperonenetwork in buffering the aggregation-prone populations.

The variability of lens phenotypes in the embryo populationexpressing the same aggregation-prone mutant must reflectgenetic or epigenetic differences in the same network thatimparts the “sensing” of the �-crystallin mutants stability. Theidentity of this network will be a subject of future investigations,although we presume it must be associated with molecularchaperones.

A prominent member of the lens chaperone network is thesmall heat shock protein ��-crystallin. Using our knock-outzebrafish line of ��-crystallin (34), we verified that reduction inits expression enhances penetrance of the lens phenotypeinduced by destabilized �D-crystallin mutants. However,increasing the available pool of �A-crystallin did not affect theexpressivity of the phenotype. This result suggests that the

aggregation of the �D-crystallin mutants is not depleting thepool of available ��-crystallin.

One of the fundamental differences between the zebrafishmodels described here and mouse models where these mutantswere identified (30, 31) is the level of expression of the mutantproteins. Whereas in the mouse the �-crystallin mutants wereunder the control of their respective native promoters, the useof the �A-crystallin (cryaa) promoter to drive the expression inthe zebrafish lens may reduce the overall concentration of theseproteins. This may allow the distinction of intrinsic differencesin the aggregation propensity of different �D-crystallin mu-tants. In the mouse it is possible that the level of expression,even of the mildly destabilized I4F mutant, overwhelms thechaperone network, hence obscuring these differences amongvarious �-crystallin mutants.

Our work establishes a new model to explore the mecha-nisms of cataract-linked mutants and provide direct evidencefor the presence and activity of a chaperone network that main-tains balanced proteostasis in the lens. Although consistentwith in vitro studies (32, 33), the lack of rescue for lens pheno-types by �A-crystallin is somewhat counterintuitive with theabundant expression of this chaperone in mammalian lenses.

FIGURE 6. Denucleation failure or apoptotic cell death was not induced by transgenic expression of human CRYGD mutants. Unlike in cloche mutantlens, an incomplete denucleation process was clearly observed; either by nuclear-localized H2B-GFP visualization (C) or by DAPI staining (F), the lens ofTg(cryaa:Hsa.CRYGD_I4F/V76D) embryos (B and E) showed no signs of denucleation failure and appeared similar to WT siblings (A and D). G and H, the TUNELstaining revealed no significant increase of apoptosis in Tg(cryaa:Hsa.CRYGD_I4F/V7D) embryos compared with WT siblings.

Mechanisms of Cataract-linked �-Crystallin Mutations

DECEMBER 2, 2016 • VOLUME 291 • NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 25393

by guest on June 24, 2020http://w

ww

.jbc.org/D

ownloaded from

Further experiments to test the modulation of the phenotypesdescribed here by the other resident chaperone �B-crystallinare underway.

Experimental Procedures

Zebrafish Maintenance and Breeding—AB wild-type strainzebrafish (Danio rerio) were used. The embryos were obtainedby natural spawning and raised at 28.5 °C on a 14/10-h light/dark cycle in 0.3 Danieau water containing 0.003% 1-phenyl-2-thiourea (w/v) to prevent pigment formation. Embryoswere staged according to their ages (dpf). The following mu-tant and transgenic fish lines were used: cryaavu532, Tg(cryaa:Rno.Cryaa,myl7:Cerulean)vu531Tg (34), Tg(cryaa:Gal4vp16)mw46Tg; Tg(UAS:GFP)kca33Tg (45), and clochem39

(58). All animal procedures were approved by the VanderbiltUniversity Institutional Animal Care and Use Committee. Thesame feeding procedures were performed on all zebrafish linesused in this study, according to their appropriate ages.

Zebrafish Transgenesis—To establish the transgenic zebra-fish expressing human (Homo sapiens) CRYGD gene (Hsa.CRYGD) specifically in the lens, Tg(cryaa:Hsa.CRYGD,myl7:Cerulean) was constructed by inserting Hsa.CRYGD cDNA(32) downstream of zebrafish cryaa promoter (1.2 kb) (59) inthe pT2HBLR vector that was also contains myl7 promoter-driven Cerulean as the selection marker. Tol2-mediated trans-genesis was performed as previously described (34) for all threeHsa.CRYGD (I4F, V76D, and I4F/V76D) transgenes. At leasttwo founder lines (F0) for each construct were screened andout-crossed to established stable F1 generations. Each F1 linewas propagated and raised into F2 and F3 generations. The lensdefects data collected were from F3 or F4 embryos, and similarpenetrance of lens defects was observed in individual stablelines that express the same �D-crystallin constructs.

Assembly of the UAS Expression Constructs—All UAS expres-sion constructs were created by MultiSite Gateway (Invitrogen)assembly reactions using protocols established previously(Tol2kit) (60). Specifically, Tol2kit vectors, p5E-UAS, andp3E-mCherry were assembled with pME-Hsa.CRYGD (wildtype and three variants: I4F, V76D, and I4F/V76D) andrecombined into pDestTol2CG2 vector, which was thenused for microinjections.

Quantitative RT-PCR—Total RNAs were isolated fromembryos at desired stages (2 and 4 dpf) by using TRIzol reagent(Invitrogen), followed by DNase treatment (Ambion). TheiScript cDNA synthesis kit (Bio-Rad) was used for reversetranscriptions and subsequent quantitative RT-PCRs (Sso-Advanced Universal SYBR Green Supermix; Bio-Rad) wereperformed on Bio-Rad CFX96 real time PCR detection systemaccording to the manufacturer’s protocols (the cycling param-eters were 95 °C for 10 min, 40 cycles of 95 °C for 15 s, and 60 °Cfor 30 s). The PCR products were analyzed by gel electrophore-sis to confirm that they were of the expected size. The CFXManager software provided with the thermocycler (Bio-Rad)was used to determine Ct values. Expression differencesbetween samples were calculated by the ���Ct (comparativeCt) method (61) and reported without Log2 conversion to foldchanges. From separate clutches of each transgenic line, 3 poolsof 10 embryos were collected, and each sample was analyzed in

triplicate. The following primers used quantitative RT-PCR areshown here: 5�-CGAGCTGTCCAACTACCGAG-3� and 5�-TCGACCTCGAGTCAGGAGAA-3� for Hsa.CRYGD (for all�D transgenes); 5�-CGAGCTGTCTTCCCATCCA-3� and 5�-TCACCAACGTAGCTGTCTTTCTG-3� for actb2 (�-actin)as an internal control for calibration of gene expression levelsbetween samples.

Cell Death Assays—Embryos were fixed overnight at 4 °C in4% paraformaldehyde in PBS, dehydrated with 100% MeOH,and stored in �20 °C. The procedures of TUNEL staining werecarried out following the manufacturer’s suggested protocol (insitu cell death detection kit, TMR red; Sigma-Aldrich catalogno. 12156792910). Acridine Orange staining was performed byimmersing live embryos in 5 �g/ml Acridine Orange for 10 minat room temperate and then imaged with a FITC filter immedi-ately after (62).

Histology and Immunohistochemistry—Plastic histologicalsections for embryonic eyes were performed as described (63),and 1 �M sections were stained by toluidine blue. The DAPIstaining was performed on cryosections as described by Uribeand Gross (64).

Microscopy and Image Processing—Lenses of live embryos in0.3 Daneau water with 1-phenyl-2-thiourea/tricane wereanalyzed by bright field microscopy (Zeiss Axiovert 200) at 4dpf and classified into three classes on the basis of the severity oflens defects as defined in our previous study (34). Class 1 con-sisted of embryos that did not appear to have visible lens defects(“WT-like”). Class 2, referred to as the “minor” lens defectgroup, showed small and sporadic mostly spherical droplets inthe lens. Class 3, referred to as “major” lens defect group, wascharacterized by large and/or numerous droplets covering largeareas of the lens. Fluorescence images were taken with a ZeissAxioZoom.V16 microscope. Differential interference con-trast (DIC) and reflectance analysis were performed by ZeissLSM510 inverted confocal microscope with �em 488 on4-dpf embryos that were embedded in 3% methylcellulose. DICimages were taken with a 20 objective lens and used as areference region of interest for reflectance analysis, which wassubsequently digitally analyzed by the image-processing pro-gram ImageJ.

iTRAQ Quantification of Protein Changes—To quantify pro-tein expression changes in transgenic zebrafish lenses, one lensfrom 15-month-old fish (F3 adults) of each type (WT, WT �D,�DI4F, and �DI4F/V76D) was homogenized in 50 ml of homog-enizing buffer (25 mM Tris, 150 mM NaCl, 1 mM DTT, 5 mM

EDTA, pH 7.5). The sample was centrifuged at 20,000 g for 30min, and pellets were washed with another 50 ml of homoge-nizing buffer followed by centrifugation at 20,000 g. Thesupernatant was pooled together as the WSF. Protein concen-trations for WSF fractions were measured by a Bradford assay(Thermo Scientific, Rockford, IL). The pellets were consideredas the WIF. WIF was washed with water twice and suspended in100 ml of water, and an aliquot was mixed with equal volume of5% SDS for a BCA assay (Thermo Scientific). 100 mg of WSFfrom each lens was reduced with 50 mM tris-(2-carboxyethyl)phosphine at 60 °C for 1 h, alkylated with 200 mM methyl meth-anethiosulfonate at room temperature for 10 min, and digestedwith sequencing grade trypsin overnight. 20 �g of each WSF

Mechanisms of Cataract-linked �-Crystallin Mutations

25394 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 291 • NUMBER 49 • DECEMBER 2, 2016

by guest on June 24, 2020http://w

ww

.jbc.org/D

ownloaded from

fraction was then labeled with iTRAQ reagents according to themanufacturer’s instructions (AB Sciex, Foster City, CA) (114 forWT, 115 for WT �D, 116 for �DI4F, and 117 for �D I4F/V76D).For WIF fractions, the entire reconstituted pellet for each WIFsample was digested with trypsin, and 18 �g was labeled withiTRAQ reagents. Reagents were reconstituted in ethanol such thateach protein sample was iTRAQ-labeled at a final concentration of90% ethanol and labeling was performed for 2 h.

The iTRAQ-labeled samples were mixed, acidified with TFA,and subsequently desalted by a modified stage tip method priorto LC-MS/MS analysis. iTRAQ-labeled samples were analyzedusing MudPIT analysis with 13 salt pulse steps (0 mM, 25 mM, 50mM, 75 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 500mM, 1 M, and 2 M ammonium acetate). The peptides were intro-duced via nanoelectrospray into a Q Exactive mass spectrome-ter (Thermo Scientific). The Q Exactive was operated in data-dependent mode acquiring higher-energy collision-activateddissociation MS/MS scans (r 17,500) after each MS1 scan(r 70,000) on the 20 most abundant ions using an MS1 iontarget of 1 106 ions and an MS2 target of 1 105 ions. Themaximum ion time for MS/MS scans was set to 100 ms, theHCD-normalized collision energy was set to 30, dynamic exclu-sion was set to 30s, and peptide match and isotope exclusionwere enabled. For iTRAQ data analysis, mass spectra were pro-cessed using the Spectrum Mill software package (versionB.04.00; Agilent Technologies). MS/MS spectra acquired onthe same precursor m/z (�0.01 m/z) within � 1 s in retentiontime were merged. MS/MS spectra of poor quality that failedthe quality filter by not having a sequence tag length �1 wereexcluded from searching. A minimum matched peak intensityrequirement was set to 50%. For peptide identification, MS/MSspectra were searched against a Uniprot zebrafish database(June 21, 2012) appended with the human CRYGD proteinsequence. Additional search parameters included trypsinenzyme specificity with a maximum of three missed cleav-ages, � 20 ppm precursor mass tolerance, � 20 ppm (HCD)product mass tolerance, and fixed modifications includingmethyl methanethiosulfonate alkylation of cysteines andiTRAQ labeling of lysines and peptide N termini. Oxidationof methionine was allowed as a variable modification.Autovalidation was performed such that peptide assign-ments to mass spectra were designated as valid following anautomated procedure during which score thresholds wereoptimized separately for each precursor charge state and themaximum target-decoy-based false discovery rate was set to1.0% (46, 65).

Statistics—Differences among groups were analyzed byStudent’s t test. The data are shown as means � S.E. Statisticalsignificance was accepted when p � 0.05.

Author Contributions—S.-Y. W. contributed to conception of theresearch, designed and performed the research, analyzed the data,and wrote the paper. P. Z., A. W. F, and S .M. performed the researchand analyzed the data. Z. W. and K. L. S. performed the research,analyzed the data, and wrote part of the paper. H. S. M. contributedto conception of the research, designed the research, and wrote thepaper. All authors reviewed the results and approved the final ver-sion of the manuscript.

Acknowledgments—We thank Dr. Derek Claxton for critical readingof the manuscript and Frances Clark and Abudi Nashabi for techni-cal assistance.

References1. Bassnett, S. (1997) Fiber cell denucleation in the primate lens. Invest. Oph-

thalmol. Vis. Sci. 38, 1678 –16872. Beebe, D. C., Vasiliev, O., Guo, J., Shui, Y. B., and Bassnett, S. (2001)

Changes in adhesion complexes define stages in the differentiation of lensfiber cells. Invest. Ophthalmol. Vis. Sci. 42, 727–734

3. Bloemendal, H., de Jong, W., Jaenicke, R., Lubsen, N. H., Slingsby, C., andTardieu, A. (2004) Ageing and vision: structure, stability and function oflens crystallins. Prog. Biophys. Mol. Biol. 86, 407– 485

4. Delaye, M., and Tardieu, A. (1983) Short-range order of crystallin proteinsaccounts for eye lens transparency. Nature 302, 415– 417

5. Tardieu, A. (1988) Eye lens proteins and transparency: from light trans-mission theory to solution X-ray structural analysis. Annu. Rev. Biophys.Biophys. Chem. 17, 47–70

6. Benedek, G. B. (1971) Theory of transparency of the eyes. Appl. Opt. 10,459 – 473

7. Caspers, G. J., Leunissen, J. A., and de Jong, W. W. (1995) The expandingsmall heat-shock protein family, and structure predictions of the con-served “�-crystallin domain.” J. Mol. Evol. 40, 238 –248

8. de Jong, W. W., Caspers, G. J., and Leunissen, J. A. (1998) Genealogy of the�-crystallin–small heat-shock protein superfamily. Int. J. Biol. Macromol.22, 151–162

9. Haslbeck, M., Franzmann, T., Weinfurtner, D., and Buchner, J. (2005)Some like it hot: the structure and function of small heat-shock proteins.Nat. Struct. Mol. Biol. 12, 842– 846

10. McHaourab, H. S., Godar, J. A., and Stewart, P. L. (2009) Structure andmechanism of protein stability sensors: chaperone activity of small heatshock proteins. Biochemistry 48, 3828 –3837

11. Van Montfort, R., Slingsby, C., and Vierling, E. (2001) Structure and func-tion of the small heat shock protein/�-crystallin family of molecular chap-erones. Adv. Protein Chem. 59, 105–156

12. Horwitz, J. (1992) �-Crystallin can function as a molecular chaperone.Proc. Natl. Acad. Sci. U.S.A. 89, 10449 –10453

13. Horwitz, J. (2003) �-Crystallin. Exp. Eye Res. 76, 145–15314. Hanson, S. R., Smith, D. L., and Smith, J. B. (1998) Deamidation and

disulfide bonding in human lens �-crystallins. Exp. Eye Res. 67,301–312

15. Hanson, S. R., Hasan, A., Smith, D. L., and Smith, J. B. (2000) The major invivo modifications of the human water-insoluble lens crystallins are disul-fide bonds, deamidation, methionine oxidation and backbone cleavage.Exp. Eye Res. 71, 195–207

16. Lampi, K. J., Ma, Z., Hanson, S. R., Azuma, M., Shih, M., Shearer, T. R.,Smith, D. L., Smith, J. B., and David, L. L. (1998) Age-related changes inhuman lens crystallins identified by two-dimensional electrophoresis andmass spectrometry. Exp. Eye Res. 67, 31– 43

17. Lampi, K. J., Shih, M., Ueda, Y., Shearer, T. R., and David, L. L. (2002) Lensproteomics: analysis of rat crystallin sequences and two-dimensional elec-trophoresis map. Invest. Ophthalmol. Vis. Sci. 43, 216 –224

18. Lampi, K. J., Amyx, K. K., Ahmann, P., and Steel, E. A. (2006) Deamidationin human lens �B2-crystallin destabilizes the dimer. Biochemistry 45,3146 –3153

19. Truscott, R. J. (2005) Age-related nuclear cataract-oxidation is the key.Exp. Eye Res. 80, 709 –725

20. Banerjee, P. R., Pande, A., Patrosz, J., Thurston, G. M., and Pande, J. (2011)Cataract-associated mutant E107A of human �D-crystallin shows in-creased attraction to �-crystallin and enhanced light scattering. Proc.Natl. Acad. Sci. U.S.A. 108, 574 –579

21. Clark, J. I., and Muchowski, P. J. (2000) Small heat-shock proteinsand their potential role in human disease. Curr. Opin. Struct. Biol. 10,52–59

22. Koteiche, H. A., and McHaourab, H. S. (2003) Mechanism of chaperonefunction in small heat-shock proteins: phosphorylation-induced acti-

Mechanisms of Cataract-linked �-Crystallin Mutations

DECEMBER 2, 2016 • VOLUME 291 • NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 25395

by guest on June 24, 2020http://w

ww

.jbc.org/D

ownloaded from

vation of two-mode binding in �B-crystallin. J. Biol. Chem. 278,10361–10367

23. Mchaourab, H. S., Dodson, E. K., and Koteiche, H. A. (2002) Mechanism ofchaperone function in small heat-shock proteins: two-mode binding ofthe excited states of T4 lysozyme mutants by �A-crystallin. J. Biol. Chem.277, 40557– 40566

24. Sharma, K. K., Kaur, H., and Kester, K. (1997) Functional elements inmolecular chaperone �-crystallin: identification of binding sites in �B-crystallin. Biochem. Biophys. Res. Commun. 239, 217–222

25. Sharma, K. K., and Santhoshkumar, P. (2009) Lens aging: effects of crys-tallins. Biochim. Biophys. Acta 1790, 1095–1108

26. Benesch, J. L., Ayoub, M., Robinson, C. V., and Aquilina, J. A. (2008) Smallheat shock protein activity is regulated by variable oligomeric substruc-ture. J. Biol. Chem. 283, 28513–28517

27. Koteiche, H. A., Claxton, D. P., Mishra, S., Stein, R. A., McDonald, E. T.,and Mchaourab, H. S. (2015) Species-specific structural and functionaldivergence of �-crystallins: zebrafish �Ba- and rodent �A(ins)-crystallinencode activated chaperones. Biochemistry 54, 5949 –5958

28. Jaya, N., Garcia, V., and Vierling, E. (2009) Substrate binding site flexibilityof the small heat shock protein molecular chaperones. Proc. Natl. Acad.Sci. U.S.A. 106, 15604 –15609

29. Stengel, F., Baldwin, A. J., Painter, A. J., Jaya, N., Basha, E., Kay, L. E.,Vierling, E., Robinson, C. V., and Benesch, J. L. (2010) Quaternary dynam-ics and plasticity underlie small heat shock protein chaperone function.Proc. Natl. Acad. Sci. U.S.A. 107, 2007–2012

30. Liu, H., Du, X., Wang, M., Huang, Q., Ding, L., McDonald, H. W., Yates,J. R., 3rd, Beutler, B., Horwitz, J., and Gong, X. (2005) Crystallin �B-I4Fmutant protein binds to �-crystallin and affects lens transparency. J. Biol.Chem. 280, 25071–25078

31. Wang, K., Cheng, C., Li, L., Liu, H., Huang, Q., Xia, C. H., Yao, K., Sun, P.,Horwitz, J., and Gong, X. (2007) �D-Crystallin associated protein aggre-gation and lens fiber cell denucleation. Invest. Ophthalmol. Vis. Sci. 48,3719 –3728

32. Mishra, S., Stein, R. A., and McHaourab, H. S. (2012) Cataract-linked�D-crystallin mutants have weak affinity to lens chaperones �-crystallins.FEBS Lett. 586, 330 –336

33. Moreau, K. L., and King, J. A. (2012) Cataract-causing defect of a mutant�-crystallin proceeds through an aggregation pathway which bypassesrecognition by the �-crystallin chaperone. PLoS One 7, e37256

34. Zou, P., Wu, S. Y., Koteiche, H. A., Mishra, S., Levic, D. S., Knapik, E.,Chen, W., and Mchaourab, H. S. (2015) A conserved role of �A-crystallinin the development of the zebrafish embryonic lens. Exp. Eye Res. 138,104 –113

35. Voisine, C., Pedersen, J. S., and Morimoto, R. I. (2010) Chaperone net-works: tipping the balance in protein folding diseases. Neurobiol. Dis. 40,12–20

36. Lampi, K. J., Kim, Y. H., Bächinger, H. P., Boswell, B. A., Lindner, R. A.,Carver, J. A., Shearer, T. R., David, L. L., and Kapfer, D. M. (2002) De-creased heat stability and increased chaperone requirement of modifiedhuman �B1-crystallins. Mol. Vis. 8, 359 –366

37. Michiel, M., Duprat, E., Skouri-Panet, F., Lampi, J. A., Tardieu, A., Lampi,K. J., and Finet, S. (2010) Aggregation of deamidated human �B2-crystal-lin and incomplete rescue by �-crystallin chaperone. Exp. Eye Res. 90,688 – 698

38. Jaenisch, R., Jähner, D., Nobis, P., Simon, I., Löhler, J., Harbers, K., andGrotkopp, D. (1981) Chromosomal position and activation of retroviralgenomes inserted into the germ line of mice. Cell 24, 519 –529

39. Wilson, C., Bellen, H. J., and Gehring, W. J. (1990) Position effects oneukaryotic gene expression. Annu. Rev. Cell Biol. 6, 679 –714

40. Rossant, J., Nutter, L. M., and Gertsenstein, M. (2011) Engineering theembryo. Proc. Natl. Acad. Sci. U.S.A. 108, 7659 –7660

41. Sandilands, A., Hutcheson, A. M., Long, H. A., Prescott, A. R., Vrensen,G., Löster, J., Klopp, N., Lutz, R. B., Graw, J., Masaki, S., Dobson, C. M.,MacPhee, C. E., and Quinlan, R. A. (2002) Altered aggregation prop-erties of mutant �-crystallins cause inherited cataract. EMBO J. 21,6005– 6014

42. Zhang, L. Y., Yam, G. H., Fan, D. S., Tam, P. O., Lam, D. S., and Pang, C. P.(2007) A novel deletion variant of �D-crystallin responsible for congenitalnuclear cataract. Mol. Vis. 13, 2096 –2104

43. Meehan, S., Berry, Y., Luisi, B., Dobson, C. M., Carver, J. A., and MacPhee,C. E. (2004) Amyloid fibril formation by lens crystallin proteins and itsimplications for cataract formation. J. Biol. Chem. 279, 3413–3419

44. Scheer, N., and Campos-Ortega, J. A. (1999) Use of the Gal4-UAS tech-nique for targeted gene expression in the zebrafish. Mech. Dev. 80,153–158

45. Hayes, J. M., Hartsock, A., Clark, B. S., Napier, H. R., Link, B. A., and Gross,J. M. (2012) Integrin �5/fibronectin1 and focal adhesion kinase are re-quired for lens fiber morphogenesis in zebrafish. Mol. Biol. Cell 23,4725– 4738

46. Wang, Z., and Schey, K. L. (2015) Proteomic analysis of lipid raft-likedetergent-resistant membranes of lens fiber cells. Invest. Ophthalmol. Vis.Sci. 56, 8349 – 8360

47. Ross, P. L., Huang, Y. N., Marchese, J. N., Williamson, B., Parker, K.,Hattan, S., Khainovski, N., Pillai, S., Dey, S., Daniels, S., Purkayastha, S.,Juhasz, P., Martin, S., Bartlet-Jones, M., He, F., et al. (2004) Multiplexedprotein quantitation in Saccharomyces cerevisiae using amine-reactiveisobaric tagging reagents. Mol. Cell. Proteomics 3, 1154 –1169

48. Li, W. C., Kuszak, J. R., Dunn, K., Wang, R. R., Ma, W., Wang, G. M.,Spector, A., Leib, M., Cotliar, A. M., and Weiss, M. (1995) Lens epithe-lial cell apoptosis appears to be a common cellular basis for non-con-genital cataract development in humans and animals. J. Cell Biol. 130,169 –181

49. Okamura, N., Ito, Y., Shibata, M. A., Ikeda, T., and Otsuki, Y. (2002) Fas-mediated apoptosis in human lens epithelial cells of cataracts associatedwith diabetic retinopathy. Med. Electron Microsc. 35, 234 –241

50. Yan, Q., Liu, J. P., and Li, D. W. (2006) Apoptosis in lens development andpathology. Differentiation 74, 195–211

51. Zhang, L., Yan, Q., Liu, J. P., Zou, L. J., Liu, J., Sun, S., Deng, M., Gong, L.,Ji, W. K., and Li, D. W. (2010) Apoptosis: its functions and control in theocular lens. Curr. Mol. Med. 10, 864 – 875

52. Harocopos, G. J., Alvares, K. M., Kolker, A. E., and Beebe, D. C. (1998)Human age-related cataract and lens epithelial cell death. Invest. Ophthal-mol. Vis. Sci. 39, 2696 –2706

53. Hinaux, H., Blin, M., Fumey, J., Legendre, L., Heuzé, A., Casane, D., andRétaux, S. (2015) Lens defects in Astyanax mexicanus cavefish: evolu-tion of crystallins and a role for �A-crystallin. Dev. Neurobiol. 75,505–521

54. De Maria, A., and Bassnett, S. (2007) DNase II� distribution and activity inthe mouse lens. Invest. Ophthalmol. Vis. Sci. 48, 5638 –5646

55. Firtina, Z., Danysh, B. P., Bai, X., Gould, D. B., Kobayashi, T., and Duncan,M. K. (2009) Abnormal expression of collagen IV in lens activates un-folded protein response resulting in cataract. J. Biol. Chem. 284,35872–35884

56. Nakahara, M., Nagasaka, A., Koike, M., Uchida, K., Kawane, K., Uchiyama,Y., and Nagata, S. (2007) Degradation of nuclear DNA by DNase II-likeacid DNase in cortical fiber cells of mouse eye lens. FEBS J. 274,3055–3064

57. Nishimoto, S., Kawane, K., Watanabe-Fukunaga, R., Fukuyama, H.,Ohsawa, Y., Uchiyama, Y., Hashida, N., Ohguro, N., Tano, Y., Mo-rimoto, T., Fukuda, Y., and Nagata, S. (2003) Nuclear cataract causedby a lack of DNA degradation in the mouse eye lens. Nature 424,1071–1074

58. Goishi, K., Shimizu, A., Najarro, G., Watanabe, S., Rogers, R., Zon, L. I.,and Klagsbrun, M. (2006) �A-Crystallin expression prevents �-crystallininsolubility and cataract formation in the zebrafish cloche mutant lens.Development 133, 2585–2593

59. Kurita, R., Sagara, H., Aoki, Y., Link, B. A., Arai, K., and Watanabe, S.(2003) Suppression of lens growth by �A-crystallin promoter-driven ex-pression of diphtheria toxin results in disruption of retinal cell organiza-tion in zebrafish. Dev. Biol. 255, 113–127

60. Kwan, K. M., Fujimoto, E., Grabher, C., Mangum, B. D., Hardy, M. E.,Campbell, D. S., Parant, J. M., Yost, H. J., Kanki, J. P., and Chien, C. B.

Mechanisms of Cataract-linked �-Crystallin Mutations

25396 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 291 • NUMBER 49 • DECEMBER 2, 2016

by guest on June 24, 2020http://w

ww

.jbc.org/D

ownloaded from

(2007) The Tol2kit: a multisite gateway-based construction kit for Tol2transposon transgenesis constructs. Dev. Dyn. 236, 3088 –3099

61. Livak, K. J., and Schmittgen, T. D. (2001) Analysis of relative gene expres-sion data using real-time quantitative PCR and the 2(���C(T)) method.Methods 25, 402– 408

62. Robu, M. E., Larson, J. D., Nasevicius, A., Beiraghi, S., Brenner, C., Farber,S. A., and Ekker, S. C. (2007) p53 activation by knockdown technologies.PLoS Genet. 3, e78

63. Nuckels, R. J., and Gross, J. M. (2007) Histological preparation of embry-onic and adult zebrafish eyes. CSH Protoc. prot4846

64. Uribe, R. A., and Gross, J. M. (2007) Immunohistochemistry oncryosections from embryonic and adult zebrafish eyes. CSH Protoc.prot4779

65. Hill, E. G., Schwacke, J. H., Comte-Walters, S., Slate, E. H., Oberg, A. L.,Eckel-Passow, J. E., Therneau, T. M., and Schey, K. L. (2008) A statisticalmodel for iTRAQ data analysis. J. Proteome Res. 7, 3091–3101

Mechanisms of Cataract-linked �-Crystallin Mutations

DECEMBER 2, 2016 • VOLUME 291 • NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 25397

by guest on June 24, 2020http://w

ww

.jbc.org/D

ownloaded from

Schey and Hassane S. MchaourabShu-Yu Wu, Ping Zou, Alexandra W. Fuller, Sanjay Mishra, Zhen Wang, Kevin L.

Proteostasis Network That Senses Protein Stability-Crystallin Variants in Zebrafish Reveals aγExpression of Cataract-linked

doi: 10.1074/jbc.M116.749606 originally published online October 21, 20162016, 291:25387-25397.J. Biol. Chem. 

  10.1074/jbc.M116.749606Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

Supplemental material:

  http://www.jbc.org/content/suppl/2016/10/21/M116.749606.DC1

  http://www.jbc.org/content/291/49/25387.full.html#ref-list-1

This article cites 63 references, 22 of which can be accessed free at

by guest on June 24, 2020http://w

ww

.jbc.org/D

ownloaded from