Altered ubiquitin causes perturbed calciumhomeostasis, hyperactivation of calpain, dysregulateddifferentiation, and cataractKe Liua,b,1,2, Lei Lyua,b,1, David Chinc,d, Junyuan Gaoe, Xiurong Sune, Fu Shanga, Andrea Caceresa, Min-Lee Changa,Sheldon Rowana, Junmin Pengc,d, Richard Mathiase, Hideko Kasaharaf, Shuhong Jiana, and Allen Taylora,g,2

aLaboratory for Nutrition and Vision Research, US Department of Agriculture Human Nutrition Research Center on Aging, Tufts University, Boston, MA 02111;bKey Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life Science, Sichuan University, Chengdu, Sichuan610064, People’s Republic of China; Departments of cStructural Biology and dDevelopmental Neurobiology, St. Jude Proteomics Facility, St. Jude Children’sResearch Hospital, Memphis, TN 38105; eDepartment of Physiology and Biophysics, State University of New York, Stony Brook, NY 11794; fPhysiology andFunctional Genomics, University of Florida College of Medicine, Gainesville, FL 32610-0274; and gDepartment of Biological Chemistry, Weizmann Institute ofScience, Rehovot 7610001, Israel

Edited* by Gregory A. Petsko, Weill Cornell Medical College, New York, NY, and approved December 10, 2014 (received for review March 6, 2014)

Although the ocular lens shares many features with other tissues,it is unique in that it retains its cells throughout life, making it idealfor studies of differentiation/development. Precipitation of pro-teins results in lens opacification, or cataract, the major blindingdisease. Lysines on ubiquitin (Ub) determine fates of Ub-proteinsubstrates. Information regarding ubiquitin proteasome systems(UPSs), specifically of K6 in ubiquitin, is undeveloped. We expressedin the lens a mutant Ub containing a K6W substitution (K6W-Ub).Protein profiles of lenses that express wild-type ubiquitin (WT-Ub)or K6W-Ub differ by only ∼2%. Despite these quantitatively minordifferences, in K6W-Ub lenses and multiple model systems we ob-served a fourfold Ca2+ elevation and hyperactivation of calpain inthe core of the lens, as well as calpain-associated fragmentation ofcritical lens proteins including Filensin, Fodrin, Vimentin, β-Crystallin,Caprin family member 2, and tudor domain containing 7. Truncationscan be cataractogenic. Additionally, we observed accumulation ofgap junction Connexin43, and diminished Connexin46 levels in vivoand in vitro. These findings suggest that mutation of Ub K6 altersUPS function, perturbs gap junction function, resulting in Ca2+ ele-vation, hyperactivation of calpain, and associated cleavage of sub-strates, culminating in developmental defects and a cataractouslens. The data show previously unidentified connections betweenUPS and calpain-based degradative systems and advance our un-derstanding of roles for Ub K6 in eye development. They alsoinform about new approaches to delay cataract and other proteinprecipitation diseases.

cataract | ubiqutin | calpain | connexin | development

Many age-related diseases such as cataracts, macular de-generation, Alzheimer’s, Parkinson’s, Huntington’s, and

several premature aging syndromes, appear to be causally asso-ciated with accumulation of abnormal proteins (1, 2). The ac-cumulation of damaged proteins in many age-related diseasesinvolves a vicious cycle of stress-induced postsynthetic mod-ifications to bulk and catalytically critical molecules and limitedcapacity to remove the damaged proteins, thus accelerating ac-cumulation of damaged proteins and protein precipitation (1–3).Clarity is essential for lens function. Age-related cataract is dueto the aggregation and precipitation of proteins from the nor-mally clear milieu and is the leading cause of adult blindnessworldwide, affecting more than 18 million people (4). Congenitalcataracts also involve protein precipitation (5).The lens is an excellent system to study specific relationships

between proteolytic pathways, stress, and maintenance of proteinquality because all of the cells are retained throughout life. Theoldest lens tissue is found at the center or core of the lens. Crys-tallins, the major gene products of the lens, are very long-livedproteins, with half-lives of decades, and their aberrant synthesis

or modification results in aggregation, insolubilization, and cat-aract (6). Common age-related stresses that confront proteins inthe lens and other tissues during aging include oxidation, glyca-tion, and methylation, as well as their sequels (3, 6–9). Effectivestress-reducing systems including antioxidants, antioxidant andrepair enzymes, and chaperone and proteolytic capacities helplimit damage and maintain solubility and function in youngertissues (3).There are three general systems for intracellular proteolysis:

lysosomal/autophagic mechanisms, calcium-activated proteases,or calpains, and the ubiquitin proteasome system (UPS). Be-cause nuclei and lysosomes are removed from most of the lenscells in regions where most cataracts form, this leaves only cy-toplasmic proteases, including the UPSs and calpains, to removedamaged proteins to retain lens function (3). There are two basicsteps to the UPS: conjugation of substrates to multiple ubiquitins(Ubs) followed by degradation of the protein substrate. Ubiq-uitin is a highly conserved protein with seven lysines (10). The

Significance

Eye lens opacification or cataract is the most prevalent age-related disease, blinding 18 million people. Cataractogenesisinvolves accumulation and precipitation of damaged proteinsfrom the normally clear lens. The ubiquitin proteolytic system isthemain cytoplasmic degradative pathway that is chargedwithselectively removing damaged proteins. Ubiquitin has sevenlysines. Although lysine 6 is involved in less than 3% of ubiq-uitin conjugates andwe find few changes in lens proteins whenlysine 6 is unavailable, we observed that mutating ubiquitinlysine 6 alters cell coupling, resulting in Ca2+ elevation, hyper-activation of calpain, and associated cleavage of substrates,culminating in developmental defects and a cataractous lens.The data show previously unidentified connections betweenubiquitin proteasome systems (UPSs) and calpain-based degra-dative systems and illuminate roles for ubiquitin lysine 6 indevelopment.

Author contributions: K.L., F.S., and A.T. designed research; K.L., L.L., D.C., J.G., X.S., F.S.,A.C., M.-L.C., J.P., R.M., H.K., and S.J. performed research; K.L., D.C., J.G., X.S., R.M., andA.T. contributed new reagents/analytic tools; K.L., L.L., D.C., J.G., X.S., F.S., A.C., M.-L.C.,S.R., J.P., R.M., and A.T. analyzed data; and K.L., F.S., M.-L.C., S.R., R.M., and A.T. wrotethe paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.1K.L. and L.L. contributed equally to this work.2To whom correspondence may be addressed. Email: allen.taylor@tufts.edu or kliu@scu.edu.cn.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1404059112/-/DCSupplemental.

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lysines are used, in the first step of the UPS, to form inter-Ublinkages that lead to Ub polymers that are conjugated to proteinsubstrates. Commonly, proteins with K48-linked Ub oligomersattached are scheduled for degradation by the 26S proteasome.The UPS is also involved at multiple critical stages of pro-liferation, differentiation, and development in most tissues.It was recently noted that only a minute proportion of Ub

conjugates use K6 on Ub (11). Thus, we were surprised to findthat expression of higher levels of K6W-Ub in the lens producedcataracts (12). Other work showed that Ub mutations in whichK6 is replaced by various amino acids, i.e., K6W-Ub, are con-jugation competent but proteolytically incompetent. Thus, cellsand tissues in which K6W-Ub is expressed accumulate Ub con-jugates (13). Exchanging K for A or R has the same effects.Although structural information about Ub conjugates built usingK6 is becoming available (14, 15), a complete understanding offeatures that render such conjugates biologically stable remain tobe elucidated.Calpain is up-regulated by increased Ca2+. Gap junction

proteins, or Connexins (Cxs), are required for maintaining Ca2+

homeostasis (16). UPS-dependent degradation of Cx has beenobserved in CHO and BWEM cells (17). The formation of cat-aracts in lenses that express K6W-Ub is compatible with novelfunctional connections between UPS activity, regulation of Cxand Ca2+, calpain activity, and lens clarity. To test the hypothesis,we targeted expression of K6W-Ub to the lens, and we monitoredstability of multiple proteins, Cx function and ubiquitination, Ca2+,calpain activity, protein integrity, solubility, and localization, aswell as lens clarity.

ResultsCataract and Altered Protein Levels in K6W-Ub Lens. K6W-Ub ex-pression in mice in the C57BL/6J×FVB or FVB backgroundresulted in cataracts (Fig. 1A) (12). With the objective of un-derstanding the consequences of expressing K6W-Ub on differ-entiation, we compared the proteome in lenses in which themutant or WT-Ub was transgenically expressed. Virtually all ofthe experiments in this work were replicated in both lines of micewith comparable results. Postnatal day 1 (P1) lenses were usedbecause differences in development between K6W-Ub and WTmice are obvious at this time (12). We identified ∼2,300 proteinsin WT- or K6W-Ub lens lysates from the total of ∼91,000 pep-tide spectral counts (Dataset S1). Replicate analyses were usedto corroborate the findings. Consistent with K6 on Ub beinginvolved in a limited proportion of conjugates, only 42 proteinswere found to have different levels (P ≤ 0.05 and false-discoveryrate < 0.20) in the WT vs. K6W-Ub lens (Tables S1 and S2). Alist of the 20 proteins (sorted by P value) that show the greatestdifferences in levels is presented in Table S3. Notably, a signifi-cant portion of these proteins (14 out of 20; highlighted in boldin Table S3) are related to lens development. Specifically,γ-Crystallins and the cytoskeleton protein Filensin are decreased∼3- and 21-fold, respectively, in K6W-Ub lens. Other cytoskel-eton proteins, Vimentin and Fodrin (spectrin α2), are elevated1.4- and 18-fold, respectively, in the mutant lens.Multiple approaches were exploited to mechanistically link

expression of K6W-Ub, the altered proteome, and cataracts.Western blots clearly verified the significant decrease in theamount of Filensin in K6W-Ub lenses in both genotypes (Fig.1B, Upper, and Fig. S1B, Upper). In the C57BL/6J×FVB/N, thecleavage products appear to have been even further degraded.Although there was a decrease of Filensin mRNA in the FVBline, this was not observed in the C57BL/6J×FVB/N mice (Fig.S1C, Lower). Thus, the difference in protein levels or spectralcounts implies an additional regulatory mechanism. The absenceof CP49 could not account for this diminution in the parentprotein or even fragments because the C57BL/6J×FVB/N is re-plete with CP49. Interestingly, in K6W-Ub tissue, the level of

full-length Vimentin, an intermediate filament protein, is com-parable or lower relative to the WT-Ub sample in both geno-types; however, the blots also indicate that native Vimentin isconverted to limited cleavage products that accumulate twofoldto threefold more in K6W-Ub than in WT-Ub lenses (Fig. 1B,Middle, asterisk, and Fig. S1 B and D). Similar findings wereobtained for Fodrin (Fig. 1C and Fig. S1F).To determine whether changes in levels, integrity, or locali-

zation of cytoskeletal proteins are associated with alterations indevelopment, lenses were evaluated immunohistochemically.Whereas F-actin is aligned with fiber cells in theWT-Ub lens (Fig.1D and Fig. S1E, panels 1 and 2), in the mutant lens the distri-bution of F-actin is more heterogeneous, often localized inclumps (Fig. 1D and Fig. S1E, panels 3 and 4). Similar disruptionsare also observed for Vimentin in the K6W-Ub lens (Fig. 1D,panels 1–4, and Fig. S1E, panels 5–8). By overlapping the F-actinand intermediate filament images, a distinct perturbation of cy-toskeletal structure in K6W-Ub lenses is revealed (Fig. 1D,compare panels 1 and 2, and panels 3 and 4).Notably, three RNA-associated proteins, Tdrd7 (tudor do-

main containing 7), Caprin2 (Caprin family member 2), andheterogeneous nuclear ribonucleoprotein A1, are also amongthe most differently expressed in K6W-Ub vs. WT-Ub lenses(Table S3). Deficiency of Tdrd7 gene causes juvenile cataract

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Fig. 1. Cataract and cleavage of cytoskeleton proteins in P1 lenses of K6W-Ub vs. WT-Ub transgenic mice. (A) Images of WT-Ub and K6W-Ub C57BL/6J×FVB/N ex vivo P1 lenses. The WT-Ub C57BL/6JxFVB/N lens (Top) is clear,whereas the K6W-Ub C57BL/6J×FVB/N lens (Middle) is opacified. In vivoimage of P30 K6W-Ub C57BL/6J×FVB/N lens showing nuclear cataract(Lower). (B and C) Pools of WT and K6W-Ub lens lysates, which containedthree to four lenses in each pool, were tested. Lysates were resolved on 10%(wt/vol) (B) or 6.5% (wt/vol) (C ) SDS gels, and proteins of interest weredetected by Western blotting. β-Actin or ubiquitin-activating enzyme E1were used as loading controls. The arrows indicate the full-length Vimentinor Fodrin, and the asterisks indicate cleavage products. For quantification ofall lens pools tested, see Fig. S1 B–D. (D) 1–4: Sagittal sections of WT-Ub andK6W-Ub P1 lenses stained for Vimentin (green), F-actin (red), and nuclei(blue). (Scale bars: 20 μm.) Bow, bow region; Epi, epithelia; Nuc, lens nucleus.5 and 6: In situ calpain activity assay of K6W-Ub and WT-Ub P1 lenses usingfluorogenic BOC-LM-CMAC substrate followed by confocal microscopy. (Scalebars: 60 μm.) The arrow indicates the calpain activation at the center (nu-cleus) of K6W-Ub lenses. 7 and 8: Immunofluorescence staining of Cx43(green) in P1 K6W-Ub and WT-Ub lenses. The filled arrow indicates area ofhigh concentration of Cx43 in K6W-Ub lens, and the asterisks indicate thecataract. Blue: DAPI staining of nuclei. (Scale bars: 20 μm.)

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(18). Caprin2 is involved in erythroblast terminal differentiation,processes with extensive similarities to lens cell development(19). Interestingly, Caprin2 is expressed at higher levels uponlens development (20) and is predicted to be a cataract-associ-ated gene (21). Mass spectrometry indicated lower levels ofTdrd7 and Caprin2 in K6W-Ub lenses (Table S3). This wascorroborated by Western blots that show robust levels of theseproteins at P1 and P15 in WT-Ub lenses, but only minimal levelsof Tdrd7 and Caprin2 in pools of K6W lenses (Fig. S1G, lanes 2and 4 vs. 1 and 3, and Fig. S1H, Upper). The similarity of mRNAlevels for Caprin2 in WT-Ub and K6W-Ub lenses suggests thatthe difference in protein levels is not primarily due to influencesof transcription (Fig. S1H, Lower). The same is probably true forFilensin and Tdrd7 despite differences in mRNA levels of theseproteins in the WT vs. Ub-mutant lens (see below).

Expressing K6W-Ub Induces Activation of Calpain and Cleavage ofβ-Crystallins, Fodrin, Filensin, Vimentin, Tdrd7, and Caprin2. The ex-tensive fragmentation of functionally different proteins sug-gested an unanticipated acceleration of proteolysis in the K6W-Ub lens. Calpain is one of the dominant proteases in mammalianlenses. Fodrin, Filensin, and Vimentin are known substrates ofcalpain in lens (22–24), and the appearance of cleavage productssuggests that there is elevated calpain activity in lenses in whichK6W-Ub is expressed. Relationships between calpain and ex-pression of K6W-Ub were examined using four different assays.MS data and corroborating Western blots indicate that Cal-

pain 3 is a dominant calpain in both WT-Ub and K6W-Ub lens,and the relative level of the full-length Calpain 3 protein is dis-tinctly lower in K6W-Ub lens (Dataset S1 and Fig. 2A, Upper).Active calpain autolyzes (25). Higher levels of calpain fragmentat the dye front and the lower levels of full-length Calpain 3 areconsistent with calpain activation in the K6W-Ub lens (Fig. 2A,Middle). By 15 d, Calpain 3 fragmentation has progressed in boththe WT-Ub and K6W-Ub lens, and by 36 d of age, Calpain 3 wasonly detected in WT-Ub lenses. Real-time PCR indicated thatlevels of Capn3 mRNA in K6W-Ub lenses were indistinguishablefrom those in WT-Ub lenses, consistent with differential post-synthetic regulation of Calpain 3 rather than decreased transcrip-tion (Fig. S2).To directly probe for calpain activity, we used a fluorescent

calpain activity assay in freshly removed lenses. In this assay,a fluorescent blue product is formed by active calpain. Greatercalpain activity, particularly in the cortex and core, was indicatedby blue calpain reaction product in K6W-Ub lenses (Fig. 1D,panels 5 and 6). These are the oldest tissues. The findings aresimilar in additional sets of P1, P3, and P6 K6W-Ub lens (whitearrows in lower panels of Fig. S3 vs. upper panels). As for fila-ment proteins, calpain activity was heterogeneously distributed

in the K6W-Ub lens. In contrast, no calpain activity was observedin the core of the WT-Ub lens, as might be expected from thelow Ca2+ in the WT-Ub lenses (see below). It is not possible tohomogenize these lenses and retest calpain activity under cell-free conditions because calpain and Ca2+ localization is lost uponhomogenization. The decreased difference in levels of calpainactivity between the K6W-Ub lens and the WT-Ub lens beyondP7 is similar to findings of limited differences in levels of majorproteins beyond this age (12).β-Crystallin is a bona fide calpain substrate and β-Crystallin

cleavage and precipitation are observed upon calpain activation(26, 27). β-Crystallin was observed in the WT-Ub and K6W-Ublenses, but cleavage of β-Crystallin was observed only in theK6W-Ub lens (Fig. 2B, lane 2 vs. 1). The extensive cleavage andinsolubilization of the β-Crystallin fragment, only in the K6W-Ublens (Fig. 2B, lane 4 vs. 3), lends additional support to the hy-pothesis that K6W-Ub–associated calpain activation is relatedto cataractogenesis.To further corroborate that Filensin (24), Tdrd7, and Caprin2

are calpain substrates in lenses from both genotypes, in vitrodegradation assays were performed using mouse lens lysate un-der conditions that enhanced or precluded calpain activation.Filensin is degraded when calpain activity is enhanced by Ca2+

addition (Fig. 3A, compare lane 3 to lane 1). Tdrd7 and Caprin2are also degraded when calpain activation is enhanced (Fig. S4,compare lane 3 to lane 1, both panels). All three proteins arestable when calpain is inactive (+EGTA; Fig. 3A and Fig. S4,compare lane 3 vs. 2). Together, the findings support the hy-pothesis that calpain is responsible for the fragmentation ofmultiple proteins in K6W-Ub lenses. In comparison, γ-Crys-tallin and GAPDH (see below) are not significantly degradedby calpain (Fig. 3A, Bottom).Finally, to corroborate that Calpain 3 is necessary for substrate

degradation, we partially or completely immunodepleted Cal-pain 3 from lens lysates. The degradation of Filensin, Tdrd7, andCaprin2 was partially or fully prevented by depletion of Calpain3 in a time- (Fig. 3B, lanes 1–4, 5–8) and dosage-dependent manner(Fig. 3B, lanes 9–12, vs. 5–8 vs. 1–4). GAPDH was also partiallydecreased over time but this was Calpain 3 independent.

Calcium Accumulation and Membrane-Coupling Disruption in K6W-UbLens. Next, we tested the hypothesis that K6W-Ub expressioncauses elevation of Ca2+ with the Ca2+-sensitive dye FURA2.The Ca2+-stimulated signal in K6W-Ub lenses was >1.6-fold thatin WT-Ub lenses in C57BL/6J×FVB/N and FVB/N mice (Fig. S5A and B). Furthermore, Ca2+ concentrations were >4.5-foldhigher in the core of the K6W-Ub lens vs. the WT-Ub lens (1,700vs. ∼350 nM, respectively) (Fig. 4). These are levels known toactivate calpain (28). In the cortical areas, there was also anapproximately fourfold difference in Ca2+ concentrations be-tween the K6W-Ub lens vs. WT-Ub lens (∼800 vs. 200 nM, re-spectively) (Fig. 4). Similar results were obtained in C57BL/6J×FVB/N mice (Fig. S5C). These data indicate that elevatedcalpain activity in the K6W-Ub lens reflects a significant increasein Ca2+ concentration due to expression of this mutant Ub.Gap junctions play critical roles in regulating intracellular Ca2+

in the lens (29). Series resistance of K6W-Ub and WT-Ub P1–P3lenses was measured immediately after the killing to determinegap junction coupling. Although the resistance at the surface ofboth K6W-Ub and WT-Ub lenses was indistinguishable, the re-sistance in the core of K6W-Ub lenses was approximately three-fold higher in the K6W-Ub compared with WT-Ub lenses (Fig.S5D). The Ca2+ gradient is 5.7-fold greater in K6W-Ub comparedwith WT-Ub lenses. Thus, an increase in gap junction-couplingresistance appears to account for a significant fraction, but not all,of the gradient in Ca2+. In support of greater disruption of gapjunctions and greater Ca2+ influx in the K6W-Ub lenses, the input

B A

Fig. 2. Activation of calpain cleavage and insolubilization of β-Crystallins inK6W-Ub lens. (A) Immunoblot detection of calpain in P1, P15, and P36 lensesidentifies calpain in these lenses and indicates that it is autolyzed more rapidlyin K6W-Ub lenses. Images were taken using short or longer exposure time tovisualize full-length protein or fragments near the dye front, respectively. (B)β-Crystallins in the soluble and insoluble fractions in WT and K6W-Ub lens.

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resistance of the K6W-Ub lenses was 2.4 ± 1.9 KΩ, whereas thatof WT-Ub lenses was 7.8 ± 1.9 KΩ.

Expressing K6W-Ub Diminishes Gap Junction Protein Cx46, but StabilizesCx43: A Mechanistic Link Between UPS and Calpain Activities. Gapjunction proteins Cxs 43, 46, and 50 regulate Ca2+ in lens. Al-though Cx46 and Cx50 are primarily responsible for membranecoupling and Ca2+ regulation in lens inner fiber cells (29), Cx43is more localized to outer lens tissue (30, 31). To investigatewhether disruption of membrane coupling, due to altered Cxlevels, might account for the accumulation of Ca2+ observed inK6W-Ub lens, we analyzed levels of each Cx. Mass spectrometrydata indicated that Cx46 and Cx50 are 1.8- and 1.5-fold lower,respectively, in K6W-Ub lens (Dataset S1). Cx43 was not detectedin our MS analysis. Consistent with MS data, Cx46 levels aresignificantly decreased by ∼70% in the K6W-Ub lens (Fig. 5A andFig. S6A, Upper). In contrast, the level of Cx43 is increased ∼1.8-fold in K6W-Ub lenses (Fig. 5B, lane 2, arrowhead, and Fig. S6B,Upper), especially in outer fibers and epithelial cells (Fig. 1D,panels 7 and 8, and Fig. S6C, arrows). mRNA levels for Cx43 andCx46 are comparable in the K6W-Ub and WT-Ub lenses (Fig. S6A and B, Lower), suggesting that the increased level of Cx43 inK6W-Ub lenses is likely due to impaired degradation rather thanmajor alterations in synthesis of Cx43 (32-34). Moreover, moreCx43 is also found as high mass moieties in K6W-Ub lens, pre-sumably Cx43 conjugated to Ub, much of which might be ex-pected to incorporate K6W-Ub (Fig. 5B, compare lane 2 to lane1). Furthermore, comparison of isolated Ub conjugates clearlyindicates more ubiquitinated Cx43 conjugates in K6W-Ub lensesvs. WT lenses (Fig. 5B, compare lane 6 to lane 5). Such conjugateswould be expected to accumulate because K6W-Ub conjugatesresist degradation (13).To verify that K6W-Ub stabilizes Cx43, we elicited the ubiq-

uitination of Cx43 by phorbol-12-myristate-13-acetate (PMA)treatment or short serum depletion (35) in human lens epithelialcells (HLECs) in vitro (Fig. 5C). Ubiquitin conjugates increasedupon PMA treatment (lanes 4, 6, and 8, vs. 3, 5, and 7, re-spectively) and brief serum depletion treatments (lanes 3, 4, 7,and 8, vs. 1, 2, 5, and 6, respectively) in WT-Ub (lanes 1–4)- andK6W-Ub (lanes 5–8)-expressing cells. In all cases, expression of

K6W-Ub also increased levels of Cx43 in its unmodified form by∼14–29%, and even more in its modified, Ub-conjugated, forms(Fig. 5D). This corroborates the in vivo data (Fig. 5B). Thesedata support the hypothesis that Cx43 is a substrate for ubiq-uitination, that K6W-Ub enters into conjugates with Cx43, andthat K6W-Ub stabilizes ubiquitinated and nonubiquitinated Cx43in lens epithelial cells.To further test the hypothesis that elevation of Cx43 is directly

related to accumulation of intracellular Ca2+, we expressed Cx43in HeLa cells (which do not express endogenous Cx43). The Insetshows that Cx43 is expressed. We observed a greater than two-fold accumulation of Ca2+ (Fig. 6). As expected, overexpressionof Cx43 also enhanced Ca2+ in HLECs (Fig. S7).

DiscussionUb pathways start with tagging of substrate proteins by covalentattachment of Ub. There have been major advances in eluci-dating structure/function of many enzymes that are required toattach ubiquitin to substrates; however, little is known about thebiologic effects of mutations in Ub per se in directing differen-tiation. Of the seven lysines on Ub, less than 1% of ubiquitinconjugates use K6. Surprisingly, eye lens cataract, perhaps themost common human disease, results when K6 on Ub is altered(Fig. 1A) (12). Here, we discovered previously unidentifiedconnections between UPS functions, Connexin levels, inter-cellular Ca2+ transport, Ca2+ accumulation, calpain activation,and unscheduled calpain-catalyzed cleavage of multiple bulk,structural, regulatory, and cytoskeletal proteins, culminating inaberrant differentiation and cataract. The profound develop-mental aberrations caused by expression of K6W-Ub indicate thateven lysines on Ub that appear to be involved in only a minority ofconjugates, and for which there is little prior functional in-formation, have critical roles with regard to development.Consistent with limited roles for K6 on Ub, we found that

levels of only 42 of >2,300 proteins were different in WT vs.K6W-Ub lenses, and we corroborated differences of critical lensproteins by Western analyses. As such, the results provide thefirst (to our knowledge) large-scale view of changes in the lensproteome that are caused by expression of a mutant Ub duringorganogenesis. Cytoskeletal proteins (Vimentin, Fodrin, Filensin),crystallins, RNA granule-associated proteins (Tdrd7, Caprin2),and proteins that control Ca2+ accumulation (Cxs) are among themoieties that differentially populate these lenses. Mislocalization

A B

Fig. 3. Stimulation of calpain enhances degradation of Filensin, but notγ-Crystallin, and inhibition or specific removal of calpain retards degradationof Filensin, Tdrd7, and Caprin2. (A) Mouse lens lysate was incubated with orwithout 10 mM EGTA to chelate Ca2+ or 10 mM Ca2+ at 37 °C for 2 h orwithout incubation as control. Filensin, GAPDH, and γ-Crystallin were ana-lyzed by Western blotting. Data for FVB/N (Top) and for C57BL/6J×FVB/N(Bottom). Results are indistinguishable. (B) In vitro degradation assay usingmouse lens lysate before (n) or after one (d) or two (dd) steps of Calpain 3depletion by immunoprecipitation. The arrows indicate full-length proteins.

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Fig. 4. Elevated calcium concentration in K6W-Ub lens. A comparison ofcalcium concentration in WT and K6W-Ub P1 lenses by using calcium-sensitivefluorescent dye FURA2. A scatterplot of calcium concentration as a functionof distance from the lens center was converted from Fig. S5B as describedin SI Materials and Methods. The data were pooled from six WT-Ub lensesand eight K6W-Ub lenses. Findings are comparable for C57BL/6J×FVB/N(Fig. S5C).

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of some proteins in the K6W-Ub lens is also indicated (Fig. 1D).The observation of elevated intracellular Ca2+ when Cx43 isexpressed in HeLa cells (that do not express endogenous Cx43)and HLECs is consistent with Cx43 accumulation and elevatedCa2+ in K6W-Ub lenses (Fig. 6).

Unscheduled Proteolysis by Calpain in K6W-Ub Lens. Far morecleavage fragments and lower levels of full length Fodrin, Fil-ensin, and Cx46 were observed in K6W-Ub lens (Table S3) (Figs.1 B and C, and 5A, and Fig. S1). There are also large decreases inlevels of native Tdrd7, Caprin2, and Calpain 3 in K6W-Ub len-ses. Because the protein levels change by as much as 21-fold,whereas the RNA levels differ by less than 2-fold (Figs. S1 C, D,and H, and S2), it would appear that the altered levels of pro-teins are primarily due to hyperactivation of calpain. A functionalconnection between expression of K6W-Ub, and accumulation ofCa2+ with hyperactivation of calpain was corroborated in assays inwhich calpain was immunodepleted, or inhibited (Fig. 3), as wellas in direct assays of calpain activity in the lens in situ (Fig. 1D andFig. S3). In contrast to our observation that perturbation ofubiquitination and UPS function is upstream of calpain activa-tion, perturbation of UPS activity upon activation of calpain hasbeen reported (36–38). Our findings are also consistent withobservations that Ca2+ accumulation and overactivation of Cal-pain 3 are linked to cataractogenesis via loss of Cxs (39, 40). Thedata regarding levels of several γ-Crystallins in vivo probably in-volve delayed synthesis in the K6W-Ub lens (12) (SI Discussion).Which of over a dozen isoforms of calpains is up-regulated in

the K6W-Ub lens? The MS and immunodepletion data indicatethat Calpain 3 is the most abundant calpain in these lenses (41),and the stabilization of Filensin, Tdrd7, and Caprin2 that isobserved upon depletion of Calpain 3 with specific Calpain 3

antibodies corroborates that Calpain 3 is activated in the K6W-Ub cataractous lens.

Ca2+ Accumulation Is Likely Induced by Disruption of Gap Junctions inK6W-Ub Lens. The 1.8-fold lower level of Cx46, in K6W-Ub lens(Dataset S1), and higher levels of Cx43, much of which is tied upin Ub conjugates, suggest dysregulation of Cxs 43 and 46 (Fig. 5).This is consistent with our observations of lower membranecoupling, Ca2+ accumulation, and calpain activation. Importantly,expression of Cx43 in HeLa cells and HLECs recapitulates theaccumulation of Ca2+ (Fig. 6 and Fig. S7). Interestingly, calpain-induced fragmentation does not appear to pertain to Cx43, themost widely expressed human Cx and an established UPS sub-strate (32, 42). It appears that an inability to clear Cx43 con-jugates that incorporate K6W-Ub results in their accumulation,a backup of this UPS, and, thus, stabilization of the parent proteinas well. This mechanism additionally suggests that expression ofK6W-Ub may also compromise and saturate the other UPSs, andit provides further rationale as to why cells in which K6W-Ub isexpressed have limited capacity to withstand various stresses,particularly proteotoxicity (8, 13). Please see SI Text for discus-sion of relations between monoubiquitination and calpain func-tion and for possible other proteolytic or nonproteolytic mechanismsregulating Cx43, Cx46.

Aberrations in RNA Granule and Structural Proteins and Cataract inK6W-Ub Lens. Interestingly, Tdrd7 levels are significantly lower inK6W-Ub lens. Another RNA granule protein, Caprin2, has alsobeen shown to be down-regulated during lens fiber cell denuclea-tion and maturation (20) as well as in K6W-Ub lenses (Fig. S1 Gand H). It will be interesting to determine how UPS and RNAgranule protein functions are related.Filensin and Vimentin are major components of the inter-

mediate filaments of the lens cytoskeleton (43). Fodrin is impor-tant in cell membrane (44). Crystallins are the predominant lensstructural proteins. Our findings of altered levels or localizationof these proteins, along with abnormal development and cataractin lenses in which K6W-Ub is expressed, indicate a critical role forubiquitin in facilitating functions of these essential proteins inlens development (Fig. 1) (22, 45). Our calpain hyperactivationhypothesis offers a parsimonious explanation of the relationshipbetween expression of K6W-Ub and a cataractous lens. As sum-marized in Fig. S8, it appears that, in the newly forming lens,expression of K6W-Ub results in dysfunctional gap junctions,accumulation of Ca2+, calpain activation, and proteolysis ofcrystallins, Vimentin, Fodrin, Filensin, Tdrd7, and Caprin2. Therole for proteolysis is emphasized by the increase in Cx43 overCx46 in lenses in vivo as well as the increase in Cx43 in HLECsthat express K6W-Ub. When fragmented, some of the proteinsbecome insoluble and cataractogenic (27). These findings also

A B

C

D

Fig. 5. Cx levels in P1 lenses are related to expression of K6W-Ub. (A)Western blotting detection of Cx46 in lens expressing WT or K6W-Ub. Thearrowheads indicate parent Cx46. (B) WT-Ub or K6W-Ub lenses were lysed,and Ub conjugates were pulled down by GST-TUBE resin (31) or GST resin asnegative control. The ubiquitination of Cx43 was monitored by anti-Cx43antibody. The arrowheads indicate parent Cx43. The bracket indicatesubiquitin conjugates. (C) Cx43 is stabilized in K6W-Ub lens epithelial cells.Western blotting detection of Cx43 in lysates of HLECs expressing WT orK6W-Ub and treated with PMA or brief serum deprivation. The density ratiosof Cxs vs. loading control are indicated at the Bottom. (D) PMA-treatedHLECs expressing WT-Ub or K6W-Ub were lysed, and Ub conjugates werepulled down by GST-TUBE. The ubiquitination of Cx43 and total ubiqui-tinated protein were monitored by anti-Cx43 antibody and anti-ubiquitinantibody, respectively. Equal levels of GST indicate that levels of beads wereindistinguishable.

Fig. 6. Expressing Cx43 in HeLa cells is associated with a marked increase inCa2+. Cells were incubated in DMEM that contained 3 μM FURA2 for 1 h at37 °C. After washing in buffer containing 1.8 mM CaCl2, A23187 was added,and the extracellular calcium concentration was increased to 21.8 mM at thetimes indicated. Inset shows that the HeLa cells were expressing the Cx43 asdetermined by recognition by anti-Cx43 Ab (green) in permeabilized cells.

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indicate a previously undescribed calpain-mediated means ofregulation of levels of RNA granule proteins that deserves furtherstudy. The finding that levels of both β- and γ-Crystallins are lowerin K6W-Ub lenses, but only β-Crystallin is a calpain substrate,indicates that the effects of expressing K6W-Ub can be bothtranscriptional and postsynthetic.

Conclusion. Combined, the data establish that a properly oper-ating UPS, specifically requiring Ub with K6 intact, is crucialfor homeostatic Ca2+ biology, specifically calpain activity, proteo-poise, organogenesis, as well as lens function. Because the UPSfunctions similarly in many tissues and organisms, it appears likelythat similar developmental abnormalities will be found upon ex-pression of this Ub variant in other cells or tissues. Thus, thefindings add a new appreciation for the importance of K6 on Ub.The essential functions of the proteins that are altered whenK6W-Ub is expressed help to rationalize why there are few reportsof diseases with mutations in Ub per se (46). The findings alsosuggest that discovery of mechanisms of—and means to—exploitthe UPS and regulate Ca2+–calpain pathways will provide newapproaches to prevent or cure human cataract and other proteinprecipitation diseases.

Materials and MethodsThe animal experiments were carried out and approved under the JeanMayer US Department of Agriculture Human Nutrition Research Center onAging at Tufts University Institutional Animal Care and Use Committee inadherence with the Association for Research in Vision and Ophthalmologystatement for the Use of Laboratory Animals in Ophthalmic and VisionResearch. Experiments were performed on transgenic animals on a FVB/Nbackground, as described previously (12), or backcrossed with C57BL/6J micefor two generations to obtain WT copies of the CP49 (Bfsp2) gene (Fig. S1A).In these animals, expression of MRGS(His)6-WT-Ub (WT-Ub) and MRGS(His)6-K6W-Ub (K6W-Ub) is under the control of the αA-Crystallin promoter. Detailsregarding mass spectrometry, immunohistochemistry, and biochemical assayscan be found in SI Materials and Methods.

ACKNOWLEDGMENTS. We thank Dr. Shinichiro Chuma (Kyoto University),Dr. Christina E. Lorén (University of British Columbia), Dr. Lena Gunhaga(Umeå University), and Dr. Paul FitzGerald (University of California, Davis)for gifting antibodies; Drs. Murry and Reinecke for Cx43 virus; and Drs. Vierstraand Kim for gifting GST-TUBE plasmid. We appreciate comments fromDr. Elizabeth Whitcomb and Andrew Kovalenko in review of this manuscript.This work was supported by US Department of Agriculture Grant 1950-510000-060-01A (to A.T.), NIH Grants R01EY13250 (to A.T.), R01EY21212 (toA.T.), R01EY06391 (to R.M.), and R21NS081571 (to J.P.), Alcon gift (to A.T.),and American Lebanese Syrian Associated Charities (J.P.).

1. Cohen E, Dillin A (2008) The insulin paradox: Aging, proteotoxicity and neuro-degeneration. Nat Rev Neurosci 9(10):759–767.

2. Taylor A (2012) Mechanistically linking age-related diseases and dietary carbohydratevia autophagy and the ubiquitin proteolytic systems. Autophagy 8(9):1404–1406.

3. Shang F, Taylor A (2012) Role of the ubiquitin-proteasome in protein quality controland signaling: Implication in the pathogenesis of eye diseases. Prog Mol Biol Transl Sci109:347–396.

4. Resnikoff S, et al. (2004) Global data on visual impairment in the year 2002. BullWorld Health Organ 82(11):844–851.

5. Francis PJ, Moore AT (2004) Genetics of childhood cataract. Curr Opin Ophthalmol15(1):10–15.

6. Talla V, Srinivasan N, Balasubramanian D (2008) Visualization of in situ intracellularaggregation of two cataract-associated human gamma-crystallin mutants: Lose a tail,lose transparency. Invest Ophthalmol Vis Sci 49(8):3483–3490.

7. Taylor A (1993) Cataract: Relationship between nutrition and oxidation. J Am CollNutr 12(2):138–146.

8. Uchiki T, et al. (2012) Glycation-altered proteolysis as a pathobiologic mechanism thatlinks dietary glycemic index, aging, and age-related disease (in nondiabetics). AgingCell 11(1):1–13.

9. Benedek GB (1997) Cataract as a protein condensation disease: The Proctor Lecture.Invest Ophthalmol Vis Sci 38(10):1911–1921.

10. Glickman MH, Ciechanover A (2002) The ubiquitin-proteasome proteolytic pathway:Destruction for the sake of construction. Physiol Rev 82(2):373–428.

11. Kaiser SE, et al. (2011) Protein standard absolute quantification (PSAQ) method forthe measurement of cellular ubiquitin pools. Nat Methods 8(8):691–696.

12. Caceres A, et al. (2010) Perturbing the ubiquitin pathway reveals how mitosis is hi-jacked to denucleate and regulate cell proliferation and differentiation in vivo. PLoSOne 5(10):e13331.

13. Shang F, et al. (2005) Lys6-modified ubiquitin inhibits ubiquitin-dependent proteindegradation. J Biol Chem 280(21):20365–20374.

14. Kulathu Y, Komander D (2012) Atypical ubiquitylation—the unexplored world ofpolyubiquitin beyond Lys48 and Lys63 linkages. Nat Rev Mol Cell Biol 13(8):508–523.

15. Hospenthal MK, Freund SM, Komander D (2013) Assembly, analysis and architectureof atypical ubiquitin chains. Nat Struct Mol Biol 20(5):555–565.

16. Gong X, Cheng C, Xia CH (2007) Connexins in lens development and cataractogenesis.J Membr Biol 218(1-3):9–12.

17. Laing JG, Beyer EC (1995) The gap junction protein connexin43 is degraded via theubiquitin proteasome pathway. J Biol Chem 270(44):26399–26403.

18. Lachke SA, et al. (2011) Mutations in the RNA granule component TDRD7 causecataract and glaucoma. Science 331(6024):1571–1576.

19. Aerbajinai W, Lee YT, Wojda U, Barr VA, Miller JL (2004) Cloning and characterizationof a gene expressed during terminal differentiation that encodes a novel inhibitor ofgrowth. J Biol Chem 279(3):1916–1921.

20. Lorén CE, Schrader JW, Ahlgren U, Gunhaga L (2009) FGF signals induce Caprin2 ex-pression in the vertebrate lens. Differentiation 77(4):386–394.

21. Lachke SA, et al. (2012) iSyTE: Integrated Systems Tool for Eye gene discovery. InvestOphthalmol Vis Sci 53(3):1617–1627.

22. Kilic F, Trevithick JR (1998) Modelling cortical cataractogenesis. XXIX. Calpain pro-teolysis of lens fodrin in cataract. Biochem Mol Biol Int 45(5):963–978.

23. Roy D, Chiesa R, Spector A (1983) Lens calcium activated proteinase: Degradation ofvimentin. Biochem Biophys Res Commun 116(1):204–209.

24. Sanderson J, Marcantonio JM, Duncan G (1996) Calcium ionophore induced pro-teolysis and cataract: Inhibition by cell permeable calpain antagonists. Biochem Bio-phys Res Commun 218(3):893–901.

25. Reed NA, Castellini MA, Ma H, Shearer TR, Duncan MK (2003) Protein expression

patterns for ubiquitous and tissue specific calpains in the developing mouse lens. Exp

Eye Res 76(4):433–443.26. Fukiage C, Nakajima E, Ma H, Azuma M, Shearer TR (2002) Characterization and

regulation of lens-specific calpain Lp82. J Biol Chem 277(23):20678–20685.27. David LL, Azuma M, Shearer TR (1994) Cataract and the acceleration of calpain-

induced beta-crystallin insolubilization occurring during normal maturation of rat lens.

Invest Ophthalmol Vis Sci 35(3):785–793.28. Murphy RM, Lamb GD (2009) Endogenous calpain-3 activation is primarily governed

by small increases in resting cytoplasmic [Ca2+] and is not dependent on stretch. J Biol

Chem 284(12):7811–7819.29. Gao J, et al. (2004) Connections between connexins, calcium, and cataracts in the lens.

J Gen Physiol 124(4):289–300.30. Mathias RT, White TW, Gong X (2010) Lens gap junctions in growth, differentiation,

and homeostasis. Physiol Rev 90(1):179–206.31. Molina SA, Takemoto DJ (2012) The role of Connexin 46 promoter in lens and other

hypoxic tissues. Commun Integr Biol 5(2):114–117.32. Banerjee D, et al. (2011) Investigation of the reciprocal relationship between the

expression of two gap junction connexin proteins, connexin46 and connexin43. J Biol

Chem 286(27):24519–24533.33. Leithe E, Rivedal E (2004) Epidermal growth factor regulates ubiquitination, in-

ternalization and proteasome-dependent degradation of connexin43. J Cell Sci 117(Pt

7):1211–1220.34. Leithe E, Rivedal E (2004) Ubiquitination and down-regulation of gap junction pro-

tein connexin-43 in response to 12-O-tetradecanoylphorbol 13-acetate treatment.

J Biol Chem 279(48):50089–50096.35. Bejarano E, et al. (2012) Autophagy modulates dynamics of connexins at the plasma

membrane in a ubiquitin-dependent manner. Mol Biol Cell 23(11):2156–2169.36. Rajakumar D, Alexander M, Oommen A (2013) Oxidative stress, NF-κB and the

ubiquitin proteasomal pathway in the pathology of calpainopathy. Neurochem Res

38(10):2009–2018.37. Nayak MK, Kumar K, Dash D (2011) Regulation of proteasome activity in activated

human platelets. Cell Calcium 49(4):226–232.38. Huang Q, Wang H, Perry SW, Figueiredo-Pereira ME (2013) Negative regulation of

26S proteasome stability via calpain-mediated cleavage of Rpn10 subunit upon mi-

tochondrial dysfunction in neurons. J Biol Chem 288(17):12161–12174.39. Baruch A, et al. (2001) Defining a link between gap junction communication, pro-

teolysis, and cataract formation. J Biol Chem 276(31):28999–29006.40. Jiang JX (2010) Gap junctions or hemichannel-dependent and independent roles of

connexins in cataractogenesis and lens development. Curr Mol Med 10(9):851–863.41. Zatz M, Starling A (2005) Calpains and disease. N Engl J Med 352(23):2413–2423.42. Leithe E, Rivedal E (2007) Ubiquitination of gap junction proteins. J Membr Biol

217(1-3):43–51.43. FitzGerald PG (2009) Lens intermediate filaments. Exp Eye Res 88(2):165–172.44. Nowak RB, Fischer RS, Zoltoski RK, Kuszak JR, Fowler VM (2009) Tropomodulin1 is

required for membrane skeleton organization and hexagonal geometry of fiber cells

in the mouse lens. J Cell Biol 186(6):915–928.45. Alizadeh A, et al. (2003) Targeted deletion of the lens fiber cell-specific intermediate

filament protein filensin. Invest Ophthalmol Vis Sci 44(12):5252–5258.46. Fischer DF, et al. (2003) Disease-specific accumulation of mutant ubiquitin as a marker

for proteasomal dysfunction in the brain. FASEB J 17(14):2014–2024.

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Correction

DEVELOPMENTAL BIOLOGYCorrection for “Altered ubiquitin causes perturbed calcium ho-meostasis, hyperactivation of calpain, dysregulated differentia-tion, and cataract,” by Ke Liu, Lei Lyu, David Chin, JunyuanGao, Xiurong Sun, Fu Shang, Andrea Caceres, Min-Lee Chang,Sheldon Rowan, Junmin Peng, Richard Mathias, Hideko Kasahara,Shuhong Jian, and Allen Taylor, which appeared in issue 4, January27, 2015, of Proc Natl Acad Sci USA (112:1071–1076; first publishedJanuary 12, 2015; 10.1073/pnas.1404059112).The authors note that the author name Shuhong Jian should

instead appear as Shuhong Jiang. The corrected author line appearsbelow. The online version has been corrected.

Ke Liu, Lei Lyu, David Chin, Junyuan Gao, Xiurong Sun,Fu Shang, Andrea Caceres, Min-Lee Chang, SheldonRowan, Junmin Peng, Richard Mathias, Hideko Kasahara,Shuhong Jiang, and Allen Taylor

www.pnas.org/cgi/doi/10.1073/pnas.1501368112

www.pnas.org PNAS | February 17, 2015 | vol. 112 | no. 7 | E817

CORR

ECTION