Cholesterol-derived bile acids enhance the chaperone activity of α-crystallins.

Human lens membranes contain the highest cholesterol concentration of any known biological membranes, but it significantly decreases with age. Oxygenation of cholesterol generates numerous forms of oxysterols (bile acids). We previously showed that two forms of the bile acid components--ursodeoxycholic acid (UDCA) and tauroursodeoxycholic acid (TUDCA)--suppressed lens epithelial cell death and alleviated cataract formation in galactosemic rat lenses. We investigated whether these compounds also suppress the thermal aggregation of human lens crystallins. Total water-soluble (WS) proteins were prepared from human lenses, and recombinant human crystallins (αA-, αB-, ßB2-, and γC-crystallin) were generated by a prokaryotic expression system and purified by liquid chromatography. The light scattering of proteins in the presence or absence of UDCA or TUDCA was measured using a spectrofluorometer set at Ex/Em = 400/400 nm. Protein blot analysis was conducted for detection of α-crystallins in the human lens WS proteins. High concentrations of UDCA and TUDCA significantly suppressed thermal aggregation of total lens WS proteins, which contained a low level of αA-/αB-crystallin. Spectroscopic analysis with each recombinant human lens crystallin indicated that the bile acids did not suppress the thermal aggregation of γC-, ßB2-, αA-, or αB-crystallin. Combination of α-crystallin and bile acid (either UDCA or TUDCA) suppressed thermal aggregation of each individual crystallin as well as a non-crystallin protein, insulin. These results suggest that UDCA or TUDCA protects the chaperone activity of α-crystallin. It is believed that these two naturally occurring intermediate waste products in the lens enhance the chaperone activity of α-crystallin. This finding may lead to the development of UDCA and TUDCA as anticataract agents.

Protein-protein interactions involving congenital cataract T5P gammaC-crystallin mutant: a confocal fluorescence microscopy study.

The human lens crystallin gene CRYGC T5P is associated with Coppock-like cataract and has a phenotype of a dust-like opacity of the fetal lens nucleus and deep cortical region. Previous in vitro mutation studies indicate that the protein has changed conformation, solubility, and stability, which may make it susceptible to aggregation, as seen in cataractous lens and cell culture expression. To investigate the mechanisms leading to these events, we studied protein-protein interactions using confocal fluorescence resonance energy transfer (FRET) microscopy. The method detects protein-protein interactions in the natural environment of living cells. Crystallin genes (CRYGC T5P, CRYGC, and CRYAA) were fused to either the green fluorescence protein (GFP) or red fluorescence protein (DsRED or RFP) vector. Each of the following GFP-RFP (donor-acceptor) plasmid pairs was cotransfected into HeLa cells: gammaC-gammaC, gammaC-gammaCT5P, gammaCT5P-gammaCT5P, alphaA-gammaC, and alphaA-gammaCT5P. After culture, confocal fluorescence cell images were taken. Protein-protein interactions in the form of net FRET were evaluated. The confocal fluorescence images show that cells expressing T5P gammaC-crystallin contain many protein aggregates, but cells co-expressing with either gammaC- or alphaA-crystallin reduce the aggregation considerably. FRET determination indicates that gammaCT5P-gammaCT5P shows less protein-protein interaction than either gammaC-gammaC or gammaC-gammaCT5P. Cotransfection with alphaA-crystallin (alphaA-gammaC or alphaA-T5PgammaC) increases nFRET compared with gammaC-gammaC or gammaC-T5PgammaC. Our results demonstrate that T5P gammaC-crystallin shows more protein aggregates and less protein-protein interaction than WT gammaC-crystallin. Chaperone alphaA-crystallin can rescue T5P gammaC-crystallin from aggregation through increased protein interaction. The formation of congenital cataract may be due to reduced protein-protein interactions and increased aggregation from an insufficient amount of alpha-crystallin for protection.

Protein-protein interactions between lens vimentin and alphaB-crystallin using FRET acceptor photobleaching.

PURPOSE: The R120G mutation of alphaB-crystallin is known to cause desmin-related myopathy, but the mechanisms underlying the formation of cataract are not clearly established. We hypothesize that alteration of protein-protein interaction between R120G alphaB-crystallin and lens intermediate filament proteins is one of the mechanisms of congenital cataract. METHODS: Protein-protein interactions were determined by confocal fluorescence resonance energy transfer (FRET) microscopy using green fluorescence protein (GFP) as the donor and red fluorescence protein (RFP) as the acceptor. The lens vimentin gene was fused into a GFP vector and the alphaB-crystallin (WT or R120G mutant) gene was fused into the RFP vector. The donor-acceptor plasmid pairs of intermediate filament (IF)-GFP and alphaB-RFP were co-transfected into HeLa cells. After incubation, confocal fluorescence images of the transfected cells were taken. FRET was estimated by the acceptor photobleaching method. Protein-protein interaction was evaluated by FRET efficiency. RESULTS: The confocal fluorescence images showed that the cells expressing vimentin and R120G alphaB-crystallin contained large amounts of protein aggregates while few vimentin fibers were observed. FRET efficiency analyses indicated that vimentin had a significantly greater protein-protein interaction with R120G alphaB-crystallin than with WT alphaB-crystallin. CONCLUSIONS: Our results show that the R120G alphaB-crystallin mutant promoted vimentin aggregation through increased protein-protein interaction. This process may contribute to the formation of congenital cataract.

Protein-protein interactions among human lens acidic and basic beta-crystallins.

Human lens beta-crystallin contains four acidic (betaA1-->betaA4) and three basic (betaB1-->betaB3) subunits. They oligomerize in the lens, but it is uncertain which subunits are involved in the oligomerization. We used a two-hybrid system to detect protein-protein interactions systematically. Proteins were also expressed for some physicochemical studies. The results indicate that all acidic-basic pairs (betaA-betaB) except betaA4-betaBs pairs show strong hetero-molecular interactions. For acidic or basic pairs, only two pairs (betaA1-betaA1 and betaA3-betaA3) show strong self-association. betaA2 and betaA4 show very weak self-association, which arises from their low solubility. Confocal fluorescence microscopy shows enormous protein aggregates in betaA2- or betaA4-crystallin transfected cells. However, coexpression with betaB2-crystallin decreased both the number and size of aggregates. Circular dichroism indicates subtle differences in conformation among beta-crystallins that may have contributed to the differences in interactions.

Confocal fluorescence resonance energy transfer microscopy study of protein-protein interactions of lens crystallins in living cells.

PURPOSE: To determine protein-protein interactions among lens crystallins in living cells. METHODS: Fluorescence resonance energy transfer (FRET) microscopy was used to visualize interactions in living cells directly. Two genes, one (alphaA-crystallin) fused with green fluorescence protein (GFP) and the other (each of the following genes: alphaB-, betaB2-, gammaC-crystallin, and R120G alphaB-crystallin mutant) fused with GFP variant red fluorescence protein (RED), were cotransfected into HeLa cells. After culture, confocal microscopy images were taken and FRET values were calculated. RESULTS: FRET occurs when the two proteins interact. The data show strong interactions between alphaA- and alphaB-crystallin and weak interactions between alphaA- and betaB2- or gammaC-crystallin, which is consistent with our previous two-hybrid system study. The R120G alphaB-crystallin mutant, however, showed significantly less FRET than wild-type alphaB-crystallin. There are also more R120G alphaB-crystallin transfected cells with protein aggregates than wild-type alphaB-crystallin transfected cells. Cotransfection with alphaA-crystallin could not rescue R120G alphaB-crystallin from aggregation. CONCLUSIONS: FRET microscopy gave excellent results on the protein-protein interactions among crystallins. It supports many previous studies and provides a novel technique for further study of protein-protein interactions among lens proteins including membrane and cytoskeletal proteins.

Domain interaction sites of human lens betaB2-crystallin.

betaB2-crystallin, the major component of beta-crystallin, is a dimer at low concentrations but can form oligomers under physiological conditions. The interaction domains have been speculated to be the beta-sheets, each of which is formed by two or more beta-strands. betaB2-crystallin consists of 16 beta-strands, 8 in the N-terminal domain and 8 in the C-terminal domain. Domain interaction sites may be removed by destroying the beta-strands, which can be done by site-specific mutations, substituting the beta-formers (Val, Phe, Leu) with Glu or Asn, strong beta-breakers. We have cloned the following beta-strand-deleted mutants, I20E, L34E, V54E, V60E, V73E, L97E, I109E, I124E, V144E, V152E, L162E, L165E, and V187E and their corresponding X --> Asn mutants. We also made two mutants, V46E and V129E, that were not on the beta-strand as controls. Disruption of protein-protein interactions was screened by a mammalian two-hybrid system assay. Protein-protein interactions decreased for all beta-strand-deleted mutants except I20E, L34E, and L162E mutants; this effect was not seen in the two mutant controls, V46E and V129E. The sequences around Val-54, Val-60, Val-73, and Leu-97 in the N-terminal region and Ile-109, Ile-124, Val-144, Val-152, Leu-165, and Val-187 in the C-terminal region that formed beta-strands appear to be important in dimerization. Some selected mutant proteins that showed strong (V46E and V129E) and reduced (V60E, V144E, V60N, and V144N) interactions were expressed in bacterial culture and were studied with spectroscopy and chromatography. The V60E and V144E mutants were found to be partially unfolded and incapable of forming a complete dimer.

Interaction and biophysical properties of human lens Q155* betaB2-crystallin mutant.

PURPOSE: Missense mutations in crystallin genes have been identified in autosomal dominant congenital cataracts. A truncation in the CRYBB2 gene (Q155*) has been associated with cerulean cataract, however its effects on biophysical properties have not been reported. We sought to determine the changes in conformation and protein-protein interactions brought about by this mutation. METHODS: Site specific mutations were performed to obtain the Q155* betaB2-crystallin mutant. Protein-protein interactions were screened by a mammalian two-hybrid system assay. Conformational changes were studied with spectroscopy (circular dichroism and fluorescence) and FPLC chromatography. RESULTS: We detected a decrease in protein-protein interactions for the Q155* betaB2-crystallin mutant. The Q155* mutant shows decreased ordered structure and stability but the partially unfolded protein retains some dimer structure. CONCLUSIONS: The Q155* mutation in betaB2-crystallin causes changes in biophysical properties that might contribute to cataract formation.

Interactions and chaperone function of alphaA-crystallin with T5P gammaC-crystallin mutant.

T5P gammaC-crystallin mutation is associated with Coppock-like cataract, one of the autosomal dominant congenital cataracts. It is not known why the abundant alpha-crystallin cannot prevent the mutation-related aggregation. Our previous studies indicate that the mutation changes conformation and reduces solubility and stability, but it is not known whether it is these events or the loss of interaction with other crystallins that causes the cataract. It is also not known whether the alpha-crystallin can protect T5P mutant as effectively from heat-induced aggregation as the wild-type (WT) gammaC-crystallin. To investigate the mechanism of interactions and chaperone function between alphaA- and gammaC-crystallin, human alphaA-crystallin and W9F mutant as well as WT gammaC-crystallin and T5P mutant were cloned. Interactions between alphaA- and gammaC-crystallin were studied with fluorescence resonance energy transfer (FRET), and chaperone activity was assessed by the suppression of heat-induced aggregation of substrate proteins. Conformational changes of substrate proteins were studied by spectroscopic measurements. The results indicate that the T5P mutant showed a slightly greater FRET than WT gammaC-crystallin with alphaA-crystallin, and alphaA-crystallin could effectively prevent both WT and T5P gammaC-crystallin from heat-induced aggregation. Spectroscopic measurements show that both alphaA-crystallin and gammaC-crystallin underwent only slight conformational change after chaperone binding. Together with previous results obtained with a two-hybrid system assay of interactions between alphaA- and gammaC-crystallin, the present FRET and chaperone results indicate that loss of interactions of T5P mutant with other crystallins may play a larger role than the protection afforded by chaperone-like activity in Coppock-like cataract.

Enhanced stability of alpha B-crystallin in the presence of small heat shock protein Hsp27.

Lens alpha-crystallin, alpha A- and alpha B-crystallin, and Hsp27 are members of the small heat shock protein family. Both alpha A- and alpha B-crystallin are expressed in the lens and serve as structural proteins and as chaperones, but alpha B-crystallin is also expressed in nonlenticular organs where Hsp27, rather than alpha A-crystallin, is expressed along with alpha B-crystallin. It is not known what additional function Hsp27 has besides as a heat shock protein, but it may serve, as alpha A-crystallin does in the lens, to stabilize alpha B-crystallin. In this study, we investigate aspects on conformation and thermal stability for the mixture of Hsp27 and alpha B-crystallin. Size exclusion chromatography, circular dichroism (CD), and light scattering measurements indicated that Hsp27 prevented alpha B-crystallin from heat-induced structural changes and high molecular weight (HMW) aggregation. The results indicate that Hsp27 indeed promotes stability of alpha B-crystallin.

Alteration of protein-protein interactions of congenital cataract crystallin mutants.

PURPOSE: A recent study demonstrated the presence of protein-protein interactions among lens crystallins in a mammalian cell two-hybrid system assay and speculated about the significance of these interactions for protein solubility and lens transparency. The current study extends those findings to the following crystallin genes involved in some congenital cataracts: CRYAA (R116C), CRYAB (R120G), and CRYGC (T5P). METHODS: A mammalian two-hybrid system was used to assay the protein-protein interactions. Congenital cataract crystallin genes were cloned and fused into the two-hybrid system vectors (target and prey proteins). Together, with the third vector containing a reporter gene, chloramphenicol acetyltransferase (CAT), they were cotransfected into human HeLa cells. The presence of protein-protein interactions and the strength of these interactions were assayed by CAT ELISA. RESULTS: The pattern of changes in protein-protein interactions of those congenital cataract gene products with the three major crystallins, alphaA- or alphaB-, betaB2-, and gammaC-crystallins, differed. For the T5P gammaC-crystallin, most of the interactions were decreased; for the R116C alphaA-crystallin, the interactions with betaB2- and gammaC-crystallin decreased and those with alphaB-crystallin and heat-shock protein (Hsp)27 increased; and for the R120G alphaB-crystallin, the interactions with alphaA- and alphaB-crystallin decreased, but those with betaB2- and gammaC-crystallin increased slightly. An attempt was made to interpret the results on the basis of conformational change and disruption of dimeric interaction involving beta-strands. CONCLUSIONS: The results clearly indicate that crystallin mutations involved in congenital cataracts altered protein-protein interactions, which may contribute to decreased protein solubility and formation of cataract.

Unfolding of human lens recombinant betaB2- and gammaC-crystallins.

betaB2- and gammaC-crystallins belong to the betagamma-crystallin superfamily and have very similar structures. Molecular spectroscopic techniques such as UV-visible absorption, circular dichroism, and fluorescence indicate they have similar biophysical properties. Their structures are characterized by the presence of two domains consisting of four Greek key motifs. The only difference is the connecting peptide of the two domains, which is flexible in gamma-crystallin but extended in beta-crystallin; thus, an intradomain association and a monomer are formed in gamma-crystallin and an interdomain association and a dimer are formed in beta-crystallin. The difference may be reflected in the thermodynamic stability. In the present study, we calculated the standard free-energy by equilibrium unfolding transition in guanidine hydrochloride using three spectroscopic parameters: absorbance at 235nm, Trp fluorescence intensity at 320nm, and far-UV circular dichroism at 223nm. Global analyses indicate that both dimeric betaB2- and monomeric gammaC-crystallins are a better fit to a three-state model than to a two-state model. In terms of standard free-energy, deltaG(0)(H(2)O,i) both betaB2-crystallin and gammaC-crystallin are stable proteins and dimeric betaB2-crystallin is more stable than the monomeric gammaC-crystallin. The significance of the thermodynamic stability for betaB2- and gammaC-crystallins may be related to their functions in the lens.

Decreased subunit exchange of heat-treated lens alpha A-crystallin.

alpha A-Crystallin high-molecular-weight (HMW) aggregates were prepared by preheating at 80-90 degrees C and studied using spectroscopic measurements. Conformational differences were suggested based on data of increased bis-ANS (4,4(')-dianilino-1,1(')-binaphthalene-5,5(')-disulfonic acid) and ThT (thioflavin T) fluorescence as well as increased far-UV and decreased near-UV circular dichroism (CD). These results indicated that HMW aggregated alpha-crystallin was more hydrophobic than the native alpha-crystallin, possibly resulting from partial unfolding of alpha-crystallin. The two cysteines in alpha A-crystallin were mostly oxidized in HMW aggregates. The effects of HMW aggregation on the dynamic structure were studied with fluorescence resonance energy transfer; subunit exchange became slower. These results strongly suggest that HMW alpha A-crystallin aggregates result from exposure of buried beta-pleated sheets and increased hydrophobic interaction.

Conformational change and destabilization of cataract gammaC-crystallin T5P mutant.

Human lens gammaC-crystallin and T5P mutant were cloned, and their biophysical properties and thermodynamic stability were studied. CRYGC (T5P) is one of the many gamma-crystallin mutant genes for autosomal dominant congenital cataracts. This mutation is associated with Coppock-like cataract, and has the phenotype of a dust-like opacity of the fetal lens nucleus. During cloning and overexpression, the majority of T5P mutant was found in the inclusion body. This property is unique among the many cataract gamma-crystallin mutant genes. It is thus worthwhile to study what factors contribute to this unique property of gammaC-crystallin. One possibility is changes in conformation and stability, which can be studied using spectroscopic measurements. In this study, conformational change was studied by circular dichroism and fluorescence measurements, and conformational stability was determined by thermal unfolding probed by Trp fluorescence and time-dependent light scattering. The T5P mutation obviously changes conformation and decreases conformational stability.

Detection of protein-protein interactions among lens crystallins in a mammalian two-hybrid system assay.

alpha-Crystallin consists of two subunits, alphaA and alphaB, and each can form an oligomer by itself or with the other. The aggregation arises from interdomain interactions. However, it is not known whether such interactions also exist among alpha-, beta-, and gamma-crystallins. This heterogeneous crystallin interaction is far weaker than the homogeneous crystallin interaction and is difficult to detect by conventional spectroscopic measurements. We used a mammalian two-hybrid system in this study. The major crystallin components, alphaA-, alphaB-, betaB2-, and gammaC-crystallin genes, were subcloned into the DNA binding domain and transcription activation domain vectors of the two-hybrid system, and they were cotransfected along with a chloramphenicol acetyltransferase (CAT) reporter vector into HeLa cells. Chloramphenicol acetyltransferase activity indicated that there were interactions between alphaA- (or alphaB-) and betaB2- or gammaC-crystallins but with an intensity of one-third that of alphaA-alphaB interactions. Hsp27, a member of the family of the small heat-shock proteins, showed a similar interaction property with alphaB-crystallin. Using the N- and C-terminal domain-truncated mutants, we demonstrated that both domains were important in the alphaA-crystallin self-interaction, but that only the C-terminal domain was important in the alphaB-crystallin self-interaction. These results show that the two-hybrid system can detect interactions among various crystallins and may be used in mapping interaction domains.