Human αA-crystallin Research Articles

Regulation of αA-crystallin via Pax6, c-Maf, CREB and a broad domain of lens-specific chromatin

Pax6 and c-Maf regulate multiple stages of mammalian lens development. Here, we identified novel distal control regions (DCRs) of the alphaA-crystallin gene, a marker of lens fiber cell differentiation induced by FGF-signaling. DCR1 stimulated reporter gene expression in primary lens explants treated with FGF2 linking FGF-signaling with alphaA-crystallin synthesis. A DCR1/alphaA-crystallin promoter (including DCR2) coupled with EGFP virtually recapitulated the expression pattern of alphaA-crystallin in lens epithelium and fibers. In contrast, the DCR3/alphaA/EGFP reporter was expressed only in 'late' lens fibers. Chromatin immunoprecipitations showed binding of Pax6 to DCR1 and the alphaA-crystallin promoter in lens chromatin and demonstrated that high levels of alphaA-crystallin expression correlate with increased binding of c-Maf and CREB to the promoter and of CREB to DCR3, a broad domain of histone H3K9-hyperacetylation extending from DCR1 to DCR3, and increased abundance of chromatin remodeling enzymes Brg1 and Snf2h at the alphaA-crystallin locus. Our data demonstrate a novel mechanism of Pax6, c-Maf and CREB function, through regulation of chromatin-remodeling enzymes, and suggest a multistage model for the activation of alphaA-crystallin during lens differentiation.

Recombinant human αA-crystallin can protect the enzymatic activity of CpUDG against thermal inactivation

α-Crystallin is one of the major protein components in mammalian lens fiber cells. It is composed of αA and αB subunits that have structural homology to the family of mammalian small heat shock proteins. Horwitz firstly characterized native α-crystallin as a molecular chaperone in vitro based on its ability to prevent heat-induced aggregation of lens proteins and enzymes. Andley et al. cloned and expressed human αA-crystallin in Escherichia coli and confirmed its chaperone activity by suppression of thermal aggregation and singlet oxygen-induced opacification. Although αA-crystallin acts as a chaperone protein, there is no report showing on its ability to protect enzymes against thermal inactivation. Here, we present data showing that αA-crystallin can prevent thermal inactivation of CpUDG that catalyzes uracil removal from DNAs.

Deamidation Affects Structural and Functional Properties of Human αA-Crystallin and Its Oligomerization with αB-Crystallin

To determine the effects of deamidation on structural and functional properties of alphaA-crystallin, three mutants (N101D, N123D, and N101D/N123D) were generated. Deamidated alphaB-crystallin mutants (N78D, N146D, and N78D/N146D), characterized in a previous study (Gupta, R., and Srivastava, O. P. (2004) Invest. Ophthalmol. Vis. Sci. 45, 206-214) were also used. The biophysical and chaperone properties were determined in (a) homoaggregates of alphaA mutants (N101D, N123D, and N101D/N123D) and (b) reconstituted heteroaggregates of alpha-crystallin containing (i) wild type alphaA (WT-alphaA): WT-alphaB crystallins, (ii) individual alphaA-deamidated mutants:WT-alphaB crystallins, and (iii) WT-alphaA:individual alphaB-deamidated mutant crystallins. Compared with the WT-alphaA, the three alphaA-deamidated mutants showed reduced levels of chaperone activity, alterations in secondary and tertiary structures, and larger aggregates. These altered properties were relatively more pronounced in the mutant N101D compared with the mutant N123D. Further, compared with heteroaggregates of WT-alphaA and WT-alphaB, the heteroaggregates containing deamidated subunits of either alphaA- or alphaB-crystallins and their counterpart WT proteins showed higher molecular mass, altered tertiary structures, lower exposed hydrophobic surfaces, and reduced chaperone activity. However, the heteroaggregate containing WT-alphaA and deamidated alphaB subunit showed lower chaperone activity, smaller oligomers, and 3-fold lower subunit exchange rate than heteroaggregate containing deamidated alphaA- and WT-alphaB subunits. Together, the results suggested that (a) both Asn residues (Asn-101 and Asn-123) are required for the structural integrity and chaperone function of alphaA-crystallin and (b) the presence of WT-alphaB in the alpha-crystallin heteroaggregate leads to packing-induced structural changes which influences the oligomerization and modulate chaperone activity.

Human αA- and αB-crystallins prevent UVA-induced apoptosis through regulation of PKCα, RAF/MEK/ERK and AKT signaling pathways

αA- and αB-crystallins are distinct antiapoptotic regulators. Regarding the antiapoptotic mechanisms, we have previously demonstrated that under staurosporine treatment, HαA- and HαB-crystallins can interact with Bax and Bcl-X S, proapoptotic members of the Bcl-2 family, to sequester their translocation into mitochondria, and thus prevent the staurosporine-induced apoptosis. In the present study, we further compared the anti-apoptotic mechanisms of HαA- and HαB-crystallin in preventing human lens epithelial cells from UVA-induced apoptosis. UVA-irradiation of human lens epithelial cells turned on the apoptotic death program. Moreover, associated with the activation of the death program, UVA also activated the RAF/MEK/ERK signaling pathway. In contrast, p38 kinase and JNK1/2 signaling pathways were not activated. Inhibition of the RAF/MEK/ERK pathway by a dominant negative mutant RAF1 greatly attenuated UVA-induced apoptosis. Expression of the exogenous human αB-crystallin prevented UVA-induced activation of RAF/MEK/ERK pathway and thus substantially abrogated UVA-induced apoptosis. In contrast, expression of the exogenous human αA-crystallin did not prevent UVA-induced activation of RAF/MEK/ERK pathway. Instead, it activated AKT kinase pathway to promote survival and thus counteracted the UVA-induced apoptosis. Together, our results for the first time reveal that by regulating multiple signaling pathways the two α-crystallins can prevent stress-induced apoptosis through different mechanisms.

Sulfur in human crystallins

Molecular models of human γ-crystallins and the ‘α-crystallin domain’ of human αA-crystallin have been built based on available related X-ray crystal structures. The accessibilities of the component cysteine, methionine and tryptophan side chains in the crystallin models have been calculated. The reactivities of these cysteines, which are oxidised in cataract, are assessed based on their known modifications and within the context of their location within the 3D models.

Expression of calpain small subunit 2 in mammalian tissues

Purpose. The purpose of the current experiments was to more closely define the distribution and the function of calpain small subunit 2 (css2). Css2 is a newly discovered regulatory protein for the calcium activated proteases, μ- and m-calpains. Methods. Tissues from rat, monkey, and man of various ages were used to determine expression patterns of css2 by relative quantitative RT-PCR using 18S rRNA as an endogenous standard. Recombinant css2 and the 80kDa catalytic subunit of m-calpain (80 kDa/css2) were co-expressed in Escherichia coli. Casein zymography was used to measure the enzymatic activity of 80 kDa/css2 proteins. Lens α-crystallin and βB1-crystallin were used as substrates to determine proteolysis by 80 kDa/css2. Computer-based homology modeling was used to predict interactions between the traditional small subunit (css1) or css2 with the 80kDa catalytic subunit. Results. Css2 appears to be a functional equivalent of css1 in vitro in that the calcium-dependent proteolytic activity of 80 kDa/css2 was similar to recombinant m-calpain (80 kDa/css1). In rat and human lens, css2 transcripts increased with age, whereas css1 transcripts decreased with age. Human βB1-crystallin and rat αA-crystallin were cleaved similarly by 80 kDa/css2 and 80 kDa/css1. Interestingly, αA-insert crystallin was not hydrolyzed when css2 was substituted for css1 in the calpain dimer, suggesting that css2 may perform different functions from css1 in terms of proteolysis of lens crystallins during maturational growth of the lens. Css2 may also assist in the proper folding of the 80kDa subunit and regulate protease activity in the absence of calcium. Conclusions. The wide distribution of css2 transcripts in rat and monkey suggested that css2 is a second, widely distributed (rather than tissue-specific) calpain small subunit, in addition to the long-recognized css1. Further studies at the protein level will indicate if css2 has unique functions apart from css1.

Quantification of the isomerization of Asp residue in recombinant human αA-crystallin by reversed-phase HPLC

A method for determining the isomerization of Asp residues in proteins is described and demonstrated by quantifying the isomerization of Asp 151 in recombinant human αA-crystallin. First, four types of dodecapeptide fragment ( 146IQTGLD 151ATHAER 157) in which the Asp residue was either l-Asp, d-Asp, l-isoAsp or d-isoAsp were synthesized, and RP-HPLC conditions were established for their separation. Next, the Asp 151-containing peptide fragments isolated from the tryptic hydrolysate of recombinant αA-crystallin were analyzed under these conditions. New peaks, the retention times of which were the same as those of peptides containing d-Asp, l-isoAsp and d-isoAsp, were generated when αA-crystallin was incubated for 140 days at 37 °C. An amino acid composition, amino acid sequence, and enantiomeric analysis revealed that two peaks with retention times identical to those of peptides containing l-isoAsp and d-isoAsp represented dodecapeptide fragments containing l-isoAsp 151 and d-isoAsp 151, respectively. RP-HPLC analysis under other condition suggested that the peak with retention time identical to that of peptide containing d-Asp represented dodecapeptide fragments containing d-Asp 151. The present method does not require acid hydrolysis, which generates further isomerization products as artifacts, and thus make possible the sensitive quantification of each type of Asp isomer individually at a specific site in a protein. In our analysis of the Asp 151 residue in human αA-crystallin, the degree of isomerization from l-Asp to d-Asp can be determined to a level as low as 0.3%.

Inversion and isomerization of Asp-58 residue in human αA-crystallin from normal aged lenses and cataractous lenses

We have previously shown that L-Asp-151 in αA-crystallin from the human lens is converted to the biologically uncommon D-isomer. This process was not simple racemization, but stereoinversion, accompanied by isomerization to form the β-Asp residue, such that L-β-Asp, D-α-Asp and D-β-Asp were formed. The present study shows that Asp-58 of human αA-crystallin is also converted to the D-isomer to a high degree to form the same isomers with age. The D/ L ratio of β-Asp-58 in aged normal lens increased to more than 3.0, showing stereoinversion by the 60 year range, then decreased to 1.0 in the 80 year range, while the isomerization of Asp-58 increased in the 80 year range. We also measured inversion and isomerization of the same residue from cataractous and normal human lenses of the 60 year range. The D/ L ratio of Asp-58 from cataractous lenses was significantly lower than that from normal lenses, while the isomerization at Asp-58 in cataractous αA-crystallin was significantly higher than that of normal αA-crystallin. These results indicate that isomerization to the β isomer of Asp-58 in cataractous αA-crystallin increased more than inversion to the D-isomer, suggesting that there are changes in the native structure of αA-crystallin in the human cataractous lens.

Changes of Secondary Structure Detected by Laser Raman Spectroscopy in Model Peptides of Human αA-Crystallin Due to Substitution of D-Aspartyl Residues for L-Aspartyl Residues

Purpose: We have found that two aspartyl (Asp-151 and Asp-58) residues of αA-crystallin are inverted and isomerized to the biologically uncommon D-β-Asp residues during aging. In order to elucidate the correlation between the formation of the D-β-Asp isomer and the environment surrounding the Asp in the protein, we performed a Raman spectroscopic study using two synthetic peptides: T6 peptide containing Asp-58, and T18 peptide, containing Asp-151, which correspond to the tryptic peptides of human αA-crystallin. Methods: Both T6 (Thr-Val-Leu-Asp 58-Ser-Gly-Ile-Ser-Glu-Val-Arg) and T18 (Ile-Gln-Thr-Gly-Leu-Asp 151-ala-thr-his-ala-Glu-Arg) peptides were synthesized with four optical isomers which have L-α-, D-α, L-β and D-β-aspartyl residues. These peptides were subjected to Raman measurement. Results: The Raman spectrum of the L-α-Asp T18 peptide measured as dry powder revealed that the secondary structure of this peptide is mainly anti-parallel β-sheet. The main structure of the D-β-Asp T18 peptide when in dry powder form was altered to an α-helix and/or random structure. The main structure of L-α-Asp T18 peptide when measured in aqueous solution also converted to an α-helix and/or random structure. The conversion of L-α-to D-β-Asp in T6 peptides when in dry powder form revealed no alteration of secondary β-sheeted structure. Conclusion: Raman spectroscopy clearly revealed a large conformational change in the secondary structure of T18 peptide caused by substitution of normal L-α-Asp to biologically uncommon Asp-isomers. This result indicates that the inversion of an amino acid in a protein greatly affects the secondary structure of the protein.

Temperature-dependent Chaperone Activity and Structural Properties of Human αA- and αB-crystallins

The chaperone activity and biophysical properties of recombinant human alphaA- and alphaB-crystallins were studied by light scattering and spectroscopic methods. While the chaperone function of alphaA-crystallin markedly improves with an increase in temperature, the activity of alphaB homopolymer appears to change very little upon heating. Compared with alphaB-crystallin, the alphaA-homopolymer is markedly less active at low temperatures, but becomes a more active species at high temperatures. At physiologically relevant temperatures, the alphaB homopolymer appears to be modestly (two times or less) more potent chaperone than alphaA homopolymer. In contrast to very similar thermotropic changes in the secondary structure of both homopolymers, alphaA- and alphaB-crystallins markedly differ with respect to the temperature-dependent surface hydrophobicity profiles. Upon heating, alphaA-crystallin undergoes a conformational transition resulting in the exposure of additional hydrophobic sites, whereas no such transition occurs for alphaB-crystallin. The correlation between temperature-dependent changes in the chaperone activity and hydrophobicity properties of the individual homopolymers supports the view that the chaperone activity of alpha-crystallin is dependent on the presence of surface-exposed hydrophobic patches. However, the present data also show that the surface hydrophobicity is not the sole determinant of the chaperone function of alpha-crystallin.

Formation of Four Isomers at the Asp-151 Residue of Aged Human αA-Crystallin by Natural Aging

Although proteins are generally composed entirely of l-amino acids, we have previously shown that Asp-151 in αA-crystallin from aged human lens is converted to the biologically uncommon d-isomer to a high degree. The formation of d-isomer was not simple racemization, but stereoinvertion. The reaction was also accompanied with isomerization to form β-Asp (isoaspartate) residue simultaneously; therefore, four isomers of Asp-151, normal l-α-Asp and biologically uncommon l-β-Asp, d-α-Asp, and d-β-Asp, are formed in αA-crystallins. In the present study, we measured the ratio of the four isomers of Asp-151 in αA-crystallins obtained from total lens proteins of human lenses of newborn and 30-, 60-, and 80-year-olds. The isomers increased with age, and the total amount of three isomers was more than that of normal l-α-Asp in the αA-crystallin of the human lenses of the 80-year-olds. These drastic changes started at birth, with about 45% of normal l-α-Asp lost by 30 years. These modifications of the Asp residue likely affect the three-dimensional packing array of the lens proteins.

D-Amino Acid Formation Induced by a Chiral Field within a Human Lens Protein during Aging

We have previously shown that Asp-151 in αA-crystallin from aged human lens are converted to the biologically uncommon D-isomer to a high degree, showing that the formation of D-isomer was not simple racemization, but stereoinvertion. This suggests that αA-crystallin has a chiral reaction field which promotes the inversion of L-Asp to D-Asp residues in the native higher order structure of αA-crystallin itself. Here, we show that when the aged human αA-crystallin, enriched at Asp-151 with the D-isomer (D/L ratio of 5.7), was unfolded by heating at 70°C or 6 M urea, the D-Asp-151 in the unfolded αA-crystallin was rapidly racemized (D/L ratio of 2.17 to 1.21). This presumably reflects a relaxation of the chiral field that was initially inducing the stereoinversion from the natural L-isomer to the D-isomer.

Cloning, Expression, and Chaperone-like Activity of Human αA-Crystallin

One of the major protein components of the ocular lens, alpha-crystallin, is composed of alphaA and alphaB chain subunits that have structural homology to the family of mammalian small heat shock proteins. Like other small heat shock proteins, alpha-crystallin subunits associate to form large oligomeric aggregates that express chaperone-like activity, as defined by the ability to suppress nonspecific aggregation of proteins destabilized by treatment with a variety of denaturants including heat, UV irradiation, and chemical modification. It has been proposed that age-related loss of sequences at the C terminus of the alphaA chain subunit may be a factor in the pathogenesis of cataract due to diminished capacity of the truncated crystallin to protect against nonspecific aggregation of lens proteins. To evaluate the functional consequences of alpha-crystallin modification, two mutant forms of alphaA subunits were prepared by site-directed mutagenesis. Like wild type (WT), aggregates of approximately 540 kDa were formed from a tryptophan-free alphaA mutant (W9F). When added in stoichiometric amounts, both WT and W9F subunits completely suppressed the heat-induced aggregation of aldose reductase. In contrast, subunits encoded by a truncation mutant in which the C-terminal 17 residues were deleted (R157STOP), despite having spectroscopic properties similar to WT, formed much larger aggregates with a marked reduction in chaperone-like activity. Similar results were observed when the chaperone-like activity was assessed through inhibition of gamma-crystallin aggregation induced by singlet oxygen. These results demonstrate that the structurally conservative substitution of Phe for Trp-9 has a negligible effect on the functional interaction of alphaA subunits, and that deletion of C-terminal sequences from the alphaA subunit results in substantial loss of chaperone-like activity, despite overall preservation of secondary structure.

Identification of New Lens Protease(s) Using Peptide Substrates Havingin VivoCleavage Sites

Bovine lens extracts were incubated in pH 7.5 buffer with succinylated VSREEKPSSAPSS, SGVDAGHS, GKPTSAPSS and GKHNERQD, the peptides having age-dependent in vivo cleavage sites in bovine and human αA-crystallin. The reaction products were analyzed to identify the initial protease cleavage sites. The results showed that bovine lens extracts contain protease activity that can cleave. Thr-Ser, Ser-Ala, Ser-Ser bonds in test substrates which correspond to peptide bonds Ser168-Ser169, Ser169-Ala170, Ser172-Ser173 in bovine αA-crystallin and Thr-Ser, Ser169-Ala170, Ser172-Ser173 in human αA-crystallin. In addition, the same extracts were also capable of hydrolyzing Asp-Ala and Asn-Glu bonds corresponding to Asp151-Ala152 and Asn101-Glu102 in αA-crystallin from bovine and human lenses. The cleavage specificity of the newly discovered protease(s) suggests that the in vivo truncation of αA-crystallin reported earlier may be due to the action of proteases. The newly discovered lens proteases(s) were resistant to inactivation by E-64 and DFP. However, prior treatment of the lens extracts with leupeptin and chymostatin resulted in partial loss of Asn-Glu hydrolytic activity. N-ethylmaleimide treatment completely abolished in the Asn-Glu hydrolyzing activity.