Preliminary X-ray crystallographic study of the turkey lens protein, δ-crystallin

The structural protein, δ-crystallin, has been purified and crystallized from adult turkey lens. The crystals are orthorhombic and normally belong to space group P2₁2₁2 with unit cell dimensions $\text{a = 99.9(2)}\text{A}\text{?}\text{, b = 133.4(3)}\text{A}\text{?}\text{and}\text{c = 69.1(2)}\text{A}\text{?}$. This corresponds to two molecules of molecular weight approximately 200,000 per unit cell. A second crystal form has also been found in which b and c increase to 135.4(3) Å and 140.0(3) Å. respectively, indicating four molecules per unit cell.     

Hypericin-mediated photooxidative damage of α-crystallin in human lens epithelial cells

St. John’s wort (Hypericum perforatum), a perennial herb native to Europe, is widely used for and seems to be effective in treatment of mild to moderate depression. Hypericin, a singlet oxygen-generating photosensitizer that absorbs in both the visible and the UVA range, is considered to be one of the bioactive ingredients of St. John’s wort, and commercial preparations are frequently calibrated to contain a standard concentration. Hypericin can accumulate in ocular tissues, including lenses, and can bind in vitro to α-crystallin, a major lens protein. α-crystallin is required for lens transparency and also acts as a chaperone to ensure its own integrity and the integrity of all lens proteins. Because there is no crystallin turnover, damage to α-crystallin is cumulative over the lifetime of the lens and can lead to cataracts, the principal cause of blindness worldwide. In this work we study hypericin photosensitization of α-crystallin and detect extensive polymerization of bovine α-crystallin exposed in vitro to hypericin and UVA. We use fluorescence confocal microscopy to visualize binding between hypericin and α-crystallin in a human lens epithelial (HLE) cell line. Further, we show that UVA irradiation of hypericin-treated HLE cells results in a dramatic decrease in α-crystallin detection concurrent with a dramatic accumulation of the tryptophan oxidation product N-formylkynurenine (NFK). Examination of actin in HLE cells indicates that this cytoskeleton protein accumulates NFK resulting from hypericin-mediated photosensitization. This work also shows that filtration of wavelengths <400 nm provides incomplete protection against α-crystallin modification and NFK accumulation, suggesting that even by wearing UV-blocking sunglasses, routine users of St. John’s wort cannot adequately shield their lenses from hypericin-mediated photosensitized damage.     

The control of δ-crystallin gene expression during lens cell development: Dissociation of cell elongation,cell division, δ-crystallin synthesis,and δ-crystallin mRNA accumulation

Previous studies have shown that freshly explanted 6-day-old embryonic chick lens epithelial cells elongate, differentially increase their synthesis of δ-crystallin, and accumulate δ-crystallin mRNA when cultured with fetal calf serum; in contrast, precultured serum-starved 6-day-old and freshly explanted 19-day-old embryonic epithelial cells divide when treated with fetal calf serum. We have explored whether the stimulation of δ-crystallin gene expression (as measured by δ-crystallin synthesis and δ-crystallin mRNA accumulation) is affected by inhibiting lens cell elongation with colchicine, and whether δ-crystallin gene expression is increased in lens epithelial cells stimulated to divide by treatment with fetal calf serum, as it is in those stimulated to elongate by treatment with serum. Three new findings were made in this study. First, the stimulation of δ-crystallin gene expression does not require elongation of the cultured lens cells. Second, a decreased proportion of δ-crystallin synthesis is observed in lens epithelial cells during normal development and during serum starvation; in neither case is this decrease associated with a reduction in the number of δ-crystallin mRNA sequences per cell. Finally, serum stimulation of lens cell division does not increase the proportion of δ-crystallin synthesis, but can promote the accumulation of δ-crystallin mRNA. Thus, the relative proportion of δ-crystallin synthesized during chick lens development is not solely a function of the number of δ-crystallin mRNA sequences in the lens cells.     

Developmental regulation of the chicken δ1-crystallin gene: analysis by transgenesis and gene dissection

We previously reviewed what we had learned about the regulation of the δ1-crystallin gene through experiments using gene transfer techniques [Kondoh et al. (1986) Cell Differ. 19, 151–160]. It was concluded then that regulatory genetic elements for the lens-specific expression are associated with the δ1-crystallin gene, and that these chicken elements properly function in mammalian cells. In the last couple of years, we have made significant progress in the understanding of lens-specific δ-crystallin expression. This is owing to success in transgenesis of mouse with the δ1-crystallin gene and in functional dissection of the gene which led us to the discovery of an intragenic enhancer as the major determinant for lens-specific expression. In this article, we summarize these recent advances.     

Developmental changes in proteins of purified membranes of chicken lenses and evidence for contamination by cytoplasmic δ-crystallin

Urea-washed membranes from embryonic chick lenses (15 days old) and from the cortical and nuclear regions of adult chicken lenses (1 year) have been prepared by repeated centrifugation through discontinuous density gradients. The protein components of the isolated membranes have been examined by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate and urea. Proteins with molecular weights of 75 000, 56 000, 54 000, 48 000, 34 000, 32 000, 25 000, and 22 000 were present in all the membrane preparations, although their proportions changed during development. One additional protein, molecular weight 70 000, was seen only in the embryonic lens membranes. The greatest developmental change was the increase in 25 000 molecular weight protein from 12% in the embryonic lens to about 45% in the adult lens. Since it has been suggested that this protein is associated with gap junctions, its increase during development may reflect a corresponding increase in the number of gap junctions in the lens.The 50 000 molecular weight protein of embryonic lens membranes and membranes of adult nuclear lens fibers consisted at least partly of δ-crystallin, since δ-crystallin peptides could be identified in tryptic pepetide maps of the isolated protein after in vitro radioiodination. Peptide maps of the 50 000 molecular weight protein of cortical lens fiber membranes contained no identifiable δ-crystallin peptides, although it is possible that modified δ-crystallin peptides may be present. The level of cytoplasmic contamination of the membrane fraction was estimated by preparing lens membranes in the presence of added δ-[³⁵S]crystallin. The results indicated that cytoplasmic contamination contributes significantly to the presence of δ-crystallin in lens membrane preparations.     

Detailed analysis of the δ-crystallin mRNA-expressing region in early development of the chick pituitary gland

Although δ-crystallin (δ-crys), also known as lens protein, is transiently expressed in Rathke's pouch (RP) of the chick embryo, detailed temporal and spatial expression patterns have been obscure. In this study, to understand the relationship between the δ-crys mRNA-expressing region and RP formation, we examined the embryonic expression pattern of δ-crys mRNA in the primordium of the adenohypophysis. δ-crys mRNA expression was initially found at stage 15 anterior to the foregut and posterior to the invaginated oral ectoderm. After RP formation, the δ-crys mRNA was expressed in the post-ventral region of RP and the anterior region of RP. δ-crys mRNA expression was then restricted to the cephalic lobe of the pituitary gland. From stage 20, the δ-crys and alpha-glycoprotein subunit (αGSU) mRNA-expressing regions were almost completely overlapping. The αGSU mRNA-expressing region is thought to be the primordium of the pars tuberalis, and these regions were overlapped with the Lhx3 mRNA-expressing region. The intensity of δ-crys mRNA expression gradually decreased with development and completely disappeared by stage 34. These results suggest that the embryonic chick pituitary gland consists of two different regions labeled with δ-crys and Lhx3.     

NMR spin–echo studies of hydration properties of the molecular chaperone α-crystallin in the bovine lens

The water-binding properties of bovine lens α-crystallin, collagen from calf skin and bovine serum albumin (BSA), were investigated with various techniques. The water absorptive capacity was obtained in high vacuum desorption experiments volumetrically, and also gravimetrically in controlled atmosphere experiments. NMR spin–echo technique was used to study the hydration of protein samples and to determine the spin–spin relaxation times (T₂) from the protons of water, absorbed on the proteins. Isolated bovine lenses were sectioned into 11–12 morphological layers (from anterior cortex through nucleus to posterior cortex). Crystallin profiles were obtained for each lens layer using thin-layer isoelectric focusing in polyacrylamide gel (IEF). The water content in relation to dry weight of proteins was measured in individual morphological lens layers. During the water vapor uptake P/P₀=0.75, α-crystallin did not absorb water, suggesting that hydrophobic regions of the protein are exposed to the aqueous solvent. At P/P₀=1.0, the absorption of water by α-crystallin was 17% with a single component decay character of spin–echo (T₂=3 ms). Addition of water to α-crystallin to about 50% of its w/w in the protein sample showed T₂=8 ms with only one single component decay of the spin–echo signal. The single component decay character of the spin–echo indicates at the tightly bound water by α-crystallin. Under a relative humidity P/P₀=1.0, collagen and BSA absorbed correspondingly 19.3% and 28% of water and showed a two-component decay curve with T₂ of about 5 and 40 ms. The findings demonstrate the presence of two water fractions in collagen and BSA which are separated in space. The IEF data suggest a tight binding of water with α-crystallin with similar distribution patterns in the lens layers. The IEF data demonstrate a possible chaperone-like function for α-crystallin in the nucleus and inner cortex of the lens, but not in the outer cortex. To conclude, it was found that α-crystallin can immobilize and bind water to a greater extent than other proteins such as collagen and BSA. These results shed new light on structural properties of α-crystallin and have important implications for understanding the mechanism of the chaperone-like action of this protein in the lens and non-ocular tissues.     

Effect of different photoperiods on the ultrastructure of the specific secretory cells and α-subunit mRNA level in the chicken pars tuberalis

The effect of different photoperiods on the specific secretory cells of the pars tuberalis was examined in male chicks. Animals were placed in one of three different photoperiod regimens: (1) normal control (light:dark = 12 h:12 h), (2) continuous light (L:D = 24 h:0), and (3) extended darkness (L:D = 1 h:23 h). The levels of common alpha-subunit mRNA in the pars tuberalis were examined by Northern blot analysis and compared with those in the pars distalis. In chicks exposed to continuous light for 1 week, alpha-subunit mRNA level in the pars tuberalis was decreased, although the level in the pars distalis was increased. Exposure to continuous light for 30 days also induced a decrease in alpha-subunit mRNA level in the pars tuberalis. On the other hand, in chicks exposed to extended darkness for 1 week, the alpha-subunit mRNA level of the pars tuberalis was markedly increased. In situ hybridization with digoxigenin-labeled common alpha-subunit cRNA probe also showed that the hybridization signals for alpha-subunit mRNA in the pars tuberalis cells become weak under continuous light for 30 days but they are very intense under extended darkness. Thus, the synthesis of alpha-subunits in the chick pars tuberalis was inhibited by continuous light but stimulated by extended darkness. These results were confirmed by semiquantitative electron-microscopic analyses. After exposure to continuous light for 30 days, many pars tuberalis (PT)-specific cells were filled with enlarged secretory granules, showing the reduction of secretory activity. On the contrary, extended darkness for 30 days induced hypertrophy of the PT-specific cells; the areas of cytoplasm and nucleus were significantly increased. In addition, secretory granules became small in size and exocytotic features were more frequent. Mitochondria and lysosomes were also increased in number. Thus, the synthetic and secretory activities of the PT-specific cells were increased under extended darkness. The data indicate that the specific cells of the pars tuberalis are responsive to photoperiodic changes in the chick.     

Amino acid sequences around the cysteine residue of calf lens α-crystallin

1. Calf lens alpha-crystallin was carboxymethylated with radioactive sodium iodoacetate to label the thiol group. 2. The protein was then digested with trypsin or alternatively fractionated in urea to obtain the acidic (A) chains, which were then digested with trypsin. Either procedure gave two radioactive peptides containing carboxymethylcysteine. 3. These two peptides were closely related: the longer form contained 28 amino acid residues, and the shorter lacked two residues at the N-terminal end of the longer form. 4. The amino acid sequence of the peptides have been determined. 5. No evidence for the presence of more than one cysteine residue/chain was found. 6. The question of the molecular weight of the chains is discussed.     

Heat-induced structural transition of α-crystallin in the eye lens tissue observed by small-angle X-ray scattering

Heat-induced structural transitions of crystallins in the eye lens tissue have been studied by small-angle X-ray scattering. It is shown that a short-time (～1 min) incubation of the bovine lens tissue at a temperature of ～60°C leads to a pronounced shift of the small-angle x-ray diffraction maximum due to the short-range order of α-crystallin oligomers. This shift indicates an increase in the molecular mass of α-crystallin oligomers. The results are evidence that, in the native surrounding and at the natural concentration of α-crystallin, heat-induced transition of α-crystallin quaternary structure takes place. Earlier, this transition of α-crystallin has been observed only in solutions and gels of this protein. The results confirm the identity of α-crystallin properties in vitro and in vivo.     

A role for γS-crystallin in the organization of actin and fiber cell maturation in the mouse lens

γS-crystallin (γS) is a highly conserved component of the eye lens. To gain insights into the functional role(s) of this protein, the mouse gene (Crygs) was deleted. Although mutations in γS can cause severe cataracts, loss of function of γS in knockout (KO) mice produced no obvious lens opacity, but was associated with focusing defects. Electron microscopy showed no major differences in lens cell organization, suggesting that the optical defects are primarily cytoplasmic in origin. KO lenses were also grossly normal by light microscopy but showed evidence of incomplete clearance of cellular organelles in maturing fiber cells. Phalloidin labeling showed an unusual distribution of F-actin in a band of mature fiber cells in KO lenses, suggesting a defect in the organization or processing of the actin cytoskeleton. Indeed, in wild-type lenses, γS and F-actin colocalize along the fiber cell plasma membrane. Relative levels of F-actin and G-actin in wild-type and KO lenses were estimated from fluorescent staining profiles and from isolation of actin fractions from whole lenses. Both methods showed a two-fold reduction in the F-actin/G-actin ratio in KO lenses, whereas no difference in tubulin organization was detected. In vitro experiments showed that recombinant mouse γS can directly stabilize F-actin. This suggests that γS may have a functional role related to actin, perhaps in 'shepherding' filaments to maintain the optical properties of the lens cytoplasm and normal fiber cell maturation.     

Thermal and acid denaturation of bovine lens α-crystallin

The chaperone-like protein α-crystallin is a ～35 subunit hetero-oligomer consisting of αA and αB subunits in a 3:1 molar ratio and has the function of maintaining eye lens transparency. We studied the thermal denaturation of α-crystallin by differential scanning calorimetry (DSC), circular dichroism (CD), and dynamic light scattering (DLS) as a function of pH. Our results show that between pH 7 and 10 the protein undergoes a reversible thermal transition. However, the thermodynamic parameters obtained by DSC are inconsistent with the complete denaturation of an oligomeric protein of the size of α-crystallin. Accordingly, the CD data suggest the presence of extensive residual secondary structure above the transition temperature. Within the pH range from 4 to 7 the increased aggregation propensity around the isoelectric point (pI ～ 6) precludes observation of a thermal transition. As pH decreases below 4 the protein undergoes a substantial unfolding. The secondary structure content of the acid-denatured state shows little sensitivity to heating. We propose that the thermal transition above pH 7 and the acid-induced transition at ambient temperature result in predominant denaturation of the αB subunit. Although the extent of denaturation of the αA subunit cannot be estimated from the current data, the existence of a native-like conformation is suggested by the preserved association of the subunits and the chaperone-like activity. A key difference between the thermal and the acid denaturation is that the latter is accompanied by dissociation of αB subunits from the remaining αA-oligomer, as supported by DLS studies.     

Human lens γ-crystallin sequences are located in the p12-qter region of chromosome 2

Summary The human -crystallin genes constitute a multigene family whose members are only expressed in the eye lens. The chromosomal location of these sequences has been determined by screening a panel of human/rodent hybrid cell lines containing overlapping subsets of human chromosomes for the presence of human -crystallin sequences. By correlating these genomic hybridization data with the chromosomal constitution of the somatic cell hybrids, all human -crystallin sequences could be assigned to chromosome 2. The use of human/hamster cell hybrids derived from human Burkitt lymphoma cells carrying a reciprocal translocation between human chromosomes 2 and 8, allowed a further localization of the sequences to the region 2p12-qter.     

Enzymic synthesis of argininosuccinate catalyzed by δ-crystallin

Summary A fast and practical procedure has been developed for the synthesis of argininosuccinate using immobilized duck lens -crystallin as catalyst.     

High-affinity binding of NADPH to camel lens ζ-crystallin

Fluorescence spectrum of camel lens ζ-crystallin, a major protein in the lens of camelids and histicomorph rodents, showed maximum emission at 315 nm. This emission maximum is blue shifted compared to most proteins, including α-crystallin, and appeared to be due to tryptophan in highly hydrophobic environment. Interaction of NADPH with ζ-crystallin quenched the protein fluorescence and enhanced the fluorescence of bound NADPH. Analysis of fluorescence quenching suggested high-affinity interaction between NADPH and ζ-crystallin with an apparent K_m<0.45 μM. This value is at least an order of magnitude lower than that suggested by activity measurements. Analysis of NADPH fluorescence showed a biphasic curve representing fluorescence of free- and bound-NADPH. The intersection between free- and bound-NADPH closely paralleled the enzyme concentration, suggesting one mole of NADPH was bound per subunit of the enzyme. Phenanthrenequinone (PQ), the substrate of ζ-crystallin, also was able to quench the fluorescence of ζ-crystallin, albeit weaker than NADPH. Quantitative analysis suggested that ζ-crystallin had low affinity for PQ in the absence of NADPH, and PQ binding induced significant conformational changes in ζ-crystallin.     

The Role of IRE1α in the Degradation of Insulin mRNA in Pancreatic β-Cells

Background

The endoplasmic reticulum (ER) is a cellular compartment for the biosynthesis and folding of newly synthesized secretory proteins such as insulin. Perturbations to ER homeostasis cause ER stress and subsequently activate cell signaling pathways, collectively known as the Unfolded Protein Response (UPR). IRE1α is a central component of the UPR. In pancreatic β-cells, IRE1α also functions in the regulation of insulin biosynthesis.

Principal Findings

Here we report that hyperactivation of IRE1α caused by chronic high glucose treatment or IRE1α overexpression leads to insulin mRNA degradation in pancreatic β-cells. Inhibition of IRE1α signaling using its dominant negative form prevents insulin mRNA degradation. Islets from mice heterozygous for IRE1α retain expression of more insulin mRNA after chronic high glucose treatment than do their wild-type littermates.

Conclusions/Significance

These results reveal a role of IRE1α in insulin mRNA expression under ER stress conditions caused by chronic high glucose. The rapid degradation of insulin mRNA could provide immediate relief for the ER and free up the translocation machinery. Thus, this mechanism would preserve ER homeostasis and help ensure that the insulin already inside the ER can be properly folded and secreted. This adaptation may be crucial for the maintenance of β-cell homeostasis and may explain why the β-cells of type 2 diabetic patients with chronic hyperglycemia stop producing insulin in the absence of apoptosis. This mechanism may also be involved in suppression of the autoimmune type 1 diabetes by reducing the amount of misfolded insulin, which could be a source of “neo-autoantigens.”     

Changes in physicochemical parameters and α-crystallin expression in the lens during cataract development in OXYS rats

The pathogenesis of cataract is associated with oxidative stress and with altered crystallin expression but it is still understood incompletely. In this study, the senescence-accelerated OXYS rats were used as a model. The first biomicro-scopic signs of cataract in OXYS rats were registered at the age of 1.5 months; at 3 months morbidity reached 90%, and at 6 months it reached 100%. Cataract manifestation progresses: at 24 months mature cataract was detected in 90% of eyes of OXYS rats, whereas in 80% of Wistar rat eyes only initial signs of this disease were detected. Analysis of lens redox-parameters has shown that in OXYS rats the intensity of tryptophan fluorescence is higher, the GSH content being higher at 2 months but during formation of mature cataract at 13, 18, and 24 months being lower than in Wistar rats. Decrease in solubility of OXYS rat lens proteins was observed at the age of 13 months. At the age of 3 months gene expression of αA-crystallin and αB-crystallin was 3-fold and 25% lower, respectively, than in Wistar rats. At the age of 14 months there was a 27-fold decrease in expression of αB-crystallin in OXYS rats and it became 21-fold lower than in control. Proteins are synthesized in lens epithelial cells and dystrophic changes in senile cataract result in decrease in structural protein expression. The changes observed in OXYS rats are evidently associated with the dystrophic changes in lens epithelium, which we have described earlier, and are consistent with the model of senile cataract.