Conformational stability of the deamidated and mutated human ßB2-crystallin.

Previous studies propose that genetic mutations and post-translational modifications in protein crystallins promote protein aggregation and are considered significant risk factors for cataract formation. The ßB2-crystallin (HßB2C) forms a high proportion of proteins in the human eye lens. Different congenital mutations and post-translational deamidations in ßB2-crystallin have been reported and linked to cataract formation. In this work, we employed extensive all-atom molecular dynamics simulations to evaluate the conformational stability of deamidated and mutated HßB2C. Our results show critical changes in the protein surface and its native contacts due to a modification in the conformational equilibrium of these proteins. The double deamidated (Q70E/Q162E) and single deamidated (Q70E) impact the well compact conformation of the HßB2C. These post-translational modifications allow the exposure of the protein hydrophobic interface, which lead to the exposure of electronegative residues. On the other hand, our mutational studies showed that the S143F mutation modifies the hydrogen-bond network of an antiparallel ß-sheet, unfolding the C-terminal domain. Interestingly, the chain termination mutation (Q155X) does not unfold the N-terminal domain. However, the resultant conformation is more compact and avoids the exposure of the hydrophobic interface. Our results provide valuable information about the first steps of HßB2C unfolding in the presence of deamidated amino acids that have been reported to appear during aging. The findings reported in this work are essential for the general knowledge of the initial steps in the cataract formation mechanism, which may be helpful for the further development of molecules with pharmacological potential against cataract disease.

A Rat Eye Lens Model of Cataract Formation.

This chapter describes the use of lenses obtained from rats as a model of cataractogenesis. At the molecular level, this is visualized as reduced activity of oxidative reductive enzymes such as aldose reductase and increased proteolysis of lens structural proteins including vimentin. In this chapter, protocols for assessment of these two pathways are presented. Specifically, this analysis shows a comparison of aldose reductase activity and vimentin cleavage in male and female rat lenses. This is because female rats are more susceptible to cataract formation compared to males.

The Major Chromophore Arising from Glucose Degradation and Oxidative Stress Occurrence during Lens Proteins Glycation Induced by Glucose.

Glucose autoxidation has been proposed as a key reaction associated with deleterious effects induced by hyperglycemia in the eye lens. Little is known about chromophores generated during glucose autoxidation. In this study, we analyzed the effect of oxidative and dicarbonyl stress in the generation of a major chromophore arising from glucose degradation (GDC) and its association with oxidative damage in lens proteins. Glucose (5 mM) was incubated with H2O2 (0.5-5 mM), Cu2+ (5-50 µM), glyoxal (0.5-5 mM) or methylglyoxal (0.5-5 mM) at pH 7.4, 5% O2, 37 °C, from 0 to 30 days. GDC concentration increased with incubation time, as well as when incubated in the presence of H2O2 and/or Cu2+, which were effective even at the lowest concentrations. Dicarbonylic compounds did not increase the levels of GDC during incubations. ¹H, 13C and FT-IR spectra from the purified fraction containing the chromophore (detected by UV/vis spectroscopy) showed oxidation products of glucose, including gluconic acid. Lens proteins solutions (10 mg/mL) incubated with glucose (30 mM) presented increased levels of carboxymethyl-lysine and hydrogen peroxide that were associated with GDC increase. Our results suggest a possible use of GDC as a marker of autoxidative reactions occurring during lens proteins glycation induced by glucose.

Advanced glycation endproducts induce photocrosslinking and oxidation of bovine lens proteins through type-I mechanism.

Advanced glycation endproducts (AGEs) have been suggested as photosensitizers that are capable of mediating eye lens photo-damage during aging. In the present work, we investigate the photo-crosslinking and oxidation of bovine lens proteins sensitized by AGEs, with special regard to low oxygen conditions. A mechanistic study was conducted using different oxygen concentrations and specific additives with the aim either to scavenge or enhance Type-I or Type-II photoprocesses. Quantum yields for Trp decomposition were determined at 5%, 20% and 100% O(2), in the presence of ferricyanide and D(2)O to elucidate the mechanism of action of AGEs. Type-I mechanism proved to be the most efficient pathway for AGE-sensitized Trp decomposition at low oxygen concentration. Photocrosslinking of lens proteins and crystallin fractions due to Type-I interaction was observed. The influence of the oxygen concentration and additives was also studied. The results show that both Type-I mechanism and oxygen-mediated reactions contribute to protein crosslinking. Carbonyl group formation due to protein photo-oxidation was detected with Oxyblot technique. The generation of high levels of hydrogen peroxide during the irradiations was detected and attributed mainly to Type-I reactions. The results support that AGEs act preferentially as Type-I sensitizers at the low oxygen concentration found in the lens and are capable of inducing protein crosslinking, oxidation and peroxide formation.

Amino acid contents along the visual and equatorial axes of a pig lens by Raman spectroscopy.

Using near infrared Raman microspectroscopy with laser light of 830 nm, the distribution of amino acids along the visual and equatorial axes of a normal pig lens was studied. The classification of pig lens Raman spectra in these axes was performed using principal component analysis and linear discriminant analysis. The analysis of the scattered light selectively collected from point to point, along the visual axis, indicated that the tyrosine and tryptophan increases and then, at approximately 4 mm position, decreases. Moreover, in the equatorial plane, the nuclear part has the highest concentration of these amino acids. However, the phenylalanine content increases from anterior to posterior cortex of the lens as long as in the equatorial axis it slightly increases and then at approximately 2-2.3 mm position, decreases. The changes in amino acid conformation along the visual axis, similarly to the changes in protein conformation, may explain the refractive gradient of the lens.

Study of the interaction between triplet riboflavin and the alpha-, betaH- and betaL-crystallins of the eye lens.

Time-resolved photolysis studies of riboflavin (RF) were carried out in the presence and absence of alpha-, betaH- and betaL-crystallins of bovine eye lens. The transient absorption spectra, recorded 5 micros after the laser pulse, reveal the presence of the absorption band (625-675 nm) of the RF neutral triplet state (tau = 42 micros) accompanied by the appearance of a long-lived absorption (tau = 320 micros) in the 500-600 nm region due to the formation of the semireduced RF radical. The RF excited state is quenched by the crystallin proteins through a mechanism that involves electron transfer from the proteins to the flavin, as shown by the decrease of the triplet RF band with the concomitant increase of the band of its semireduced form. Tryptophan loss on RF-sensitized photooxidation of the crystallins when irradiated with monochromatic visible light (450 nm) in a 5% oxygen atmosphere was studied. A direct correlation was found between the triplet RF quenching rate constants by the different crystallin fractions and the decomposition rate constants for the exposed and partially buried tryptophans in the proteins. The RF-sensitized photooxidation of the crystallins is accompanied by the decrease of the low molecular weight constituents giving rise to its multimeric forms. A direct correlation was observed between the initial rate of decrease of the low molecular weight bands corresponding to the irradiated alpha-, betaH- and betaL-crystallins and the quenching constant values of triplet RF by the different crystallins. The correlations found in this study confirm the importance of the Type-I photosensitizing mechanism of the crystallins, when RF acts as a sensitizer at low oxygen concentration, as can occur in the eye lens.

Antioxidant properties of alpha-crystallin.

alpha-Crystallin is a major chaperone lens protein to which has been ascribed antioxidant functions. In the present work we have evaluated the antioxidant and free radical scavenging properties of bovine alpha-crystallin in a series of in vitro models: zimosan-induced, luminol-enhanced chemiluminescence response of polymorphonuclear leukocytes, the autoxidation of brain homogenate, bleaching of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)-derived radical cations, trapping of peroxyl radicals, and reactivity toward hypochloric acid. In all these systems, the reactivity of alpha-crystallin is higher than or similar to that of bovine serum albumin. It is concluded that, given the high concentrations of ol-crystallin in the lenses, its capacity to interact with free radicals and to remove hypochlorous acid could contribute to the maintenance of the lens functionality.

Properties of chicken lens MIP channels reconstituted into planar lipid bilayers.

Membrane fractions highly enriched in chicken lens MIP (MIP28) were found to form ion channels when incorporated into planar lipid bilayers. The channels displayed prominent unitary conductances of about 60 and 290 pS in symmetric 150 mm KCl solution and were slightly anion selective. For both depolarizing and hyperpolarizing voltages, voltage sensitivity of the MIP28-induced conductance could be fit by a Boltzmann relation, symmetric around zero mV, with V0 = 18.5 mV, n = 4.5 and gmin/gmax = 0.17. Channel properties were not appreciably altered by pH in the range of 5.8 to 7, although channel incorporation was observed to occur more frequently at lower pH values. Calcium, at millimolar concentrations, decreased the channel mean open time. Partial proteolysis of MIP28 to yield MIP21 did not appreciably affect single-channel conductance or voltage sensitivity of the reconstituted channels. MIP28 was not phosphorylated by cAMP dependent protein kinase (PKA). Although unitary conductance and selectivity of the chicken MIP channel are similar to those reported for the bovine MIP (MIP26), the voltage sensitivity of MIP28 was higher than that of the bovine homologue, and voltage sensitivity of MIP28 was not modulated by treatments previously shown to affect MIP26 voltage gating (partial proteolysis and protein phosphorylation by PKA: (Ehring et al., 1990). The existence of such strikingly different functional properties in highly homologous channel isoforms may provide a useful system for exploration of the structure-function relations of MIP channels.

Melanins and lens pigments: a comparative study.

Based on previous findings that lens pigments and melanins share many physicochemical properties, human lens pigments and natural (hair) and synthetic melanins were submitted to oxidation with permanganate under strong acidic conditions. This procedure has been utilized for the characterization of melanins and results in the well defined products, thiazole-4,5-dicarboxylic acid (TDCA) and pyrrole-2,3,5-tricarboxylic acid (PTCA), which can be quantitated by high performance liquid chromatography (HPLC). PTCA is regarded as a marker of black eumelanins and was therefore a main component of synthetic DOPA-eumelanin and dark hair. Its identity was established by synthesis from 5-hydroxyindole-2-carboxylic acid. TDCA derives from pheomelanins and was therefore an important component of red hair and synthetic GSH-pheomelanin. TDCA was identified by its retention time relative to PTCA. The analysis of a series of cataract digests of increasing pigmentation (type I < type IV < type V) and a purified fraction of lens pigments (DE52 pigment) revealed the presence in these preparations of both PTCA and TDCA. The concentration of TDCA significantly increased with the degree of pigmentation of the digests and reached a maximum in the DE52 pigment. The TDCA/PTCA ratio was high in the lens preparations and comparable to that given by hair pheomelanin. These findings support that pheomelanin is an integral part of lens pigments. By comparing the yields of TDCA in GSH-pheomelanin and in the purified lens pigment, a 9% contribution of pheomelanin to the lens pigment was estimated.

Melanins and lens pigments: a comparative study

Based on previous findings that lens pigments and melanins share many physicochemical properties, human lens pigments and natural (hair) and synthetic melanins were submitted to oxidation with permanganate under strong acidic conditions. This procedure has been utilized for the characterization of melanins and results in the well defined products, thiazole-4,5-dicarboxylic acid (TDCA) and pyrrole-2,3,5-tricarboxylic acid (PTCA), which can be quantitated by high performance liquid chromatography (HPLC). PTCA is regarded as a marker of black eumelanins and was therefore a main component of synthetic DOPA-eumelanin and dark hair. Its identity was established by synthesis from 5-hydroxyindole-2-carboxylic acid. TDCA derives from pheomelanins and was therefore an important component of red hair and synthetic GSH-pheomelanin. TDCA was identified by its retention time relative to PTCA. The analysis of a series of cataract digests of increasing pigmentation (type I < type IV < type V) and a purified fraction of lens pigments (DE52 pigment) revealed the presence in these preparations of both PTCA and TDCA. The concentration of TDCA significantly increased with the degree of pigmentation of the digests and reached a maximum in the DE52 pigment. The TDCA/PTCA ratio was high in the lens preparations and comparable to that given by hair pheomelanin. These findings support that pheomelanin is an integral part of lens pigments. By comparing the yields of TDCA in GSH-pheomelanin and in the purified lens pigment, a 9 per cent contribution of pheomelanin to the lens pigment was estimated

An improved method combining two electrophoretic procedures: application to the separation of lens alpha-crystallin isoforms.

Polypeptides having different net electric charges and very similar molecular weights, visualized as one single band in sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE), can be readily analyzed by an improved method combining two electrophoretic procedures. The methodology consists of the identification and isolation of selected protein bands from SDS-PAGE, their equilibration in an isoelectric focusing (IEF) sample buffer, and their casting and separation in an IEF flat-bed gel. This method requires no extra equipment, is highly reproducible, is suitable for quantitative and comparative studies, and is especially useful in the case of small samples. As a particular example, we analyze here the subunit composition of alpha-crystallins of young and embryonic quail lenses.