Identification of the preferentially targeted proteins by carbamylation during whole lens incubation by using radio-labelled potassium cyanate and mass spectrometry.

Aim: To attempt to identify the primary targets of carbamylation in bovine lenses incubated under physiological condition.

Methods: Fresh intact bovine lenses were incubated with [(14)C]-labelled potassium cyanate for seven days. The water-soluble proteins (WSP) of both cortex and nucleus lens were isolated by size-exclusion chromatography on a Sephacryl S-300HR column.

Effect of a combination of carnosine and aspirin eye drops on streptozotocin -- induced diabetic cataract in rats.

Purpose: To investigate the effect of a combination of carnosine and aspirin eye drops on the progression of diabetic cataract formation induced by streptozotocin (STZ).

Methods: Rats were made diabetic with STZ. Animals in the treated groups received carnosine, aspirin, or a combination of carnosine and aspirin as drops to the eyes.

Identification of the primary targets of carbamylation in bovine lens proteins by mass spectrometry.

Purpose: Carbamylation, an important post-translational modification of proteins, inevitably causes conformational changes of lens proteins. It may increase aggregation between crystallin molecules and disrupt the close packing required for transparency thus leading to cataract. The aim of this study was to isolate the primary targets of carbamylation in the lens and identify them by mass spectrometry.

X-ray- and neutron-scattering studies of alpha-crystallin and evidence that the target protein sits in the fenestrations of the alpha-crystallin shell.

Purpose: Alpha-crystallin, a ubiquitous molecular chaperone, is found in high concentrations in the lens. Its structure and precise mechanism of action, however, are unknown. The purpose of these experiments was to further the understanding of the chaperone function of alpha-crystallin.

Revival of glutathione reductase in human cataractous and clear lens extracts by thioredoxin and thioredoxin reductase, in conjunction with alpha-crystallin or thioltransferase.

Glutathione reductase (GR) plays a key role in maintaining thiol groups in the lens, and its activity decreases with aging and cataract formation. Mammalian thioredoxin (Trx) and thioredoxin reductase (TrxR), or the Trx/TrxR system, participates in the repair of oxidatively damaged lens proteins and enzymes. Alpha-crystallin, a molecular chaperone, prevents the aggregation of partially denatured proteins under various stress conditions.

Thioredoxin, thioredoxin reductase, and alpha-crystallin revive inactivated glyceraldehyde 3-phosphate dehydrogenase in human aged and cataract lens extracts.

Purpose: To investigate whether mammalian thioredoxin (Trx) and thioredoxin reductase (TrxR), with or without alpha-crystallin can revive inactivated glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in both the cortex and nucleus of human aged clear and cataract lenses.

Methods: The lens cortex (including capsule-epithelium) and the nucleus were separated from human aged clear and cataract lenses (grade II and grade IV) with similar average age. The activity of GAPDH in the water-soluble fraction after incubation with or without Trx or/and TrxR for 60 min at 30 degrees C was measured spectrophotometrically.

Protection against glycation and similar post-translational modifications of proteins.

Glycation and other non-enzymic post-translational modifications of proteins have been implicated in the complications of diabetes and other conditions. In recent years there has been extensive progress in the search for ways to prevent the modifications and prevent the consequences of the modifications. These areas are covered in this review together with newer ideas on possibilities of reversing the chemical modifications.

The effect of the presence of globular proteins and elongated polymers on enzyme activity.

We have studied the effect of a crowded (macromolecular) solution on reaction rates of the decarboxylating enzymes urease, pyruvate decarboxylase and glutamate decarboxylase. A variety of crowding agents were used including haemoglobin, lysozyme, various dextrans and polyethylene glycol. Enzyme reaction rates of all three enzymes show two different types of effect that separate the globular proteins from the polysaccharides/polymers.

Carnosine inhibits modifications and decreased molecular chaperone activity of lens alpha-crystallin induced by ribose and fructose 6-phosphate.

Purpose: Alpha-crystallin, a major structural protein in the lens, prevents heat- and oxidative stress-induced aggregation of proteins and inactivation of enzymes by acting as a molecular chaperone. Modification of alpha-crystallin by some posttranslational modifications results in conformational changes and decreases in chaperone activity, which may contribute to cataractogenesis in vivo. Carnosine (beta-alanyl-L-histidine), an endogenous histidine dipeptide, prevents protein modifications including glycation and oxidation.

Glutathione-related enzymes and the eye.

Glutathione and the related enzymes belong to the defence system protecting the eye against chemical and oxidative stress. This review focuses on GSH and two key enzymes, glutathione reductase and glucose-6-phosphate dehydrogenase in lens, cornea, and retina. Lens contains a high concentration of reduced glutathione, which maintains the thiol groups in the reduced form.

Glutathione reductase from human cataract lenses can be revived by reducing agents and by a molecular chaperone, alpha-crystallin.

Purpose: The aim of this study was to investigate how glutathione reductase (GR) loses its activity during cataract formation and whether it is possible to revive it back to the normal levels.

Method: In this study, endogenous as well as synthetic reducing systems (GSH, TTase, DTT, captopril) and alpha-crystallin at different concentrations were incubated with the soluble fraction of human cataract lens protein. The activity of glutathione reductase with or without the reducing agents and alpha-crystallin was tested, and the difference in activity gained was calculated.

Carnosine protects against the inactivation of esterase induced by glycation and a steroid.

Carnosine, an endogenous histidine-containing dipeptide, protects protein from oxidation and glycation, which may contribute to a potential treatment for some conformational diseases including cataract. Glycation, the non-enzymic reaction of sugars with proteins, promotes cross-linking and further aggregation. Prolonged use of glucocorticoids is a risk factor for cataract, as is diabetes.

Trehalose and 6-aminohexanoic acid stabilize and renature glucose-6-phosphate dehydrogenase inactivated by glycation and by guanidinium hydrochloride.

A number of naturally occurring small organic molecules, primarily involved in maintaining osmotic pressure in the cell, display chaperone-like activity, stabilizing the native conformation of proteins and protecting them from various kinds of stress. Most of them are sugars, polyols, amino acids or methylamines. In addition to their intrinsic protein-stabilizing activity, these small organic stress molecules regulate the activity of some molecular chaperones, and may stabilize the folded state of proteins involved in unfolding or in misfolding diseases, such as Alzheimer's and Parkinson's diseases, or alpha1-antitrypsin deficiency and cystic fibrosis, respectively.

Revival of inactive glyceraldehyde 3-phosphate dehydrogenase in human cataract lenses by reduction.

In this study, endogenous as well as synthetic reducing systems were shown to reduce the disulphide bonds formed in glyceraldehyde 3-phosphate dehydrogenase, an important glycolytic enzyme previously reported to have lost its activity in human cataract lenses, resulting in reviving the activity of this enzyme. Disulphide bond formation is a non-specific posttranslational modification of proteins, which leads to a loss of function of the affected protein. When an enzyme is targeted, this harmful effect can be easily detected by monitoring the change of activity.

The identification of a reaction site of glutathione mixed-disulphide formation on gammaS-crystallin in human lens.

The glutathionylation of human lens proteins was examined by Western-blot analysis with an anti-GSH antibody and scanning. Several different glutathionylated proteins were observed, and a 47 kDa band was of particular interest. This band did not appear after SDS/PAGE under reducing conditions, suggesting that it was a glutathionylated fraction.

The molecular chaperone, alpha-crystallin, protects against loss of antigenicity and activity of esterase caused by sugars, sugar phosphate and a steroid.

Previously we showed that glycation-induced inactivation and loss of antigenicity of enzymes occur simultaneously. Alpha-crystallin, a major structural protein of the mammalian lens, prevents the aggregation of other proteins and protects enzyme function against post-translational modification in vitro. However, it is not known whether alpha-crystallin can also protect against loss of antigenicity of enzymes.

Gamma III-crystallin is the primary target of glycation in the bovine lens incubated under physiological conditions.

Several mechanisms have been proposed for the way in which glucose and its metabolites cause cataract, retinopathy and other complications of diabetes, the most convincing being glycation. Glycation, the reaction of sugars with free amino groups of proteins, is one of a variety of non-enzymic post-translational modifications. The aim of the present study was to identify some of the most reactive proteins in the lens when incubated under physiological conditions.

The molecular chaperone alpha-crystallin incorporated into red cell ghosts protects membrane Na/K-ATPase against glycation and oxidative stress.

Alpha-crystallin, a molecular chaperone and lens structural protein protects soluble enzymes against heat-induced aggregation and inactivation by a variety of molecules. In this study we investigated the chaperone function of alpha-crystallin in a more physiological system in which alpha-crystallin was incorporated into red cell 'ghosts'. Its ability to protect the intrinsic membrane protein Na/K-ATPase from external stresses was studied.

Enzyme activity after resealing within ghost erythrocyte cells, and protection by alpha-crystallin against fructose-induced inactivation.

The role of alpha-crystallin as a molecular chaperone has been shown in many in vitro studies. In the present paper, we report on the chaperone function of alpha-crystallin within resealed erythrocyte ghosts. Eight enzymes were individually resealed within erythrocyte ghosts and assayed at zero time and at 24 h.

Viewing molecular mechanisms of ageing through a lens.

Many late-life diseases are conformational diseases in tissues where there are unfolded or misfolded proteins which can form aggregates. These diseases have other common features in their aetiology. Cataract is one such disease and post-translational modifications of proteins in the lens during cataract formation are described as a possible guide to the changes in other age-related conditions.

The effect of modification of alpha-crystallin by prednisolone-21-hemisuccinate and fructose 6-phosphate on chaperone activity.

The major lenticular protein alpha-crystallin has chaperone activity. With increasing age this chaperone function is compromised. Diabetes and glucocorticoid therapy are risk factors for cataract and are associated with raised sugar and glucocorticoid levels, respectively.

Effects of modifications of alpha-crystallin on its chaperone and other properties.

The role of alpha-crystallin, a small heat-shock protein and chaperone, may explain how the lens stays transparent for so long. alpha-Crystallin prevents the aggregation of other lens crystallins and proteins that have become unfolded by 'trapping' the protein in a high-molecular-mass complex. However, during aging, the chaperone function of alpha-crystallin becomes compromised, allowing the formation of light-scattering aggregates that can proceed to form cataracts.

Protective effects of ibuprofen and its major metabolites against in vitro inactivation of catalase and fumarase: relevance to cataract.

Ibuprofen and its major metabolites were incubated with catalase and fumarase, in the presence of protein-modifying biomolecules, to explore the mode of action of ibuprofen in protection against cataract. Both 2 and 10 mM ibuprofen/metabolites protected catalase against fructose-, cyanate- and prednisolone-induced inactivation; the carboxy-metabolite gave the highest protection (31%). The 2 mM ibuprofen/metabolites protected fumarase against fructose- and cyanate-induced inactivation by up to 26%, but had no effect on prednisolone-induced inactivation.