Protein-bound kynurenine is a photosensitizer of oxidative damage (original) (raw)
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Journal of Biological Chemistry, 2014
Background: Human lens proteins accumulate pigmented, protein-cross-linked AGE adducts during cataract formation, and the mechanisms of their formation are poorly understood. Results: UVA-excited kynurenines can oxidize ascorbate under anoxic conditions and promote synthesis of AGEs. Conclusion: UVA light-excited kynurenines promote AGE synthesis and contribute to cataract formation. Significance: This study provides a mechanism for UVA light-mediated damage to lens proteins during cataract formation. Advanced glycation end products (AGEs) contribute to lens protein pigmentation and cross-linking during aging and cataract formation. In vitro experiments have shown that ascorbate (ASC) oxidation products can form AGEs in proteins. However, the mechanisms of ASC oxidation and AGE formation in the human lens are poorly understood. Kynurenines are tryptophan oxidation products produced from the indoleamine 2,3-dioxygenase (IDO)-mediated kynurenine pathway and are present in the human lens. This study investigated the ability of UVA lightexcited kynurenines to photooxidize ASC and to form AGEs in lens proteins. UVA light-excited kynurenines in both free and protein-bound forms rapidly oxidized ASC, and such oxidation occurred even in the absence of oxygen. High levels of GSH inhibited but did not completely block ASC oxidation. Upon UVA irradiation, pigmented proteins from human cataractous lenses also oxidized ASC. When exposed to UVA light (320-400 nm, 100 milliwatts/cm 2 , 45 min to 2 h), young human lenses (20-36 years), which contain high levels of free kynurenines, lost a significant portion of their ASC content and accumulated AGEs. A similar formation of AGEs was observed in UVA-irradiated lenses from human IDO/human sodium-dependent vitamin C transporter-2 mice, which contain high levels of kynurenines and ASC. Our data suggest that kynurenine-mediated ASC oxidation followed by AGE formation may be an important mechanism for lens aging and the development of senile cataracts in humans.
Glutathione and NADH, but not ascorbate, protect lens proteins from modification by UV filters
Experimental Eye …, 2002
Age-dependent human lens colouration and¯uorescence may stem primarily from the covalent binding of UV ®lters to crystallins. The tendency of the kynurenine (Kyn) UV ®lters to deaminate at neutral pH, with the generation of reactive a,b-ketoalkenes, underlies this phenomenon. In this study the authors examined the ability of small molecular weight antioxidants, which are known to be present in the lens, to inhibit this process. Crystallins were incubated with Kyn at pH 7 in the presence of glutathione (GSH), ascorbate or NADH. Ascorbate, even at high (15 mM) levels, was not found to signi®cantly retard the time-dependent covalent binding of Kyn to the proteins. GSH, and to a lesser extent NADH, however, had a major impact in preventing this modi®cation. The increase in protein UV absorbance and¯uorescence was inhibited by GSH intercepting the reactive ketone intermediate, to form a GSH±Kyn adduct. NADH seemed to protect by both reduction of the reactive ketone intermediate and by competing with Kyn for presumably hydrophobic sites on the crystallins. This may indicate that the covalent attachment of aromatic Kyn molecules could be facilitated by initial hydrophobic interactions. Since GSH is present at far greater concentrations than NADH, these results show that in primate lenses, GSH is the key agent responsible for protecting the crystallins from covalent modi®cation.
oxidative changes in lens proteins
1. Proteins from the cortex and nucleus of the human lens were studied to determine if any changes could be detected in their amino acids during senile cataract formation.
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 2000
In humans, the crystallin proteins of the ocular lens become yellow-coloured and fluorescent with ageing. With the development of senile nuclear cataract, the crystallins become brown and additional fluorophores are formed. The mechanism underlying crystallin colouration is not known but may involve interaction with kynurenine-derived UV filter compounds. We have recently identified a sulphur-linked glutathionyl-3-hydroxykynurenine glucoside adduct in the lens and speculated that kynurenine may also form adducts with GSH and possibly with nucleophilic amino acids of the crystallins (e.g. Cys). Here we show that kynurenine modifies calf lens crystallins non-oxidatively to yield coloured (365 nm absorbing), fluorescent (Ex 380 nm/Em 450^490 nm) protein adducts. Carboxymethylation and succinylation of crystallins inhibited kynurenine-mediated modification by approx. 90%, suggesting that Cys, Lys and possibly His residues may be involved. This was confirmed by showing that kynurenine formed adducts with GSH as well as with poly-His and poly-Lys. NMR studies revealed that the novel poly-Lys-kynurenine covalent linkage was via the O-amino group of the Lys side chain and the LC of the kynurenine side chain. Analysis of tryptic peptides of kynurenine-modified crystallins revealed that all of the coloured peptides contained either His, Cys or an internal Lys residue. We propose a novel mechanism of kynurenine-mediated crystallin modification which does not require UV light or oxidative conditions as catalysts. Rather, we suggest that the side chain of kynurenine-derived lens UV filters becomes deaminated to yield an K,L-unsaturated carbonyl which is highly susceptible to attack by nucleophilic amino acid residues of the crystallins. The inability of the lens fibre cells to metabolise 0167-4838 / 00 / $^see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 -4 8 3 8 ( 9 9 ) 0 0 2 3 4 -4 their constituent proteins results in the accumulation of coloured/fluorescent crystallins with age. ß
Novel protein modification by kynurenine in human lenses
Journal of Biological …, 2002
It is known that human lenses increase in color and fluorescence with age, but the molecular basis for this is not well understood. We demonstrate here that proteins isolated from human lenses contain significant levels of the UV filter kynurenine covalently bound to histidine and lysine residues. Identification was confirmed by synthesis of the kynurenine amino acid adducts and comparison of the chromatographic retention times and mass spectra of these authentic standards with those of corresponding adducts isolated from human lenses following acid hydrolysis. Using calf lens proteins as a model, covalent binding of kynurenine to lens proteins has been shown to proceed via side chain deamination in a manner analogous to that observed for the related UV filter, 3-hydroxykynurenine O--D-glucoside. Levels of histidylkynurenine and lysylkynurenine were low in human lenses in subjects younger than 30, but thereafter increased in concentration with the age of the individual. Post-translational modification of lens proteins by tryptophan metabolites therefore appears to be responsible, at least in part, for the age-dependent increase in coloration and fluorescence of the human lens, and this process may also be important in other tissues in which up-regulation of tryptophan catabolism occurs. The lens of the eye plays a crucial role in vision. Its chemical composition is unusual in that proteins represent ϳ38% of the wet mass. The high concentration of protein is needed to achieve the refractive index necessary for focusing (1). Crystallins constitute more than 90% of the lens protein and comprise three main classes, ␣, , and ␥, based on their aggregation behavior and sequence homology (2). The tightly packed and ordered distribution of the crystallins is essential for maintaining lens transparency and therefore vision (3). The lenses of humans and other primates contain low molecular weight compounds that act as intraocular filters by absorbing UV light in the 300-400 nm region (4, 5), thus preventing UV-induced photodamage to the retina (6). These filters are produced through the catabolism of tryptophan. The
The Generation of Hydrogen Peroxide by the Uva Irradiation of Human Lens Proteins
Photochemistry and Photobiology, 1995
The water-insoluble proteins from aged human lens are known to contain protein-bound chromophores that act as UVA sensitizers. The irradiation of a sonication-solubilized, water-insoluble fraction from human lenses (55-75 years) with UVA light (1.5 kJ/cm2, X > 338 nm) caused an oxygendependent photolysis of tryptophan, not seen when either a-crystallin or lysozyme were irradiated. The suggested requirement for active oxygen species was consistent with a linear increase in hydrogen peroxide formation, which was also observed. A final concentration of 55 p M H202 was attained, with no H20, being detected in either dark-incubated controls or in irradiated samples of native proteins. The UVA-dependent H20, formation was increased 50% by superoxide dismutase (SOD) and abolished by catalase, arguing for the initial generation of superoxide anion. A linear photolysis of histidine and tryptophan was also seen; however, the addition of SOD or SOD and catalase had no effect on the photolytic destruction of either amino acid. Superoxide dismutase increased the oxidation of protein SH groups implicating H202, but SOD and catalase caused a decrease in SH oxidation only at later time periods. The direct addition of H202 to a water-insoluble sonicate supernatant fraction caused only a slight oxidation of SH groups, but this was increased four-to eight-fold when the protein was denatured in 4.0 M guanidine hydrochloride. Overall, the data suggest a UVA-dependent oxidation of protein SH groups via H202 generated within the large protein aggregates of the water-insoluble fraction. These data also provide a mechanism for oxidation of the sulfur-containing amino acids in vivo-a process that is known to accompany the formation of age-onset cataracts.
Experimental Eye Research, 1995
Using photochemically induced oxidative stress and rat lenses in organ culture with 4% O2 and 4 μm riboflavin, it has been found that the observed changes in lens parameters are, in most cases, irreversible. This has made possible the elucidation of the sequence of biological changes leading to cataract. The earliest detectable changes in lens cell biology are observed in the epithelial cell redox set point and at the DNA level in terms of DNA integrity and 3H-thymidine incorporation followed by decreased membrane transport and changes in gene expression. Significant modification in classical cataract parameters such as hydration, steady state non-protein thiol, glyceraldehyde-phosphate-dehydrogenase activity and transparency occur at later times. The data suggest a definitive pattern of lens breakdown resulting in opacity starting at the epithelial cell level and leading to subsequent fibre cell involvement.
Photochemistry and photobiology, 2015
UVA-visible light has been proposed as a risk factor in the photo-aging of the human eye lens, as well as in the etiology of cataract disease. There is accumulating evidence indicating that photosensitizing reactions mediated by endogenous chromophores, which are generated during human eye lens aging, can play an important role in the generation of these processes. These reactions can lead to protein impairment by inducing non-enzymatic post-translational modifications such as protein oxidation and crosslinking. Although numerous chromophores have been characterized as both bound to human eye lens proteins and as unbound low-molecular-mass compounds, their contribution to eye lens photoaging and cataract disease is not completely understood. In this article we discuss the photochemical contribution of UV-filters derived from tryptophan catabolism and advanced glycation end products (AGEs) to human eye lens aging and cataract disease. We also discuss the recently described photosensi...