Nicole R Parker

University of Sydney, Sydney, New South Wales, Australia

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Publications (4)12.15 Total impact

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    ABSTRACT: Human ultraviolet light (UV) filters, such as kynurenine (Kyn), readily deaminate to reactive unsaturated ketones that covalently modify proteins in older human lenses. The aim of this study was to examine in vitro rates of formation and decomposition of the three major Kyn-amino acid adducts and possible consequences for the lens. The t-Boc-protected Kyn-His, Kyn-Lys, and Kyn-Cys adducts and Kyn-Cys were synthesized from the corresponding amino acids and Kyn. Calf lens proteins were modified with Kyn by incubation at pH 7. Stability and competition studies of the adducts were conducted under physiological conditions. Kyn-amino acids and their decomposition products were quantified using HPLC. At physiological pH, Kyn-Cys adducts formed more rapidly than either Lys or His adducts, but they also decomposed readily. By contrast, His adducts were stable. Cysteine (Cys) residues in beta-crystallins were major sites of modification. The Kyn moiety, initially bound to Cys residues, was found to transfer to other amino acids. Glutathione promoted the breakdown of Kyn-Cys. These data may help explain why proteins in young lenses are not modified by UV filters in situ. The initial phase of the modification of proteins in the human lens by UV filters may be a dynamic process. In lenses, Cys residues of crystallins modify preferentially, but these adducts also decompose to release deaminated Kyn. This can then potentially react with other amino acids. Glutathione, which is present in high concentrations in the lenses of young people, may play a vital role in keeping proteins free from modification by intercepting reactive deaminated kynurenines formed by the spontaneous breakdown of free UV filters, promoting the decomposition of Kyn-Cys residues, and sequestering the unsaturated ketones once they are released from modified proteins.
    Investigative Ophthalmology &amp Visual Science 09/2007; 48(8):3705-13. · 3.44 Impact Factor
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    ABSTRACT: Human lens proteins become progressively modified by tryptophan-derived UV filter compounds in an age-dependent manner. One of these compounds, kynurenine, undergoes deamination at physiological pH, and the product binds covalently to nucleophilic residues in proteins via a Michael addition. Here we demonstrate that after covalent attachment of kynurenine, lens proteins become susceptible to photo-oxidation by wavelengths of light that penetrate the cornea. H2O2 and protein-bound peroxides were found to accumulate in a time-dependent manner after exposure to UV light (lambda > 305-385 nm), with shorter-wavelength light giving more peroxides. Peroxide formation was accompanied by increases in the levels of the protein-bound tyrosine oxidation products dityrosine and 3,4-dihydroxyphenylalanine, species known to be elevated in human cataract lens proteins. Experiments using D2O, which enhances the lifetime of singlet oxygen, and azide, a potent scavenger of this species, are consistent with oxidation being mediated by singlet oxygen. These findings provide a mechanistic explanation for UV light-mediated protein oxidation in cataract lenses, and also rationalize the occurrence of age-related cataract in the nuclear region of the lens, as modification of lens proteins by UV filters occurs primarily in this region.
    Free Radical Biology and Medicine 11/2004; 37(9):1479-89. · 5.27 Impact Factor
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    ABSTRACT: Posttranslational modification by UV filters is a key event in human lenses that appears to be largely responsible for normal age-dependent yellowing. It has been proposed that subsequent reactions of these covalently bound UV filters may also be involved in the genesis of age-related nuclear cataract. To examine this hypothesis, the levels of kynurenine-lysine and kynurenine-histidine were measured in both normal and cataractous human lenses. Proteins isolated from the nuclei of normal lenses and lenses with and types I to IV nuclear cataract were hydrolyzed in 6 M HCl, and the levels of kynurenine-lysine and kynurenine-histidine were determined by HPLC. The content of kynurenine-lysine and kynurenine-histidine decreased substantially with the progression of age-related nuclear cataract. On average, levels of both kynurenine adducts were four times lower in advanced cataract (type IV) than in normal lenses. Simple autoxidation of the derivatives did not appear to be responsible for this decrease, because incubation in the presence of oxygen or H(2)O(2) did not affect adduct stability. Although protein-bound kynurenine accumulates over time in normal lenses, the levels attached to the proteins decrease significantly with the progression of age-related nuclear cataract. This finding suggests that in cataract there is a breakdown of the protein-bound adducts. Such further reactions of bound UV filters may contribute to the etiology of age-related nuclear cataract.
    Investigative Ophthalmology &amp Visual Science 04/2004; 45(3):879-83. · 3.44 Impact Factor
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    Nicole R Parker
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    ABSTRACT: Human lens proteins become progressively modified by tryptophan-derived UV filter compounds in an age-dependent manner. Kynurenine, for example, undergoes deamination at physiological pH, and the product binds covalently to the nucleophilic residues in protein via a Michael addition. Key sites of kynurenine modification in human lens proteins include cysteine, histidine and lysine residues. The factors determining the levels of Kyn-amino acid adducts in vivo are not known. An aim of this study was to determine the rate of reaction of Kyn with nucleophilic amino acids (His, Cys and Lys) under physiological conditions (pH 7.2, 37°C) and to evaluate the stability of the Kyn-amino acid adducts under these conditions. Kinetic and stability studies were performed using both free amino acids and modified calf lens protein, and reactions analysed by high performance liquid chromatography and mass spectrometry. Kinetic studies using free amino acids revealed that Cys reacted with Kyn at approximately three times the rate of His and four times the rate of Lys. Kyn-t-Boc-His was found to be the most stable of all the Kyn-amino acid adducts under physiological conditions, followed by Kyn-t-Boc-Cys and Kyn-t-Boc-Lys. Kyn-t-Boc-Lys and Kyn-Cys decomposed under physiological conditions, releasing deaminated Kyn. Kyn-Cys was the least stable of the Kyn-amino acid adducts. Incubation of Kyn-Cys in the presence of excess of t-Boc-L-His resulted in a decrease in Kyn-Cys and a corresponding increase in Kyn-t-Boc-His, due to the transfer of deaminated Kyn. Addition of a t-Boc group to the -amino group of Cys increased the stability of the Kyn-Cys adduct by a factor of three. Oxidation of Kyn-t-Boc-Cys to a sulfoxide derivative decreased the stability by a factor of three. Kinetic studies performed with calf lens protein incubated with Kyn showed that Cys was the preferred site of Kyn modification, followed by His and Lys, repectively. Following 14 days of incubation, Kyn-Cys was present at 12- and 17-fold greater levels than Kyn-His and Kyn-Lys. Protein-bound Kyn-Cys appeared more stable than the free Kyn-Cys adduct and was similar in stability to the free Kyn-t-Boc-Cys adduct. Under these conditions, protein-bound Kyn-His levels increased by 38%, whereas Kyn-Lys decreased by 28% with incubation time. Protein-bound Kyn-Lys was also relatively more stable compared the free Kyn-amino acid adduct. Both the free amino acid and calf lens protein studies confirmed that Cys was the best nucleophile at physiological pH, followed by His and Lys. Kyn-His, however, was the most stable modification. The final pattern of Kyn-modification was shown to be a factor of amino acid reactivity and stability rather than amino acid abundance. Another aim of this study was to explore the hypothesis that protein-bound Kyn is oxidised in the cataractous lens, as the levels of Kyn-His and Kyn-Lys decrease by a factor of 4 with increasing severity of age-related nuclear (ARN) cataract. Model studies were performed in which Kyn-t-Boc-His and Kyn-t-Boc-Lys were incubated in the presence of an equimolar concentration of hydrogen peroxide. These studies showed that hydrogen peroxide did not affect the decomposition of either adduct under physiological conditions, suggesting that decomposition of these species in the cataractous lens may be the result of other factors, for example, stronger oxidising agents. The final aim of this study was to investigate the photochemistry of proteinbound Kyn. Previous studies have shown that Kyn, when free in solution, is an inefficient sensitizer of oxidative damage. However, the photochemistry of proteinbound UV filter molecules has not been investigated and this may be of significance, especially for the older human lens, as a result of the decline in free Kyn and an increase in the bound form. Lens proteins covalently modified with kynurenine were susceptible to photooxidation by wavelengths of light that penetrate the cornea (UVA light λ>305, >345 and >385 nm). These wavelengths were chosen because light in the 300-400 nm band are absorbed by the lens. Hydrogen peroxide and protein-bound peroxides were found to accumulate in a time-dependent manner after exposure to UV light (λ 305-385 nm), with shorter wavelength light generating more peroxides. Peroxide formation was accompanied by increases in the levels of protein-bound tyrosine oxidation products dityrosine and 3,4- ihydroxyphenylalanine, species known to be elevated in human cataract lens proteins. Experiments using D2O, which enhances the lifetime of singlet oxygen, and azide, a potent scavenger of this species, are consistent with oxidation being mediated by singlet oxygen. These findings provide a mechanistic explanation of UV light-mediated protein oxidation in cataract lenses, and also rationalise the occurrence of age-related cataract in the nuclear region of the lens, as modification of lens protein by UV filters occurs primarily in this region.
    University of Wollongong Thesis Collection.

Publication Stats

68 Citations
12.15 Total Impact Points

Institutions

  • 2007
    • University of Sydney
      • Save Sight Institute
      Sydney, New South Wales, Australia
  • 2004
    • University of Wollongong
      • School of Chemistry
      Wollongong, New South Wales, Australia