Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0086543 (cataract)
 29,165 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Oxidative damage of the lens causes disulfide bonds between cysteinyl residues of lens proteins and thiols such as glutathione and cysteine, which may lead to cataract. The effect of H2O2 oxidation was determined by comparing bovine lenses incubated with and without 30 mM H2O2. The H2O2 treatment decreased the glutathione and increased the protein-glutathione and protein-cysteine disulfides in the lens. The molecular mass of the gammaB-crystallin isolated from lenses, not treated with H2O2, agreed with the published sequence (Mr 20,966). Some lenses also had a less abundant gammaB-crystallin component 305 Da higher (Mr 21,270), suggesting the presence of a glutathione adduct. The gammaB-crystallins from H2O2 treated lenses had three components, the major one with one GSH adduct, another one with the mass of unmodified gammaB-crystallin, and a third with a mass consistent with addition of two GSH adducts. Mass spectrometric analysis of tryptic peptides of gammaB-crystallins from different lenses indicated that the +305 Da modifications were not at a specific cysteine. For the lenses incubated without H2O2, there was evidence of adducts at Cys-41 and in peptide 10-31, which includes 3 cysteines. Analysis of modified peptide 10-31 by tandem mass spectrometry showed GSH adducts at Cys-15, Cys-18, and Cys-22. In addition, gammaB-crystallins from H2O2-treated lenses had an adduct at Cys-109, partial oxidation at all 7 Met residues, and evidence for two disulfide bonds.
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PMID:Thiolation of the gammaB-crystallins in intact bovine lens exposed to hydrogen peroxide. 998 10

Oxidation of cysteine, glutathione and ascorbate by photoexcited proteins from normal and cataractous lenses was investigated using electron paramagnetic resonance in combination with spin trapping. We report that illumination of these proteins in pH 7 buffer with light > 300 nm in the presence of thiols (RSH) and a spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), afforded DMPO/S-cysteine and DMPO/SG adducts, suggesting the formation of the corresponding thiyl radicals. In a nonbuffered aqueous solution, illumination of the proteins and glutathione also produced superoxide detected as a DMPO/O2H adduct. Irradiation of these proteins in the presence of ascorbate generated ascorbate radical. We conclude that chromophores present in the natural normal and cataractous lenses are capable of initiating photooxidative processes involving endogenous thiols and ascorbic acid. This observation may be pertinent to UV-induced development of cataract.
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PMID:Electron paramagnetic resonance and spin trapping investigations of the photoreactivity of human lens proteins. 1004 18

The purpose of this study was to characterize Lp82 calpain in normal mouse. Lp82 is a lens-specific, calcium-activated isozyme from the calpain super family of cysteine proteases (EC 34.22.17). RT-PCR and molecular cloning were performed on total RNA from 12 day-old mice. Lp82 and m-calpain protein levels and proteolytic activities in lenses were measured by casein zymography, immunoblotting, and ELISA after partial purification by DEAE-HPLC. The 2334-bp cDNA encoding for mouse Lp82 contained a single large open reading frame encoding a protein of 709 amino acid residues with a calculated molecular weight of 82.2 kDa and a predicted pI of 5.8. The amino acid sequence of mouse lens Lp82 was 99% homologous to rat lens Lp82. As in rat, mouse lens Lp82 showed a unique N -terminus and deletion of the IS1 and IS2 regions. In contrast to rat, Lp82 was the dominant calpain in young mouse lens. Lp82 was lens-specific, and the lens nucleus contained the highest specific activity of Lp82 and very little m-calpain. Endogenous Lp82 in lens soluble proteins was activated by addition of calcium and caused limited proteolysis of crystallins even in the presence of large amounts of recombinant domain I from the natural calpain inhibitor calpastatin. Loss of Lp82 protein accompanied aging of mouse lens. Lp82 may be responsible for a major portion of crystallin proteolysis occurring during normal lens development and maturation, or during cataract formation in young mice.
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PMID:Lp82 is the dominant form of calpain in young mouse lens. 1019 2

Human lens nuclei were collected during routine cataract surgery and used to study the role of oxidation in cataract formation and brunescence. This study focused on the comparison of the intensities of nuclear opacity and pigmentation (brunescence) with the changes in free glutathione (GSH) and the three species of protein-thiol mixed disulfides: protein-S-S-glutathione (PSSG), protein-S S-cysteine (PSSC) and protein-S-S-gamma-glutamylcysteine (PSSGC). Eighty-one freshly excised human lens nuclei from a population with a mean age of 77 were used. The nuclear color was graded using the CCRG system, ranging from yellow to dark brown. The nuclear cataract opalescence of these lenses was also graded using the LOCS II system, ranging from LOCS II NO-1 to NO-4. Three normal human lenses (average age of 88 yr) were also included in the study as controls. The nuclear samples were each analyzed for free GSH and protein-thiol mixed disulfides, respectively. It was found that nuclear GSH decreased as the nuclear color increased from yellow to dark brown (from 0.73+/-0.13 to 0.13+/-0.03 micromole g wet wt-1) and as the nuclear opalescence increased from NO.1 to NO.4 (from 0. 80+/-0.19 to 0.20+/-0.01 micromole g wet wt-1). All these values were lower than that of GSH in normal controls (1.43+/-0.59 micromole g wet wt-1). Levels of both PSSG and PSSC progressively increased, however, as the nuclear color intensified. PSSG increased from 0.29+/-0.05 to 0.91+/-0.11 micromole g wet wt-1while PSSC increased from 0.13+/-0.04 to 0.41+/- 0.06 micromole g wet wt-1. PSSGC concentration progressively increased with increases in both nuclear pigmentation (from 0.05+/-0.01 to 0.23+/-0.05 micromole g wet wt-1) and nuclear opacity (from 0.02+/-0.00 to 0.20+/-0.02 micromole g wet wt-1). In comparison, normal controls had lower levels of all three mixed disulfide species: PSSG, 0.22+/-0.06; PSSC, 0.08+/-0.02; PSSGC, 0.02+/-0.06 micromole g wet wt-1, respectively. The correlation of lens nuclear color and opalescence intensity with nuclear protein S-thiolation indicates that protein-thiol mixed disulfides may play an important role in cataractogenesis and development of brunescence in human lenses.
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PMID:Correlation of nuclear color and opalescence with protein S-thiolation in human lenses. 1032 68

Protein S-thiolation is a process in which under oxidative stress, vulnerable sulfhydryl groups of proteins are conjugated to non-protein thiols such as glutathione (GSH) or cysteine resulting in the formation of protein-thiol mixed disulfides, protein-S-S-glutathione (PSSG) and protein-S-S-cysteine (PSSC). This process spontaneously disrupts the redox homeostasis of the cells, which in turn leads to functional disturbances in the respective tissue. In the ocular lens, such modification of proteins may trigger a cascade of events starting with the alteration of protein conformation, protein/enzyme deactivation, protein-S-S-protein aggregation and eventually lens opacification or cataract. Generally, the first line of defense system in the cells protects the lens proteins against such damage. Recent studies in our laboratory have shown that in addition to this defense system, lens cells also possess a well developed system to repair the oxidative damage to the lens proteins. We have identified this repair system as thioltransferase (TTase) and have proved that TTase by its dethiolase activity reverses the protein S-thiolation process which returns the oxidatively damaged lens proteins/enzymes to their original reduced state and restores their physiological functions. We investigated if this repair mechanism was mediated by enzymes other than TTase. We studied glutathione S-transferase (GST) and report here for the first time the cloning, high level expression, and purification of human lens mu and pi isoforms of GST. A comparative study of recombinant human lens TTase and GST (mu and pi) on their dethiolating abilities using lens crystallin-thiol mixed disulfides showed that the lens TTase is 60-70% more efficient in the dethiolation/repair process than GST. When TTase and GST were tested in conjunction for the dethiolation of thiol mixed disulfides, there was no significant enhancement of dethiolase activity. These findings suggest that TTase by itself is an efficient enzyme in the dethiolation/repair process and hence can be considered a crucial system to counteract oxidative stress in the lens.
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PMID:Does glutathione-S-transferase dethiolate lens protein-thiol mixed disulfides?-A comparative study with thioltransferase. 1037 35

In a recent paper, patients with a progressive juvenile-onset hereditary cataract have been reported to have a point mutation in the human gammaD crystallin gene (Stephan, D. A., Gillanders, E., Vanderveen, D., Freas-Lutz, D., Wistow, G., Baxevanis, A. D., Robbins, C. M., VanAuken, A., Quesenberry, M. I., Bailey-Wilson, J., et al. (1999) Proc. Natl. Acad. Sci. USA 96, 1008-1012). This mutation results in the substitution of Arg-14 in the native protein by a Cys residue. It is not understood how this mutation leads to cataract. We have expressed recombinant wild-type human gammaD crystallin (HGD) and its Arg-14 to Cys mutant (R14C) in Escherichia coli and show that R14C forms disulfide-linked oligomers, which markedly raise the phase separation temperature of the protein solution. Eventually, R14C precipitates. In contrast, HGD slowly forms only disulfide-linked dimers and no oligomers. These data strongly suggest that the observed cataract is triggered by the thiol-mediated aggregation of R14C. The aggregation profiles of HGD and R14C are consistent with our homology modeling studies that reveal that R14C contains two exposed cysteine residues, whereas HGD has only one. Our CD, fluorescence, and differential scanning calorimetric studies show that HGD and R14C have nearly identical secondary and tertiary structures and stabilities. Thus, contrary to current views, unfolding or destabilization of the protein is not necessary for cataractogenesis.
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PMID:Molecular basis of a progressive juvenile-onset hereditary cataract. 1068 88

The high content of glutathione (GSH) in the lens is believed to protect the thiols in structural proteins and enzymes for proper biological functions. The lens has both biosynthetic and regenerating systems for GSH to maintain its large pool size (4-6 mM). However, we have observed that, in aging lenses or lenses under oxidative stress, the size of GSH pool is diminished; and some protein thiols are being S-thiolated by oxidized nonprotein thiols to form protein-thiol mixed disulfides, either as protein-S-S-glutathione (PSSG) or protein-S-S-cysteine (PSSC). We have shown in an H2O2-induced cataract model that PSSG formation precedes a cascade of events starting with protein disulfide crosslinks, protein solubility loss, and eventual lens opacification. Recently, we discovered that this early oxidative damage in protein thiols could be spontaneously reversed in H2O2 pretreated lenses if the oxidant was removed in time. This dethiolation process is likely mediated through a redox regulating enzyme, thioltransferase (TTase), which has been discovered recently in the lens. To understand if the role of oxidative defense and repair is the physiological function of TTase in the lens, we cloned the TTase gene and purified the recombinant human lens TTase. Although TTase required GSH for its activity, TTase was far more efficient in dethiolating lens proteins than GSH alone. It favored PSSG over PSSC and dethiolated gamma-crystallin-S-S-G better than the alpha-crystallin counterparts. Furthermore, TTase showed a remarkable resistance to oxidation (H2O2) in cultured rabbit lens epithelial cells when GSH peroxidase, GSH reductase, and glyceraldehyde-3-phosphate dehydrogenase were severely inactivated. We further showed that activity loss in those SH sensitive enzymes could be attributed to S-thiolation, but reactivation via dethiolation could be attributed to TTase. We conclude that TTase can regulate and repair the thiols in lens proteins and enzymes through its dethiolase activity, thus contributing to the maintenance of the function of the lens.
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PMID:Thiol regulation in the lens. 1080 24

SPARC (secreted protein, acidic and rich in cysteine) is a matricellular glycoprotein that regulates morphogenesis, cellular proliferation, and differentiation. SPARC is a critical factor in the development and maintenance of lens transparency in mice. SPARC-null mice develop lenticular opacity at an early age that progresses gradually to mature cataract. Despite the high level of homology between the mouse and human genes, little is known about SPARC in the human lens. We have studied the expression of SPARC protein in human lens and surrounding ocular tissues from normal human donors (60-70 years old). Immunohistochemical and immunoblot analyses were conducted on lens, aqueous humor, vitreous, ciliary epithelium, pigment epithelium, cornea and retina. The epithelia and capsule of the lens contained SPARC, whereas the cortical and nuclear fibers did not. In contrast, the aqueous humor and vitreous, which provide nutrients to the lens and regulate its development and function, contained significant amounts of SPARC. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracts of various ocular tissues revealed bands of 43 and 29 kD after disulfide bond reduction that were reactive with anti-SPARC IgG. Despite the presence of protease inhibitors during sample preparation, we observed cleavage of intact SPARC to a 29 kD fragment, a peptide reported in other tissues and attributed to endogenous proteolysis. In addition, bands of molecular mass 150 and 200 kD were present that appeared to be disulfide-bonded complexes of SPARC monomers. Human cornea, ciliary epithelium, pigment epithelium and retina also contained SPARC. The presence of SPARC in the aqueous humor and vitreous, as well as in the lens, indicates a functional importance of SPARC in adult human eye as well as in lens development.
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PMID:Expression and characterization of SPARC in human lens and in the aqueous and vitreous humors. 1088 Feb 78

Explanted cultures of crystalline lenses have been used to investigate mechanisms of xenobiotic-induced cataract formation. However, very few studies have utilized mechanistic information to predict the cataractogenic potential of structurally diverse xenobiotics. The present investigation outlines how visual assessment of lens clarity, biochemical endpoints of toxicity, and mechanisms of lenticular opacity formation can be used to select compounds with a lower probability of causing cataract formation in vivo. The rat lens explant culture system has been used to screen thiazolidinediones against ciglitazone for their direct cataractogenic potential in vitro. The two compounds that were selected as development candidates (englitazone and darglitazone) did not produce cataracts in rats exposed daily for 3 months. The culture system has also been used to illustrate that the lens is capable of metabolizing compounds to reactive intermediates. In this example, the toxicity of S-(1,2-dichlorovinyl)-L-cysteine (DCVC), a model cataractogen, was attenuated by inhibiting lenticular cysteine conjugate beta-lyase metabolism using aminooxyacetic acid. Finally, this model was used retrospectively to investigate the cataractogenic potential of CJ-12,918 and CJ-13,454 in rats. These compounds showed differences in the incidence of cataract formation in vivo based on differences in hepatic metabolism and penetration of parent drug and metabolites into the lens. The rank order of cataractogenic potential in vitro correlated better with in vivo results when an induced S9 microsomal fraction was added to the culture media. However, the model did not correctly predict the cataractogenic potential of ZD2138, a structurally similar compound. These studies illustrate the use of explant culture to assess mechanisms of cataract formation and outline its use and limitations for predicting cataractogenic potential in vivo.
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PMID:The use of explant lens culture to assess cataractogenic potential. 1108 7

Protein-bound 3,4-dihydroxyphenylalanine (DOPA) can be generated in mammalian cells by both controlled enzymatic pathways, and by uncontrolled radical reactions. Protein-bound DOPA (PB-DOPA) has reducing activity and the capacity to inflict secondary damage on other important biomolecules such as DNA. This may be mediated through replenishment of transition metals or from catechol-quinone-catechol redox cycles in the presence of cellular components such as ascorbate or cysteine, resulting in amplification of radical damaging events. The generation of PB-DOPA confers on protein the ability to chelate transition metals generating protein 'oxychelates'; this may be amongst the factors, which localise such damage. Tissue levels of PB-DOPA are increased in a number of age-related pathologies such as atherosclerosis and cataract formation. We discuss the detoxification, and the subsequent proteolysis and excretion of components of PB-DOPA. We contrast the fact that in marine organisms, and particularly in extracellular proteins, PB-DOPA and other DOPA-polymers can play important functional roles in adhesion and the provision of tensile properties.
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PMID:Metabolism of protein-bound DOPA in mammals. 1108 74


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