Thermodynamics of thermal and athermal denaturation of .gamma.-crystallins: changes in conformational stability upon glutathione reaction

Kono, Masahiro; Sen, Ashish; Chakrabarti, Buddhapriya

doi:10.1021/bi00454a022

Cited by 56 publications

(14 citation statements)

References 39 publications

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“…Perhaps the initial thiolation in peptide 10 -31 or at Cys-41 caused a conformational change that exposed the previously buried Cys-78, making it more accessible for disulfide bond formation. This suggestion of a thiolation-induced conformational change, along with previous evidence that GSH adducts induce conformational changes (5,6,45), further supports the hypothesis (16) that oxidative stress-induced thiolation plays a pivotal role in the mechanism of cataract formation.…”

Section: Discussionsupporting

confidence: 88%

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Thiolation of the γB-Crystallins in Intact Bovine Lens Exposed to Hydrogen Peroxide

Hanson¹,

Chen

Smith³

et al. 1999

Journal of Biological Chemistry

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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 H 2 O 2 oxidation was determined by comparing bovine lenses incubated with and without 30 mM H 2 O 2 . The H 2 O 2 treatment decreased the glutathione and increased the protein-glutathione and proteincysteine disulfides in the lens. The molecular mass of the ␥B-crystallin isolated from lenses, not treated with H 2 O 2 , agreed with the published sequence (M r 20,966). Some lenses also had a less abundant ␥B-crystallin component 305 Da higher (M r 21,270), suggesting the presence of a glutathione adduct. The ␥B-crystallins from H 2 O 2 treated lenses had three components, the major one with one GSH adduct, another one with the mass of unmodified ␥B-crystallin, and a third with a mass consistent with addition of two GSH adducts. Mass spectrometric analysis of tryptic peptides of ␥B-crystallins from different lenses indicated that the ؉305 Da modifications were not at a specific cysteine. For the lenses incubated without H 2 O 2 , 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, ␥B-crystallins from H 2 O 2 -treated lenses had an adduct at Cys-109, partial oxidation at all 7 Met residues, and evidence for two disulfide bonds.Age-related cataract is one of the major causes of blindness in humans. The pathology of such a condition involves opacification and decreased transparency of the lens, which can lead to loss of vision. Although the mechanisms for age-related cataractogenesis are not understood, oxidation of the lens proteins, known as crystallins, is associated with cataract formation in humans (1, 2). Protein thiolation, which involves the formation of disulfide bonds between the cysteinyl residues of lens proteins and other low molecular weight thiols in the lens, is one of the modifications caused by oxidative stress of the lens (3). Thiols in the lens that participate in this reaction are GSH and cysteine, which form protein-S-S-glutathione (PSSG) 1 and protein-S-S-cysteine (PSSC), respectively (4). These proteinthiol products are referred to as mixed disulfides. The conformational changes caused by protein thiolation (5, 6) may allow some of the buried functional groups to be exposed and modified. This may cause proteins to aggregate and disrupt the close packing of the crystallins, decreasing their solubility (7) and leading to cataract formation. In the H 2 O 2 -induced cataract model, the progression of cataract is associated with sequential events involving first the formation of PSSG, followed by protein-protein disulfide cross-links, decreased protein solubility, and finally an increase in the formation of high molecular weight aggregates (8). Protein-thiol mixed disulfides accumulate in older human lenses (9) and in all types of human cataractous lenses ...

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Section: Discussionsupporting

confidence: 88%

“…These proteinthiol products are referred to as mixed disulfides. The conformational changes caused by protein thiolation (5,6) may allow some of the buried functional groups to be exposed and modified. This may cause proteins to aggregate and disrupt the close packing of the crystallins, decreasing their solubility (7) and leading to cataract formation.…”

mentioning

confidence: 99%

Thiolation of the γB-Crystallins in Intact Bovine Lens Exposed to Hydrogen Peroxide

Hanson¹,

Chen

Smith³

et al. 1999

Journal of Biological Chemistry

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“…These values are well within the range of those observed for all native bovine ␥-crystallins ( max from 324-335 nm; ref. 28). The small red shift in the max for R14C could be caused by the loss of hydrogen bonds (typically formed by the guanidinium group of Arg) after the substitution of Arg-14 by Cys.…”

Section: Resultsmentioning

confidence: 99%

“…Data were taken at pH 2 for comparison with previously published work on bovine ␥B crystallin (28,29). The increased sensitivity of our instrument enabled us to use 10-fold lower protein concentrations (0.1-0.2 mg͞ml) than those used by Kono et al (28) and Rudolf et al (29).…”

Section: Resultsmentioning

confidence: 99%

Molecular basis of a progressive juvenile-onset hereditary cataract

Pande

Asherie

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

130

184

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We have expressed recombinant wild-type human ␥D 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 thiolmediated 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.I n the hereditary, juvenile-onset cataract described by Stefan et al. (1), the lens, which is clear at birth, develops punctate opacities progressively, such that by two years of age the cataract is readily detectable, and matures by early childhood or adolescence. The punctate opacities seen in this cataract are in the nucleus and inner cortex, regions of the lens that are enriched in the ␥-crystallins. In the human lens, only two members of the ␥-crystallin family, ␥C and ␥D, are expressed in appreciable amounts, and only ␥D crystallin continues to be expressed until late childhood (2, 3). In affected individuals, a single point mutation has been identified in the ␥D crystallin gene that corresponds to the substitution of Arg-14 by a Cys. The identification of this mutation and the parallel between the time course of the pathology and the physiological expression of human ␥D crystallin strongly implicate the Arg-14 3 Cys mutant of ␥D in the development of this cataract. However, the molecular mechanism invoked to explain the observed opacity has been speculative (1).In the past, it has not been possible to conduct detailed studies on human ␥D crystallin because of the difficulty of obtaining sufficient quantities of pure protein from young, normal human lenses (4). Therefore, to characterize the normal protein thoroughly and investigate the mechanism by which the Arg-14 3 Cys mutation in ␥D could lead to cataract, we cloned and expressed human ␥D crystallin and its Arg-14 3 Cys mutant in Escherichia coli. Both the wild-type recombinant ␥D crystallin (HGD) and its Arg-14 3 Cys mutant (R14C) folded efficiently in E. coli and accumulated as soluble proteins. We isolated and purified the HGD and R14C proteins and determined their solution properties. Our results suggest that the disulfidecrosslinked oligomerization of R14C is responsible for the observed cataract. Furthermore, such oligomerization occurs without significant change in protein structure, conformation, and stability. Materials and MethodsCloning, Expression, and Isolation of Proteins. The human ␥D crystallin coding sequence was amplif...

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“…The extra charge and mass of the -SG group that S-conjugates onto a protein may cause conformational change (55) and/or destabilization of lens crystallin proteins (56,57). These types of reactions may allow multiple modified lens proteins to aggregate and become water-insoluble.…”

Section: Discussionmentioning

confidence: 99%

Glutaredoxin 2 (Grx2) Gene Deletion Induces Early Onset of Age-dependent Cataracts in Mice

David

et al. 2014

Journal of Biological Chemistry

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Background:Mitochondrial glutaredoxin 2 is a member of the oxidoreductase family with disulfide reductase activity that repairs thiol oxidation-damaged proteins. Results: Glutaredoxin 2 null mice develop early cataracts during aging. Conclusion: Glutaredoxin 2 deletion causes mitochondrial malfunction and cataract formation and is a good model for studying the mechanism of human age-related cataracts. Significance: Thiol oxidation repair system is essential for lens transparency.

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Thermodynamics of thermal and athermal denaturation of .gamma.-crystallins: changes in conformational stability upon glutathione reaction

Cited by 56 publications

References 39 publications

Thiolation of the γB-Crystallins in Intact Bovine Lens Exposed to Hydrogen Peroxide

Thiolation of the γB-Crystallins in Intact Bovine Lens Exposed to Hydrogen Peroxide

Molecular basis of a progressive juvenile-onset hereditary cataract

Glutaredoxin 2 (Grx2) Gene Deletion Induces Early Onset of Age-dependent Cataracts in Mice

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