Altered heat-lability of a fraction of glutathione reductase in aging human lens.
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ABSTRACT:
SUBMITTER: Harding JJ
PROVIDER: S-EPMC1177907 | biostudies-other | 1973 Aug
REPOSITORIES: biostudies-other
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Project description:Glutathione (GSH) is the archetypal antioxidant, and plays a central role in the protection of the ocular lens from cataract formation. High levels of GSH are maintained in the transparent lens, but with advancing age, GSH levels fall in the lens nucleus relative to outer cortical cells, thereby exposing the nucleus of the lens to the damaging effects of oxygen radicals, which ultimately leads to age-related nuclear (ARN) cataract. Under normal conditions, GSH also forms endogenous conjugates to detoxify the lens of reactive cellular metabolites and to maintain cell homeostasis. Due to the intrinsic gradient of lens fibre cell age, the lens contains distinct regions with different metabolic requirements for GSH. To investigate the impact of fibre cell and lens aging on the varied roles that GSH plays in the lens, we have utilised high mass resolution MALDI mass spectrometry profiling and imaging analysis of lens tissue sections. High Dynamic Range (HDR)-MALDI FTICR mass spectrometry was used as an initial screening method to detect regional differences in lens metabolites from normal bovine lenses and in those subjected to hyperbaric oxygen as a model of lens aging. Subsequent MALDI imaging analysis was used to spatially map GSH and its endogenous conjugates throughout all lenses. Accurate mass measurement by MALDI FTICR analysis and LC-MS/MS mass spectrometry of lens region homogenates were subsequently used to identify endogenous GSH conjugates. While the distribution and relative abundance of GSH-related metabolic intermediates involved in detoxification pathways remained relatively unchanged upon HBO treatment, those involved in its antioxidant function were altered under conditions of oxidative stress. For example, reduced glutathione levels were decreased in the lens cortex while oxidised glutathione levels were elevated in the lens outer cortex upon HBO treatment. Interestingly, cysteineglutathione disulfide, was detected in the inner cortex of the normal lens, but was greatly decreased in the HBO-treated lenses. These results contribute to our understanding of the multiple roles that GSH plays in maintenance of lens transparency and in the age-related metabolic changes that lead to lens cataract formation.
| S-EPMC5359059 | biostudies-literature
Project description:Dehydroalanine (DHA) and dehydrobutyrine (DHB) intermediates, formed through ?-elimination, induce protein irreversible glutathionylation and protein-protein crosslinking in human lens fiber cells. In total, irreversible glutathionylation was detected on 52 sites including cysteine, serine and threonine residues in 18 proteins in human lenses. In this study, the levels of GSH modification on three serine residues and four cysteine residues located in seven different lens proteins isolated from different regions and different aged lenses were quantified. The relative levels of modification (modified/nonmodified) were site-specific and age-related, ranging from less than 0.05% to about 500%. The levels of modification on all of the sites quantified in the lens cortex increased with age and GSH modification also increased from cortex to outer nucleus region suggesting an age-related increase of modification. The levels of modification on sites located in stable regions of the proteins such as Cys117 of ?A3, Cys80 of ?B1 and Cys27 of ?S, continued increasing in inner nucleus, but modification on sites located in regions undergoing degradation with age decreased in the inner nucleus suggesting GSH modified proteins were more susceptible to further modification. Irreversible GSH modification in cataract lenses was typically higher than in age-matched normal lenses, but the difference did not reach statistical significance for a majority of sites, with the exception Cys117 of ?A3 crystallin in WSF. Except for S59 of ?A and ?B crystallins, GSH modification did not induce protein insolubility suggesting a possible role for this modification in protection from protein-protein crosslinking.
| S-EPMC6167147 | biostudies-literature
Project description:Efficient enzyme catalysis depends on exquisite details of structure beyond those resolvable in typical medium- and high-resolution crystallographic analyses. Here we report synchrotron-based cryocrystallographic studies of natural substrate complexes of the flavoenzyme human glutathione reductase (GR) at nominal resolutions between 1.1 and 0.95 A that reveal new aspects of its mechanism. Compression in the active site causes overlapping van der Waals radii and distortion in the nicotinamide ring of the NADPH substrate, which enhances catalysis via stereoelectronic effects. The bound NADPH and redox-active disulfide are positioned optimally on opposite sides of the flavin for a 1,2-addition across a flavin double bond. The new structures extend earlier observations to reveal that the redox-active disulfide loop in GR is an extreme case of sequential peptide bonds systematically deviating from planarity--a net deviation of 53 degrees across five residues. But this apparent strain is not a factor in catalysis, as it is present in both oxidized and reduced structures. Intriguingly, the flavin bond lengths in oxidized GR are intermediate between those expected for oxidized and reduced flavin, but we present evidence that this may not be due to the protein environment but instead due to partial synchrotron reduction of the flavin by the synchrotron beam. Finally, of more general relevance, we present evidence that the structures of synchrotron-reduced disulfide bonds cannot generally be used as reliable models for naturally reduced disulfide bonds.
| S-EPMC2593804 | biostudies-literature
Project description:Purpose: To better understand the effects of glutathione (GSH)-deficiency on lens homeostasis and cataractogenesis. Methods: The transcriptome of lens epithelia and fiber cells was obtained from C57BL/6 LEGSKO (lens GSH synthesis knockout) and buthionine sulfoximine (BSO)-treated LEGSKO mice and compared to C57BL/6 wild-type mice using RNA-Seq. Results: RNA-Seq results were in excellent agreement with qPCR (correlation coefficients between 0.87-0.94 and p<5E-6 for a subset of 36 mRNAs). Of the 24,415 transcripts mapped to the mouse genome, 441 genes showed significantly modulated expression. Pathway analysis indicated major changes in EMT signaling, visual cycle, small molecule biochemistry, and lipid metabolism. GSH-deficient lenses showed upregulation of genes relating to detoxification, including Aldh1a1, Aldh3a1 (aldehyde dehydrogenases), Mt1, Mt2 (metallothioneins), Ces1g (carboxylesterase), and Slc14a1 (urea transporter UT-B). These proteins share substrate specificity with GSH or glutathione-S-transferase and may protect GSH-deficient lenses. Genes associated with canonical EMT pathways, including Wnt10a, Egf, and Syk, showed upregulation in lens epithelia samples. Severely GSH-deficient lens epithelia showed a broad downregulation of vision-related genes (including Cryge, Crygf, and Rho). The BSO-treated LEGSKO lens epithelia transcriptome has significant correlation (r=0.63,P<0.005) to that of lens epithelia undergoing EMT. Conclusions: These results show that GSH depletion of the lens leads to expression of detoxifying genes and activation of EMT signaling, in addition to changes in transport systems and lipid homeostasis. These data give new insight into the adaptation and consequences of GSH-deficiency in the lens and suggest that supplementation of GSH or a precursor after cataract surgery could potentially reduce the incidence of EMT-mediated posterior subcapsular opacification.
2017-05-18 | GSE95688 | GEO
Project description:Two new desoxo molybdenum(V) complexes have been synthesized and characterized as models for the paramagnetic high-g split intermediate observed in the catalytic cycle of dimethyl sulfoxide reductase (DMSOR). Extended X-ray absorption fine structure (EXAFS) and electron paramagnetic resonance (EPR) data are used to provide new insight into the geometric and electronic structures of high-g split and other EPR-active type II/III DMSOR family enzyme forms. The results support a 6-coordinate [(PDT)2Mo(OH)(OSer)]- structure (PDT = pyranopterin dithiolene) for a high-g split with four S donors from two PDT ligands, a coordinated hydroxyl ligand, and a serinate O donor. This geometry orients the redox orbital toward the substrate access channel for the two-electron reduction of substrates.
| S-EPMC9070986 | biostudies-literature
Project description:This study aims to investigate the effect of mild heating on lens epithelial cells and to explore its possibility as an in vitro model for lens aging. Human lens epithelial cells (LECs) were heated at 50?°C for a cellular lens aging study. Analysis of the head group order of lens membranes was performed using Laurdan labeling. Immunofluorescence was performed to detect changes in ?-crystallin expression and its cellular distribution. The chaperone-like activity of ?-crystallin was also assessed. After mild heating, ?-crystallin in LECs showed a tendency towards accumulation around the nucleus. The membrane head group environment of lens epithelial cells became more fluid with increasing time of exposure to mild heating, as indicated by increased water penetration. Furthermore, the chaperone activity of ?-crystallin decreased, and suggests a relatively lower protective effect on other functional proteins in LECs. Thus, compared to the mild heating model based on lens tissue, this cellular model could provide a more convenient and accurate method for studying lens aging in vitro, including changes in membrane head group order in each cell, the real-time observation of crystallin distribution, and the monitoring of functional changes in the chaperone activity of crystallins as a result of aging.
| S-EPMC5057073 | biostudies-literature
Project description:ObjectiveKlotho is an aging-suppressor gene which leads to accelerated aging when disrupted. This study was designed to investigate whether glutathione reductase (GR), a critical intracellular antioxidant enzyme, is involved in the pathogenesis of kidney damages associated with accelerated aging in Klotho-haplodeficient (KL+/-) mice.Methods and resultsKlotho-haplodeficient (KL+/-) mice and WT mice were used. We found that Klotho haplodeficiency impaired kidney function as evidenced by significant increases in plasma urea and creatinine and a decrease in urinary creatinine in KL+/- mice. The expression and activity of GR was decreased significantly in renal tubular epithelial cells of KL+/- mice, suggesting that Klotho deficiency downregulated GR. We constructed adeno-associated virus 2 (AAV2) carrying GR full-length cDNA (AAV-GR). Interestingly, in vivo AAV-GR delivery significantly improved Klotho deficiency-induced renal functional impairment and structural remodeling. Furthermore, in vivo expression of GR rescued the downregulation of the reduced glutathione/oxidized glutathione (GSH/GSSG) ratio, which subsequently diminished oxidative damages in kidneys, as evidenced by significant decreases in renal 4-HNE expression and urinary 8-isoprostane levels in KL mice.ConclusionThis study provides the first evidence that Klotho deficiency-induced kidney damage may be partly attributed to downregulation of GR expression. In vivo delivery of AAV-GR may be a promising therapeutic approach for aging-related kidney damage.
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Project description:The lens capsule of the eye is important in focusing light onto the retina during the process of accommodation and, in later life, housing a prosthetic lens implanted during cataract surgery. Though considerable modeling work has characterized the mechanics of accommodation, little has been done to understand the mechanics of the lens capsule after cataract surgery. As such, we present the first 3-D finite element model of the post-surgical human lens capsule with an implanted tension ring and, separately, an intraocular lens to characterize the altered stress field compared to that in a model of the native lens capsule. All finite element models employed a Holzapfel hyperelastic constitutive model with regional variations in anisotropy. The post-surgical lens capsule demonstrated a dramatic perturbation to the stress field with mostly large reductions in stresses (except at the equator where the implant contacts the capsule) compared to native, wherein maximal changes in Cauchy stress were -100% and -145% for the tension ring and intraocular lens, respectively. However, implantation of the tension ring produced a more uniform stress field compared to the IOL. The magnitudes and distribution of the perturbed stress field may be an important driver of the fibrotic response of inhabiting lens epithelial cells and associated lens capsule remodeling after cataract surgery. Thus, the mechanical effects of an implant on the lens capsule could be an essential consideration in the design of intraocular lenses, particularly those with an accommodative feature.
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Project description:Cells evolved robust homeostatic mechanisms to protect against oxidation or alkylation by electrophilic species. Glutathione (GSH) is the most abundant intracellular thiol, protects cellular components from oxidation and is maintained in a reduced state by glutathione reductase (GR). Nitro oleic acid (NO2-OA) is an electrophilic fatty acid formed under digestive and inflammatory conditions that both reacts with GSH and induces its synthesis upon activation of Nrf2 signaling. The effects of NO2-OA on intracellular GSH homeostasis were evaluated. In addition to upregulation of GSH biosynthesis, we observed that NO2-OA increased intracellular GSSG in an oxidative stress-independent manner. NO2-OA directly inhibited GR in vitro by covalent modification of the catalytic Cys61, with kon of (3.45 ± 0.04) × 103 M-1 s-1, koff of (4.4 ± 0.4) × 10-4 s-1, and Keq of (1.3 ± 0.1) × 10-7 M. Akin to NO2-OA, the electrophilic Nrf2 activators bardoxolone-imidazole (CDDO-Im), bardoxolone-methyl (CDDO-Me) and dimethyl fumarate (DMF) also upregulated GSH biosynthesis while promoting GSSG accumulation, but without directly inhibiting GR activity. In vitro assays in which GR was treated with increasing GSH concentrations and GSH depletion experiments in cells revealed that GR activity is finely regulated via product inhibition, an observation further supported by theoretical (kinetic modeling of cellular GSSG:GSH levels) approaches. Together, these results describe two independent mechanisms by which electrophiles modulate the GSH/GSSG couple, and provide a novel conceptual framework to interpret experimentally determined values of GSH and GSSG.
| S-EPMC6348771 | biostudies-literature
Project description:Selective suicide inhibitors represent a seductively attractive approach for inactivation of therapeutically relevant enzymes since they are generally devoid of off-target toxicity in vivo. While most suicide inhibitors are converted to reactive species at enzyme active sites, theoretically bioactivation can also occur in ectopic (secondary) sites that have no known function. Here, we report an example of such an "ectopic suicide inhibition", an unprecedented bioactivation mechanism of a suicide inhibitor carried out by a non-catalytic site of thioredoxin glutathione reductase (TGR). TGR is a promising drug target to treat schistosomiasis, a devastating human parasitic disease. Utilizing hits selected from a high throughput screening campaign, time-resolved X-ray crystallography, molecular dynamics, mass spectrometry, molecular modeling, protein mutagenesis and functional studies, we find that 2-naphtholmethylamino derivatives bound to this novel ectopic site of Schistosoma mansoni (Sm)TGR are transformed to covalent modifiers and react with its mobile selenocysteine-containing C-terminal arm. In particular, one 2-naphtholmethylamino compound is able to specifically induce the pro-oxidant activity in the inhibited enzyme. Since some 2-naphtholmethylamino analogues show worm killing activity and the ectopic site is not conserved in human orthologues, a general approach to development of novel and selective anti-parasitic therapeutics against schistosoma is proposed.
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