scholarly journals Cataract-associated deamidations on the surface of γS-crystallin increase protein unfolding and flexibility at distant regions

2019 ◽  
Author(s):  
Heather M. Forsythe ◽  
Calvin Vetter ◽  
Kayla Ann Jara ◽  
Patrick N. Reardon ◽  
Larry L. David ◽  
...  
Keyword(s):  
Hydrogen Exchange ◽  
Protein Unfolding ◽  
Structural Changes ◽  
Human Lens ◽  
Relaxation Dynamics ◽  
Amide Hydrogen Exchange ◽  
Terminal Domain ◽  
Age Related ◽  
Surface Loops

AbstractDeamidation is a major age-related modification in the human lens that is highly prevalent in crystallins isolated from cataractous lenses. However, the mechanism by which deamidation causes proteins to become insoluble is not known, because of only subtle structural changes observed in vitro. We have identified Asn14 and Asn76 of γS-crystallin as highly deamidated in insoluble proteins. These sites are on the surface of the N-terminal domain and were mimicked by replacing the Asn with Asp residues. We used heteronuclear NMR spectroscopy to measure their amide hydrogen exchange and 15N relaxation dynamics to identify regions with significantly increased dynamics compared to wildtype-γS. Changes in dynamics were localized to the C-terminal domain, particularly to helix and surface loops distant from the mutation sites. Thus, a potential mechanism for γS deamidation-induced insolubilization in cataractous lenses is altered dynamics due to local regions of unfolding and increased flexibility.

scholarly journals Nanoceria Prevents Glucose-Induced Protein Glycation in Eye Lens Cells

Nanomaterials ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1473
Author(s):  
Belal I. Hanafy ◽  
Gareth W. V. Cave ◽  
Yvonne Barnett ◽  
Barbara K. Pierscionek
Keyword(s):  
Protein Glycation ◽  
Human Lens ◽  
Lens Epithelial Cells ◽  
Oxide Nanoparticles ◽  
Lens Protein ◽  
Oxygen Species ◽  
Human Lens Epithelial Cells ◽  
Age Related ◽  
Age Related Changes

Cerium oxide nanoparticles (nanoceria) are generally known for their recyclable antioxidative properties making them an appealing biomaterial for protecting against physiological and pathological age-related changes that are caused by reactive oxygen species (ROS). Cataract is one such pathology that has been associated with oxidation and glycation of the lens proteins (crystallins) leading to aggregation and opacification. A novel coated nanoceria formulation has been previously shown to enter the human lens epithelial cells (HLECs) and protect them from oxidative stress induced by hydrogen peroxide (H2O2). In this work, the mechanism of nanoceria uptake in HLECs is studied and multiple anti-cataractogenic properties are assessed in vitro. Our results show that the nanoceria provide multiple beneficial actions to delay cataract progression by (1) acting as a catalase mimetic in cells with inhibited catalase, (2) improving reduced to oxidised glutathione ratio (GSH/GSSG) in HLECs, and (3) inhibiting the non-enzymatic glucose-induced glycation of the chaperone lens protein α-crystallin. Given the multifactorial nature of cataract progression, the varied actions of nanoceria render them promising candidates for potential non-surgical therapeutic treatment.

scholarly journals Garlic Extract: Inhibition of Biochemical and Biophysical Changes in Glycated HSA

2021 ◽  
Vol 11 (22) ◽  
pp. 11028
Author(s):  
Mohd W. A. Khan ◽  
Ahmed A. Otaibi ◽  
Arwa F. M. Alhumaid ◽  
Abdulmohsen K. D. Alsukaibi ◽  
Asma K. Alshamari ◽  
...  
Keyword(s):  
Structural Changes ◽  
Therapeutic Potential ◽  
Inhibitory Effect ◽  
Significant Inhibition ◽  
Garlic Extract ◽  
Diabetic Patients ◽  
Age Related ◽  
Health Complications ◽  
Biophysical Changes

Glycation of various biomolecules contributes to structural changes and formation of several high molecular weight fluorescent and non-fluorescent, advanced glycation end products (AGEs). AGEs and glycation are involved in various health complications. Synthetic medicines, including metformin, have several adverse effects. Natural products and their derivatives are used in the treatment of various diseases due to their significant therapeutic qualities. Allium sativum (garlic) is used in traditional medicines because of its antioxidant, anti-inflammatory, and anti-diabetic properties. This study aimed to determine the anti-glycating and AGEs inhibitory activities of garlic. Biochemical and biophysical analyses were performed for in vitro incubated human serum albumin (HSA) with 0.05 M of glucose for 1, 5, and 10 weeks. Anti-glycating and AGEs inhibitory effect of garlic was investigated in glycated samples. Increased biochemical and biophysical changes were observed in glycated HSA incubated for 10 weeks (G-HSA-10W) as compared to native HSA (N-HSA) as well as glycated HSA incubated for 1 (G-HSA-1W) and 5 weeks (G-HSA-5W). Garlic extract with a concentration of ≥6.25 µg/mL exhibited significant inhibition in biophysical and biochemical changes of G-HSA-10W. Our findings demonstrated that garlic extract has the ability to inhibit biochemical and biophysical changes in HSA that occurred due to glycation. Thus, garlic extract can be used against glycation and AGE-related health complications linked with chronic diseases in diabetic patients due to its broad therapeutic potential.

scholarly journals Increased Association of Deamidated αA-N101D with Lens Membrane of Transgenic αAN101D vs. Wild Type αA Mice: Potential Effects on Intracellular Ionic Imbalance and Membrane Disorganization

2020 ◽  
Author(s):  
Om Srivast ◽  
Kiran Srivast ◽  
Roy Joseph ◽  
Landon Wilson
Keyword(s):  
Epithelial Cells ◽  
Western Blot ◽  
Immunogold Labeling ◽  
Human Lens ◽  
Membrane Association ◽  
Related Protein ◽  
Wild Type ◽  
Age Related ◽  
Ionic Imbalance

Abstract We have generated two mouse models, in one by inserting the human lens αAN101D transgene in CRYαAN101D mice, and in the other by inserting human wild-type αA-transgene in CRYαAWT mice. The CRYαAN101D mice developed cortical cataract at about 7-months of age relative to CRYαAWT mice. The objective of the study was to determine the following relative changes in the lenses of CRYαAN101D- vs. CRYαAWT mice: age-related changes with specific emphasis on protein insolubilization, relative membrane-association of αAN101D vs. WTαA proteins, and changes in intracellular ionic imbalance and membrane organization. Methods: Lenses of varying ages from CRYαAWT and CRYαAN101D mice were compared for an age-related protein insolubilization. The relative lens membrane-association of the αAN101D- and WTαA proteins in the two types of mice was determined by immunohistochemical-, immunogold-labeling-, and western blot analyses. The relative levels of membrane-binding of recombinant αAN101D- and WTαA proteins was determined by an in vitro assay, and the levels of intracellular Ca2+ uptake and Na, K-ATPase mRNA were determined in the cultured epithelial cells from lenses of the two types of mice.Results: Compared to the lenses of CRYαAWT, the lenses of CRYαAN101D mice exhibited: (A) An increase in age-related protein insolubilization beginning at about 4-months of age. (B) A greater lens membrane-association of αAN101D- relative to WTαA protein during immunogold-labeling- and western blot analyses, including relatively a greater membrane swelling in the CRYαAN101D lenses. (C) During in vitro assay, the greater levels of binding αAN101D- relative to WTαA protein to membranes was observed. (D) The 75% lower level of Na, K-ATPase mRNA but 1.5X greater Ca2+ uptake were observed in cultured lens epithelial cells of CRYαAN101D- than those of CRYαAWT mice. Conclusions: The results show that an increased lens membrane association of αAN101D--relative WTαA protein in CRYαAN101D mice than CRYαAWT mice occurs, which causes intracellular ionic imbalance, and in turn, membrane swelling that potentially leads to cortical opacity.

scholarly journals Loss of D2 Receptor Binding with Age in Rhesus Monkeys: Importance of Correction for Differences in Striatal Size

1999 ◽  
Vol 19 (2) ◽  
pp. 218-229 ◽  
Author(s):  
Evan D. Morris ◽  
Svetlana I. Chefer ◽  
Mark A. Lane ◽  
Raymond F. Muzic ◽  
Dean F. Wong ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Rhesus Monkeys ◽  
Structural Changes ◽  
D2 Receptor ◽  
Binding Potential ◽  
Age Related ◽  
Positron Emission ◽  
Dopamine Receptor Binding

The relation between striatal dopamine D2 receptor binding and aging was investigated in rhesus monkeys with PET. Monkeys (n = 18, 39 to 360 months of age) were scanned with 11C-raclopride; binding potential in the striatum was estimated graphically. Because our magnetic resonance imaging analysis revealed a concomitant relation between size of striatum and age, the dynamic positron emission tomography (PET) data were corrected for possible partial volume (PV) artifacts before parameter estimation. The age-related decline in binding potential was 1% per year and was smaller than the apparent effect if the age-related change in size was ignored. This is the first in vivo demonstration of a decline in dopamine receptor binding in nonhuman primates. The rate of decline in binding potential is consistent with in vitro findings in monkeys but smaller than what has been measured previously in humans using PET. Previous PET studies in humans, however, have not corrected for PV error, although a decline in striatal size with age has been demonstrated. The results of this study suggest that PV correction must be applied to PET data to accurately detect small changes in receptor binding that may occur in parallel with structural changes in the brain.

scholarly journals Age-dependent effect of oxidative stress on cardiac sarcoplasmic reticulum vesicles

2008 ◽  
pp. S49-S54
Author(s):  
E Babušíková ◽  
M Jeseňák ◽  
D Dobrota ◽  
N Tribulová ◽  
P Kaplán
Keyword(s):  
Oxidative Stress ◽  
Sarcoplasmic Reticulum ◽  
Oxidative Damage ◽  
Structural Changes ◽  
Membrane Surface ◽  
Thiobarbituric Acid ◽  
Surface Hydrophobicity ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Age Related

The oxidative stress hypothesis of aging suggests that accumulation of oxidative damage is a key factor of the alterations in physiological function during aging. We studied age-related sensitivity to oxidative modifications of proteins and lipids of cardiac sarcoplasmic reticulum (SR) isolated from 6-, 15- and 26-month-old rats. Oxidative stress was generated in vitro by exposing SR vesicles to 0.1 mmol/l FeSO4/EDTA + 1 mmol/l H2O2 at 37 degrees C for 60 min. In all groups, oxidative stress was associated with decreased membrane surface hydrophobicity, as detected by 1-anilino-8-naphthalenesulfonate as a probe. Structural changes in SR membranes were accompanied by degradation of tryptophan and significant accumulation of protein dityrosines, protein conjugates with lipid peroxidation products, conjugated dienes and thiobarbituric acid reactive substances. The sensitivity to oxidative damage was most pronounced in SR of 26-month-old rat. Our results indicate that aging and oxidative stress are associated with accumulation of oxidatively damaged proteins and lipids and these changes could contribute to cardiovascular injury.

scholarly journals MicroRNA-182-5p protects human lens epithelial cells against oxidative stress-induced apoptosis by inhibiting NOX4 and p38 MAPK signalling

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Zhao-Na Li ◽  
Ming-Xu Ge ◽  
Zhong-Fang Yuan
Keyword(s):  
Epithelial Cells ◽  
P38 Mapk ◽  
Mapk Signaling ◽  
Luciferase Reporter ◽  
Human Lens ◽  
Lens Epithelial Cells ◽  
Luciferase Reporter Gene ◽  
Age Related ◽  
Induced Apoptosis

Abstract Background MicroRNAs (miRNAs) are abnormally expressed in various ocular diseases, including age-related cataract. However, the role of miR-182-5p in the progression of age-related cataract remains unclear. Methods The expression of miR-182-5p in HLE-B3 cells was detected by qRT-PCR. HLE-B3 cells were transfected with miR-182-5p mimics. CCK-8, EdU, flow cytometry, 2′,7′-dichlorodihydrofluorescein diacetate, JC-1 kit, and western blot were used to assess the cell viability, proliferation, apoptosis, reactive oxygen species (ROS) level, mitochondrial membrane potential (MMP), and protein expression, respectively, in vitro. The relationship between miR-182-5p and NOX4 was confirmed using the dual-luciferase reporter gene analysis. Results We found that miR-182-5p expression was significantly decreased by the H2O2 exposure. Overexpression of miR-182-5p promoted cell proliferation and inhibited ROS production and apoptosis in H2O2-induced HLE-B3 cells. Moreover, p-p-38, p-ERK, and p-JNK were up-regulated in H2O2-treated HLE-B3 cells, and overexpression of miR-182-5p reversed the effects of H2O2 on HLE-B3 cells. In addition, dual-luciferase reporter assay substantiated that NOX4 was a direct target and downregulated by miR-182-5p. Conclusions We concluded that miR-182-5p inhibited lens epithelial cells apoptosis through regulating NOX4 and p38 MAPK signaling, providing a novel biomarker for treatment of age-related cataract.

scholarly journals MicroRNA-182-5p protects human lens epithelial cells against oxidative stress-induced apoptosis by inhibiting NOX4 and p38 MAPK signalling

2020 ◽  
Author(s):  
Zhao-Na Li ◽  
Ming-Xu Ge ◽  
Zhong-Fang Yuan
Keyword(s):  
Epithelial Cells ◽  
P38 Mapk ◽  
Mapk Signaling ◽  
Luciferase Reporter ◽  
Human Lens ◽  
Lens Epithelial Cells ◽  
Luciferase Reporter Gene ◽  
Age Related ◽  
Induced Apoptosis

Abstract Background: MicroRNAs (miRNAs) are abnormally expressed in various ocular diseases, including age-related cataract. However, the role of miR-182-5p in the progression of age-related cataract remains unclear.Methods: The expression of miR-182-5p in HLE-B3 cells was detected by qRT-PCR. HLE-B3 cells were transfected with miR-182-5p mimics. CCK-8, EdU, flow cytometry, 2',7'-dichlorodihydrofluorescein diacetate, JC-1 kit, and western blot were used to assess the cell viability, proliferation, apoptosis, reactive oxygen species (ROS) level, mitochondrial membrane potential (MMP), and protein expression, respectively, in vitro. The relationship between miR-182-5p and NOX4 was confirmed using the dual-luciferase reporter gene analysis.Results: We found that miR-182-5p expression was significantly decreased by the H2O2 exposure. Overexpression of miR-182-5p promoted cell proliferation and inhibited ROS production and apoptosis in H2O2-induced HLE-B3 cells. Moreover, p-p-38, p-ERK, and p-JNK were up-regulated in H2O2-treated HLE-B3 cells, and overexpression of miR-182-5p reversed the effects of H2O2 on HLE-B3 cells. In addition, dual-luciferase reporter assay substantiated that NOX4 was a direct target and downregulated by miR-182-5p.Conclusions: We concluded that miR-182-5p inhibited lens epithelial cells apoptosis through regulating NOX4 and p38 MAPK signaling, providing a novel biomarker for treatment of age-related cataract.

Elastin modulation and modification by homocysteine: a key factor in the pathogenesis of Pseudoexfoliation syndrome?

2018 ◽  
Vol 103 (7) ◽  
pp. 985-992 ◽  
Author(s):  
Manohar Rebecca ◽  
Ramakrishnan Gayathri ◽  
Renganathan Bhuvanasundar ◽  
Krishnamoorthy Sripriya ◽  
Balekudaru Shantha ◽  
...  
Keyword(s):  
Structural Changes ◽  
Systemic Disease ◽  
Mrna Level ◽  
Pseudoexfoliation Syndrome ◽  
Human Lens ◽  
Amyloid Formation ◽  
Homocysteine Thiolactone ◽  
Key Factor ◽  
Α Helix

BackgroundPseudoexfoliation syndrome (PXF) is an idiopathic, elastogenesis-associated systemic disease characterised by amyloid-like material aggregates in the eye. Elevated plasma and aqueous humour (aqH) homocysteine (Hcy) is reportedly associated with PXF. This study is aimed to probe Hcy-mediated alterations in elastin expression.MethodologyLens level of Hcy (total Hcy (tHcy)), mRNA expression of Eln, CBS and MTR in lens capsule, protein expression of elastin in aqH were estimated by enzyme immunoassay, quantitative PCR and western blot, respectively in PXF, PXF with glaucoma (PXF-G) cases, in comparison with cataract-alone disease controls. Human lens epithelial cells (hLECs) were exposed to Hcy and homocysteine thiolactone (HCTL) to evaluate elastin expression in vitro. Furthermore, elastin recombinant protein was incubated with Hcy and HCTL to assess secondary and tertiary structural modifications based on circular dichroism spectroscopy, spectrophotometric and SEM studies.ResultsThe lens tHcy was significantly high in PXF (p=0.02) and PXF-G (p=0.009). Eln expression was elevated in PXF and PXF-G (p=0.0007). Elastin level in aqH was elevated in PXF (p=0.01) and PXF-G (p=0.002). Hcy (200 µM) and HCTL (1 µM) promoted elastin expression at mRNA level by 36-fold (p=0.02) and 10-fold (p=0.05), respectively, and at protein level by nearly two-fold in cultured hLECs. Secondary structure changes in elastin protein caused by Hcy were evident from 34.11% drop in α-helix and 6.17% gain in β-sheet. Fluorescence, spectral assays and SEM analyses showed aggregation and amyloid formation of elastin with homocysteinylation.ConclusionThe study reveals that lens accumulation of Hcy associated with hyperhomocysteinaemia is characteristic of PXF that augments elastin expression. Hcy causes structural changes promoting elastin aggregation, thereby contributing to defective elastin in PXF and PXF-G.

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