ABSTRACT
Proteins in the eye lens have negligible turnover and therefore progressively accumulate chemical modifications during aging. Carbonyls and oxidative stresses, which are intricately linked to one another, predominantly drive such modifications. Oxidative stress leads to the loss of glutathione (GSH) and ascorbate degradation; this in turn leads to the formation of highly reactive dicarbonyl compounds that react with proteins to form advanced glycation end products (AGEs). The formation of AGEs leads to the crosslinking and aggregation of proteins contributing to lens aging and cataract formation. To inhibit AGE formation, we developed a disulfide compound linking GSH diester and mercaptoethylguanidine, and we named it carboxitin. Bovine lens organ cultured with carboxitin showed higher levels of GSH and mercaptoethylguanidine in the lens nucleus. Carboxitin inhibited erythrulose-mediated mouse lens protein crosslinking, AGE formation and the formation of 3-deoxythreosone, a major ascorbate-derived AGE precursor in the human lens. Carboxitin inhibited the glycation-mediated increase in stiffness in organ-cultured mouse lenses measured using compressive mechanical strain. Delivery of carboxitin into the lens increases GSH levels, traps dicarbonyl compounds and inhibits AGE formation. These properties of carboxitin could be exploited to develop a therapy against the formation of AGEs and the increase in stiffness that causes presbyopia in aging lenses.
Subject(s)
Glutathione/analogs & derivatives , Glutathione/chemical synthesis , Lens, Crystalline/drug effects , Animals , Cattle , Glycation End Products, Advanced , Glycosylation , Lens, Crystalline/physiology , Mice , Mice, Inbred C57BL , Protein Binding , Tetroses/metabolism , Tumor Cells, CulturedABSTRACT
Oxidative cleavage of docosahexaenoate (DHA) in retinal pigmented epithelial (RPE) cells produces 4-hydroxy-7-oxohept-5-enoic acid (HOHA) esters of 2-lysophosphatidylcholine (PC). HOHA-PC spontaneously releases a membrane-permeant HOHA lactone that modifies primary amino groups of proteins and ethanolamine phospholipids to produce 2-(ω-carboxyethyl)pyrrole (CEP) derivatives. CEPs have significant pathological relevance to age-related macular degeneration (AMD) including activation of CEP-specific T-cells leading to inflammatory M1 polarization of macrophages in the retina involved in "dry AMD" and TLR2-dependent induction of angiogenesis that characterizes "wet AMD". RPE cells accumulate DHA from shed rod photoreceptor outer segments through phagocytosis and from plasma lipoproteins secreted by the liver through active uptake from the choriocapillaris. As a cell model of light-induced oxidative damage of DHA phospholipids in RPE cells, ARPE-19â¯cells were supplemented with DHA, with or without the lipofuscin fluorophore A2E. In this model, light exposure, in the absence of A2E, promoted the generation HOHA lactone-glutathione (GSH) adducts, depletion of intracellular GSH and a competing generation of CEPs. While DHA-rich RPE cells exhibit an inherent proclivity toward light-induced oxidative damage, photosensitization by A2E nearly doubled the amount of lipid oxidation and expanded the spectral range of photosensitivity to longer wavelengths. Exposure of ARPE-19â¯cells to 1⯵M HOHA lactone for 24â¯h induced massive (50%) loss of lysosomal membrane integrity and caused loss of mitochondrial membrane potential. Using senescence-associated ß-galactosidase (SA ß-gal) staining that detects lysosomal ß-galactosidase, we determined that exposure to HOHA lactone induces senescence in ARPE-19â¯cells. The present study shows that products of light-induced oxidative damage of DHA phospholipids in the absence of A2E can lead to RPE cell dysfunction. Therefore, their toxicity may be especially important in the early stages of AMD before RPE cells accumulate lipofuscin fluorophores.
Subject(s)
Docosahexaenoic Acids/pharmacology , Light/adverse effects , Macular Degeneration/metabolism , Oxidative Stress/radiation effects , Retinal Pigment Epithelium/metabolism , Cells, Cultured , Humans , Lipid Peroxidation , Lysosomes/metabolism , Lysosomes/radiation effects , Macular Degeneration/pathology , Membrane Potential, Mitochondrial/drug effects , Membrane Potential, Mitochondrial/radiation effects , Oxidation-Reduction , Oxidative Stress/drug effects , Retinal Pigment Epithelium/drug effectsABSTRACT
PURPOSE: The current study aimed to develop a three-dimensional (3D) dynamic oxygen-17 (17 O) MR imaging method with high temporal and spatial resolution to delineate the kinetics of 17 O water uptake and washout in the brains of mice with glioblastoma (GBM). METHODS: A 3D imaging method with a stack-of-stars golden-ratio-based radial sampling scheme was employed to acquire 17 O signal in vivo. A k-space-weighted image reconstruction method was used to improve the temporal resolution while preserving spatial resolution. Simulation studies were performed to validate the method. Using this method, the kinetics of 17 O water uptake and washout in the brains of mice with GBM were delineated after an intravenous bolus injection of 17 O water. RESULTS: The proposed 17 O imaging method achieved an effective temporal resolution of 7.56 s with a nominal voxel size of 5.625 µL in the mouse brain at 9.4 T. Reduced uptake and prolonged washout of 17 O water were observed in tumor tissue, suggesting compromised cerebral perfusion. CONCLUSION: This study demonstrated a promising dynamic 17 O imaging approach that can delineate 17 O water kinetics in vivo with high temporal and spatial resolution. It can also be used to image cerebral oxygen consumption rate in oxygen-17 inhalation studies. Magn Reson Med 79:256-263, 2018. © 2017 International Society for Magnetic Resonance in Medicine.
Subject(s)
Brain Neoplasms/diagnostic imaging , Brain/diagnostic imaging , Glioblastoma/diagnostic imaging , Image Processing, Computer-Assisted , Oxygen Isotopes/chemistry , Water/chemistry , Algorithms , Animals , Computer Simulation , Contrast Media , Image Enhancement , Image Interpretation, Computer-Assisted , Imaging, Three-Dimensional , Kinetics , Magnetic Resonance Imaging , Male , Mice , Mice, Nude , Phantoms, ImagingABSTRACT
We previously discovered that oxidative cleavage of docosahexaenoate (DHA), which is especially abundant in the retinal photoreceptor rod outer segments and retinal pigmented endothelial (RPE) cells, generates 4-hydroxy-7-oxo-5-heptenoate (HOHA) lactone, and that HOHA lactone can enter RPE cells that metabolize it through conjugation with glutathione (GSH). The consequent depletion of GSH results in oxidative stress. We now find that HOHA lactone induces upregulation of the antioxidant transcription factor Nrf2 in ARPE-19 cells. This leads to expression of GCLM, HO1, and NQO1, three known Nrf2-responsive antioxidant genes. Besides this protective response, HOHA lactone also triggers a countervailing inflammatory activation of innate immunity. Evidence for a contribution of the complement pathway to age-related macular degeneration (AMD) pathology includes the presence of complement proteins in drusen and Bruch's membrane from AMD donor eyes, and the identification of genetic susceptibility loci for AMD in the complement pathway. In eye tissues from a mouse model of AMD, accumulation of complement protein in Bruch's membrane below the RPE suggested that the complement pathway targets this interface, where lesions occur in the RPE and photoreceptor rod outer segments. In animal models of AMD, intravenous injection of NaIO3 to induce oxidative injury selectively destroys the RPE and causes secretion of factor C3 from the RPE into areas directly adjacent to sites of RPE damage. However, a molecular-level link between oxidative injury and complement activation remained elusive. We now find that sub-micromolar concentrations of HOHA lactone foster expression of C3, CFB, and C5 in ARPE-19 cells and induce a countervailing upregulation of CD55, an inhibitor of C3 convertase production and complement cascade amplification. Ultimately, HOHA lactone causes membrane attack complex formation on the plasma membrane. Thus, HOHA lactone provides a molecular-level connection between free-radical-induced oxidative cleavage of DHA and activation of the complement pathway in AMD pathology.
Subject(s)
Complement System Proteins/drug effects , Lactones/toxicity , Retinal Pigment Epithelium/drug effects , Animals , Cell Line , Complement System Proteins/metabolism , Disease Models, Animal , Dose-Response Relationship, Drug , Glutathione/metabolism , Humans , Macular Degeneration/metabolism , Mice , NF-E2-Related Factor 2/metabolism , Oxidative Stress/drug effects , Retinal Pigment Epithelium/cytology , Retinal Pigment Epithelium/metabolismABSTRACT
4-Hydroxy-7-oxo-5-heptenic acid (HOHA)-lactone is a biologically active oxidative truncation product released (t1/2 = 30 min at 37 °C) by nonenzymatic transesterification/deacylation from docosahexaenoate lipids. We now report that HOHA-lactone readily diffuses into retinal pigmented epithelial (RPE) cells where it is metabolized. A reduced glutathione (GSH) Michael adduct of HOHA-lactone is the most prominent metabolite detected by LC-MS in both the extracellular medium and cell lysates. This molecule appeared inside of ARPE-19 cells within seconds after exposure to HOHA-lactone. The intracellular level reached a maximum concentration at 30 min and then decreased with concomitant increases in its level in the extracellular medium, thus revealing a unidirectional export of the reduced GSH-HOHA-lactone adduct from the cytosol to extracellular medium. This metabolism is likely to modulate the involvement of HOHA-lactone in the pathogenesis of human diseases. HOHA-lactone is biologically active, e.g., low concentrations (0.1-1 µM) induce secretion of vascular endothelial growth factor (VEGF) from ARPE-19 cells. HOHA-lactone is also a precursor of 2-(ω-carboxyethyl)pyrrole (CEP) derivatives of primary amino groups in proteins and ethanolamine phospholipids that have significant pathological and physiological relevance to age-related macular degeneration (AMD), cancer, and wound healing. Both HOHA-lactone and the derived CEP can contribute to the angiogenesis that defines the neovascular "wet" form of AMD and that promotes the growth of tumors. While GSH depletion can increase the lethality of radiotherapy, because it will impair the metabolism of HOHA-lactone, the present study suggests that GSH depletion will also increase levels of HOHA-lactone and CEP that may promote recurrence of tumor growth.
Subject(s)
Lactones/metabolism , Retinal Pigment Epithelium/metabolism , Cell Line , Chromatography, High Pressure Liquid , Gas Chromatography-Mass Spectrometry , Glutathione/metabolism , Humans , Mass Spectrometry , Retinal Pigment Epithelium/cytologyABSTRACT
Isolevuglandins (isoLGs) are stereo and structurally isomeric γ-ketoaldehydes produced through free radical-induced oxidation of arachidonates. Some isoLG isomers are also generated through enzymatic cyclooxygenation. Post-translational modification of proteins by isoLGs is associated with loss-of-function, cross-linking and aggregation. We now report that a low level of modification by one or two molecules of isoLG has a profound effect on the activity of a multi subunit protease, calpain-1. Modification of one or two key lysyl residues apparently suffices to abolish catalytic activity. Covalent modification of calpain-1 led to intersubunit cross-linking. Hetero- and homo-oligomers of the catalytic and regulatory subunits of calpain-1 were detected by SDS-PAGE with Western blotting. N-Acetyl-glycyl-lysine methyl ester and ß-amyloid(11-17) peptide EVHHQKL were used as models for characterizing the cross-linking of protein lysyl residues resulting from adduction of iso[4]LGE2. Aminal, bispyrrole, and trispyrrole cross-links of these two peptides were identified and fully characterized by mass spectrometry. Aminal and bispyrrole dimers were both detected. Furthermore, a complex mixture of derivatives of the bispyrrole cross-link containing one or more additional atoms of oxygen was found. Interesting differences are evident in the predominant cross-link type generated in the reaction of iso[4]LGE2 with these peptides. More aminal cross-links versus bispyrrole are formed during the reaction of the dipeptide with iso[4]LGE2. In contrast, more bispyrrole versus aminal cross-links are formed during the reaction of EVHHQKL with iso[4]LGE2. It is tempting to speculate that the EVHHQKL peptide-pyrrole modification forms noncovalent aggregates that favor the production of covalent bispyrrole cross-links because ß-amyloid(11-17) tends to spontaneously oligomerize.
Subject(s)
Calpain/chemistry , Cross-Linking Reagents/chemistry , Fatty Acids, Unsaturated/chemistry , Animals , Calpain/metabolism , Cross-Linking Reagents/metabolism , Fatty Acids, Unsaturated/metabolism , Humans , Molecular StructureABSTRACT
Oxidative stress and angiogenesis have been implicated not only in normal phenomena such as tissue healing and remodeling but also in many pathological processes. However, the relationships between oxidative stress and angiogenesis still remain unclear, although oxidative stress has been convincingly demonstrated to influence the progression of angiogenesis under physiological and pathological conditions. The retina is particularly susceptible to oxidative stress because of its intensive oxygenation and high abundance of polyunsaturated fatty acyls. In particular, it has high levels of docosahexanoates, whose oxidative fragmentation produces 4-hydroxy-7-oxo-5-heptenoic acid lactone (HOHA-lactone). Previously, we found that HOHA-lactone is a major precursor of 2-(ω-carboxyethyl)pyrrole (CEP) derivatives, which are tightly linked to age-related macular degeneration (AMD). CEPs promote the pathological angiogenesis of late-stage AMD. We now report additional mechanisms by which HOHA-lactone promotes angiogenesis. Using cultured ARPE-19 cells, we observed that HOHA-lactone induces secretion of vascular endothelial growth factor (VEGF), which is correlated to increases in reactive oxygen species and decreases in intracellular glutathione (GSH). Wound healing and tube formation assays provided, for the first time, in vitro evidence that HOHA-lactone induces the release of VEGF from ARPE-19 cells, which promotes angiogenesis by human umbilical vein endothelial cells (HUVEC) in culture. Thus, HOHA-lactone can stimulate vascular growth through a VEGF-dependent pathway. In addition, results from MTT and wound healing assays as well as tube formation experiments showed that GSH-conjugated metabolites of HOHA-lactone stimulate HUVEC proliferation and promote angiogenesis in vitro. Previous studies demonstrated that HOHA-lactone, through its CEP derivatives, promotes angiogenesis in a novel Toll-like receptor 2-dependent manner that is independent of the VEGF receptor or VEGF expression. The new studies show that HOHA-lactone also participates in other angiogenic signaling pathways that include promoting the secretion of VEGF from retinal pigmented epithelial cells.
Subject(s)
Lactones/pharmacology , Neovascularization, Pathologic/prevention & control , Neovascularization, Physiologic/drug effects , Cell Line , Glutathione/metabolism , Humans , Oxidative Stress , Retinal Pigment Epithelium/cytology , Retinal Pigment Epithelium/drug effects , Retinal Pigment Epithelium/metabolism , Toll-Like Receptor 2/metabolism , Vascular Endothelial Growth Factor A/metabolism , Wound HealingABSTRACT
Advanced glycation end products (AGEs) contribute to lens protein pigmentation and cross-linking during aging and cataract formation. In vitro experiments have shown that ascorbate (ASC) oxidation products can form AGEs in proteins. However, the mechanisms of ASC oxidation and AGE formation in the human lens are poorly understood. Kynurenines are tryptophan oxidation products produced from the indoleamine 2,3-dioxygenase (IDO)-mediated kynurenine pathway and are present in the human lens. This study investigated the ability of UVA light-excited kynurenines to photooxidize ASC and to form AGEs in lens proteins. UVA light-excited kynurenines in both free and protein-bound forms rapidly oxidized ASC, and such oxidation occurred even in the absence of oxygen. High levels of GSH inhibited but did not completely block ASC oxidation. Upon UVA irradiation, pigmented proteins from human cataractous lenses also oxidized ASC. When exposed to UVA light (320-400 nm, 100 milliwatts/cm(2), 45 min to 2 h), young human lenses (20-36 years), which contain high levels of free kynurenines, lost a significant portion of their ASC content and accumulated AGEs. A similar formation of AGEs was observed in UVA-irradiated lenses from human IDO/human sodium-dependent vitamin C transporter-2 mice, which contain high levels of kynurenines and ASC. Our data suggest that kynurenine-mediated ASC oxidation followed by AGE formation may be an important mechanism for lens aging and the development of senile cataracts in humans.
Subject(s)
Ascorbic Acid/metabolism , Cataract/metabolism , Crystallins/metabolism , Glycation End Products, Advanced/metabolism , Kynurenine/radiation effects , Lens, Crystalline/radiation effects , Ultraviolet Rays , Animals , Humans , Indoleamine-Pyrrole 2,3,-Dioxygenase/genetics , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Kynurenine/metabolism , Lens, Crystalline/growth & development , Lens, Crystalline/metabolism , Mice , Oxidation-ReductionABSTRACT
2-(ω-Carboxyethyl)pyrrole (CEP) derivatives of proteins were previously shown to have significant pathological and physiological relevance to age-related macular degeneration, cancer and wound healing. Previously, we showed that CEPs are generated in the reaction of ε-amino groups of protein lysyl residues with 1-palmityl-2-(4-hydroxy-7-oxo-5-heptenoyl)-sn-glycero-3-phosphatidylcholine (HOHA-PC), a lipid oxidation product uniquely generated by oxidative truncation of docosahexanenate-containing phosphatidylcholine. More recently, we found that HOHA-PC rapidly releases HOHA-lactone and 2-lyso-PC (t1/2 = 30 min at 37 °C) by nonenzymatic transesterification/deacylation. Now we report that HOHA-lactone reacts with Ac-Gly-Lys-OMe or human serum albumin to form CEP derivatives in vitro. Incubation of human red blood cell ghosts with HOHA-lactone generates CEP derivatives of membrane proteins and ethanolamine phospholipids. Quantitative analysis of the products generated in the reaction HOHA-PC with Ac-Gly-Lys-OMe showed that HOHA-PC mainly forms CEP-dipeptide that is not esterified to 2-lysophosphatidycholine. Thus, the HOHA-lactone pathway predominates over the direct reaction of HOHA-PC to produce the CEP-PC-dipeptide derivative. Myleoperoxidase/H2O2/NO2(-) promoted in vitro oxidation of either 1-palmityl-2-docosahexaneoyl-sn-glycero-3-phosphatidylcholine (DHA-PC) or docosahexaenoic acid (DHA) generates HOHA-lactone in yields of 0.45% and 0.78%, respectively. Lipid oxidation in human red blood cell ghosts also releases HOHA-lactone. Oxidative injury of ARPE-19 human retinal pigmented epithelial cells by exposure to H2O2 generated CEP derivatives. Treatment of ARPE-19 cells with HOHA-lactone generated CEP-modified proteins. Low (submicromolar), but not high, concentrations of HOHA-lactone promote increased vascular endothelial growth factor (VEGF) secretion by ARPE-19 cells. Therefore, HOHA-lactone not only serves as an intermediate for the generation of CEPs but also is a biologically active oxidative truncation product from docosahexaenoate lipids.
Subject(s)
Erythrocytes/metabolism , Lactones/metabolism , Phosphatidylethanolamines/metabolism , Pyrroles/metabolism , Retinal Pigment Epithelium/cytology , Serum Albumin/metabolism , Cell Line , Cell Proliferation , Docosahexaenoic Acids/chemistry , Docosahexaenoic Acids/metabolism , Erythrocytes/chemistry , Erythrocytes/cytology , Humans , Lactones/chemistry , Oxidation-Reduction , Phosphatidylethanolamines/chemistry , Pyrroles/chemistry , Retinal Pigment Epithelium/chemistry , Retinal Pigment Epithelium/metabolism , Serum Albumin/chemistry , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Oxidation of docosahexaenoate phospholipids produces 4-hydroxy-7-oxo-hept-5-eonyl phospholipids (HOHA-PLs) that react with protein lysyl ε-amino residues to generate 2-ω-carboxyethylpyrrole (CEP) derivatives, endogenous factors that induce angiogenesis in the retina and tumors. It seemed likely, but remained unproven, that HOHA-PLs react with ethanolamine phospholipids (EPs) in vivo to generate CEP-EPs. We now show that CEP-EPs are present in human blood at 4.6-fold higher levels in age-related macular degeneration plasma than in normal plasma. We also show that CEP-EPs are pro-angiogenic, inducing tube formation by human umbilical vein endothelial cells by activating Toll-like receptor 2. CEP-EP levels may be a useful biomarker for clinical assessment of AMD risk and CEP-associated tumor progression and a tool for monitoring the efficacy of therapeutic interventions.
Subject(s)
Phosphatidylethanolamines/blood , Phospholipids/blood , Chromatography, Liquid , Human Umbilical Vein Endothelial Cells , Humans , Macular Degeneration/blood , Magnetic Resonance Spectroscopy , Phospholipids/physiology , Tandem Mass SpectrometryABSTRACT
α-Crystallin is a major protein in the human lens that is perceived to help to maintain the transparency of the lens through its chaperone function. In this study, we demonstrate that many lens proteins including αA-crystallin are acetylated in vivo. We found that K70 and K99 in αA-crystallin and, K92 and K166 in αB-crystallin are acetylated in the human lens. To determine the effect of acetylation on the chaperone function and structural changes, αA-crystallin was acetylated using acetic anhydride. The resulting protein showed strong immunoreactivity against a N(ε)-acetyllysine antibody, which was directly related to the degree of acetylation. When compared to the unmodified protein, the chaperone function of the in vitro acetylated αA-crystallin was higher against three of the four different client proteins tested. Because a lysine (residue 70; K70) in αA-crystallin is acetylated in vivo, we generated a protein with an acetylation mimic, replacing Lys70 with glutamine (K70Q). The K70Q mutant protein showed increased chaperone function against three client proteins compared to the Wt protein but decreased chaperone function against γ-crystallin. The acetylated protein displayed higher surface hydrophobicity and tryptophan fluorescence, had altered secondary and tertiary structures and displayed decreased thermodynamic stability. Together, our data suggest that acetylation of αA-crystallin occurs in the human lens and that it affects the chaperone function of the protein.
Subject(s)
Lens, Crystalline/chemistry , Lens, Crystalline/metabolism , Molecular Chaperones/chemistry , Molecular Chaperones/metabolism , alpha-Crystallin A Chain/chemistry , alpha-Crystallin A Chain/metabolism , Acetic Anhydrides/metabolism , Acetylation , Crystallins , Glutamine/metabolism , Humans , Hydrophobic and Hydrophilic Interactions , Lysine/metabolism , Middle Aged , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Protein Structure, Secondary , Protein Structure, Tertiary , Spectrometry, Fluorescence/methods , Structure-Activity Relationship , Thermodynamics , Tryptophan/metabolism , alpha-Crystallin B Chain/chemistry , alpha-Crystallin B Chain/metabolismABSTRACT
γ-Hydroxyalkenals, 4-hydroxynonenal (HNE) and phospholipid esters of 4-hydroxy-8-oxooctenoic acid (HOOA-PL), are produced from the alkyl and carboxyl termini of arachidonyl phospholipids by radical-induced oxidative cleavage. Metabolism of HNE by Michael addition of glutathione (GSH) followed by reduction of the aldehyde carbonyl produces a GSH derivative of 1,4-dihydroxynonane (DHN)-GSH. Analogous biochemistry was anticipated to produce a GSH derivative of 5,8-dihydroxyoctanoic acid (DHOA-GSH) that has structural and functional similarity to the cysteinyl leukotriene (LT)C4. We now report that exposure of human retinal pigment epithelial cells to CoCl2, an in vitro model of hypoxia-induced oxidative stress, generates DHOA-GSH and two products of its peptidolysis, DHOA-CysGly and DHOA-Cys that resemble LTD4 and LTE4. Identification of these metabolites was confirmed by unambiguous chemical syntheses that also provided a heavy isotope labeled quantitative standard 13C2 15N-DHOA-GSH. The availability of pure samples of these arachidonate metabolites will enable assessment of their biological activities, and testing the hypothesis that øLTs promote pathological inflammation by serving as LT receptor agonists. Because LT biosynthetic enzymes, e.g., 5-lipoxygenase, are not involved in the generation of øLTs in vivo, inhibitors of LT biosynthesis, e.g., Zileuton, are not expected to prevent the generation of øLTs. On the other hand, if øLTs are leukotriene receptor agonists, then the therapeutic effects of leukotriene receptor antagonist drugs, e.g., Montelukast, may include inhibition not only of LT-induced but also øLT-induced LT receptor activation and signaling.
ABSTRACT
The proteins of the human eye are highly susceptible to the formation of advanced glycation end products (AGEs) from the reaction of sugars and carbonyl compounds. AGEs progressively accumulate in the aging lens and retina and accumulate at a higher rate in diseases that adversely affect vision such as, cataract, diabetic retinopathy and age-related macular degeneration. In the lens AGEs induce irreversible changes in structural proteins, which lead to lens protein aggregation and formation of high-molecular-weight aggregates that scatter light and impede vision. In the retina AGEs modify intra- and extracellular proteins that lead to an increase in oxidative stress and formation of pro-inflammatory cytokines, which promote vascular dysfunction. This review outlines recent advances in AGE research focusing on the mechanisms of their formation and their role in cataract and pathologies of the retina. The therapeutic action and pharmacological strategies of anti-AGE agents that can inhibit or prevent AGE formation in the eye are also discussed.
Subject(s)
Aging/metabolism , Eye Diseases/metabolism , Glycation End Products, Advanced/metabolism , Animals , Eye Diseases/pathology , Glycation End Products, Advanced/chemistry , Humans , Maillard Reaction , Molecular Structure , Retina/metabolismABSTRACT
Human lens proteins (HLP) become chemically modified by kynurenines and advanced glycation end products (AGEs) during aging and cataractogenesis. We investigated the effects of kynurenines on AGE synthesis in HLP. We found that incubation with 5 mM ribose or 5 mM ascorbate produced significant quantities of pentosidine, and this was further enhanced in the presence of two different kynurenines (200-500 microM): N-formylkynurenine (Nfk) and kynurenine (Kyn). Another related compound, 3-hydroxykynurenine (3OH-Kyn), had disparate effects; low concentrations (10-200 microM) promoted pentosidine synthesis, but high concentrations (200-500 microM) inhibited it. 3OH-Kyn showed similar effects on pentosidine synthesis from Amadori-enriched HLP or ribated lysine. Chelex-100 treatment of phosphate buffer reduced pentosidine synthesis from Amadori-enriched HLP by approximately 90%, but it did not inhibit the stimulating effect of 3OH-Kyn and EDTA. 3OH-Kyn (100-500 microM) spontaneously produced copious amounts of H(2)O(2) (10-25 microM), but externally added H(2)O(2) had only a mild stimulating effect on pentosidine but had no effect on N(epsilon)-carboxymethyl lysine (CML) synthesis in HLP from ribose and ascorbate. Further, human lens epithelial cells incubated with ribose and 3OH-Kyn showed higher intracellular pentosidine than cells incubated with ribose alone. CML synthesis from glycating agents was inhibited 30 to 50% by 3OH-Kyn at concentrations of 100-500 microM. Argpyrimidine synthesis from 5mM methylglyoxal was slightly inhibited by all kynurenines at concentrations of 100-500 microM. These results suggest that AGE synthesis in HLP is modulated by kynurenines, and such effects indicate a mode of interplay between kynurenines and carbohydrates important for AGE formation during lens aging and cataract formation.
Subject(s)
Crystallins/metabolism , Glycation End Products, Advanced/biosynthesis , Kynurenine/pharmacology , Arginine/analogs & derivatives , Arginine/biosynthesis , Arginine/chemistry , Buffers , Cell Line , Crystallins/chemistry , Edetic Acid , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Glycation End Products, Advanced/chemistry , Humans , Hydrogen Peroxide/metabolism , Hydrogen Peroxide/pharmacology , In Vitro Techniques , Kynurenine/analogs & derivatives , Kynurenine/metabolism , Lens, Crystalline/cytology , Lens, Crystalline/drug effects , Lens, Crystalline/metabolism , Lysine/analogs & derivatives , Lysine/biosynthesis , Lysine/chemistry , Maillard Reaction , Models, Biological , Resins, SyntheticABSTRACT
AlphaA-crystallin is a molecular chaperone; it prevents aggregation of denaturing proteins. We have previously demonstrated that upon modification by a metabolic alpha-dicarbonyl compound, methylglyoxal (MGO), alphaA-crystallin becomes a better chaperone. AlphaA-crystallin also assists in refolding of denatured proteins. Here, we have investigated the effect of mild modification of alphaA-crystallin by MGO (with 20-500 microM) on the chaperone function and its ability to refold denatured proteins. Under the conditions used, mildly modified protein contained mostly hydroimidazolone modifications. The modified protein exhibited an increase in chaperone function against thermal aggregation of beta(L)- and gamma-crystallins, citrate synthase (CS), malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) and chemical aggregation of insulin. The ability of the protein to assist in refolding of chemically denatured beta(L)- and gamma-crystallins, MDH and LDH, and to prevent thermal inactivation of CS were unchanged after mild modification by MGO. Prior binding of catalytically inactive, thermally denatured MDH or the hydrophobic probe, 2-p-toluidonaphthalene-6-sulfonate (TNS) abolished the ability of alphaA-crystallin to assist in the refolding of denatured MDH. However, MGO modification of chaperone-null TNS-bound alphaA-crystallin resulted in partial regain of the chaperone function. Taken together, these results demonstrate that: 1) hydroimidazolone modifications are sufficient to enhance the chaperone function of alphaA-crystallin but such modifications do not change its ability to assist in refolding of denatured proteins, 2) the sites on the alphaA-crystallin responsible for the chaperone function and refolding are the same in the native alphaA-crystallin and 3) additional hydrophobic sites exposed upon MGO modification, which are responsible for the enhanced chaperone function, do not enhance alphaA-crystallin's ability to refold denatured proteins.
Subject(s)
Crystallins/chemistry , Imidazoles/chemistry , Molecular Chaperones/chemistry , Protein Folding , Crystallins/metabolism , Humans , Hydrophobic and Hydrophilic Interactions , Molecular Chaperones/metabolism , Protein Denaturation , Protein Structure, Tertiary/physiologyABSTRACT
Dideoxyosones (DDOs) are intermediates in the synthesis of advanced glycation endproducts (AGEs), such as pentosidine and glucosepane. Although the formation of pentosidine and glucosepane in the human lens has been firmly established, the formation of DDOs has not been demonstrated. The aim of this study was to develop a reliable method to detect DDOs in lens proteins. A specific DDO trapping agent, biotinyl-diaminobenzene (3,4-diamino-N-(3-[5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoyl]aminopropyl)benzamide) (BDAB) was added during in vitro protein glycation or during protein extraction from human lenses. In vitro glycated human lens protein showed strong reaction in monomeric and polymeric crosslinked proteins by Western blot and ELISA. Glycation of BSA in the presence of BDAB resulted in covalent binding of BDAB to the protein and inhibited pentosidine formation. Mass spectrometric analysis of lysozyme glycated in the presence of BDAB showed the presence of quinoxalines at lysine residues at positions K1, K33, K96, and K116. The ELISA results indicated that cataractous lens proteins contain significantly higher levels of DDO than non-cataractous lenses (101.9±67.8 vs. 31.7±19.5AU/mg protein, p<0.0001). This study provides first direct evidence of DDO presence in human tissue proteins and establishes that AGE crosslink synthesis in the human lens occurs via DDO intermediates.
Subject(s)
Cataract/metabolism , Crystallins/chemistry , Glycation End Products, Advanced/analysis , Lens, Crystalline/chemistry , Adult , Amino Acid Sequence , Animals , Blotting, Western/methods , Cattle , Crystallins/metabolism , Enzyme-Linked Immunosorbent Assay/methods , Glycation End Products, Advanced/metabolism , Glycosylation , Humans , Lens, Crystalline/metabolism , Lysine/chemistry , Lysine/metabolism , Mass Spectrometry/methods , Molecular Sequence Data , Reproducibility of Results , Young AdultABSTRACT
Oxidation of docosahexaenoate (DHA)-containing phospholipids in the cell plasma membrane leads to release of the α,ß-unsaturated aldehyde 4-hydroxy-7-oxo-5-heptenoic acid (HOHA) lactone which is capable of inducing retinal pigmented epithelial (RPE) cell dysfunction. Previously, HOHA lactone was shown to induce apoptosis and angiogenesis, and to activate the alternative complement pathway. RPE cells metabolize HOHA lactone through enzymatic conjugation with glutathione (GSH). Competing with this process is the adduction of HOHA lactone to protein lysyl residues generating 2-(ω-carboxyethyl)pyrrole (CEP) derivatives that have pathological relevance to age-related macular degeneration (AMD). We now find that HOHA lactone induces mitochondrial dysfunction. It decreases ATP levels, mitochondrial membrane potentials, enzymatic activities of mitochondrial complexes, depletes GSH and induces oxidative stress in RPE cells. The present study confirmed that pyridoxamine and other primary amines, which have been shown to scavenge γ-ketoaldehydes formed by carbohydrate or lipid peroxidation, are ineffective for scavenging the α,ß-unsaturated aldehydes. Histidyl hydrazide (HH), that has both hydrazide and imidazole nucleophile functionalities, is an effective scavenger of HOHA lactone and it protects ARPE-19 cells against HOHA lactone-induced cytotoxicity. The HH α-amino group is not essential for this electrophile trapping activity. The Nα-acyl L-histidyl hydrazide derivatives with 2- to 7-carbon acyl groups with increasing lipophilicities are capable of maintaining the effectiveness of HH in protecting ARPE-19 cells against HOHA lactone toxicity, which potentially has therapeutic utility for treatment of age related eye diseases.
Subject(s)
Lactones , Retinal Pigment Epithelium , Epithelial Cells , Lactones/metabolism , Lactones/toxicity , Mitochondria , Oxidative Stress , Retinal Pigment Epithelium/metabolismABSTRACT
Retinal pigment epithelial (RPE) cell dysfunction and death play vital roles in age-related macular degeneration (AMD) pathogenesis. Previously we showed that oxidative cleavage of docosahexenoate (DHA) phospholipids generates an α,ß-unsaturated aldehyde, 4-hydroxy-7-oxohept-4-enoic acid (HOHA) lactone, that forms ω-carboxyethylpyrrole (CEP) derivatives through adduction to proteins and ethanolamine phospholipids. CEP derivatives and autoantibodies accumulate in the retinas and blood plasma of individuals with AMD and are a biomarker of AMD. They promote the choroidal neovascularization of "wet AMD". Immunization of mice with CEP-modified mouse serum albumin induces "dry AMD"-like lesions in their retinas as well as interferon-gamma and interleukin-17 production by CEP-specific T cells that promote inflammatory M1 polarization of macrophages. The present study confirms that oxidative stress or inflammatory stimulus produces CEP in both the primary human ARPE-19 cell line and hRPE cells. Exposure of these cells to HOHA lactone fosters production of reactive oxygen species. Thus, HOHA lactone participates in a vicious cycle, promoting intracellular oxidative stress leading to oxidative cleavage of DHA to produce more HOHA lactone. We now show that HOHA lactone is cytotoxic, inducing apoptotic cell death through activation of the intrinsic pathway. This suggests that therapeutic interventions targeting HOHA lactone-induced apoptosis may prevent the loss of RPE cells during the early phase of AMD. We also discovered that ARPE-19 cells are more susceptible than hRPE cells to HOHA lactone cytotoxicity. This is consistent with the view that, compared to normal RPE cells, ARPE-19 cells exhibit a diseased RPE phenotype that also includes elevated expression of the mesenchymal indicator vimentin, elevated integrin a5 promotor strength and deficient secretion of the anti-VEGF molecule pigment-epithelium-derived factor fostering weaker tight junctions.
Subject(s)
Lactones , Retinal Pigment Epithelium , Animals , Apoptosis , Epithelial Cells , Mice , Oxidative Stress , Retinal PigmentsABSTRACT
Retinal degeneration is a form of neurodegenerative disease and is the leading cause of vision loss globally. The Toll-like receptors (TLRs) are primary components of the innate immune system involved in signal transduction. Here we show that TLR2 induces complement factors C3 and CFB, the common and rate-limiting factors of the alternative pathway in both retinal pigment epithelial (RPE) cells and mononuclear phagocytes. Neutralization of TLR2 reduces opsonizing fragments of C3 in the outer retina and protects photoreceptor neurons from oxidative stress-induced degeneration. TLR2 deficiency also preserves tight junction expression and promotes RPE resistance to fragmentation. Finally, oxidative stress-induced formation of the terminal complement membrane attack complex and Iba1+ cell infiltration are strikingly inhibited in the TLR2-deficient retina. Our data directly implicate TLR2 as a mediator of retinal degeneration in response to oxidative stress and present TLR2 as a bridge between oxidative damage and complement-mediated retinal pathology.
Subject(s)
Oxidative Stress/physiology , Retinal Degeneration/metabolism , Toll-Like Receptor 2/metabolism , Animals , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Retinal Degeneration/genetics , Retinal Degeneration/pathology , Toll-Like Receptor 2/deficiency , Toll-Like Receptor 2/geneticsABSTRACT
Previous studies from this laboratory have shown that there are striking similarities between the yellow chromophores, fluorophores and modified amino acids released by proteolytic digestion from calf lens proteins ascorbylated in vitro and their counterparts isolated from aged and cataractous lens proteins. The studies reported in this communication were conducted to further investigate whether ascorbic acid-mediated modification of lens proteins could lead to the formation of lens protein aggregates capable of scattering visible light, similar to the high molecular aggregates found in aged human lenses. Ascorbic acid, but not glucose, fructose, ribose or erythrulose, caused the aggregation of calf lens proteins to proteins ranging from 2.2 x 10(6) up to 3.0 x 10(8 )Da. This compared to proteins ranging from 1.8 x 10(6) up to 3.6 x 10(8 )Da for the water-soluble (WS) proteins isolated from aged human lenses. This aggregation was likely due to the glycation of lens crystallins because [U-(14)C] ascorbate was incorporated into the aggregate fraction and because NaCNBH(3), which reduces the initial Schiff base, prevented any protein aggregation. Reactions of ascorbate with purified crystallin fractions showed little or no aggregation of alpha-crystallin, significant aggregation of beta(H)-crystallin, but rapid precipitation of purified beta(L)- and gamma-crystallin. The aggregation of lens proteins can be prevented by the binding of damaged crystallins to alpha-crystallin due to its chaperone activity. Depending upon the ratios between the components of the incubation mixtures, alpha-crystallin prevented the precipitation of the purified beta(L)- and gamma-crystallin fractions during ascorbylation. The addition of at least 20% of alpha-crystallin by weight into glycation mixtures with beta(L)-, or gamma-crystallins completely inhibited protein precipitation, and increased the amount of the high molecular weight aggregates in solution. Static and dynamic light scattering measurements of the supernatants from the ascorbic acid-modified mixtures of alpha- and beta(L)-, or gamma-crystallins showed similar molar masses (up to 10(8 )Da) and hydrodynamic diameter (up to 80( )nm). These data support the hypothesis, that if the lens reducing environment is compromised, the ascorbylation of lens crystallins can significantly change the short range interactions between different classes of crystallins leading to protein aggregation, light scattering and eventually to senile cataract formation.