Senile cataracts are associated with progressive oxidation, fragmentation, cross-linking, insolubilization, and yellow pigmentation of lens crystallins. We hypothesized that the Maillard reaction, which leads browning and aroma development during the baking of foods, would occur between the lens proteins and the highly reactive oxidation products of vitamin C. To test this hypothesis, we engineered a mouse that selectively overexpresses the human vitamin C transporter SVCT2 in the lens. Consequently, lenticular levels of vitamin C and its oxidation products were 5-to 15-fold elevated, resulting in a highly compressed aging process and accelerated formation of several protein-bound advanced Maillard reaction products identical with those of aging human lens proteins. These data strongly implicate vitamin C in lens crystallin aging and may serve as a model for protein aging in other tissues particularly rich in vitamin C, such as the hippocampal neurons and the adrenal gland. The hSVCT2 mouse is expected to facilitate the search for drugs that inhibit damage by vitamin C oxidation products.advanced glycation end products ͉ ascorbic acid ͉ cataract ͉ diabetes ͉ oxidation

Low glutathione levels are associated with crystallin oxidation in age-related nuclear cataract. To understand the role of cysteine residue oxidation, we used the novel approach of comparing human cataracts with glutathionedepleted LEGSKO mouse lenses for intra-versus intermolecular disulfide crosslinks using 2D-PAGE and proteomics, and then systematically identified in vivo and in vitro all disulfide forming sites using ICAT labeling method coupled with proteomics. Crystallins rich in intramolecular disulfides were abundant at young age in human and WT mouse lens but shifted to multimeric intermolecular disulfides at older age. The shift was 4x accelerated in LEGSKO lens. Most cysteine disulfides in β-crystallins (except βA4 in human) were highly conserved in mouse and human and could be generated by oxidation with H 2 O 2 , whereas γ-crystallin oxidation selectively affected γC23/42/79/80/154, γD42/33, and γS83/115/130 in human cataracts, and γB79/80/110, γD19/109, γF19/79, γE19, γS83/130, and γN26/128 in mouse. Analysis based on available crystal structure suggests that conformational changes are needed to expose Cys42, Cys79/80, Cys154 in γC; Cys42, Cys33 in γD, and Cys83, Cys115, and Cys130 in γS. In conclusion, the β-crystallin disulfidome is highly conserved in age-related nuclear cataract and LEGSKO mouse, and reproducible by in vitro oxidation, whereas some of the disulfide formation sites in γ-crystallins necessitate prior conformational changes. Overall, the LEGSKO mouse model is closely reminiscent of age- Aging lens crystallins accumulate post-synthetic modifications that can be broadly classified into three categories, namely (1) protein backbone changes, such as racemization and truncation (1-3), (2) conversion of one amino acid into another, such as deamidation of asparagine into aspartate or deguanidination of arginine into ornithine, deamination of lysine into allysine and 2-aminoadipic acid (4 -6), and (3) amino acid residue damage from reactive carbonyls and reactive oxygen species (7,8). Carbonyl damage results from the Maillard Reaction by glucose, methylglyoxal, or oxidation products of ascorbate, tryptophan or lipids which form adducts and crosslinks with nucleophilic group of lysine, arginine and cysteine. Examples include carboxymethyl-lysine, pentosidine, methylglyoxal hydroimidazolones, HNE-cysteine adducts and kynurenine (7,9 -12). Oxidative damage results from reactive oxygen species that directly damage amino acid residues, e.g. oxidizing tryptophan into N-formyl kynurenine and kynurenine, methionine into its sulfoxide, and cysteine into cysteine disulfides or cysteic acid (13)(14)(15).Because of their relevance to age-related cataract, the impact of each of these modifications on crystallin structure and stability is the subject of intense investigation. Importantly, Benedek proposed that high molecular weight (HMW) 1 crystallin aggregates the size of 50 million daltons are needed in order for lens opacification to be visible (16,17). Crystallin aggregation conceivably occurs by o...

Background/Aims: Circular RNAs (circRNAs) are key regulators in the development and progression of human cancers, however its role in non-small cell lung cancer (NSCLC) tumorigenesis is not well understood. The aim of this study is to identify the expression level of circPVT1 in NSCLC and further investigated its functional relevance with NSCLC progression both in vitro and in vivo. Methods: Quantative real-time PCR was used for the measurement of circPVT1 in NSCLC specimens and cell lines. Fluorescence in situ hybridization analysis (FISH) assay was used for the identification of sublocation of circPVT1 in NSCLC cells. Bioinformatics analysis, luciferase reporter assay and RNA immunoprecipitation (RIP) were performed to verify the binding of c-Fos at circPVT1 promoter region, and the direct interaction between circPVT1 and miR-125b. Gain- or loss-function assays were performed to evaluate the effects of circPVT1 on cell proliferation and invasion. Western blot and immunohistochemistry assays were performed to detect the protein levels involved in E2F2 pathway. Results: We found that circPVT1 was upregulated in NSCLC specimens and cells. The transcription factor c-Fos binded to the promoter region of circPVT1, resulting in the overexpression of circPVT1 in NSCLC. Knockdown of circPVT1 suppressed NSCLC cell proliferation, migration and invasion, and increased apoptosis. In addition, circPVT1 mediated NSCLC progression via the regulation of E2F2 signaling pathway. More importantly, circPVT1 was predominantly abundant in the cytoplasm of NSCLC cells, and circPVT1 could serve as a competing endogenous RNA to regulate E2F2 expression and tumorigenesis in a miR-125b-dependent manner, which is further verified by using an in vivo xenograft model. Conclusion: circPVT1 promotes NSCLC cell growth and invasion, and may serve as a promising therapeutic target for NSCLC patients. Therefore, silence of circPVT1 could be a future direction to develop a novel treatment strategy.

Cysteine (Cys) residues are major causes of crystallin disulfide formation and aggregation in aging and cataractous human lenses. We recently found that disulfide linkages are highly and partly conserved in β- and γ-crystallins, respectively, in human age-related nuclear cataract and glutathione depleted LEGSKO mouse lenses, and could be mimicked by in vitro oxidation. Here we determined which Cys residues are involved in disulfide-mediated crosslinking of recombinant human γD-crystallin (hγD). In vitro diamide oxidation revealed dimer formation by SDS-PAGE and LC-MS analysis with Cys 111-111 and C111-C19 as intermolecular disulfides and Cys 111-109 as intramolecular sites. Mutation of Cys111 to alanine completely abolished dimerization. Addition of αB-crystallin was unable to protect Cys 111 from dimerization. However, Cu-induced hγD-crystallin aggregation was suppressed up to 50% and 80% by mutants C109A and C111A, respectively, as well as by total glutathionylation. In contrast to our recently published results using ICAT-labeling method, manual mining of the same database confirmed the specific involvement of Cys111 in disulfides with no free Cys111 detectable in γD-crystallin from old and cataractous human lenses. Surface accessibility studies show that Cys111 in hγD is the most exposed Cys residue (29%), explaining thereby its high propensity toward oxidation and polymerization in the aging lens.

Background: Human lens proteins accumulate pigmented, protein-cross-linked AGE adducts during cataract formation, and the mechanisms of their formation are poorly understood. Results: UVA-excited kynurenines can oxidize ascorbate under anoxic conditions and promote synthesis of AGEs. Conclusion: UVA light-excited kynurenines promote AGE synthesis and contribute to cataract formation. Significance: This study provides a mechanism for UVA light-mediated damage to lens proteins during cataract formation.