Evaluation of structure, chaperone-like activity and protective ability of peroxynitrite modified human α-Crystallin subunits against copper-mediated ascorbic acid oxidation

Abstract

The copper-catalyzed oxidation of ascorbic acid (ASA) to dehydroascorbate (DHA) and hydrogen peroxide plays a central role in pathology of cataract diseases during ageing and in diabetic patients. In the current study, the structural feature, chaperone-like activity and protective ability of peroxynitrite (PON) modified αA- and αB-Crystallin (Cry) against copper-mediated ASA oxidation were studied using different spectroscopic measurements and gel mobility shift assay. Upon PON modification, additional to protein structural alteration, the contents of nitrotyrosine, nitrotryptophan, dityrosine and carbonyl groups were significantly increased. Moreover, αB-Cry demonstrates significantly larger capacity for PON modification than αA-Cry. Also, based on the extent of PON modification, these proteins may display an improved chaperone-like activity and enhanced protective ability against copper-mediated ASA oxidation. In the presence of copper ions, chaperone-like activity of both native and PON-modified α-Cry subunits were appreciably improved. Additionally, binding of copper ions to native and PON-modified proteins results in the significant reduction of their solvent exposed hydrophobic patches. Overall, the increase in chaperone-like activity/ASA protective ability of PON-modified α-Cry and additional enhancement of its chaperoning action with copper ions appear to be an important defense mechanism offered by this protein.

The Double-Edged Sword of αB-Crystallin Chaperone Activity: Insights from P39L Mutant at N-terminal region

“…For catalase and γ-Cry, incubation was performed at 60°C to induce the client protein aggregation. Then the light scattering spectra was recorded at 400 nm to measure the extent of aggregation using a Carry 100 Bio UV-Vis spectrophotometer (Varian, Australia) 45 .…”

“…To mimic the chaperone activity inside cells, we also carried out an in cellulo chaperone activity assay by evaluating the growth rescue of E. coli cells expressing the chaperones under thermal stress 45 .…”

Simple method for O-GlcNAc sensitive detection based on graphene quantum dots

“…47 In addition, the up regulation of a-crystallin has been associated with ischemic heart, Parkinson's disease, multiple sclerosis and so on. 48 It is particularly difficult to detect O-GlcNAc on a-crystallin because of the presence of only one major modication site and its low stoichiometry of glycosylation. 49,50 Herein, to further study the potential practical applications of this method, puried a-crystallin protein was used as a model sample to analyze the O-GlcNAc amount in the protein.…”

Preventive role of lens antioxidant defense mechanism against riboflavin-mediated sunlight damaging of lens crystallins

“…The protein samples were diluted to 0.4 mg/ml in 100 mM NaPi buffer and the change of absorption spectra was recorded between 200-700 nm. In order to distinguish between aggregation and chromophore formation, the modified protein samples were centrifuged at 10000 g for 20 min to remove any existed protein aggregates [43]. Then, the absorption spectra were recorded between 200-700 nm.…”