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Folami Lamoke, Srinivas Rao Sripathi, Priyanka Thakur, Michael Duncan, Wan Jin Jahng, Manuela Bartoli; Identification and Functional Characterization of TXNIP’s Phosphoproteome in the Human Diabetic Retina. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4928.
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Hyperglycemia-induced up-regulation of the thioredoxin-inhibitory protein (TXNIP) has been shown to play a key role in the pro-oxidative and pro-inflammatory events leading to neurovascular injury and progression of diabetic retinopathy (DR). We have previously determined that TXNIP undergoes serine phosphorylation (TXNIP-SerP) in the diabetic milieu. Here, we have conducted studies to specifically identify and characterize TXNIP-SerP in the human diabetic retina.
Human post-mortem retinas were obtained from Georgia Eye Bank. Mass spectrometry analysis of phospho-enriched TXNIP immunoprecipitates was used to identify specific phosphoserine residues. Site-directed mutagenesis was used to generate TXNIP serine to alanine mutants (S-A) that were subsequently transfected in cultured retinal endothelial cells (REC) to assess their functional properties. Cell fractionation was used to determine the effects of TXNIP-S-A mutants and its intracellular redistribution in response to oxidative stimuli. Co-immunoprecipitation was used to assess TXNIP interaction with thioredoxin-1 (Trx-1) and the cryopyrin NALP3.
Four most frequent sites of TXNIP SerP were identified in human diabetic retinas and these were Ser298, Ser300, Ser304 and Ser305. In in vitro assays using retinal endothelial cells (RECs) we have found that overexpression of all 4 TXNIP-S-A mutants significantly decreased TXNIP overexpression-induced production of free radicals, as demonstrated by loss of DCF-positive fluorescence. Overexpression of the inactive mutants S300A, S304A and S305A also blunted TXNIP-interaction with Trx-1, whereas S298A did not. However, overexpression of TXNIP S298A mutant demonstrated its selective ability to block TXNIP interaction with NALP3 in response to different stimuli including high glucose (25mM D-glucose).
The results of our studies provide new insights in the understanding of TXNIP pathological function in the diabetic human retina and its contribution to the pro-oxidative and pro-inflammatory mechanisms involved in DR pathogenesis.
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