The Thioredoxin-like Protein Rod-derived Cone Viability Factor (RdCVFL) Interacts with TAU and Inhibits Its Phosphorylation in the Retina

Ram Fridlich,Thérèse Cronin,Ludivine Perrocheau,Céline Jaillard,Jose-Alain Sahel,Marie-Laure Niepon,Olivier Poch,Arne Holmgren,Thierry Léveillard,Alain Van Dorsselaer,Géraldine Millet-Puel,Laetitia Poidevin,Jun Lu,Francois Delalande

doi:10.1074/mcp.m800406-mcp200

Abstract

Rod-derived cone viability factor (RdCVF) is produced by the Nxnl1 gene that codes for a second polypeptide, RdCVFL, by alternative splicing. Although the role of RdCVF in promoting cone survival has been described, the implication of RdCVFL, a putative thioredoxin enzyme, in the protection of photoreceptors is presently unknown. Using a proteomics approach we identified 90 proteins interacting with RdCVFL including the microtubule-binding protein TAU. We demonstrate that the level of phosphorylation of TAU is increased in the retina of the Nxnl1(-/-) mice as it is hyperphosphorylated in the brain of patients suffering from Alzheimer disease, presumably in some cases through oxidative stress. Using a cell-based assay, we show that RdCVFL inhibits TAU phosphorylation. In vitro, RdCVFL protects TAU from oxidative damage. Photooxidative stress is implicated in retinal degeneration, particularly in retinitis pigmentosa, where it is considered to be a contributor to secondary cone death. The functional interaction between RdCVFL and TAU described here is the first characterization of the RdCVFL signaling pathway involved in neuronal cell death mediated by oxidative stress.

Highlights

Rod-derived cone viability factor (RdCVF) is produced by the nucleoredoxin-like 1 (Nxnl1) gene that codes for a second polypeptide, RdCVFL, by alternative splicing
These results show that the interaction of RdCVFL with TAU protects this protein from oxidative damage, and this protection assists in the reduction of TAU by the thioredoxin system
RdCVF is a trophic factor belonging to the thioredoxin family

Summary

EXPERIMENTAL PROCEDURES

For the estimation of the false positive rate, a target-decoy database search was performed [26, 27] In this approach peptides are matched against a database consisting of the native protein sequences found in the database (target) and of the sequence-reversed entries (decoy). The evaluations were performed using the peptide validation software Scaffold (Proteome Software) This strategy was used to obtain a final catalogue of proteins with an estimated false positive rate below 1%. GST, GST-RdCVFL (in PBS), and the recombinant human TAU protein samples (Sigma, catalogue number T9392) in 50 mM MOPS, pH 6.8, 100 mM NaCl, 1 mM EDTA, 5 mM DTT, 1 mM PMSF were dialyzed against 50 mM Tris-HCl, pH 7.5, 1 mM EDTA overnight with DispoBiodialyzer devices (molecular mass cutoff, 10 kDa) (Sigma). Recombinant protein sequences used in the study are provided in supplemental Table III

RESULTS

B Da Da BDa Da Da Da BDa Aa ABDa

DISCUSSION