![]() Moreover, in agreement with previous studies, we see that UV irradiation causes a translocation of La protein to the cytoplasm ( Figure 1j–l). These data show that the intracellular oxidative conditions caused by UV irradiation are sufficient to oxidize La protein in living cells whereby the epitope recognized by the anti-La mAb 7B6 becomes accessible. Already, a short irradiation with UV light prior to the fixation with methanol of analytical grade results in nuclear staining with the anti-La mAb 7B6 without any washing with H 2O 2 prior to the staining ( Figure 1g–i). If the fixed cells, however, are washed with PBS containing H 2O 2 (30 μM) the epitope becomes accessible ( Figure 1d–f). In agreement with recent studies, human cells fixed with methanol lacking peroxides are not stained by the anti-La mAb 7B6 ( Figure 1a–c). Accessibility of the Epitope Recognized by the Anti-La mAb 7B6 after UV Irradiation In summary, we show that La protein is a redox sensor that translocalizes to the cytoplasm in a redox-dependent manner. In line with this, we show that translocation of La protein to the cytoplasm can be triggered in a TLR ligand-/receptor-/redox-dependent manner. It is well known that toll-like receptor (TLR) signaling results in intracellular oxidative stress. Finally, we tested whether or not ligand-/receptor-dependent induced intracellular oxidative stress can also lead to shuttling of La protein. However, deprivation of intracellular reducing agents in endothelial cells turns them sensitive to a redox-dependent shuttling of La protein. While all analyzed oxidative stress conditions lead to the shuttling of La protein in a wide variety of tested cell types, cultured endothelial cells appeared to be insensitive to the shuttling of La protein. ![]() The shuttling of La protein depends on the redox status of the respective cell type. After oxidation, La protein shuttles to the cytoplasm. Here we show that La protein undergoes redox-dependent conformational changes in living cells. According to previous studies, this region is part of the nuclear retention element of La protein. The anti-La mAb 7B6 recognizes the amino acid sequence EKEALKKIIEDQQESLNK (aa311-328 of human La protein), which includes the α3 helix in the RRM2 domain of La protein. Most importantly, the anti-La mAb 7B6 can be used to detect the oxidized form of La protein by immunofluorescence microscopy (IF). Vice versa, the epitope recognized by the anti-La mAb 7B6 is cryptic in human La protein but becomes accessible when La protein becomes oxidized. We found that anti-La mAbs directed to the La motif recognizes the reduced but not the oxidized form of La protein. In order to differentiate between the oxidized and reduced forms of La protein, we recently established anti-La monoclonal Abs (anti-La mAbs), which bind to either the reduced or oxidized form of La protein. Whether or not oxidoreduction and conformational changes also occur in living cells remained unclear. So far, oxidoreduction of La protein was experimentally achieved in vitro by exposure of La protein to H 2O 2 or CuSO 4. The primary sequence of La protein contains three cysteine residues being involved in these redox-dependent conformational changes. Recently, we showed that La protein undergoes redox-dependent conformational changes. However, the deprivation of intracellular reducing agents in endothelial cells makes endothelial cells sensitive to a redox-dependent shuttling of La protein. Endothelial cells are usually resistant to the shuttling of La protein, while dendritic cells are highly sensitive. We show that ligands of toll-like receptors lead to a redox-dependent shuttling of La protein. Moreover, we show that translocation of La protein to the cytoplasm can be triggered in a ligand/receptor-dependent manner under physiological conditions. Using these tools, here we show that redox-dependent conformational changes are the driving force for the shuttling of La protein. Moreover, we developed anti-La monoclonal antibodies (anti-La mAbs), which are specific for either the reduced form of La protein or the oxidized form. ![]() ![]() Moreover, the driving mechanism for the shuttling of La protein remains unclear. Even until today, the shuttling and the cytoplasmic function of La/SS-B is controversially discussed. Unfortunately, these harsh conditions could also cause an artificial release of La protein. ![]() All of these conditions are somehow related to oxidative stress. This shuttling of La protein was seen after UV irradiation, virus infections, hydrogen peroxide exposure and the Fenton reaction based on iron or copper ions. Decades ago, we and many other groups showed a nucleo-cytoplasmic translocation of La protein in cultured cells. ![]()
0 Comments
Leave a Reply. |