DNAJB12 and DNJB14 are non-redundant Hsp40 redox chaperones involved in endoplasmic reticulum protein reflux.

BACKGROUND: The endoplasmic reticulum (ER) transmembrane chaperones DNAJB12(B12) and DNAJB14(B14) are cofactors that cooperate with cytosolic Heat Shock-70 protein (HSC70) facilitating folding/degradation of nascent membrane proteins and supporting the ER-membrane penetration of viral particles. Here, we assessed structural/functional features of B12/B14 with respect to their regulation by ER stress and their involvement in ER stress-mediated protein reflux. METHODS: We investigated the effect of Unfolded Protein Response(UPR)-eliciting drugs on the expression/regulation of B12-B14 and their roles in ER-to-cytosol translocation of Protein Disulfide Isomerase-A1(PDI). RESULTS: We show that B12 and B14 are similar but do not seem redundant. They share predicted structural features and show high homology of their cytosolic J-domains, while their ER-lumen DUF1977 domains are quite dissimilar. Interactome analysis suggested that B12/B14 associate with different biological processes. UPR activation did not significantly impact on B12 gene expression, while B14 transcripts were up-regulated. Meanwhile, B12 and B14 (33.4 kDa isoform) protein levels were degraded by the proteasome upon acute reductive challenge. Also, B12 degradation was impaired upon sulfenic-acid trapping by dimedone. We originally report that knockdown of B12/B14 and their cytosolic partner SGTA in ER-stressed cells significantly impaired the amount of the ER redox-chaperone PDI in a cytosolic-enriched fraction. Additionally, B12 but not B14 overexpression increased PDI relocalization in non-stressed cells. CONCLUSIONS AND GENERAL SIGNIFICANCE: Our findings reveal that B12/B14 regulation involves thiol redox processes that may impact on their stability and possibly on physiological effects. Furthermore, we provide novel evidence that these proteins are involved in UPR-induced ER protein reflux.

Novel application of Macrolampis sp2 firefly luciferase for intracellular pH-biosensing in mammalian cells.

Bioluminescence is widely used in biosensors. Firefly luciferase-based bioluminescent sensors are among the most popular ones. Firefly luciferases are pH-sensitive, displaying a large red shift at acidic pH, a property that has been considered undesirable for most applications. Currently, biosensors that can detect intracellular pH are in demand, and some fluorescent biosensors are available. However, pH sensors using bioluminescence have not been used yet. Thus, we decided to harness a firefly luciferase to measure the intracellular pH in mammalian cells. For this purpose, we engineered the luciferase derived from Macrolampis sp2 firefly to localize it on the cytosol or nucleus, in order to observe pH variation in these compartments during biological activities. We first calibrated the emission ratios (R = Igreen/Ired) at different pH values. As expected, we observed a red shift of light emission under acidic conditions when the cells were subjected to different pH conditions in the presence of the K+/H+ ionophore, nigericin. Based on these results, we concluded that this firefly luciferase can be used as a diagnostic tool for measuring the intracellular pH variation in pathogenic cells or in cells during apoptosis. This is the first example of real time-monitoring of pH change using color tuning luciferase.