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Background Polyphenolic compounds isolated from pomegranate fruit possess several pharmacological activities including anti-inflammatory, hepatoprotective, antigenotoxic and anticoagulant activities. The present work focuses the attention on PDIA3 interaction with punicalagin and ellagic acid, the most predominant components of pomegranate extracts. PDIA3, a member of the protein disulfide isomerase family involved in several cellular functions, is associated with different human diseases and it has the potential to be a pharmacological target. Methods The interaction of polyphenols with PDIA3 purified protein was explored by fluorescence quenching and calorimetric techniques and their effect on PDIA3 activity was investigated. Results A higher affinity was observed for punicalagin which also strongly affects PDIA3 reductase activity in vitro as a non-competitive inhibitor. Isothermal titration calorimetry confirmed the high affinity of punicalagin for PDIA3. Considering the PDIA3 involvement in oxidative cellular stress response observed in neuroblastoma cells after treatment with hydrogen peroxide, a comparative study was conducted to evaluate the effect of punicalagin on wild type and PDIA3-silenced cells. Punicalagin increases the cell sensitivity to hydrogen peroxide in neuroblastoma cells, but this effect is drastically reduced in PDIA3-silenced cells treated in the same experimental conditions. Conclusions Punicalagin binds PDIA3 and inhibits its redox activity. Comparative experiments conducted on unsilenced and PDIA3-silenced neuroblastoma cells suggest the potential of punicalagin to modulate PDIA3 reductase activity also in a biological model. General significance Punicalagin can be used as a new PDIA3 inhibitor and this can provide information on the molecular mechanisms underlying the biological activities of PDIA3 and punicalagin. [ABSTRACT FROM AUTHOR]

The glucocorticoid receptor (GR) acts as a ubiquitous cortisol-dependent transcription factor (TF). To identify co-factors, we used protein-fragment complementation assays and found that GR recognizes FLI1 and additional ETS family proteins, TFs relaying proliferation and/or migration signals. Following steroid-dependent translocation of FLI1 and GR to the nucleus, the FLI1-specific domain (FLS) binds with GR and strongly enhances GR's transcriptional activity. This interaction has functional consequences in Ewing sarcoma (ES), childhood and adolescence bone malignancies driven by fusions between EWSR1 and FLI1. In vitro , GR knockdown inhibited the migration and proliferation of ES cells, and in animal models, antagonizing GR (or lowering cortisol) retarded both tumor growth and metastasis from bone to lung. Taken together, our findings offer mechanistic rationale for repurposing GR-targeting drugs for the treatment of patients with ES. • Glucocorticoids activate GR, an inducible TF regulating growth and metabolism • Protein complementation assays identified FLI1 and ERG as GR binders and activators • EWS-FLI1, a fusion protein, drives Ewing sarcoma (ES) and binds with activated GR • Pharmacological inhibition of GR activation retards progression in ES animal models The single oncogene of Ewing sarcoma (ES), a childhood cancer, encodes a FLI1 fusion protein. Srivastava et al. report physical interactions between the fusion protein and the glucocorticoid receptor. Drug-induced inhibition of these interactions retards the progression of ES in mouse models. [ABSTRACT FROM AUTHOR]

The unfolded protein response plays an evolutionarily conserved role in homeostasis, and its dysregulation often leads to human disease, including diabetes and cancer. IRE1a is a major transducer that conveys endoplasmic reticulum stress via biochemical signals, yet major gaps persist in our understanding of how the detection of stress is converted to one of several molecular outcomes. It is known that, upon sensing unfolded proteins via its endoplasmic reticulum luminal domain, IRE1a dimerizes and then oligomerizes (often visualized as clustering). Once assembled, the kinase domain trans-autophosphorylates a neighboring IRE1a, inducing a conformational change that activates the RNase effector domain. However, the full details of how the signal is transmitted are not known. Here, we describe a previously unrecognized role for helix aK, located between the kinase and RNase domains of IRE1a, in conveying this critical conformational change. Using constructs containing mutations within this interdomain helix, we show that distinct substitutions affect oligomerization, kinase activity, and the RNase activity of IRE1a differentially. Furthermore, using both biochemical and computational methods, we found that different residues at position 827 specify distinct conformations at distal sites of the protein, such as in the RNase domain. Of importance, an RNase-inactive mutant, L827P, can still dimerize with wildtype monomers, but this mutation inactivates the wildtype molecule and renders leukemic cells more susceptible to stress. We surmise that helix aK is a conduit for the activation of IRE1a in response to stress. [ABSTRACT FROM AUTHOR]