The Keap1-Nrf2 system is central for mammalian cytoprotection against various stresses and a drug target for disease prevention and treatment. One model for the molecular mechanisms leading to Nrf2 activation is the Hinge-Latch model, where the DLGex-binding motif of Nrf2 dissociates from Keap1 as a latch, while the ETGE motif remains attached to Keap1 as a hinge. To overcome the technical difficulties in examining the binding status of the two motifs during protein-protein interaction (PPI) simultaneously, we utilized NMR spectroscopy titration experiments. Our results revealed that latch dissociation is triggered by low-molecular-weight Keap1-Nrf2 PPI inhibitors and occurs during p62-mediated Nrf2 activation, but not by electrophilic Nrf2 inducers. This study demonstrates that Keap1 utilizes a unique Hinge-Latch mechanism for Nrf2 activation upon challenge by non-electrophilic PPI-inhibiting stimuli, and provides critical insight for the pharmacological development of next-generation Nrf2 activators targeting the Keap1-Nrf2 PPI.

No abstract

Recurrent laryngeal nerve palsy is a complication that should be avoided but does not seem to be severe enough to affect patient survival after surgery. Although bilateral recurrent laryngeal nerve lymph node dissection can induce recurrent laryngeal nerve palsy in patients who undergo transthoracic oesophagectomy, this procedure did not correlate with aspiration and pneumonia.

A microwave dielectric measurement was performed to study the hydration properties of proteins in solution with a precision network analyzer with high reproducibility within the errors of 0.02 in relative dielectric constant over 2 to 10 GHz. A measurement was carried out for catalase, chymotrypsinogen A, cytochrome C, hemoglobin, peroxidase, lysozyme, myoglobin, ovalbumin, and bovine serum albumin at 20.0 ( 0.01 °C. The hydration properties of protein molecules were evaluated based on the Wagner mixture theory combined with single Debye approximation to the complex dielectric constant of hydrated solutes in the above frequency range. This was used to evaluate the loosely bound water number N w from the single Debye fitting, which gives the relaxation frequency (f c ) of the hydration shell loosely bound and tightly bound water number N s with lower relaxation frequencies than f c . Those hydration numbers were compared with the monolayer water numbers accessible to hydrophobic and hydrophilic exposed atoms, respectively, obtained from the calculation of accessible surface area of each protein structure based on the protein database. The result showed a good agreement both in the ratio and numbers. * To whom correspondence should be addressed. ap * ) a *{2(1φ) a * + (1 + 2 φ) q *}/{(2 + φ) a * +(1φ) q *} (1)

The quantitative sensitivity and dynamic range of conventional immunohistochemistry (IHC) with 3,3′-diaminobenzidine (IHC-DAB) used in pathological diagnosis in hospitals are poor, because enzyme activity can affect the IHC-DAB chromogenic reaction. Although fluorescent IHC can effectively increase the quantitative sensitivity of conventional IHC, tissue autofluorescence interferes with the sensitivity. Here, we created new fluorescent nanoparticles called phosphor-integrated dots (PIDs). PIDs have 100-fold greater brightness and a more than 300-fold greater dynamic range than those of commercially available fluorescent nanoparticles, quantum dots, whose fluorescence intensity is comparable to tissue autofluorescence. Additionally, a newly developed image-processing method enabled the calculation of the PID particle number in the obtained image. To quantify the sensitivity of IHC using PIDs (IHC-PIDs), the IHC-PIDs method was compared with fluorescence-activated cell sorting (FACS), a method well suited for evaluating total protein amount, and the two values exhibited strong correlation (R = 0.94). We next applied IHC-PIDs to categorize the response to molecular target-based drug therapy in breast cancer patients. The results suggested that the PID particle number estimated by IHC-PIDs of breast cancer tissues obtained from biopsy before chemotherapy can provide a score for predicting the therapeutic effect of the human epidermal growth factor receptor 2-targeted drug trastuzumab.