Structural basis for Ccd1 auto-inhibition in the Wnt pathway through homomerization of the DIX domain

Wnt signaling plays an important role in governing cell fate decisions. Coiled-coil-DIX1 (Ccd1), Dishevelled (Dvl), and Axin are signaling proteins that regulate the canonical pathway by controlling the stability of a key signal transducer β-catenin. These proteins contain the DIX domain with a ubiquitin-like fold, which mediates their interaction in the β-catenin destruction complex through dynamic head-to-tail polymerization. Despite high sequence similarities, mammalian Ccd1 shows weaker stimulation of β-catenin transcriptional activity compared with zebrafish (z) Ccd1 in cultured cells. Here, we show that the mouse (m) Ccd1 DIX domain displays weaker ability for homopolymerization than that of zCcd1. Furthermore, X-ray crystallographic analysis of mCcd1 and zCcd1 DIX domains revealed that mCcd1 was assembled into a double-helical filament by the insertion of the β1-β2 loop into the head-to-tail interface, whereas zCcd1 formed a typical single-helical polymer similar to Dvl1 and Axin. The mutation in the contact interface of mCcd1 double-helical polymer changed the hydrodynamic properties of mCcd1 so that it acquired the ability to induce Wnt-specific transcriptional activity similar to zCcd1. These findings suggest a novel regulatory mechanism by which mCcd1 modulates Wnt signaling through auto-inhibition of dynamic head-to-tail homopolymerization

Rapid HER2 cytologic fluorescence in situ hybridization for breast cancer using noncontact alternating current electric field mixing

Background: Human epidermal growth factor receptor 2-in situ hybridization (HER2-ISH) is widely approved for diagnostic, prognostic biomarker testing of formalin- fixed paraffin-embedded tissue blocks. However, cytologic ISH analysis has a potential advantage in tumor samples such as pleural effusion and ascites that are difficult to obtain the histological specimens. Our aim was to evaluate the clinical reliability of a novel rapid cytologic HER2 fluorescence ISH protocol (rapid-CytoFISH). Materials and Methods: Using a new device, we applied a high-voltage/frequency, noncontact alternating current electric field to tissue imprints and needle rinses, which mixed the probe within microdroplets as the voltage was switched on and off (AC mixing). Cytologic samples (n = 143) were collected from patients with immunohistochemically identified HER2 breast cancers. The specimens were then tested using standard dual-color ISH using formalin-fixed paraffin-embedded tissue (FFPE-tissue DISH) for HER2-targeted therapies, CytoFISH, and rapid-CytoFISH (completed within 4 h). Results: All 143 collected cytologic specimens (50 imprinted cytology specimens from resected tumors and 93 liquid-based cytology specimens from needle rinses) were suitable for FISH analysis. The HER2/chromosome enumeration probe (CEP) 17 ratios did not significantly differ between FFPE-tissue DISH and either CytoFISH protocol. Based on HER2 scoring criteria, we found 95.1% agreement between FFPEtissue DISH and CytoFISH (Cohen\u27s kappa coefficient = 0.771 and 95% confidence interval (CI): 0.614–0.927). Conclusion: CytoFISH could potentially serve as a clinical tool for prompt determination of HER2 status in breast cancer cytology. Rapid-CytoFISH with AC mixing will enable cancer diagnoses and HER2 status to be determined on the same day a patient comes to a clinic or hospital

The adaptor protein p40(phox) as a positive regulator of the superoxide-producing phagocyte oxidase

Activation of the superoxide-producing phagocyte NADPH oxidase, crucial in host defense, requires the cytosolic proteins p67(phox) and p47(phox). They translocate to the membrane upon cell stimulation and activate flavocytochrome b(558), the membrane-integrated catalytic core of this enzyme system. The activators p67(phox) and p47(phox) form a ternary complex together with p40(phox), an adaptor protein with unknown function, comprising the PX/PB2, SH3 and PC motif- containing domains: p40(phox) associates with p67(phox) via binding of the p40(phox) PC motif to the p67(phox) PB1 domain, while p47(phox) directly interacts with p67(phox) but not with p40(phox). Here we show that p40(phox) enhances membrane translocation of p67(phox) and p47(phox) in stimulated cells, which leads to facilitated production of superoxide. The enhancement cannot be elicited by a mutant p40(phox) carrying the D289A substitution in PC or a p67(phox) with the K355A substitution in PB1, each being defective in binding to its respective partner. Thus p40(phox) participates in activation of the phagocyte oxidase by regulating membrane recruitment of p67(phox) and p47(phox) via the PB1–PC interaction with p67(phox)

なぜB氏は特別支援学校教員として定年退職まで働き続けることができたのか : 複線径路等至性モデリング（TEM）による分析

The purpose of this study was to focus on Mr. B, a man in his 70s who worked as a teacher at a special needs school until retirement, and to identify the process by which he worked without considering an earlier retirement. By clarifying this, we can examine Mr. B’s experiences since he was a student himself, which have changed his sense of self and why he was able to work for so long. The study used semi-structured interviews followed by trajectory equifinality modeling to analyze the data. We identified three factors that enabled Mr. B to work as a special-needs teacher without thinking about early retirement. First, he chose an academic field in which he could contribute to society, rather than a field based solely on his interests. Second, for Mr. B, fully demonstrating his values was linked to his desire to work. Third, after becoming a special-needs teacher, he was able to maintain an environment in which he was continuously involved with children and their parents, and his work always left him feeling that he was performing a service of value. This has allowed him to see any unforeseen events that happened to his students as his own concern, rather than as a daunting setback. He also enjoyed watching children grow up and continued to support them and their parents as a volunteer after retiring

Expression, purification and characterization of hepatitis B virus X protein BH3-like motif-linker-Bcl-xL fusion protein for structural studies

Hepatitis B virus X protein (HBx) is a multifunctional protein that interacts directly with many host proteins. For example, HBx interacts with anti-apoptotic proteins, Bcl-2 and Bcl-xL, through its BH3-like motif, which leads to elevated cytosolic calcium levels, efficient viral DNA replication and the induction of apoptosis. To facilitate sample preparation and perform detailed structural characterization of the complex between HBx and Bcl-xL, we designed and purified a recombinant HBx BH3-like motif-linker-Bcl-xL fusion protein produced in E. coli. The fusion protein was characterized by size exclusion chromatography, circular dichroism and nuclear magnetic resonance experiments. Our results show that the fusion protein is a monomer in aqueous solution, forms a stable intramolecular complex, and likely retains the native conformation of the complex between Bcl-xL and the HBx BH3-like motif. Furthermore, the HBx BH3-like motif of the intramolecular complex forms an α-helix. These observations indicate that the fusion protein should facilitate structural studies aimed at understanding the interaction between HBx and Bcl-xL at the atomic level

Observation of Protein and Lipid Membrane Structures in a Model Mimicking the Molecular Crowding Environment of Cells Using Neutron Scattering and Cell Debris

The interior of living cells is a molecular-crowding environment, where large quantities of various molecules coexist. Investigations into the nature of this environment are essential for an understanding of both the elaborate biological reactions and maintenance of homeostasis occurring therein. The equilibrium states of biological macromolecular systems are affected by molecular crowding environments unmatched by in vitro diluted environments; knowledge about crowding effects is still insufficient due to a lack of relevant experimental studies. Recent developments in the techniques of in-cell NMR and large-scale molecular dynamics simulation are providing new insights into the structure and dynamics of biological molecules inside cells. This study focused on a new experimental technique to directly observe the structure of a specific protein or membrane in condensed crowder solutions using neutron scattering. Deuterated whole-cell debris was used to reproduce an environment that more closely mimics the interior of living cells than models used previously. By the reduction of the background-scattering from large amounts of cell debris, we successfully extracted structure information for both a small globular protein and a small unilamellar vesicle (SUV) from the concentrated cell-debris solution up to a weight ratio of 1/60 for protein/crowder and 1/40 for SUV/crowder