Regulation of the tubulin polymerization-promoting protein by Ca2+/S100 proteins

To elucidate S100 protein-mediated signaling pathways, we attempted to identify novel binding partners for S100A2 by screening protein arrays carrying 19,676 recombinant glutathione S-transferase (GST)-fused human proteins with biotinylated S100A2. Among newly discovered putative S100A2 interactants, including TMLHE, TRH, RPL36, MRPS34, CDR2L, OIP5, and MED29, we identified and characterized the tubulin polymerization-promoting protein (TPPP) as a novel S100A2-binding protein. We confirmed the interaction of TPPP with Ca2+/S100A2 by multiple independent methods, including the protein array method, S100A2 overlay, and pulldown assay in vitro and in transfected COS-7 cells. Based on the results from the S100A2 overlay assay using various GST-TPPP mutants, the S100A2-binding region was identified in the C-terminal (residues 111-160) of the central core domain of a monomeric form of TPPP that is involved in TPPP dimerization. Chemical cross-linking experiments indicated that S100A2 suppresses dimer formation of His-tagged TPPP in a dosedependent and a Ca2+-dependent manner. In addition to S100A2, TPPP dimerization is disrupted by other multiple S100 proteins, including S100A6 and S100B, in a Ca2+-dependent manner but not by S100A4. This is consistent with the fact that S100A6 and S100B, but not S100A4, are capable of interacting with GST-TPPP in the presence of Ca2+. Considering these results together, TPPP was identified as a novel target for S100A2, and it is a potential binding target for other multiple S100 proteins, including S100A6 and S100B. Direct binding of the S100 proteins with TPPP may cause disassembly of TPPP dimer formation in response to the increasing concentration of intracellular Ca2+, thus resulting in the regulation of the physiological function of TPPP, such as microtubule organization

Fundamental physics activities with pulsed neutron at J-PARC(BL05)

"Neutron Optics and Physics (NOP/ BL05)" at MLF in J-PARC is a beamline for studies of fundamental physics. The beamline is divided into three branches so that different experiments can be performed in parallel. These beam branches are being used to develop a variety of new projects. We are developing an experimental project to measure the neutron lifetime with total uncertainty of 1 s (0.1%). The neutron lifetime is an important parameter in elementary particle and astrophysics. Thus far, the neutron lifetime has been measured by several groups; however, different values are obtained from different measurement methods. This experiment is using a method with different sources of systematic uncertainty than measurements conducted to date. We are also developing a source of pulsed ultra-cold neutrons (UCNs) produced from a Doppler shifter are available at the unpolarized beam branch. We are developing a time focusing device for UCNs, a so called "rebuncher", which can increase UCN density from a pulsed UCN source. At the low divergence beam branch, an experiment to search an unknown intermediate force with nanometer range is performed by measuring the angular dependence of neutron scattering by noble gases. Finally the beamline is also used for the research and development of optical elements and detectors. For example, a position sensitive neutron detector that uses emulsion to achieve sub-micrometer resolution is currently under development. We have succeeded in detecting cold and ultra-cold neutrons using the emulsion detector.Comment: 9 pages, 5 figures, Proceedings of International Conference on Neutron Optics (NOP2017

Rapid Generation of Monoclonal Antibodies from Single B Cells by Ecobody Technology

Single B cell sampling following to direct gene amplification and transient expression in animal cells has been recognized as powerful monoclonal antibodies (mAbs) screening strategies. Here we report Ecobody technology which allows mAbs screening from single B cells in two days This technology uses Escherichia coli cell-free protein synthesis (CFPS) for mAb expression. In the CFPS step, we employed our original techniques: (1) ‘Zipbody’ as a modified Fab (fragment of antigen binding) format, in which the active Fab formation is facilitated by adhesive leucine zipper peptides fused at the C-termini of the light and heavy chains; and (2) an N-terminal SKIK peptide tag that can markedly increase protein production. By the Ecobody technology, we demonstrated rapid screening of antigen specific mAbs from immunized rabbits and Epstein-Barr Virus infected human B cells. We further obtained rabbit mAbs in E. coli expression system yielding to 8.5 mg of purified proteins from 1 L bacterial culture

De Novo Macrocyclic Peptide Inhibitors of Hepatitis B Virus Cellular Entry

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