In silico methods in enzyme screening and gene expression

INTMSAlign is a software to assign consensus residues of target protein utilizing large amount of their family sequences. We generated three protein sequences with S-selective hydroxynitrile lyase (S-HNL) activity, which we call designed S-HNLs; these proteins folded as efficiently as the native S-HNL (1). a-Amino-e-caprolactam (ACL) racemase from Achromobacter obae has been shown to be an effective catalyst for the dynamic kinetic resolution of amino acid amide and a-aminonitriles to form chiral amino acids. We searched for ACL racemase in silico with INTMSAlign software. By fixing Lys 241 as one of the key residues, we discovered thirteen ACL racemase genes from 413 fold type-I PLP genes (2). Insolubility of proteins expressed in Escherichia coli expression hinders the progress of both basic and applied research. Insoluble proteins contain residues that decrease their solubility (aggregation hotspots). We discovered a phenomenon of soluble expression of HNL from Manihot esculenta, in E. coli. By random mutagenesis, we found that a single point mutation H103L, and mutation with alterations at three positions (Lys-Pro mutations at positions 176, 199 and 224) cause total solubility in E. coli even when grown at 37°C (3). If a relationship between soluble expression and mutation points could be established, it will become very easy to generate a mutant for correctly folded expression in E. coli. Using a combination of approaches involving directed evolution and primary sequence analysis, we found two rules of thumb to help identify hotspots: one focuses on the hydrophobicity of amino acids in the a-helix structure, and another one focuses the difference in hydrophobicity relative to the corresponding amino acid in the consensus protein. Using these two relationships together, we succeeded in developing methods to improve the solubility of expressed proteins in E. coli (4). References: (1) S. Nakano and Y. Asano, Sci. Rep., 5, 8193 (2015). (2) W. Payoungkiattikun, S. Okazaki, S. Nakano, A. Ina, A. H-Kittikun, and Y. Asano, Appl. Biochem. Biotechnol., 176 (5), 1303-1314 (2015). (3) Y. Asano, M. Dadashipour, M. Yamazaki, N. Doi, and H. Komeda. Prot. Eng. Des. Sel., 24 (8), 607-616 (2011). (4) D. Matsui, S. Nakano, M. Dadashipour, and Y. Asano, submitted

Reconstruction of ancestral L-amino acid oxidases to broaden substrate selectivity

Characteristic functions of enzymes, such as high thermal stability and substrate specificity, are attained during the evolutionary process. Ancestral sequence reconstruction (ASR) is applied to infer the process by designing artificial enzymes which are located on ancestral node of phylogenetic tree; here, the inferred enzymes called ancestral enzymes. Ancestral enzymes often exhibit substrate promiscuity and high thermal stability of which functions are suitable to perform enzyme engineering. In addition, applicability of the ASR is high because the method requires only sequence data to design ancestral enzymes. Thus, we believe that artificial enzymes contributing to progress in enzyme engineering can be designed by ASR. Please click Additional Files below to see the full abstract

Proliferation and cell death of human glioblastoma cells after carbon-ion beam exposure: Morphologic and morphometric analyses

Histological analyses of glioblastoma cells after carbon-ion exposure are still limited and ultrastructural characteristics have not been investigated in detail. Here we report the results of morphological and morphometric analyses of a human glioblastoma cell line, CGNH-89, after ionizing radiation to characterize the effect of a carbon-beam on glioblastoma cells. Using CGNH-89 cells exposed to 0–10 Gy of X-ray (140kVp) or carbon-ions (18.3 MeV/nucleon, LET = 108 keV/μm), we performed conventional histology and immunocytochemistry with MIB-1 antibody, transmission electron microscopy, and computer-assisted, nuclear size measurements. CGNH-89 cells with a G to A transition in codon 280 in exon 8 of the TP53 gene had nuclei with pleomorphism, marked nuclear atypia and brisk mitotic activity. After carbon-ion and X-ray exposure, living cells showed decreased cell number, nuclear condensation, increased atypical mitotic figures, and a tendency of cytoplasmic enlargement at the level of light microscopy. The deviation of the nuclear area size increased during 48 hours after irradiation, while the small cell fraction increased in 336 hours. In glioblastoma cells of the control, 5 Gy carbon-beam, and 10 Gy carbon-beam, and MIB-1 labeling index decreased in 24 hours (12%, 11%, 7%, respectively) but increased in 48 hours (10%, 20%, 21%, respectively). Ultrastructurally, cellular enlargement seemed to depend on vacuolation, swelling of mitochondria, and increase of cellular organelles, such as the cytoskeleton and secondary lysosome. We could not observe apoptotic bodies in the CGNH-89 cells under any conditions. We conclude that carbon-ion irradiation induced cell death and senescence in a glioblastoma cell line with mutant TP53. Our results indicated that the increase of large cells with enlarged and bizarre nuclei, swollen mitochondria, and secondary lysosome occurred in glioblastoma cells after carbon-beam exposure.学位記番号：医博甲1096, 学位の種類：博士（医）, 学位授与年月日：平成20年3月25

AirID, a novel proximity biotinylation enzyme, for analysis of protein–protein interactions

Proximity biotinylation based on Escherichia coli BirA enzymes such as BioID (BirA*) and TurboID is a key technology for identifying proteins that interact with a target protein in a cell or organism. However, there have been some improvements in the enzymes that are used for that purpose. Here, we demonstrate a novel BirA enzyme, AirID (ancestral BirA for proximity-dependent biotin identification), which was designed de novo using an ancestral enzyme reconstruction algorithm and metagenome data. AirID-fusion proteins such as AirID-p53 or AirID-IκBα indicated biotinylation of MDM2 or RelA, respectively, in vitro and in cells, respectively. AirID-CRBN showed the pomalidomide-dependent biotinylation of IKZF1 and SALL4 in vitro. AirID-CRBN biotinylated the endogenous CUL4 and RBX1 in the CRL4CRBN complex based on the streptavidin pull-down assay. LC-MS/MS analysis of cells that were stably expressing AirID-IκBα showed top-level biotinylation of RelA proteins. These results indicate that AirID is a novel enzyme for analyzing protein–protein interactions

Structural implication for the impaired binding of W150A mutant LOX-1 to oxidized low density lipoprotein, OxLDL

Lectin-like oxidized lipoprotein (OxLDL) receptor 1, LOX-1, is the major OxLDL receptor expressed on vascular endothelial cells. We have previously reported the ligand-recognition mode of LOX-1 based on the crystal structure of the ligand binding domain (C-type lectin-like domain, CTLD) and surface plasmon resonance analysis, which suggested that the functional significance of the CTLD dimer (the 'canonical' dimer) is to harbor the characteristic "basic spine" on its surface. In this study, we have identified the key inter-domain interactions in retaining the canonical CTLD dimer by X-ray structural analysis of the inactive mutant W150A CTLD. The canonical CTLD dimer forms through tight hydrophobic interactions, in which W150 engages in a lock-and-key manner and represents the main interaction. The loss of the Trp ring by mutation to Ala prevents the formation of the canonical dimer, as elucidated from docking calculations using the crystal structure of W150A CTLD. The results emphasize that the canonically formed CTLD dimer is essential for LOX-1 to bind to OxLDL, which supports our proposed view that the basic spine surface present in the correctly formed dimer plays a primal role in OxLDL recognition. This concept provides insight into the pathogenic pattern recognized by LOX-1 as a member of the pattern recognition receptors

Near-IR imaging polarimetry toward a bright-rimmed cloud: Magnetic field in SFO 74

We have made near-infrared (JHKs) imaging polarimetry of a bright-rimmed cloud (SFO 74). The polarization vector maps clearly show that the magnetic field in the layer just behind the bright rim is running along the rim, quite different from its ambient magnetic field. The direction of the magnetic field just behind the tip rim is almost perpendicular to that of the incident UV radiation, and the magnetic field configuration appears to be symmetric as a whole with respect to the cloud symmetry axis. We estimated the column and number densities in the two regions (just inside and far inside the tip rim) and then derived the magnetic field strength, applying the Chandrasekhar-Fermi method. The estimated magnetic field strength just inside the tip rim, ~90 ?G, is stronger than that far inside, ~30 ?G. This suggests that the magnetic field strength just inside the tip rim is enhanced by the UV-radiation-induced shock. The shock increases the density within the top layer around the tip and thus increases the strength of the magnetic field. The magnetic pressure seems to be comparable to the turbulent one just inside the tip rim, implying a significant contribution of the magnetic field to the total internal pressure. The mass-to-flux ratio was estimated to be close to the critical value just inside the tip rim. We speculate that the flat-topped bright rim of SFO 74 could be formed by the magnetic field effect

Application of First-Principles-Based Artificial Neural Network Potentials to Multiscale-Shock Dynamics Simulations on Solid Materials

The use of artificial neural network (ANN) potentials trained with first-principles calculations has emerged as a promising approach for molecular dynamics (MD) simulations encompassing large space and time scales while retaining first-principles accuracy. To date, however, the application of ANN-MD has been limited to near-equilibrium processes. Here we combine first-principles-trained ANN-MD with multiscale shock theory (MSST) to successfully describe far-from-equilibrium shock phenomena. Our ANN-MSST-MD approach describes shock-wave propagation in solids with first-principles accuracy but a 5000 times shorter computing time. Accordingly, ANN-MD-MSST was able to resolve fine, long-time elastic deformation at low shock speed, which was impossible with first-principles MD because of the high computational cost. This work thus lays a foundation of ANN-MD simulation to study a wide range of far-from-equilibrium processes