Catalytic residues and substrate specificity of scytalidoglutamic peptidase, the first member of the eqolisin in family (G1) of peptidases

Scytalidoglutamic peptidase (SGP) is the first-discovered member of the eqolisin family of peptidases with a unique structure and a presumed novel catalytic dyad (E136 and Q53) [Fujinaga et al., PNAS 101 (2004) 3364-3369]. Mutants of SGP, E136A, Q53A, and Q53E lost both the autoprocessing and enzymatic activities of the wild-type enzyme. Coupled with the results from the structural analysis of SGP, Glu136 and Gln53 were identified as the catalytic residues. The substrate specificity of SGP is unique, particularly, in the preference at the P3 (basic amino acid), P1' (small a.a.), and P3' (basic a.a.) positions. Superior substrates and inhibitors have been synthesized for kinetic studies based on the results reported here. kcat, Km, and kcat/Km of SGP for D-Dap(MeNHBz)-GFKFF*ALRK(Dnp)-D-R-D-R were 34.8 s-1, 0.065 microM, and 535 microM-1 s-1, respectively. Ki of Ac-FKF-(3S,4S)-phenylstatinyl-LR-NH2 for SGP was 1.2x10(-10) M. Taken together, we can conclude that SGP has not only structural and catalytic novelties but also a unique subsite structure.     

Effect of conformational states on protein dynamical transition

In order to examine the properties specific to the folded protein, the effect of the conformational states on protein dynamical transition was studied by incoherent elastic neutron scattering for both wild type and a deletion mutant of staphylococcal nuclease. The deletion mutant of SNase which lacks C-terminal 13 residues takes a compact denatured structure, and can be regarded as a model of intrinsic unstructured protein. Incoherent elastic neutron scattering experiments were carried out at various temperature between 10 K and 300 K on IN10 and IN13 installed at ILL. Temperature dependence of mean-square displacements was obtained by the q-dependence of elastic scattering intensity. The measurements were performed on dried and hydrated powder samples. No significant differences were observed between wild type and the mutant for the hydrated samples, while significant differences were observed for the dried samples. A dynamical transition at ∼ 140 K observed for both dried and hydrated samples. The slopes of the temperature dependence of MSD before transition and after transition are different between wild type and the mutant, indicating the folding induces hardening. The hydration water activates a further transition at ∼ 240 K. The behavior of the temperature dependence of MSD is indistinguishable for wild type and the mutant, indicating that hydration water dynamics dominate the dynamical properties.     

Local mechanical properties measured by atomic force microscopy for cultured bovine endothelial cells exposed to shear stress

Morphology and mechanical properties of cultured endothelial cells were measured, using a novel atomic force microscope (AFM) system, developed in our laboratory, in conjunction with an inverted confocal laser scanning microscope. We used this system to examine endothelial cell both in static cultures and exposed to a shear stress of 2 Pa. Initially, the three-dimensional topography of a cell was measured by the AFM and a location was selected for the subsequent measurement of the mechanical response of the cell. The surface of statically cultured cell was smooth. The cell height was not altered by the exposed duration of shear stress. A relationship between external force, F, and the indentation depth, delta, was obtained for several different locations on a cell. This force-indentation response was modelled using a quadratic equation, F = adelta2 + bdelta, indicating that two parameters, a and b, will be constants which are representative of the mechanical response. Endothelial cells cultured at static conditions demonstrated a polygonal shape and less stiff mechanical characteristics around the nucleus compared to those at peripheral regions. The stiffness of the endothelial cells exposed to shear stress increased with the duration time of exposure. At 6-h exposures, the stiffness was higher at upstream side of the cell than the downstream side. However, after 24-h exposure, the stiffness was similar on both sides of the cell. These changes in the stiffness of endothelial cells when exposed to shear stress were suggested to correspond with the distribution of stress fibers in the cell.     

Stimulation of Ras guanine nucleotide exchange activity of Ras-GRF1/CDC25(Mm) upon tyrosine phosphorylation by the Cdc42-regulated kinase ACK1

Ras-GRF1 is a brain-specific guanine nucleotide exchange factor (GEF) for Ras, whose activity is regulated in response to Ca(2+) influx and G protein-coupled receptor signals. In addition, Ras-GRF1 acts as a GEF for Rac when tyrosine-phosphorylated following G protein-coupled receptor stimulation. However, the mechanisms underlying the regulation of Ras-GRF1 functions remain incompletely understood. We show here that activated ACK1, a nonreceptor tyrosine kinase that belongs to the focal adhesion kinase family, causes tyrosine phosphorylation of Ras-GRF1. On the other hand, kinase-deficient ACK1 exerted no effect. GEF activity of Ras-GRF1 toward Ha-Ras, as defined by in vitro GDP binding and release assays, was augmented after tyrosine phosphorylation by ACK1. In contrast, GEF activity toward Rac1 remained latent, implying that ACK1 does not represent a tyrosine kinase that acts downstream of G protein-coupled receptors. Consistent with enhanced Ras-GEF activity, accumulation of the GTP-bound form of Ras within the cell was shown through the use of Ras-binding domain pull-down assays. Furthermore, Ras-dependent activation of ERK2 by Ras-GRF1 was enhanced following co-expression of activated ACK1. These results implicate ACK1 as an upstream modulator of Ras-GRF1 and suggest a signaling cascade consisting of Cdc42, ACK1, Ras-GRF1, and Ras in neuronal cells.     

Inorganic ion compositions in brown algae,with special reference to sulfuric acid ion accumulations

Hydrobiologia - Cellular pH estimated from cell extract pH and the ion compositions of major inorganic ions (Na+, NH4 +, K+, Mg2+, Ca2+,Cl−, Br−, NO3 −, SO4 2−) were studied...     

The reversible change in the redox state of type I Cu in Myrothecium verrucaria bilirubin oxidase depending on pH

The redox state of type I Cu in Myrothecium verrucaria bilirubin oxidase (BO), a multicopper oxidase utilized in the clinical investigation of liver, is an equilibrium state of the oxidized and reduced forms, reflected in the reversible absorption and electron paramagnetic resonance (EPR) spectral changes depending on pH.     

Age-related decrease of miRNA-92a levels in human CD8+ T-cells correlates with a reduction of naïve T lymphocytes

MicroRNA (miR)-17-92a expression plays a crucial role in lymphocyte ontogeny. We therefore set out to determine miR-92a expression levels in peripheral blood lymphocytes from healthy subjects to ascertain any association between these levels and ageing. We found a positive correlation between the miR-92a expression level and the percentages of RO-CD8⁺CD27⁺ (P = 0.0046) and CD3⁺CD8⁺CD62L⁺ (P = 0.0011). This suggests that the majority of miR-92a of CD8⁺ T cells is derived from naïve cells, and the miR-92a expression level in CD8⁺ T cells declines progressively with age. These results indicate that the age-related attrition of naïve T cells is linked to a reduction of miR-92a in human T -lymphocytes. Therefore, we should careful attention when evaluating human miRNA levels in T lymphocytes to use normal control values.     

Improved synthesis with high yield and increased molecular weight of poly(alpha,beta-malic acid) by direct polycondensation

The development of synthetic biodegradable polymers, such as poly(alpha-hydroxy acid), is particularly important for constructing medical devices, including scaffolds and sutures, and has attracted growing interest in the biomedical field. Here, we report a novel approach to preparing high molecular weight poly(malic acid) (HMW--PMA) as a biodegradable and bioabsorbable water-soluble polymer. We investigated in detail the reaction conditions for the simple direct polycondensation of l-malic acid, including the reaction times, temperatures, and catalysts. The molecular weight of synthesized alpha,beta-PMA is dependent on both the reaction temperature and time. The optimum reaction condition to obtain alpha,beta-PMA by direct polycondensation using tin(II) chloride as a catalyst was thus determined to be 110 degrees C for 45 h with a molecular weight of 5300. The method for alpha,beta-PMA synthesis established here will facilitate production of alpha,beta-PMA of various molecular weights, which may have a potential utility as biomaterials.     

Identification and characterization of a novel alternative splice variant of mouse GMx33alpha/GPP34

We have cloned and characterized a novel splice variant of mouse GMx33alpha/Golgi-associated protein of 34 kDa (GPP34), hereby designated GMx33alphaV/GPP34V. This splice variant skips the second and third exons, and the resulting frame shift generates a stop codon in the fourth exon. GMx33alphaV/GPP34V is comprised of 81 amino acid residues derived from the N-terminal end of the full length protein and corresponds to approximately one-third of the full length GMx33alpha/GPP34 sequence with a calculated molecular mass of 8900. In contrast to GMx33alpha/GPP34 mRNA which is expressed at similar levels in various tissues, GMx33alphaV/GPP34V mRNA was differentially expressed when examined by RT-PCR. Compared to other tissues, skeletal muscle showed relatively strong expression of GMx33alphaV/GPP34V mRNA. This splice variant cDNA was also detected in a human cell line.     

Induced pluripotent stem cell‐derived melanocyte precursor cells undergoing differentiation into melanocytes

Induced pluripotent stem cell (iPSC) technology offers a novel approach for conversion of human primary fibroblasts into melanocytes. During attempts to explore various protocols for differentiation of iPSCs into melanocytes, we found a distinct and self‐renewing cell lineage that could differentiate into melanocytes, named as melanocyte precursor cells (MPCs). The MPCs exhibited a morphology distinctive from that of melanocytes, in lacking either the melanosomal structure or the melanocyte‐specific marker genes MITF, TYR, and SOX10. In addition, gene expression studies in the MPCs showed high‐level expression of WNT5A, ROR2, which are non‐canonical WNT pathway markers, and its related receptor TGFβR2. In contrast, MPC differentiation into melanocytes was achieved by activating the canonical WNT pathway using the GSK3β inhibitor. Our data demonstrated the distinct characteristic of MPCs' ability to differentiate into melanocytes, and the underlying mechanism of interfacing between canonical WNT signaling pathway and non‐canonical WNT signaling pathway.