Crystal structure of the serine protease domain of prophenoloxidase activating factor-I

A family of serine proteases (SPs) mediates the proteolytic cascades of embryonic development and immune response in invertebrates. These proteases, called easter-type SPs, consist of clip and chymotrypsin-like SP domains. The SP domain of easter-type proteases differs from those of typical SPs in its primary structure. Herein, we report the first crystal structure of the SP domain of easter-type proteases, presented as that of prophenoloxidase activating factor (PPAF)-I in zymogen form. This structure reveals several important structural features including a bound calcium ion, an additional loop with a unique disulfide linkage, a canyon-like deep active site, and an exposed activation loop. We subsequently show the role of the bound calcium and the proteolytic susceptibility of the activation loop, which occurs in a clip domain-independent manner. Based on biochemical study in the presence of heparin, we suggest that PPAF-III, highly homologous to PPAF-I, contains a surface patch that is responsible for enhancing the catalytic activity through interaction with a nonsubstrate region of a target protein. These results provide insights into an activation mechanism of easter-type proteases in proteolytic cascades, in comparison with the well studied blood coagulation enzymes in mammals.     

Poliovirus entry into human brain microvascular cells requires receptor-induced activation of SHP-2

Viruses use specific receptor molecules to bind selectively to target cells. Receptors have often been considered as mere docking sites, but they may also possess intrinsic signaling capacities that serve to prime the cell for entry and infection. Poliovirus (PV) initiates infection by binding to the PV receptor (PVR) and causes paralytic poliomyelitis by replicating within motor neurons of the brain and spinal cord. We have examined the process by which PV enters cultured human brain microvascular endothelial cells (HBMEC), an in vitro model of the blood-brain barrier. We found that PV enters HBMEC by dynamin-dependent caveolar endocytosis, and that entry depends on intracellular signals triggered by virus attachment to PVR. Tyrosine kinase and RhoA GTPase activation initiated by PVR ligation were both essential. Virus attachment also induced tyrosine phosphorylation of PVR; this permitted the association of PVR with SHP-2, a protein tyrosine phosphatase whose activation was required for entry and infection. The results indicate that receptor-induced signals promote virus entry and suggest a role for tyrosine phosphatases in viral pathogenesis.     

Central beta-amyloid peptide-induced peripheral interleukin-6 responses in mice

beta-Amyloid peptides (Abetas) share with lipopolysaccharide, a potent pro-inflammatory agent, the property of stimulating glial cells or macrophages to induce various inflammatory mediators. We recently reported that central administration of lipopolysaccharide induces peripheral interleukin-6 responses via both the central and peripheral norepinephrine system. In this study, the effect of intracerebroventricular injection of various synthetic Abetas on plasma interleukin-6 levels was examined in mice. Abeta(1-42) dose-dependently increased plasma interleukin-6 levels: 'aged' Abeta(1-42) was more effective than fresh, whereas Abeta(42-1) had no effect. 'Aged' Abeta(1-42) (205 pmol/mouse i.c.v.)-induced plasma interleukin-6 peaked at 2 h post injection, which is earlier than the peak time of the Abeta(1-42)-induced brain interleukin-6, tumor necrosis factor-alpha and interleukin-1beta levels, which was 4, 4 and 24 h, respectively. Among various peripheral organs, Abeta(1-42) (205 pmol/mouse i.c.v.) significantly increased interleukin-6 mRNA expression in lymph nodes and liver. Abeta(1-42) (205 pmol/mouse i.c.v.) significantly increased norepinephrine turnover in both hypothalamus and spleen. Either central or peripheral norepinephrine depletion effectively inhibited the Abeta(1-42)-induced peripheral interleukin-6 response. Pretreatment with prazosin (alpha(1)-adrenergic antagonist), yohimbine (alpha(2)-adrenergic antagonist), and ICI-118,551 (beta(2)-adrenergic antagonist), but not with betaxolol (beta(1)-adrenergic antagonist), inhibited Abeta(1-42)-induced plasma interleukin-6 levels. These results demonstrate that centrally administered Abeta(1-42) effectively induces the systemic interleukin-6 response which is mediated, in part, by central Abeta(1-42)-induced activation of the central and the peripheral norepinephrine systems.     

An apoA-I mimetic peptibody generates HDL-like particles and increases alpha-1 HDL subfraction in mice

The aim of this study is to investigate the capability of an apoA-I mimetic with multiple amphipathic helices to form HDL-like particles in vitro and in vivo. To generate multivalent helices and to track the peptide mimetic, we have constructed a peptibody by fusing two tandem repeats of 4F peptide to the C terminus of a murine IgG Fc fragment. The resultant peptidbody, mFc-2X4F, dose-dependently promoted cholesterol efflux in vitro, and the efflux potency was superior to monomeric 4F peptide. Like apoA-I, mFc-2X4F stabilized ABCA1 in J774A.1 and THP1 cells. The peptibody formed larger HDL particles when incubated with cultured cells compared with those by apoA-I. Interestingly, when administered to mice, mFc-2X4F increased both pre-β and α-1 HDL subfractions. The lipid-bound mFc-2X4F was mostly in the α-1 migrating subfraction. Most importantly, mFc-2X4F and apoA-I were found to coexist in the same HDL particles formed in vivo. These data suggest that the apoA-I mimetic peptibody is capable of mimicking apoA-I to generate HDL particles. The peptibody and apoA-I may work cooperatively to generate larger HDL particles in vivo, either at the cholesterol efflux stage and/or via fusion of HDL particles that were generated by the peptibody and apoA-I individually.     

Molecular characterization of flavonol synthase from poplar and its application to the synthesis of 3-<Emphasis Type="Italic">O</Emphasis>-methylkaempferol

Biosynthesis of flavonoid derivatives requires enzyme(s) having high reactivity as well as regioselectivity. We have synthesized 3-O-kaempferol from naringenin using two enzymes. The first reaction, in which naringenin is converted to kaempferol, is mediated by flavonol synthase (FLS). An FLS (PFLS) with strong catalytic activity was cloned and characterized from the genome sequence of the poplar (Populus deltoides). PFLS consists of a 1,008 bp ORF encoding a 38 kDa protein. PFLS was expressed in Escherichia coli with a glutathione-S-transferase (GST) tagging. The purified recombinant PFLS was characterized. Catalytically, it was more efficient than the previously characterized FLSs. A mixture of two E. coli transformants harboring either PFLS or ROMT9 (a kaempferol 3-O-methyltransferase) converted naringenin into 3-O-methylkaempferol.     

Solid phase synthesis of the proteinase of bovine leukemia virus. Comparison of its specificity to that of HIV-2 proteinase.

The 126-residue proteinase (PR) of bovine leukemia virus (BLV) was synthesized by solid-phase peptide synthesis and its activity was shown using various oligopeptide substrates representing cleavage sites in BLV, human T-cell leukemia virus type 1 (HTLV-1), murine leukemia virus (MuLV) and human immunodeficiency virus type 1 (HIV-1). The specificity of the BLV PR was also compared to that of chemically synthesized human immunodeficiency virus type 2 (HIV-2) PR. Many of the peptides were cleaved at the expected site, however, 6 out of 15 were hydrolyzed only by one of the PRs. Furthermore, one BLV peptide was processed differently by the two enzymes. These results, together with the relative activities and the lack of inhibition of BLV PR by two HIV-1 PR inhibitors, suggest that the BLV PR specificity is substantially different from that of HIV PRs.     

(4-Piperidinylphenyl)aminoethyl amides as a novel class of non-covalent cathepsin K inhibitors

A series of (4-piperidinylphenyl)aminoethyl amides based on dipeptide anilines were synthesized and tested against cathepsin K, cathepsin L and cathepsin B. These new non-covalent inhibitors exhibited single-digit nM inhibition of the cysteine proteases. Compounds 3 and 7 demonstrated potency in both mouse and human osteoclast resorption assays.     

Variations in the behaviors to novel objects among five inbred strains of mice

Novelty stimuli cause various behavioral responses, such as exploration and avoidance, and contextual variables may contribute to the behavioral outputs. Here, we tried to compare the behavioral responses to novel objects of five inbred strains of mice (C57BL/6J, 129/svJae, C3H/HeJ, BALB/cJ and DBA/2J) by using a modified novel object test where a small light-weight cube wrapped with paper was presented to mice in a home cage without beddings. In response to these objects, the C57BL/6J, 129/svJae and C3H/HeJ mice showed mild exploratory behaviors, such as approaching, sniffing or brief contact. In striking contrast, the BALB/cJ and DBA/2J mice, which have been known to show high avoidance/low exploration in other behavioral paradigms, exhibited play-like secondary reactions toward the objects after initial primary exploratory behaviors. Specifically, DBA/2J mice would move the object around in the cage, holding it with their mouths, and BALB/cJ mice would gnaw the object, eventually stripping off the wrapping paper. Such behaviors decreased when similar objects were presented repeatedly. The present results suggest that active manipulations of novel objects may be a relevant parameter for measuring novelty-induced behaviors in mice and appear to be strongly influenced by genetic factors.