Kaori Wakamatsu

The adaptor protein p40phox 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 p67phox and p47phox. They translocate to the membrane upon cell stimulation and activate flavocytochrome b558, the membrane‐integrated catalytic core of this enzyme system. The activators p67phox and p47phox form a ternary complex together with p40phox, an adaptor protein with unknown function, comprising the PX/PB2, SH3 and PC motif‐ containing domains: p40phox associates with p67phox via binding of the p40phox PC motif to the p67phox PB1 domain, while p47phox directly interacts with p67phox but not with p40phox. Here we show that p40phox enhances membrane translocation of p67phox and p47phox in stimulated cells, which leads to facilitated production of superoxide. The enhancement cannot be elicited by a mutant p40phox carrying the D289A substitution in PC or a p67phox with the K355A substitution in PB1, each being defective in binding to its respective partner. Thus p40phox participates in activation of the phagocyte oxidase by regulating membrane recruitment of p67phox and p47phox via the PB1–PC interaction with p67phox.

Release of cytokines/chemokines and cell death in UVB-irradiated human keratinocytes, HaCaT

Ultraviolet (UV) B can lead to inflammatory responses such as sunburn, which involves the production of various inflammatory cytokines and chemokines, and the induction of cell death. Keratinocytes in the skin has one of the highest risks of exposure to UV. However, the detailed mechanisms underlying UVB irradiation‐induced inflammation and cell death are not well known. Thus, we investigated the effect of UVB irradiation on the production of various cytokines/chemokines and the induction of cell death in UVB‐irradiated human keratinocytes (HaCaT cells). We evaluated 11 cytokines/chemokines in cell culture supernatants from HaCaT cells exposed to 0–400 mJ/cm2 UVB irradiation. UVB at a dose 400 mJ/cm2 induced the release of various cytokines; interleukin (IL)‐1β, IL‐6, IL‐8, interferon (IFN)‐γ, granulocyte‐colony stimulating factor (G‐CSF), macrophage inflammatory protein (MIP)‐1β, and tumor necrosis factor (TNF)‐α. These results suggest that UVB irradiation‐induced the release of several cytokines/chemokines and led to cell death in human keratinocytes. UV exposure may be associated with multiple physiological events in the human skin.

A new general method for the biosynthesis of stable isotope-enriched peptides using a decahistidine-tagged ubiquitin fusion system: An application to the production of mastoparan-X uniformly enriched with 15N and 15N/13C

A new strategy is described for the production of peptides enriched with stable isotopes. Peptides of interest are expressed in Escherichia coli (E. coli) cells as recombinant fusion proteins with Saccharomyces cerevisiae ubiquitin. This method yields as much as 30–100 mg/l of isotope-enriched fusion proteins in minimal media. A decahistidine tag attached to the N-terminus of ubiquitin enables a one-step purification of the fusion protein via Ni2+-chelating affinity chromatography. The ubiquitin moiety is then easily and specifically cleaved off by a protease, yeast ubiquitin hydrolase. Since this enzyme is also expressed at a high level in E. coli cells and can be purified in one step, the presented strategy has an advantage in view of costs over others that use commercially available proteases. In addition, since ubiquitin fusion proteins easily refold, the fusion protein can be expressed either in a soluble form or as inclusion bodies. This flexibility enables us to prepare peptides that are unstable in a soluble state in E. coli cells. As an example, the expression and the uniform stable isotope enrichment with 15N and/or13 C are described for mastoparan-X, a tetradecapeptide known to activate GTP-binding regulatory proteins. An amide group at the C-terminus of this peptide can also be formed by our method. The presented system is considered powerful for the stable isotope enrichment of short peptides with proton resonances that are too severely overlapped to be analyzed solely by proton NMR.

Effects of peptide dimerization on pore formation: Antiparallel disulfide-dimerized magainin 2 analogue

To elucidate the effects of peptide dimerization on pore formation by magainin 2 (MG2), a covalently linked antiparallel dimer of the MG2 analogue [(F5Y, L6C, F16W, I20C-MG2)(2): II] was synthesized based on the dimer structure revealed by our NMR study. The interactions of the dimer with lipid bilayers were investigated by CD and fluorescence in comparison with a monomer analogue (F5Y, F16W-MG2: I). Similar to I, II was found to form a peptide-lipid supramolecular complex pore accompanied with lipid flip-flop and peptide translocation. The pore formed by II was characterized by a slightly larger pore diameter and a threefold longer lifetime than that of I, although the pore formation rate of the dimer was lower than that of the monomer. The coexistence of the dimer and the monomer exhibited slight but significant synergism in membrane permeabilization, which was maximal at a monomer/dimer ratio of 3. Therefore, we concluded that a pentameric pore composed of one pore-stabilizing dimer and three monomers maximized the overall leakage activity in keeping with our kinetic prediction.

Discovery of Mitocryptide-1, a Neutrophil-activating Cryptide from Healthy Porcine Heart

Although neutrophils are known to migrate in response to various chemokines and complement factors, the substances involved in the early stages of their transmigration and activation have been poorly characterized to date. Here we report the discovery of a peptide isolated from healthy porcine hearts that activated neutrophils. Its primary structure is H-Leu-Ser-Phe-Leu-Ile-Pro-Ala-Gly-Trp-Val-Leu-Ser-His-Leu-Asp-His-Tyr-Lys-Arg-Ser-Ser-Ala-Ala-OH, and it was indicated to originate from mitochondrial cytochrome c oxidase subunit VIII. This peptide caused chemotaxis at concentrations lower than that inducing β-hexosaminidase release. Such responses were observed in neutrophilic/granulocytic differentiated HL-60 cells but not in undifferentiated cells, and Gi2-type G proteins were suggested to be involved in the peptide signaling. Moreover the peptide activated human neutrophils to induce β-hexosaminidase secretion. A number of other amphipathic neutrophil-activating peptides presumably originating from mitochondrial proteins were also found. The present results suggest that neutrophils monitor such amphipathic peptides including the identified peptide as an initiation signal for inflammation at injury sites.

Mastoparan as a G Protein Activator

Mastoparan was isolated from the venom of wasp, Vespula lewisii, as a peptide that induces histamine release from rat peritoneal mast cells (Hirai et al., 1979). It is a C-terminally amidated tetradecapeptide comprising primarily basic and hydrophobic amino acid residues but no acidic residues (Tab. 1). Structurally similar peptides that also induce the histamine release have been isolated from venom of other wasps and hornets, and are collectively termed mastoparans (Nakajima et al., 1985). Mastoparans were also found to induce exocytosis from a variety of cells: serotonin from platelets (Ozaki et al., 1990), insulin from pancreatic β cells (Yokokawa et al., 1989), surfactant from pulmonary alveolar type 2 epithelial cells (Joyce-Brady et al., 1991), and catecholamine from chromaffin cells (Kuroda et al., 1980; Vitale et al., 1993). This peptide also elevates intracellular Ca2+ concentration in neutrophils (Perianin and Snyderman, 1989) and promotes proliferation of Swiss 3T3 cells (Gil et al., 1991). Table 1 Primary structure of mastoparan and its derivatives. Peptide Primary structurea Commercial sourceb notes mastoparan Ile Asn Leu Lys Ala Leu Ala Ala Leu Ala LysLyslIe Leu NH2 1,2,3,4,5,6 c Mas7 Iie Asn Leu LysAla Leu Ala Ala Leu Ala Lys Ala Leu Leu NH2 1,2,3,4 d Mas8 Iie Asn Leu LysAla Leu Ala Ala Leu Ala Lys Arg Leu Leu NH2 1,3 d Mas11 Iie Asn Leu LysAla Leu Ala Ala Leu Ala Lys Ala Leu Leu NH2 none d Mas17 Ile Asn Leu Lys Ala Ala Ala Leu Ala Lys Lys Leu Leu NH2 1,2,3,4 d a Amino acid residues that differ from those of mastoparan are shown underlined. b Commercial source: 1, Peninsula Laboratories; 2, Bachem; 3, Phoenix Pharmaceuticals; 4, Biomolecular Research Laboratories; 5, Peptide Institute; 6, Sigma. cfrom (Hirai et al., 1979) dfrom (Higashijima et al., 1990)

Enhancement of antibody production by the addition of Coenzyme-Q10

Recently, there has been a growing demand for therapeutic monoclonal antibodies (MAbs) on the global market. Because therapeutic MAbs are more expensive than low-molecular-weight drugs, there have been strong demands to lower their production costs. Therefore, efficient methods to minimize the cost of goods are currently active areas of research. We have screened several enhancers of specific MAb production rate (SPR) using a YB2/0 cell line and found that coenzyme-Q10 (CoQ10) is a promising enhancer candidate. CoQ10 is well known as a strong antioxidant in the respiratory chain and is used for healthcare and other applications. Because CoQ10 is negligibly water soluble, most studies are limited by low concentrations. We added CoQ10 to a culture medium as dispersed nanoparticles at several concentrations (Q-Media) and conducted a fed-batch culture. Although the Q-Media had no effect on cumulative viable cell density, it enhanced SPR by 29%. In addition, the Q-Media had no effect on the binding or cytotoxic activity of MAbs. Q-Media also enhanced SPR with CHO and NS0 cell lines by 30%. These observations suggest that CoQ10 serves as a powerful aid in the production of MAbs by enhancing SPR without changing the characteristics of cell growth, or adversely affecting the quality or biological activity of MAbs.

Peptide fragment of the m3 muscarinic acetylcholine receptor activates Gq but not Gi2

Gq, a heterotrimeric guanine nucleotide‐binding protein, plays important roles such as the regulation of calcium mobilization and cell proliferation. This protein is considered as a promising drug target for the treatment of cardiac hypertrophy. Selective activation of Gq would be quite useful for analyzing the role of Gq in signaling pathways. We synthesized m3i3c—a peptide with 16 amino acid residues that corresponds to the junction between the C‐terminus of the third intracellular loop and the sixth transmembrane helix (TM‐VI) of human m3 muscarinic acetylcholine receptor, which couples to Gq but not Gi2. At micromolar concentrations, this peptide was found to activate Gq but not Gi2. This peptide is the first small compound that selectively activates Gq but not Gi2. Copyright

Interaction of anti-aggregation agent dimethylethylammonium propane sulfonate with acidic fibroblast growth factor

Prevention of aggregation is critical for analyzing protein structure. Non-detergent sulfobetaines (NDSBs) are known to prevent protein aggregation, but the molecular mechanisms of their anti-aggregation effect are poorly understood. To elucidate the underlying mechanisms, we analyzed the effects of dimethylethylammonium propane sulfonate (NDSB-195) on acidic fibroblast growth factor (aFGF). NDSB-195 (0.5M) increased both aggregation and denaturation temperatures of aFGF by 4 degrees C. Chemical shift perturbation analyses indicated that many affected residues were located at the junction between a beta-strand (or 3(10)-helix) and a loop, irrespective of the chemical properties of the residue. The apparent dissociation constants of the interaction ranged from 0.04 to 3M, indicating weak interactions between NDSB and protein molecules.

Crystallization and preliminary X-ray crystallographic analysis of the receptor-uncoupled mutant of Gαi1

In order to understand the molecular mechanisms by which G-protein-coupled receptors (GPCRs) activate G proteins, the K349P mutant of Gαi1 (K349P), which is unable to couple to the muscarinic acetylcholine receptor, was prepared and its crystals were grown along with those of wild-type Gαi1 protein (WT). The two proteins were crystallized under almost identical conditions, thus enabling a detailed structural comparison. The crystallization conditions performed well irrespective of the identity of the bound nucleotide (GDP or GTPγS) and the crystals diffracted to resolutions of 2.2 A (WT·GDP), 2.8 A (WT·GTPγS), 2.6 A (K349P·GDP) and 3.2 A (K349P·GTPγS).