Cytoplasmic Antiproteinase 2 (PI8) and Bomapin (PI10) Map to the Serpin Cluster at 18q21.3

High-molecular-weight serine proteinase inhibitors (serpins) regulate a diverse set of intracellular and extracellular processes such as complement activation, fibrinolysis, coagulation, cellular differentiation, tumor suppression, apoptosis, and cell migration. The ov-serpins are a subset of the serpin superfamily and are characterized by their high degree of homology to chicken ovalbumin, the lack of N- and C-terminal extensions, the absence of a signal peptide, and aSerrather than anAsnresidue at the penultimate position. Recently, we mapped four members of the family [SCCA1, SCCA2, PAI2, and PI5 (maspin)] to a 300-kb region within 18q21.3. Using a panel of 18q21.3 YAC clones, PCR, and DNA blotting, we mapped two additional ov-serpins, cytoplasmic antiproteinase 2 [CAP2 (PI8)] and bone marrow-associated serpin [bomapin (PI10)], to the same region. Three of the serpins, PI8, PI10, and PAI2 mapped to the same YACs, yA27D8 and yA24E4. We estimated that the size of the 18q21.3 serpin cluster spanned ∼500 kb and contained at least six serpin genes. The order wascen–PI5, SCCA2, SCCA1, PAI2, PI10, PI8–tel.The clustering of serpins at 18q21 provides new opportunities to study coordinate gene regulation and the evolution of gene families.     

Energetic feasibility of hydrogen abstraction and recombination in coenzyme B(12)-dependent diol dehydratase reaction.

Coenzyme B(12) serves as a cofactor for enzymatic radical reactions. The essential steps in all the coenzyme B(12)-dependent rearrangements are two hydrogen abstraction steps: hydrogen abstraction of the adenosyl radical from substrates, and hydrogen back-abstraction (recombination) of a product-derived radical from 5'-deoxyadenosine. The energetic feasibility of these hydrogen abstraction steps in the diol dehyratase reaction was examined by theoretical calculations with a protein-free, simplified model at the B3LYP/6-311G* level of density functional theory. Activation energies for the hydrogen abstraction and recombination with 1,2-propanediol as substrate are 9.0 and 15.1 kcal/mol, respectively, and essentially not affected by coordination of the substrate and the radical intermediate to K+. Since these energies can be considered to be supplied by the substrate-binding energy, the computational results with this simplified model indicate that the hydrogen abstraction and recombination in the coenzyme B(12)-dependent diol dehydratase reaction are energetically feasible.     

Development of vaccines against pertussis caused by Bordetella holmesii using a mouse intranasal challenge model

Bordetella holmesii is recognized as the third causative agent of pertussis (whooping cough) in addition to Bordetella pertussis and Bordetella parapertussis. Pertussis caused by B. holmesii is not rare around the world. However, to date, there is no effective vaccine against B. holmesii. We examined the protective potency of pertussis vaccines available in Japan and vaccines prepared from B. holmesii. A murine model of respiratory infection was exploited to evaluate protective potency. No Japanese commercial pertussis vaccines were effective against B. holmesii. In contrast, a wBH vaccine and an aBH vaccine prepared from B. holmesii were both protective. Passive immunization with sera from mice immunized with aBH vaccine established protection against B. holmesii, indicating that B. holmesii‐specific serum antibodies might play an important role in protection. Immuno‐proteomic analysis with sera from mice immunized with aBH vaccine revealed that the sera recognized a BipA‐like protein of B. holmesii. An aBH vaccine prepared from a BipA‐like protein‐deficient mutant strain did not have a protective effect against B. holmesii. Taken together, our results suggest that the BipA‐like protein plays an important role in the protective efficacy of aBH vaccine.     

Biochemical characterization of a N-terminal fragment (p5) cleaved from fibroblast growth factor-binding protein (FGF-BP) in bovine milk in vitro

By means of successive gel filtration on a Superdex 30 pg column and Mono S column chromatography, a 5-kDa polypeptide (p5) was highly purified from the low molecular weight (LMW) fraction separated from the partially purified lactoferrin (bLF) fraction of bovine milk, and biochemically characterized as a phosphate acceptor for two protein kinases [cAMP-dependent protein kinase (PKA) and casein kinase 1delta (CK1delta)] in vitro. Purified p5 was identified as a fragment (N-terminal positions 24-51, 28 amino acid residues) cleaved from fibroblast growth factor-binding protein (FGF-BP, p37). Both purified p5 and synthetic p5 (sp5) were effectively phosphorylated by PKA, and also phosphorylated by CK1delta in the presence of two sulfated lipids [sulfatide or cholesterol-3-sulfate (CH-3S), SCS] in vitro. A novel phosphorylation site (RNRRGS) for CK1delta and a potent SCS-binding site (RNRR) on p5 were identified. The PKA-mediated phosphorylation of p5 was highly stimulated when incubated with either acidic FGF (aFGF) or bLF in vitro, but this phosphorylation was more sensitive to SCS than H-89 (a specific PKA inhibitor). Immunoprecipitate experiments revealed p5, but not the phosphorylated p5, to be directly bound to aFGF in vitro. These results show that (i) p5 has a high binding affinity with aFGF as well as bLF; (ii) the binding of SCS to p5 results in the selective inhibition of its phosphorylation by PKA; and (iii) SCS functions as an effective stimulator for the phosphorylation of p5 by CK1delta in vitro. In addition, p5 may play an important physiological role as a trafficking factor for the physiological interaction between aFGF group including endothelial cell growth factors and their binding proteins in vivo.     

Biochemical characterization and structural insight into aliphatic β‐amino acid adenylation enzymes IdnL1 and CmiS6

Macrolactam antibiotics such as incednine and cremimycin possess an aliphatic β‐amino acid as a starter unit of their polyketide chain. In the biosynthesis of incednine and cremimycin, unique stand‐alone adenylation enzymes IdnL1 and CmiS6 select and activate the proper aliphatic β‐amino acid as a starter unit. In this study, we describe the enzymatic characterization and the structural basis of substrate specificity of IdnL1 and CmiS6. Functional analysis revealed that IdnL1 and CmiS6 recognize 3‐aminobutanoic acid and 3‐aminononanoic acid, respectively. We solved the X‐ray crystal structures of IdnL1 and CmiS6 to understand the recognition mechanism of these aliphatic β‐amino acids. These structures revealed that IdnL1 and CmiS6 share a common recognition motif that interacts with the β‐amino group of the substrates. However, the hydrophobic side‐chains of the substrates are accommodated differently in the two enzymes. IdnL1 has a bulky Leu220 located close to the terminal methyl group of 3‐aminobutanoate of the trapped acyl‐adenylate intermediate to construct a shallow substrate‐binding pocket. In contrast, CmiS6 possesses Gly220 at the corresponding position to accommodate 3‐aminononanoic acid. This structural observation was supported by a mutational study. Thus, the size of amino acid residue at the 220 position is critical for the selection of an aliphatic β‐amino acid substrate in these adenylation enzymes. Proteins 2017; 85:1238–1247. © 2017 Wiley Periodicals, Inc.     

Herquline A,produced by Penicillium herquei FKI-7215, exhibits anti-influenza virus properties

In the course of screening for new anti-influenza virus antibiotics, we isolated herquline A from a culture broth of the fungus, Penicillium herquei FKI-7215. Herquline A inhibited replication of influenza virus A/PR/8/34 strain in a dose-dependent manner without exhibiting cytotoxicity against several human cell lines. It did not inhibit the viral neuraminidase.     

Efficient production of genetically engineered,male-sterile <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> using anther-specific promoters and genes derived from <Emphasis Type="Italic">Brassica oleracea</Emphasis> and <Emphasis Type="Italic">B. rapa</Emphasis>

Prevention of transgene flow from genetically modified crops to food crops and wild relatives is of concern in agricultural biotechnology. We used genes derived from food crops to produce complete male sterility as a strategy for gene confinement as well as to reduce the food purity concerns of consumers. Anther-specific promoters (A3, A6, A9, MS2, and MS5) were isolated from Brassica oleracea and B. rapa and fused to the β-glucuronidase (GUS) reporter gene and candidate genes for male sterility, including the cysteine proteases BoCysP1 and BoCP3, and negative regulatory components of phytohormonal responses involved in male development. These constructs were then introduced into Arabidopsis thaliana. GUS analyses revealed that A3, A6, and A9 had tapetum-specific promoter activity from the anther meiocyte stage. Male sterility was confirmed in tested constructs with protease or gibberellin insensitive (gai) genes. In particular, constructs with BoCysP1 driven by the A3 or A9 promoter most efficiently produced plants with complete male sterility. The tapetum and middle layer cells of anthers expressing BoCysP1 were swollen and excessively vacuolated when observed in transverse section. This suggests that the ectopic expression of cysteine protease in the meiocyte stage may inhibit programmed cell death. The gai gene also induced male sterility, although at a low frequency. This is the first report to show that plant cysteine proteases and gai from food crops are available as a novel tool for the development of genetically engineered male-sterile plants. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Evaluation of the toxicity of stress-related aldehydes to photosynthesis in chloroplasts

Aldehydes produced under various environmental stresses can cause cellular injury in plants, but their toxicology in photosynthesis has been scarcely investigated. We here evaluated their effects on photosynthetic reactions in chloroplasts isolated from Spinacia oleracea L. leaves. Aldehydes that are known to stem from lipid peroxides inactivated the CO₂ photoreduction to various extents, while their corresponding alcohols and carboxylic acids did not affect photosynthesis. α,β-Unsaturated aldehydes (2-alkenals) showed greater inactivation than the saturated aliphatic aldehydes. The oxygenated short aldehydes malondialdehyde, methylglyoxal, glycolaldehyde and glyceraldehyde showed only weak toxicity to photosynthesis. Among tested 2-alkenals, 2-propenal (acrolein) was the most toxic, and then followed 4-hydroxy-(E)-2-nonenal and (E)-2-hexenal. While the CO₂-photoreduction was inactivated, envelope intactness and photosynthetic electron transport activity (H₂O → ferredoxin) were only slightly affected. In the acrolein-treated chloroplasts, the Calvin cycle enzymes phosphoribulokinase, glyceraldehyde-3-phosphate dehydrogenase, fructose-1,6-bisphophatase, sedoheptulose-1,7-bisphosphatase, aldolase, and Rubisco were irreversibly inactivated. Acrolein treatment caused a rapid drop of the glutathione pool, prior to the inactivation of photosynthesis. GSH exogenously added to chloroplasts suppressed the acrolein-induced inactivation of photosynthesis, but ascorbic acid did not show such a protective effect. Thus, lipid peroxide-derived 2-alkenals can inhibit photosynthesis by depleting GSH in chloroplasts and then inactivating multiple enzymes in the Calvin cycle.