Analysis of insertions and extensions in the functional evolution of the ribonucleotide reductase family

Ribonucleotide reductases (RNRs) are used by all free‐living organisms and many viruses to catalyze an essential step in the de novo biosynthesis of DNA precursors. RNRs are remarkably diverse by primary sequence and cofactor requirement, while sharing a conserved fold and radical‐based mechanism for nucleotide reduction. In this work, we expand on our recent phylogenetic inference of the entire RNR family and describe the evolutionarily relatedness of insertions and extensions around the structurally homologous catalytic barrel. Using evo‐velocity and sequence similarity network (SSN) analyses, we show that the N‐terminal regulatory motif known as the ATP‐cone domain was likely inherited from an ancestral RNR. By combining SSN analysis with AlphaFold2 predictions, we also show that the C‐terminal extensions of class II RNRs can contain folded domains that share homology with an Fe‐S cluster assembly protein. Finally, using sequence analysis and AlphaFold2, we show that the sequence motif of a catalytically essential insertion known as the finger loop is tightly coupled to the catalytic mechanism. Based on these results, we propose an evolutionary model for the diversification of the RNR family.     

Structural requirements of lipid A species in activation of clotting enzymes from the horseshoe crab, and the human complement cascade

The structure/activity relationship of lipid A, a bioactive center of endotoxic lipopolysaccharides, in the activation of the clotting enzyme cascade of a horseshoe crab amoebocyte lysate (Limulus activity) and the complement system in human serum, was examined using synthetic lipids A and related compounds. Regarding Limulus activity, a newly developed colorimetric method, which utilizes a mixture of recombined clotting factors and a chromogenic substance, was much more sensitive for detecting changes in the chemical structure of test compounds than the conventional gelation method using the amoebocyte whole lysate. (beta 1-6)-D-Glucosamine disaccharide bisphosphates, which had neither 3-hydroxyacyl nor 3-acyloxyacyl groups, and acylglucosamine phosphates, which in structure correspond or are analogous to the non-reducing or reducing moieties of lipids A and biosynthetic disaccharide lipid A precursors showed only negligible activity in the colorimetric tests, but they exhibited a distinct though much weaker gelation activity than the parent disaccharide molecules. The assay results obtained by the colorimetric Limulus test correlate better with the pyrogenicity of the test synthetic compounds than those given by the gelation method, although the dependence of pyrogenicity on chemical structure is greater. The presence of 3-hydroxyacyl groups on the bisphosphorylated (beta 1-6)-D-glucosamine disaccharide backbone is the prerequisite for effective activation of the clotting enzyme cascade of horseshoe crab amoebocyte lysate, while the presence of an adequate number (one or two) of 3-acyloxyacyl groups on the disaccharide bisphosphate backbone is needed for full pyrogenicity. Complement activation, on the other hand, showed structural requirements quite different from those for the colorimetric Limulus activity and the pyrogenicity. The disaccharide compounds that had only non-hydroxylated acyl groups, acylated glucosamine phosphates that had the structure of the non-reducing portion of lipids A and biosynthetic disaccharide precursors, which were scarcely active in the colorimetric Limulus test, caused complement activation comparable to or sometimes stronger than that of the parent disaccharide molecules. Acylglucosamine phosphates, corresponding in structure to the reducing moiety of disaccharide compounds, however, showed little activity.     

Expression and purification of a small cytokine growth-blocking peptide from armyworm Pseudaletia separata by an optimized fermentation method using the methylotrophic yeast Pichia pastoris

A small multifunctional cytokine, growth-blocking peptide (GBP), from the armyworm Pseudaletia separata larvae was expressed as a soluble and active recombinant peptide in the methylotrophic yeast Pichia pastoris. An expression vector for GBP secretion was constructed using vector pPIC9, and GBP was expressed under the control of the alcohol oxidase (AOX1) promoter. Although we first tried to cultivate GBP in shake flask cultures, the yield was low, probably due to proteolysis of the recombinant protein. To overcome this problem, we utilized a high-density fermentation method. The pH of the medium in the fermenter was kept at 3.0, and the medium was collected within 48h post methanol shift to minimize exposure of the target peptide to proteases. Recombinant GBP was purified through three reverse-phase HPLC columns. We characterized the 25 amino acid GBP by molecular mass spectrometry and amino acid sequencing. Plasmatocyte spreading, one of the activities of GBP, was similar between chemically synthesized GBP and purified recombinant GBP. Up to 50mg GBP was recovered per 1L of yeast culture supernatant.     

Transcriptome-based single nucleotide polymorphism markers for genome mapping in Japanese pear (Pyrus pyrifolia Nakai)

The development of single nucleotide polymorphism (SNP) markers in Japanese pear (Pyrus pyrifolia Nakai) offers the opportunity to use DNA markers for marker-assisted selection in breeding programs because of their high abundance, codominant inheritance, and potential for automated high-throughput analysis. We developed a 1,536-SNP bead array without a reference genome sequence from more than 44,000 base changes on the basis of a large-scale expressed sequence tag (EST) analysis combined with 454 genome sequencing data of Japanese pear ‘Housui’. Among the 1,536 SNPs on the array, 756 SNPs were genotyped, and 609 SNP loci were mapped to linkage groups on a genetic linkage map of ‘Housui’, based on progeny of an interspecific cross between European pear (Pyrus communis L.) ‘Bartlett’ and ‘Housui’. The newly constructed genetic linkage map consists of 951 loci, comprising 609 new SNPs, 110 pear genomic simple sequence repeats (SSRs), 25 pear EST–SSRs, 127 apple SSRs, 61 pear SNPs identified by the “potential intron polymorphism” method, and 19 other loci. The map covers 22 linkage groups spanning 1341.9 cM with an average distance of 1.41 cM between markers and is anchored to reference genetic linkage maps of European pears and apples. A total of 514 contigs containing mapped SNP loci showed significant similarity to known proteins by functional annotation analysis.     

A novel serine protease highly expressed in the pancreas is expressed in various kinds of cancer cells

We have isolated a cDNA that encodes a novel serine protease, prosemin, from human brain. The cDNA of human prosemin is 1306 bp, encoding 317 amino acids. It showed significant homology with the sequence of a chromosome 16 cosmid clone (accession no. NT_037887.4). The prosemin gene contains six exons and five introns. The amino acid sequence of prosemin shows significant homology to prostasin, gamma-tryptase, and testisin (43%, 41%, and 38% identity, respectively), the genes of which are also located on chromosome 16. Northern hybridization showed that prosemin is expressed predominantly in the pancreas and weakly in the prostate and cerebellum. However, western blot and RT-PCR analyses showed that prosemin is expressed and secreted from various kinds of cancer cells, such as glioma, pancreas, prostate, and ovarian cell lines. Prosemin is secreted in the cystic fluid of clinical ovarian cancers. Furthermore, immunohistochemistry showed prosemin protein localized in the apical parts of ovarian carcinomas. Recombinant prosemin was expressed in COS cells and was purified by immunoaffinity chromatography. Recombinant prosemin preferentially cleaved benzyloxycarbonyl (Z)-His-Glu-Lys-methylcoumaryl amidide (MCA) and t-butyloxycarbonyl (Boc)-Gln-Ala-Arg-MCA. Our results suggest that prosemin is a novel serine protease of the chromosome 16 cluster that is highly expressed in the pancreas. The usefulness of this serine protease as a candidate tumor marker should be further examined.     

Microbial Transformation of Squalene: Terminal Methyl Group Oxidation by Corynebacterium sp

Corynebacterium sp. strain SY-79 was isolated from soil, using squalene as a carbon source. Microbiological properties of this bacterium are described. The metabolic product of this bacterium from squalene was identified as 2,6,10,15,19, 23-hexamethyl-2,6,10,14,18,22-tetracosahexaenedioic acid (squalenedioic acid).     

Viral FLIP enhances Wnt signaling downstream of stabilized beta-catenin, leading to control of cell growth

Death receptor-mediated apoptosis is potently inhibited by viral FLIP (FLICE/caspase 8 inhibitory protein), which is composed of two tandemly repeated death effector domains (DEDs), through reduced activation of procaspase 8. Here, we show that equine herpesvirus 2-encoded viral FLIP E8 enhances Wnt/beta-catenin signaling in a variety of cell lines. E8 was shown to strikingly augment Wnt3a signaling, as shown both in a luciferase assay for T-cell factor/beta-catenin and through induction of endogenous cyclin D1. The effect of E8 was independent of its direct binding activity with DED-containing signaling molecules, including caspase 8 and FADD, in death receptor-mediated apoptosis. E8 enhanced Wnt signaling downstream of stabilized beta-catenin, while a long form of cellular FLIP (c-FLIP(L)) enhanced stabilization of beta-catenin in 293T cells. Consequently, coexpression of E8 and c-FLIP(L) synergistically increased Wnt signaling in 293T cells. Moreover, E8-mediated stimulation of Wnt signaling induced dramatic growth retardation in untransformed cell lines but not in transformed cell lines. Thus, viral FLIP E8 not only inhibits death receptor-mediated apoptosis but also enhances Wnt signaling pathways that are closely related to those of both ontogenesis and oncogenesis.     

Phylogenies using mtDNA and SRY provide evidence for male-mediated introgression in Asian domestic cattle

Using nucleotide sequences of the mitochondrial DNA (mtDNA) cytochrome b and SRY genes, we examined the genetic status of two major groups of domestic cattle, the humpless taurine (Bos taurus) and humped zebu (B. indicus), using 10 cattle populations in Asia. Several sequence polymorphisms specific for each major group were found, although the frequency of these polymorphisms varied in each population. Six major mtDNA-SRY composite types were observed. The Mishima, Mongolian, Korean, Chinese Yellow and Sri Lanka cattle populations had a full match between the mtDNA and SRY sequences, specifically the taurine/taurine type or zebu/zebu type. A non-match type (zebu/taurine type) was found at a high frequency in the Bangladesh (83.4%) and Nepal populations (83.3%). Our results suggest that these non-match type populations developed from genetic hybridization of different strains. Also, the domestication history of modern Asian domestic cattle could be explained by male-mediated introgression. Additionally, our results suggest the occurrence of introgression of mtDNA from other Bibos or Poephagus species into native cattle populations. The existence of other mtDNA-SRY composite types, such as the Bali-zebu and yak-zebu types in Indonesia (85.7%) and Nepal (16.7%), respectively, suggests that genetic introgression also occurred from other genera into domestic cattle during the process of domestication.