Purification and molecular cloning of an inducible gram-negative bacteria-binding protein from the silkworm, Bombyx mori.

A 50-kDa hemolymph protein, having strong affinity to the cell wall of Gram(-) bacteria, was purified from the hemolymph of the silkworm, Bombyx mori. The cDNA encoding this Gram(-) bacteria-binding protein (GNBP) was isolated from an immunized silkworm fat body cDNA library and sequenced. Comparison of the deduced amino acid sequence with known sequences revealed that GNBP contained a region displaying significant homology to the putative catalytic region of a group of bacterial beta-1,3 glucanases and beta-1,3-1,4 glucanases. Silkworm GNBP was also shown to have amino acid sequence similarity to the vertebrate lipopolysaccharide receptor CD14 and was recognized specifically by a polygonal anti-CD14 antibody. Northern blot analysis showed that GNBP was constitutively expressed in fat body, as well as in cuticular epithelial cells of naive silkworms. Intense transcription was, however, rapidly induced following a cuticular or hemoceolien bacterial challenge. An mRNA that hybridized with GNBP cDNA was also found in the l(2)mbn immunocompetent Drosophila cell line. These observations suggest that GNBP is an inducible acute phase protein implicated in the immune response of the silkworm and perhaps other insects.

Multiprotein bridging factor 1 (MBF1) is an evolutionarily conserved transcriptional coactivator that connects a regulatory factor and TATA element-binding protein

Multiprotein bridging factor 1 (MBF1) is a transcriptional cofactor that bridges between the TATA box-binding protein (TBP) and the Drosophila melanogaster nuclear hormone receptor FTZ-F1 or its silkworm counterpart BmFTZ-F1. A cDNA clone encoding MBF1 was isolated from the silkworm Bombyx mori whose sequence predicts a basic protein consisting of 146 amino acids. Bacterially expressed recombinant MBF1 is functional in interactions with TBP and a positive cofactor MBF2. The recombinant MBF1 also makes a direct contact with FTZ-F1 through the C-terminal region of the FTZ-F1 DNA-binding domain and stimulates the FTZ-F1 binding to its recognition site. The central region of MBF1 (residues 35–113) is essential for the binding of FTZ-F1, MBF2, and TBP. When the recombinant MBF1 was added to a HeLa cell nuclear extract in the presence of MBF2 and FTZ622 bearing the FTZ-F1 DNA-binding domain, it supported selective transcriptional activation of the fushi tarazu gene as natural MBF1 did. Mutations disrupting the binding of FTZ622 to DNA or MBF1, or a MBF2 mutation disrupting the binding to MBF1, all abolished the selective activation of transcription. These results suggest that tethering of the positive cofactor MBF2 to a FTZ-F1-binding site through FTZ-F1 and MBF1 is essential for the binding site-dependent activation of transcription. A homology search in the databases revealed that the deduced amino acid sequence of MBF1 is conserved across species from yeast to human.

Role of the integument in insect defense: pro-phenol oxidase cascade in the cuticular matrix.

The cuticle of the silkworm Bombyx mori was demonstrated to contain pro-phenol oxidase [zymogen of phenol oxidase (monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1)] and its activating cascade. The activating cascade contained at least one serine proteinase zymogen (latent form of pro-phenol oxidase activating enzyme). When the extracted cascade components were incubated with Ca2+, the latent form of pro-phenol oxidase activating enzyme was itself activated and, in turn, converted through a limited proteolysis of pro-phenol oxidase to phenol oxidase. Immuno-gold localization of prophenol oxidase in the cuticle using a cross-reactive hemolymph anti-pro-phenol oxidase antibody revealed a random distribution of this enzyme in the nonlamellate endocuticle and a specific orderly arrayed pattern along the basal border of the laminae in the lamellate endocuticle of the body wall. Furthermore, prophenol oxidase was randomly distributed in the taenidial cushion of the tracheal cuticle. At the time of pro-phenol oxidase accumulation in the body wall cuticle, no pro-phenol oxidase mRNA could be detected in the epidermal tissue, whereas free-circulating hemocytes contained numerous transcripts of pro-phenol oxidase. Our results suggest that the pro-phenol oxidase is synthesized in the hemocytes and actively transported into the cuticle via the epidermis.

Molecular cloning of insect pro-phenol oxidase: a copper-containing protein homologous to arthropod hemocyanin.

Pro-phenol oxidase [pro-PO; zymogen of phenol oxidase (monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1)] is present in the hemolymph plasma of the silkworm Bombyx mori. Pro-PO is a heterodimeric protein synthesized by hemocytes. A specific serine proteinase activates both subunits through a limited proteolysis. The amino acid sequences of both subunits were deduced from their respective cDNAs; amino acid sequence homology between the subunits was 51%. The deduced amino acid sequences revealed domains highly homologous to the copper-binding site sequences (copper-binding sites A and B) of arthropod hemocyanins. The overall sequence homology between silkworm pro-PO and arthropod hemocyanins ranged from 29 to 39%. Phenol oxidases from prokaryotes, fungi, and vertebrates have sequences homologous to only the copper-binding site B of arthropod hemocyanins. Thus, silkworm pro-PO DNA described here appears distinctive and more closely related to arthropod hemocyanins. The pro-PO-activating serine proteinase was shown to hydrolyze peptide bonds at the carboxyl side of arginine in the sequence-Asn-49-Arg-50-Phe-51-Gly-52- of both subunits. Amino groups of N termini of both subunits were indicated to be N-acetylated. The cDNAs of both pro-PO subunits lacked signal peptide sequences. This result supports our contention that mature pro-PO accumulates in the cytoplasm of hemocytes and is released by cell rupture, as for arthropod hemocyanins.

The FEZ1 gene at chromosome 8p22 encodes a leucine-zipper protein, and its expression is altered in multiple human tumors

Alterations of human chromosome 8p occur frequently in many tumors. We identified a 1.5-Mb common region of allelic loss on 8p22 by allelotype analysis. cDNA selection allowed isolation of several genes, including FEZ1. The predicted Fez1 protein contained a leucine-zipper region with similarity to the DNA-binding domain of the cAMP-responsive activating-transcription factor 5. RNA blot analysis revealed that FEZ1 gene expression was undetectable in more than 60% of epithelial tumors. Mutations were found in primary esophageal cancers and in a prostate cancer cell line. Transcript analysis from several FEZ1-expressing tumors revealed truncated mRNAs, including a frameshift. Alteration and inactivation of the FEZ1 gene may play a role in various human tumors.

Tertiary hypothyroidism and hyperglycemia in mice with targeted disruption of the thyrotropin-releasing hormone gene

Thyrotropin-releasing hormone (TRH) is a brain hypothalamic hormone that regulates thyrotropin (TSH) secretion from the anterior pituitary and is ubiquitously distributed throughout the brain and other tissues including pancreas. To facilitate studies into the role of endogenous TRH, we have used homologous recombination to generate mice that lack TRH. These TRH−/− mice are viable, fertile, and exhibit normal development. However, they showed obvious hypothyroidism with characteristic elevation of serum TSH level and diminished TSH biological activity. Their anterior pituitaries exhibited an apparent decrease in TSH immunopositive cells that was not due to hypothyroidism. Furthermore, this decrease could be reversed by TRH, but not thyroid hormone replacement, suggesting a direct involvement of TRH in the regulation of thyrotrophs. The TRH−/− mice also exhibited hyperglycemia, which was accompanied by impaired insulin secretion in response to glucose. These findings indicate that TRH−/− mice provide a model of exploiting tertiary hypothyroidism, and that TRH gene abnormalities cause disturbance of insulin secretion resulting in marked hyperglycemia.

Neural restrictive silencer factor recruits mSin3 and histone deacetylase complex to repress neuron-specific target genes

Accumulative evidence suggests that more than 20 neuron-specific genes are regulated by a transcriptional cis-regulatory element known as the neural restrictive silencer (NRS). A trans-acting repressor that binds the NRS, NRSF [also designated RE1-silencing transcription factor (REST)] has been cloned, but the mechanism by which it represses transcription is unknown. Here we show evidence that NRSF represses transcription of its target genes by recruiting mSin3 and histone deacetylase. Transfection experiments using a series of NRSF deletion constructs revealed the presence of two repression domains, RD-1 and RD-2, within the N- and C-terminal regions, respectively. A yeast two-hybrid screen using the RD-1 region as a bait identified a short form of mSin3B. In vitro pull-down assays and in vivo immunoprecipitation-Western analyses revealed a specific interaction between NRSF-RD1 and mSin3 PAH1-PAH2 domains. Furthermore, NRSF and mSin3 formed a complex with histone deacetylase 1, suggesting that NRSF-mediated repression involves histone deacetylation. When the deacetylation of histones was inhibited by tricostatin A in non-neuronal cells, mRNAs encoding several neuronal-specific genes such as SCG10, NMDAR1, and choline acetyltransferase became detectable. These results indicate that NRSF recruits mSin3 and histone deacetylase 1 to silence neural-specific genes and suggest further that repression of histone deacetylation is crucial for transcriptional activation of neural-specific genes during neuronal terminal differentiation.

Roles of SecG in ATP- and SecA-dependent protein translocation

SecA, the translocation ATPase in Escherichia coli, undergoes cycles of conformational changes (insertion/deinsertion) in response to ATP and a preprotein. The membrane-embedded portion of protein translocase, SecYEG, has crucial roles in the SecA-driven preprotein translocation reaction. We previously identified a secY mutation (secY205) that did not allow an ATP- and preprotein-dependent (productive) insertion of SecA as well as secA mutations that suppressed the secY205 translocation defect. One of the suppressor mutations, secA36, also suppressed the cold-sensitive phenotype of the secG deletion mutant. In vitro experiments at 20°C showed that inverted membrane vesicles lacking SecG were almost inactive in combination with the wild-type SecA protein in translocation of proOmpA as well as in the accompanying ATP hydrolysis. In contrast, the SecA36 mutant protein was found to be able to execute the translocation activity fully at this temperature, even in the absence of SecG. A SecG requirement and its alleviation by the SecA36 alteration also were shown for the SecA insertion reaction. The finding that the SecA36 protein no longer requires assistance from SecG in its insertion and in its catalysis of protein translocation agrees with the idea that SecG normally assists in the functioning of SecA. In agreement with this notion, when the intrinsic SecA function was compromised by a lowered ATP concentration, SecG became essential even at 37°C and even for the SecA36 protein. We propose that in the normal translocase, SecG cooperates with SecA to facilitate efficient movement of preprotein in each catalytic cycle of SecA.

Endoplasmic Reticulum Stress-induced mRNA Splicing Permits Synthesis of Transcription Factor Hac1p/Ern4p That Activates the Unfolded Protein Response

An intracellular signaling from the endoplasmic reticulum (ER) to the nucleus, called the unfolded protein response (UPR), is activated when unfolded proteins are accumulated in the ER under a variety of stress conditions (“ER stress”). We and others recently identified Hac1p/Ern4p as a transcription factor responsible for the UPR in Saccharomyces cerevisiae. It was further reported that Hac1p (238 aa) is detected only in ER-stressed cells, and its expression is mediated by unconventional splicing of HAC1 precursor mRNA. The splicing replaces the C-terminal portion of Hac1p; it was proposed that precursor mRNA is also translated but the putative product of 230 aa is rapidly degraded by the ubiquitin–proteasome pathway. We have identified and characterized the same regulated splicing and confirmed its essential features. Contrary to the above proposal, however, we find that the 238-aa product of mature mRNA and the 230-aa-type protein tested are highly unstable with little or no difference in stability. Furthermore, we demonstrate that the absence of Hac1p in unstressed cells is due to the lack of translation of precursor mRNA. We conclude that Hac1p is synthesized as the result of ER stress-induced mRNA splicing, leading to activation of the UPR.

Mammalian Transcription Factor ATF6 Is Synthesized as a Transmembrane Protein and Activated by Proteolysis in Response to Endoplasmic Reticulum Stress

The unfolded protein response (UPR) controls the levels of molecular chaperones and enzymes involved in protein folding in the endoplasmic reticulum (ER). We recently isolated ATF6 as a candidate for mammalian UPR-specific transcription factor. We report here that ATF6 constitutively expressed as a 90-kDa protein (p90ATF6) is directly converted to a 50-kDa protein (p50ATF6) in ER-stressed cells. Furthermore, we showed that the most important consequence of this conversion was altered subcellular localization; p90ATF6 is embedded in the ER, whereas p50ATF6 is a nuclear protein. p90ATF6 is a type II transmembrane glycoprotein with a hydrophobic stretch in the middle of the molecule. Thus, the N-terminal half containing a basic leucine zipper motif is oriented facing the cytoplasm. Full-length ATF6 as well as its C-terminal deletion mutant carrying the transmembrane domain is localized in the ER when transfected. In contrast, mutant ATF6 representing the cytoplasmic region translocates into the nucleus and activates transcription of the endogenous GRP78/BiP gene. We propose that ER stress-induced proteolysis of membrane-bound p90ATF6 releases soluble p50ATF6, leading to induced transcription in the nucleus. Unlike yeast UPR, mammalian UPR appears to use a system similar to that reported for cholesterol homeostasis.

d-β-Hydroxybutyrate protects neurons in models of Alzheimer's and Parkinson's disease

The heroin analogue 1-methyl-4-phenylpyridinium, MPP+, both in vitro and in vivo, produces death of dopaminergic substantia nigral cells by inhibiting the mitochondrial NADH dehydrogenase multienzyme complex, producing a syndrome indistinguishable from Parkinson's disease. Similarly, a fragment of amyloid protein, Aβ1–42, is lethal to hippocampal cells, producing recent memory deficits characteristic of Alzheimer's disease. Here we show that addition of 4 mM d-β-hydroxybutyrate protected cultured mesencephalic neurons from MPP+ toxicity and hippocampal neurons from Aβ1–42 toxicity. Our previous work in heart showed that ketone bodies, normal metabolites, can correct defects in mitochondrial energy generation. The ability of ketone bodies to protect neurons in culture suggests that defects in mitochondrial energy generation contribute to the pathophysiology of both brain diseases. These findings further suggest that ketone bodies may play a therapeutic role in these most common forms of human neurodegeneration.

Activation of target-tissue immune-recognition molecules by double-stranded polynucleotides

Abnormal expression of major histocompatibility complex (MHC) class I and class II in various tissues is associated with autoimmune disease. Autoimmune responses can be triggered by viral infections or tissue injuries. We show that the ability of a virus or a tissue injury to increase MHC gene expression is duplicated by any fragment of double-stranded (ds) DNA or dsRNA introduced into the cytoplasm of nonimmune cells. Activation is sequence-independent, is induced by ds polynucleotides as small as 25 bp in length, and is not duplicated by single-stranded polynucleotides. In addition to causing abnormal MHC expression, the ds nucleic acids increase the expression of genes necessary for antigen processing and presentation: proteasome proteins (e.g., LMP2), transporters of antigen peptides; invariant chain, HLA-DM, and the costimulatory molecule B7.1. The mechanism is different from and additive to that of γ-interferon (γIFN), i.e., ds polynucleotides increase class I much more than class II, whereas γIFN increases class II more than class I. The ds nucleic acids also induce or activate Stat1, Stat3, mitogen-activated protein kinase, NF-κB, the class II transactivator, RFX5, and the IFN regulatory factor 1 differently from γIFN. CpG residues are not responsible for this effect, and the action of the ds polynucleotides could be shown in a variety of cell types in addition to thyrocytes. We suggest that this phenomenon is a plausible mechanism that might explain how viral infection of tissues or tissue injury triggers autoimmune disease; it is potentially relevant to host immune responses induced during gene therapy.

Cloning of Nicotianamine Synthase Genes, Novel Genes Involved in the Biosynthesis of Phytosiderophores

Nicotianamine synthase (NAS), the key enzyme in the biosynthetic pathway for the mugineic acid family of phytosiderophores, catalyzes the trimerization of S-adenosylmethionine to form one molecule of nicotianamine. We purified NAS protein and isolated the genes nas1, nas2, nas3, nas4, nas5-1, nas5-2, and nas6, which encode NAS and NAS-like proteins from Fe-deficient barley (Hordeum vulgare L. cv Ehimehadaka no. 1) roots. Escherichia coli expressing nas1 showed NAS activity, confirming that this gene encodes a functional NAS. Expression of nas genes as determined by northern-blot analysis was induced by Fe deficiency and was root specific. The NAS genes form a multigene family in the barley and rice genomes.

Dynamics of mobile element activity in chalcone synthase loci in the common morning glory (Ipomoea purpurea)

Mobile element dynamics in seven alleles of the chalcone synthase D locus (CHS-D) of the common morning glory (Ipomoea purpurea) are analyzed in the context of synonymous nucleotide sequence distances for CHS-D exons. By using a nucleotide sequence of CHS-D from the sister species Ipomoea nil (Japanese morning glory [Johzuka-Hisatomi, Y., Hoshino, A., Mori, T., Habu, Y. & Iida, S. (1999) Genes Genet. Syst. 74, 141–147], it is also possible to determine the relative frequency of insertion and loss of elements within the CHS-D locus between these two species. At least four different types of transposable elements exist upstream of the coding region, or within the single intron of the CHS-D locus in I. purpurea. There are three distinct families of miniature inverted-repeat transposable elements (MITES), and some recent transpositions of Activator/Dissociation (Ac/Ds)-like elements (Tip100), of some short interspersed repetitive elements (SINEs), and of an insertion sequence (InsIpCHSD) found in the neighborhood of this locus. The data provide no compelling evidence of the transposition of the mites since the separation of I. nil and I. purpurea roughly 8 million years ago. Finally, it is shown that the number and frequency of mobile elements are highly heterogeneous among different duplicate CHS loci, suggesting that the dynamics observed at CHS-D are locus-specific.

An essential amino acid residue in the protein translocation channel revealed by targeted random mutagenesis of SecY

The SecY/Sec61α family of membrane proteins are the central subunits of the putative protein translocation channel. We introduced random mutations into a segment of Escherichia coli SecY within its cytoplasmic domain 5, which was shown previously to be important for the SecA-dependent translocation activity. Mutations were classified into those retaining function and those gaining a dominant-interfering ability caused by a loss of function. These analyses showed that Arg-357, Pro-358, Gly-359, and Thr-362 are functionally important; Arg-357, conserved in almost all organisms, was identified as an indispensable residue.