A non-radioactive assay for selenophosphate synthetase activity using recombinant pyruvate pyrophosphate dikinase from Thermus thermophilus HB8

Biosynthesis of selenocysteine-containing proteins requires monoselenophosphate, a selenium-donor intermediate generated by selenophosphate synthetase (Sephs). A non-radioactive assay was developed as an alternative to the standard [8-¹⁴C] AMP-quantifying assay. The product, AMP, was measured using a recombinant pyruvate pyrophosphate dikinase from Thermus thermophilus HB8. The K_M and k_cat for Sephs2-Sec60Cys were determined to be 26 μM and 0.352 min^?1, respectively.     

Delivery of selenium to selenophosphate synthetase for selenoprotein biosynthesis

Background

Selenophosphate, the key selenium donor for the synthesis of selenoprotein and selenium-modified tRNA, is produced by selenophosphate synthetase (SPS) from ATP, selenide, and H₂O. Although free selenide can be used as the in vitro selenium substrate for selenophosphate synthesis, the precise physiological system that donates in vivo selenium substrate to SPS has not yet been characterized completely.

Phosphatases in the rice stem borer, Chilo suppressalis Walker (Lepidoptera; Pyralidae): Some properties and changes of the activities during hibernation

To elucidate the regulation of glycerol synthesis from α-glycerophosphate (α-GP) in larvae of the rice stem borer, Chilo suppressalis Walker, phosphatase activities were examined during hibernation. The presence of alkaline and acid phosphatase was demonstrated in hibernating larvae. The activity of the alkaline type decreased during diapause entry while the activity of the acid type increased at the initiation of diapause and then decreased during postdiapause stage. This pattern of acid type activity change coincided with that of glycerol accumulation in hemolymph during hibernation. Thus, the acid type phosphatase may largely be involved in glycerol synthesis.     

Adjustment of Cell-Type Composition Minimizes Systematic Bias in Blood DNA Methylation Profiles Derived by DNA Collection Protocols

Differences in DNA collection protocols may be a potential confounder in epigenome-wide association studies (EWAS) using a large number of blood specimens from multiple biobanks and/or cohorts. Here we show that pre-analytical procedures involved in DNA collection can induce systematic bias in the DNA methylation profiles of blood cells that can be adjusted by cell-type composition variables. In Experiment 1, whole blood from 16 volunteers was collected to examine the effect of a 24 h storage period at 4°C on DNA methylation profiles as measured using the Infinium HumanMethylation450 BeadChip array. Our statistical analysis showed that the P-value distribution of more than 450,000 CpG sites was similar to the theoretical distribution (in quantile-quantile plot, λ = 1.03) when comparing two control replicates, which was remarkably deviated from the theoretical distribution (λ = 1.50) when comparing control and storage conditions. We then considered cell-type composition as a possible cause of the observed bias in DNA methylation profiles and found that the bias associated with the cold storage condition was largely decreased (λ_adjusted = 1.14) by taking into account a cell-type composition variable. As such, we compared four respective sample collection protocols used in large-scale Japanese biobanks or cohorts as well as two control replicates. Systematic biases in DNA methylation profiles were observed between control and three of four protocols without adjustment of cell-type composition (λ = 1.12–1.45) and no remarkable biases were seen after adjusting for cell-type composition in all four protocols (λ_adjusted = 1.00–1.17). These results revealed important implications for comparing DNA methylation profiles between blood specimens from different sources and may lead to discovery of disease-associated DNA methylation markers and the development of DNA methylation profile-based predictive risk models.     

Tenascin-C Induction in Whitlock-Witte Culture: A Relevant Role of the Thiol Moiety in Lymphoid-Lineage Differentiation

The extracellular matrix (ECM) glycoprotein tenascin-C is expressed in a temporally and spatially restricted pattern during embryogenesis and carcinogenesis in association with stromal-epithelial interactions. First, we investigated the production of tenascin-C and other ECM glycoproteins in the established in vitro model system specific for the lymphoid-lineage hemopoiesis, i.e., the Whitlock-Witte (W-W) culture system. In murine primary long-term bone marrow cultures, tenascin-C was produced constitutively and was expressed significantly in higher amounts in this system than in the other established in vitro model system specific for the myeloid-lineage hemopoiesis, i.e., the Dexter culture system. 2-Mercaptoethanol (2-ME), a component of the W-W system, induced the secretion of tenascin-C and upregulated the expression of its mRNA. Furthermore, the reduced glutathione, which, like 2-ME, contains a thiol moiety, induced tenascin-C glycoprotein and its mRNA. By contrast, hydrocortisone (HC), a component of the Dexter system, inhibited the secretion of ECM glycoproteins. 2-ME and TGF-β1, the latter of which is known as an inducer of ECM glycoproteins, had an additive effect on the induction of tenascin-C when they were simultaneously added to the W-W system. The TGF-β receptor binding analysis demonstrated that this induction by 2-ME was not mediated by the cell-surface TGF-β receptors, suggesting that it was regulated independently of TGF-β1. Then, the role of thiol compounds in the lymphoid-lineage differentiation was examined. The omission of 2-ME from the W-W system completely eliminated its ability to support the lymphoid-lineage differentiation. Glutathione, which, unlike 2-ME, does not passively permeate through the plasma membrane, did not support the development of a lymphoid lineage. These results indicate that 2-ME, essential for the lymphoid-lineage differentiation in the W-W culture system, is a potent inducer of tenascin-C expression in vitro.     

Global Identification of Genes Affecting Iron-Sulfur Cluster Biogenesis and Iron Homeostasis

Iron-sulfur (Fe-S) clusters are ubiquitous cofactors that are crucial for many physiological processes in all organisms. In Escherichia coli, assembly of Fe-S clusters depends on the activity of the iron-sulfur cluster (ISC) assembly and sulfur mobilization (SUF) apparatus. However, the underlying molecular mechanisms and the mechanisms that control Fe-S cluster biogenesis and iron homeostasis are still poorly defined. In this study, we performed a global screen to identify the factors affecting Fe-S cluster biogenesis and iron homeostasis using the Keio collection, which is a library of 3,815 single-gene E. coli knockout mutants. The approach was based on radiolabeling of the cells with [2-¹⁴C]dihydrouracil, which entirely depends on the activity of an Fe-S enzyme, dihydropyrimidine dehydrogenase. We identified 49 genes affecting Fe-S cluster biogenesis and/or iron homeostasis, including 23 genes important only under microaerobic/anaerobic conditions. This study defines key proteins associated with Fe-S cluster biogenesis and iron homeostasis, which will aid further understanding of the cellular mechanisms that coordinate the processes. In addition, we applied the [2-¹⁴C]dihydrouracil-labeling method to analyze the role of amino acid residues of an Fe-S cluster assembly scaffold (IscU) as a model of the Fe-S cluster assembly apparatus. The analysis showed that Cys37, Cys63, His105, and Cys106 are essential for the function of IscU in vivo, demonstrating the potential of the method to investigate in vivo function of proteins involved in Fe-S cluster assembly.     

GAPDH-A Recruits a Plant Virus Movement Protein to Cortical Virus Replication Complexes to Facilitate Viral Cell-to-Cell Movement

The formation of virus movement protein (MP)-containing punctate structures on the cortical endoplasmic reticulum is required for efficient intercellular movement of Red clover necrotic mosaic virus (RCNMV), a bipartite positive-strand RNA plant virus. We found that these cortical punctate structures constitute a viral replication complex (VRC) in addition to the previously reported aggregate structures that formed adjacent to the nucleus. We identified host proteins that interacted with RCNMV MP in virus-infected Nicotiana benthamiana leaves using a tandem affinity purification method followed by mass spectrometry. One of these host proteins was glyceraldehyde 3-phosphate dehydrogenase-A (NbGAPDH-A), which is a component of the Calvin-Benson cycle in chloroplasts. Virus-induced gene silencing of NbGAPDH-A reduced RCNMV multiplication in the inoculated leaves, but not in the single cells, thereby suggesting that GAPDH-A plays a positive role in cell-to-cell movement of RCNMV. The fusion protein of NbGAPDH-A and green fluorescent protein localized exclusively to the chloroplasts. In the presence of RCNMV RNA1, however, the protein localized to the cortical VRC as well as the chloroplasts. Bimolecular fluorescence complementation assay and GST pulldown assay confirmed in vivo and in vitro interactions, respectively, between the MP and NbGAPDH-A. Furthermore, gene silencing of NbGAPDH-A inhibited MP localization to the cortical VRC. We discuss the possible roles of NbGAPDH-A in the RCNMV movement process.     

Crystal Structure of a Homolog of Mammalian Serine Racemase from Schizosaccharomyces pombe

d-Serine is an endogenous coagonist for the N-methyl-d-aspartate receptor and is involved in excitatory neurotransmission in the brain. Mammalian pyridoxal 5′-phosphate-dependent serine racemase, which is localized in the mammalian brain, catalyzes the racemization of l-serine to yield d-serine and vice versa. The enzyme also catalyzes the dehydration of d- and l-serine. Both reactions are enhanced by Mg·ATP in vivo. We have determined the structures of the following three forms of the mammalian enzyme homolog from Schizosaccharomyces pombe: the wild-type enzyme, the wild-type enzyme in the complex with an ATP analog, and the modified enzyme in the complex with serine at 1.7, 1.9, and 2.2 Å resolution, respectively. On binding of the substrate, the small domain rotates toward the large domain to close the active site. The ATP binding site was identified at the domain and the subunit interface. Computer graphics models of the wild-type enzyme complexed with l-serine and d-serine provided an insight into the catalytic mechanisms of both reactions. Lys-57 and Ser-82 located on the protein and solvent sides, respectively, with respect to the cofactor plane, are acid-base catalysts that shuttle protons to the substrate. The modified enzyme, which has a unique “lysino-d-alanyl” residue at the active site, also exhibits catalytic activities. The crystal-soaking experiment showed that the substrate serine was actually trapped in the active site of the modified enzyme, suggesting that the lysino-d-alanyl residue acts as a catalytic base in the same manner as inherent Lys-57 of the wild-type enzyme.d-Serine, which is present at a high level in the mammalian brain, serves as an endogenous coagonist for the N-methyl-d-aspartate (NMDA)⁵ receptor selectively localized on the postsynaptic membrane of the excitatory synapse (1–5) and is involved in excitatory neurotransmission and higher brain functions such as learning and memory (3, 6, 7). Stimulation of the NMDA receptor requires the binding of d-serine as well as the agonist l-glutamate. The major enzyme for d-serine synthesis from l-serine in the brain is considered to be pyridoxal 5′-phosphate (PLP)-dependent serine racemase (SR) (8–10). d-Serine and SR are localized on protoplasmic astrocytes that have the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor. Glutamate released from presynaptic neurons approaches and activates the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor, which in turn induces SR to produce d-serine and is followed by d-serine release from astrocytes that act on the NMDA receptor. Recently, it was shown that not only glia but also neurons synthesize and release d-serine involved in signaling (11). SR also catalyzes α,β-elimination of water from d- or l-serine to form pyruvate and ammonia as well as the conversion of l-serine into d-serine and vice versa and is presumed to link d-serine synthesis and energy metabolism of astrocytes (12) and to control the d-serine level (13). Mg·ATP, which is fully bound to SR under physiological conditions, stimulates racemization and the α,β-elimination reaction catalyzed by SR (12, 14).SR was first discovered in pupae of the silkworm Bombyx mori (15), which was followed by purification of the enzyme from a rat brain and cloning of the mouse and human genes (8, 9). The primary structure of mammalian SR is distinct from those of racemases from prokaryotes but is similar to those of fold-type II PLP-dependent enzymes (16–18). We have cloned and expressed the Schizosaccharomyces pombe gene homologous to human and mouse SRs, the sequence identities being 35.1 and 37.4%, respectively, in Escherichia coli. The protein product is a bifunctional enzyme that catalyzes racemization and the α,β-elimination reaction of D, l-serine as mammalian SR does. SR from S. pombe (spSR) comprises 322 residues (the N-terminal Met is removed in the purified enzyme) and one PLP per subunit, the subunit molecular weight being 34,917. The mammalian SR homolog, spSR, is an interesting target enzyme for the development of a novel therapeutic compound controlling the d-serine level because d-serine is the product of an SR-catalyzed reaction. In our recent report, the active site of spSR was shown to be modified with its natural substrate serine by mass spectroscopic and x-ray studies (19). Interestingly, the catalytic lysine, which originally forms a Schiff base with PLP, is converted to a lysino-d-alanyl residue through the reaction with the substrate, serine (Fig. 1). The modified enzyme exhibits racemase (54% of the wild-type enzyme) and α,β-elimination (68% of the wild-type enzyme) activities with the amino group of the d-alanyl moiety of the lysinoalanyl residue forming a Schiff base with PLP in place of the lysine (19). In addition, the mammalian SR seems to be possibly modified to have a lysinoalanyl residue at the active site, as observed in spSR (20).Open in a separate windowFIGURE 1.Covalent modification of the active site. The catalytic Lys-57 in spSRw is converted to lysino-d-alanyl residue. The α-amino group (indicated with “α”) of the d-alanyl moiety in the residue acts as a catalytic base in spSRm. The circled P is a phosphate group.Although the structure of modified spSR (spSRm) has been determined (19), the structure-function relationship of essential wild-type spSR (spSRw), the binding mode of activator Mg·ATP, the catalytic base to shuttle protons to the substrate d-serine, and the substrate recognition of the modified enzyme have not yet been uncovered. We now report the three-dimensional structures of unliganded spSRw in the open form, spSRw·AMP-PCP in the open form, and spSRm·serine in the closed form.     

Brain-derived Neurotrophic Factor Enhances the Basal Rate of Protein Synthesis by Increasing Active Eukaryotic Elongation Factor 2 Levels and Promoting Translation Elongation in Cortical Neurons

The constitutive and activity-dependent components of protein synthesis are both critical for neural function. Although the mechanisms controlling extracellularly induced protein synthesis are becoming clear, less is understood about the molecular networks that regulate the basal translation rate. Here we describe the effects of chronic treatment with various neurotrophic factors and cytokines on the basal rate of protein synthesis in primary cortical neurons. Among the examined factors, brain-derived neurotrophic factor (BDNF) showed the strongest effect. The rate of protein synthesis increased in the cortical tissues of BDNF transgenic mice, whereas it decreased in BDNF knock-out mice. BDNF specifically increased the level of the active, unphosphorylated form of eukaryotic elongation factor 2 (eEF2). The levels of active eEF2 increased and decreased in BDNF transgenic and BDNF knock-out mice, respectively. BDNF decreased kinase activity and increased phosphatase activity against eEF2 in vitro. Additionally, BDNF shortened the ribosomal transit time, an index of translation elongation. In agreement with these results, overexpression of eEF2 enhanced protein synthesis. Taken together, our results demonstrate that the increased level of active eEF2 induced by chronic BDNF stimulation enhances translational elongation processes and increases the total rate of protein synthesis in neurons.The synthesis and post-translational modification of proteins play key roles in neural development, synaptic plasticity, and cognitive brain functions such as learning and memory (1, 2). Recent studies have revealed that activity-dependent regulation of translation affects neural plasticity (3, 4). Previously, we reported that BDNF,² a critical molecule for neural plasticity (5–7), enhances protein synthesis and activates the translational machinery in central nervous system neurons (8). In addition, neurotransmitters such as glutamate (9, 10), dopamine (11), and serotonin (12) are also reported to facilitate translation in neurons. These observations indicate that endogenous molecules can acutely modulate neuronal translation in response to neural activity. Translation of an mRNA molecule comprises three steps: initiation, elongation, and release (or termination) (13). In the first step, mRNA and methionyl-tRNA_i^Met are recruited to a ribosome. During elongation, aminoacyl-tRNAs are sequentially recruited and the nascent peptide chain lengthens incrementally as amino acids are covalently attached via peptide bonds. Finally, the polypeptide chain is released from the ribosome. Each step is regulated by a variety of factors. The activities of these regulatory proteins are predominantly controlled by phosphorylation and GTP binding. BDNF activates both initiation and elongation by modulating these processes (8, 14, 15).In addition to these acute, stimulation-induced changes in the translation rate, the long term regulation of translation plays important roles in developing and mature brains. In fact, recent studies have shown that genetic disruption or overexpression of translation factors or modulator genes alters synaptic plasticity and behavior as well as the basal rate of protein synthesis. Mice lacking the gene encoding GCN2, a kinase that phosphorylates eIF2α, exhibited enhanced translation as well as aberrant long term potentiation and spatial learning (16). Similar phenotypes have been observed in mice carrying a constitutively active mutant variant (Ser⁵² to Ala) of eIF2α (17). Mice lacking eIF4E-binding protein 2 (4EBP2) exhibited increased cap-dependent translation and altered long-term potentiation, long-term depression (LTD), and learning (18, 19). Mice expressing a transgene encoding a dominant-negative version of MEK, which inhibits the phosphorylation of eIF4E and protein synthesis, were found to have learning deficits (20). Thus, modifying the rate of protein synthesis can produce deleterious effects on synaptic plasticity and brain function.Although genetic modifications can affect translation, the mechanisms by which the basal translation rate is controlled in normal neurons are unknown. Here, we demonstrate that chronic treatment of primary cortical neurons with BDNF increases the level of active, unphosphorylated eukaryotic elongation factor 2 (eEF2) and enhances the rates of elongation and protein synthesis. Analysis of BDNF mutant mice supports a role for this neurotrophin in regulating the basal rate of protein synthesis.