Herpes Simplex Virus 1 VP22 Inhibits AIM2-Dependent Inflammasome Activation to Enable Efficient Viral Replication

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A comprehensive analysis of allelic methylation status of CpG islands on human chromosome 11q: comparison with chromosome 21q.

It was generally believed that autosomal CpG islands (CGIs) escape methylation. However, our comprehensive analysis of allelic methylation status of 149 CGIs on human chromosome 21q revealed that a sizable fraction of them are methylated on both alleles even in normal blood cells. Here, we performed a similar analysis of 656 CGIs on chromosome 11q, which is gene-rich in contrast with 21q. The results indicate that 11q contains less methylated CGIs, especially those with tandem repeats and those in the coding or 3'-untranslated regions (UTRs), than 21q. Thus, methylation status of CGIs may substantially differ from one chromosome to another.     

Time lag between the increase of IL-6 with fever and NF-kappaB activation in the peripheral blood in inflammatory myofibroblastic tumor

We describe a case of inflammatory myofibroblastic tumor (IMT) that occurred in the retroperitoneum. The patient manifested systemic symptoms, such as intermittent fever, anemia, thrombocytosis, and hypergammaglobulinemia. In order to elucidate the mechanism of intermittent fever in IMT, we analyzed nuclear factor-kappa B (NF-kappaB) activation in peripheral blood mononuclear cells (PBMCs) using flow cytometry, and serum cytokine levels. NF-kappaB activation was observed in the peripheral blood T cells and monocytes/macrophages. Among the measured cytokines, only interleukin (IL)-6 levels were elevated. IL-6 levels during pyrexia in the afternoon were higher than those during apyrexia in the morning. In contrast to IL-6, NF-kappaB activation in PBMCs was lower during pyrexia than during apyrexia; this is considered to be because the activation is subject to negative feedback. The time lag between the increase of IL-6 in the serum and NF-kappaB activation in the PBMCs at the onset of intermittent fever in IMT may provide further insight into the role of cytokines and NF-kappaB activation in febrile inflammatory diseases.     

Synthesis and absolute configuration of hormone alpha1

An important biological event in phytopathogens of the genus Phytophthora is sexual reproduction, which is conducted by two mating types, A1 and A2. A factor known as hormone alpha1 is secreted by the A1 mating type and induces the formation of sexual spores (oospores) in the A2 mating type. Here we describe the asymmetric synthesis and assignment of the absolute configuration of hormone alpha1 by oospore-inducing assays of the synthesized isomers.     

A rapid, simple, and effective method of constructing a randomly mutagenized plasmid library free from ligation

The QuikChange site-directed mutagenesis methodology was applied to constructing a randomly mutagenized plasmid library simply by adding manganese to the reaction mixture. This method is superior to the normally employed Pol I-type polymerase-based error-prone PCR in that (i) it does not require a subsequent ligation reaction, and (ii) there is no accumulation of mutations at the same site. alpha-Complementation analysis and subsequent sequence analyses of the lacZ alpha genes in the mutated library revealed that the mutations occurred randomly within the target gene and involved all possible base substitutions.     

Prolyl 4-Hydroxylation of ��-Fibrinogen: A NOVEL PROTEIN MODIFICATION REVEALED BY PLASMA PROTEOMICS*

Plasma proteome analysis requires sufficient power to compare numerous samples and detect changes in protein modification, because the protein content of human samples varies significantly among individuals, and many plasma proteins undergo changes in the bloodstream. A label-free proteomics platform developed in our laboratory, termed “Two-Dimensional Image Converted Analysis of Liquid chromatography and mass spectrometry (2DICAL),” is capable of these tasks. Here, we describe successful detection of novel prolyl hydroxylation of α-fibrinogen using 2DICAL, based on comparison of plasma samples of 38 pancreatic cancer patients and 39 healthy subjects. Using a newly generated monoclonal antibody 11A5, we confirmed the increase in prolyl-hydroxylated α-fibrinogen plasma levels and identified prolyl 4-hydroxylase A1 as a key enzyme for the modification. Competitive enzyme-linked immunosorbent assay of 685 blood samples revealed dynamic changes in prolyl-hydroxylated α-fibrinogen plasma level depending on clinical status. Prolyl-hydroxylated α-fibrinogen is presumably controlled by multiple biological mechanisms, which remain to be clarified in future studies.For comprehensive analysis of plasma proteins, it is necessary to compare a sufficient number of blood samples to avoid simple interindividual heterogeneity, because the protein content of human samples varies significantly among individuals. Also, the provision of sufficient power is needed to detect protein modification because many plasma proteins undergo changes in the bloodstream (1). Even though the proteomic technologies have advanced (2, 3), there remains room for improvement. Different isotope labeling and identification-based methods have been developed for quantitative proteomics technologies (4–6), but the number of samples that can be compared by the current isotope-labeling methods is limited, and identification-based proteomics is unable to capture information regarding unknown modifications.A label-free proteomics platform developed in our laboratory, termed “Two-Dimensional Image Converted Analysis of Liquid chromatography and mass spectrometry (2DICAL)² (7), simply compares the liquid chromatography and mass spectrometry (LC-MS) data and detects a protein modification by finding changes in the mass to charge ratio (m/z) and retention time (RT). Enhanced methods for accurate MS peak alignment across multiple LC runs have enabled the successful implementation of clinical studies requiring comparison of a large number of samples (8, 9). Using 2DICAL to analyze plasma samples of pancreatic cancer patients and healthy controls, novel prolyl hydroxylation of α-fibrinogen was successfully discovered.Fibrinogen and its modification has been investigated because of its clinical importance (10, 11). On the other hand, prolyl hydroxylation has attracted attention after the discovery of the hypoxia-inducible factor 1α (HIF1α) prolyl-hydroxylase and its role in switching of HIF1α functions (12). Prolyl hydroxylation in other proteins has been energetically sought, but only a few such proteins have been identified (13). Only one study has reported prolyl hydroxylation of fibrinogen at the β chain (14).Here, we report the detection of prolyl 4-hydroxylated α-fibrinogen by plasma proteome analysis, a protein modification that dynamically changes in plasma depending on the clinical status and is a candidate plasma biomarker.     

The Magnitude of the Light-induced Conformational Change in Different Rhodopsins Correlates with Their Ability to Activate G Proteins

Light converts rhodopsin, the prototypical G protein-coupled receptor, into a form capable of activating G proteins. Recent work has shown that the light-activated state of different rhodopsins can possess different molecular properties, especially different abilities to activate G protein. For example, bovine rhodopsin is ∼20-fold more effective at activating G protein than parapinopsin, a non-visual rhodopsin, although these rhodopsins share relatively high sequence similarity. Here we have investigated possible structural aspects that might underlie this difference. Using a site-directed fluorescence labeling approach, we attached the fluorescent probe bimane to cysteine residues introduced in the cytoplasmic ends of transmembrane helices V and VI in both rhodopsins. The fluorescence spectra of these probes as well as their accessibility to aqueous quenching agents changed dramatically upon photoactivation in bovine rhodopsin but only moderately so in parapinopsin. We also compared the relative movement of helices V and VI upon photoactivation of both rhodopsins by introducing a bimane label and the bimane-quenching residue tryptophan into helices VI and V, respectively. Both receptors showed movement in this region upon activation, although the movement appears much greater in bovine rhodopsin than in parapinopsin. Together, these data suggest that a larger conformational change in helices V and VI of bovine rhodopsin explains why it has greater G protein activation ability than other rhodopsins. The different amplitude of the helix movement may also be responsible for functional diversity of G protein-coupled receptors.Rhodopsin, the photosensitive G protein-coupled receptor (GPCR),³ is responsible for transmitting a light signal into an intracellular signaling cascade through activation of G protein in visual and non-visual photoreceptor cells. Rhodopsin consists of a protein moiety (opsin, comprising seven transmembrane α-helical segments) combined with a chromophore (11-cis retinal) that acts as the light-sensitive ligand. Photoisomerization of the 11-cis retinal to the all-trans form induces structural changes in the protein moiety that then enable it to couple with and activate the G protein.The crystal structure of inactive bovine rhodopsin has been extensively investigated (1–3). Recently, a crystal structure of inactive invertebrate squid rhodopsin was also solved (4), and crystal structures of the inactive form of β-adrenergic receptors and A2 adenosine receptor have been reported (5–7). Remarkably, all of these crystal structures exhibit a very similar arrangement for the seven transmembrane helices (4, 8). Together, these facts suggest that the architecture for the inactive form is conserved among rhodopsin-like GPCRs.The structural features of an activated GPCR are much less defined. Thus, a variety of biochemical and biophysical methods, including cross-linking methods (9, 10) and site-directed spin and fluorescence labeling methods (10–13), have been employed to identify the dynamic and structural changes involved in forming the activated state. The data from these studies consistently suggest that some kind of movement of helix VI is involved in the formation of the active state of the rhodopsins. In particular, the cytoplasmic end of helix VI has been proposed to rotate and/or tilt toward helix V (10–13). Remarkably, the recent crystal structures of bovine opsin are consistent with the widely accepted helix motion model. Both the structures of opsin (the ligand-free form of rhodopsin that has partial G protein activation ability) and a complex of opsin with a peptide derived from the G protein C terminus show a movement of helix VI toward helix V, compared with the dark state rhodopsin structure (14, 15). Studies of β-adrenergic and muscarinic receptors also show that agonist binding promotes movement of helix VI toward helix V in these receptors (16, 17). Because the region between the cytoplasmic ends of helices V and VI in various GPCRs is a main site of interaction with G proteins (18), it is possible that movement of helices V and VI leads to formation of a conformation capable of interacting with G protein (19).Together, these studies imply that the active state conformation of GPCRs may be similar. However, a detailed comparison of the active-state conformation for two different GPCRs has never been precisely undertaken in the same laboratory using the same methods.In this context we have been investigating rhodopsins with different functional properties to determine whether their active states have different conformations. Our goal was to determine whether any functional or structural differences in the active states of these GPCRs could be detected under the exact same experimental conditions.Previously, we have found that several rhodopsins, such as an invertebrate rhodopsin and a vertebrate non-visual rhodopsin parapinopsin (20, 21), can be activated not only by light but also by exogenous all-trans retinal acting as a full agonist (22). This is in contrast to vertebrate visual rhodopsins, including bovine rhodopsin, which cannot fully form the active state by direct binding of all-trans retinal (23), although all-trans retinal can fully activate some rhodopsin mutants (24). Other invertebrate rhodopsin (25) and the circadian photoreceptor melanopsin (26) can also bind all-trans retinal directly.Interestingly, the active form of the all-trans retinal-activated rhodopsins exhibit some striking differences in their spectroscopic and biochemical properties compared with vertebrate visual rhodopsins (27). In particular, the efficiency of bovine rhodopsin for activating G protein is ∼20∼50-fold higher than that of parapinopsin and invertebrate rhodopsin. This difference could be related to the difference in position of a specific amino acid residue counterion that is essential for rhodopsin to absorb visible light, namely one at position 113 or 181 (28).⁴ Further biochemical analyses using chimeric mutants combining rhodopsins with lower and higher G protein activation abilities suggested that the difference in G protein activation ability was because of a structural difference in transmembrane helices in the active states but not because of difference in amino acid sequence of G protein interaction site (29) (Fig. 1, A–C). In addition, the active states of parapinopsin and the invertebrate rhodopsin are thermally stable and can be reconverted to the inactive state by subsequent light absorption, showing photo-regenerable or bistable nature (21, 28), unlike the active state of bovine rhodopsin, which is thermally unstable and cannot revert to the inactive state by subsequent light absorption (30).Open in a separate windowFIGURE 1.Molecular properties and sites of fluorescent probe attachment for bovine rhodopsin and parapinopsin. A, sequence alignment of bovine rhodopsin and parapinopsin. Amino acid residues to which cysteine and fluorescence label were introduced are marked with red. The amino acid residues identical and similar between bovine rhodopsin and parapinopsin are shown with white characters with black and gray background, respectively. Bovine rhodopsin and parapinopsin show 41% sequence identity and 61% similarity. In this paper the residue number of parapinopsin is described by using the bovine rhodopsin numbering system. B and C, comparison of G protein activation ability of rhodopsin and parapinopsin wild type (WT) proteins and loop-replaced mutants. In these mutants the second and/or third cytoplasmic loop was swapped between the two receptors. ParaL2 and ParaL3 indicate mutants of bovine rhodopsin in which second and third loops were replaced with the corresponding loop of parapinopsin, respectively. RhoL2 and RhoL3 indicate mutants of parapinopsin in which the second and third loops were replaced with the corresponding loops of bovine rhodopsin, respectively. ParaL2L3 and RhoL2L3 are mutants of bovine rhodopsin and parapinopsin in which both the second and third loops were swapped, respectively. See Terakita et al. (29) for more details. Data are presented as the means ± S.E. of three separate experiments except for mutants RhoL3, RhoL2L3, and ParaL2L3 (n = 2). D, model of bovine rhodopsin. Amino acid residues which were mutated to cysteine to enable attachment of the fluorescent probe bimane or mutated to tryptophan are indicated. Positions 226, 227, 244, 250, and 251 in the crystal structure of the dark state of bovine rhodopsin (PDB code 1GZM) are shown. E, reaction of the mBBr label with a sulfhydryl group. The mutants labeled with mBBr are named by the number of the residue and the suffix B₁. F, reaction of the PDT-bimane with a sulfhydryl group. The mutants labeled with PDT-bimane are named by the number of the residue and the suffix B₂. The disulfide linkage between the label and protein can be cleaved using Tris(2-carboxyethyl)phosphine (32).In this study we used site-directed fluorescence labeling (13, 31) to compare the structural features of active states of bovine rhodopsin with lamprey parapinopsin, a UV-sensitive non-visual pigment in the pineal organs (21). Parapinopsin shows relatively high sequence similarity (∼60%) to bovine rhodopsin, yet it has a greatly reduced ability to activate G protein (see Fig. 1, A–C) (21, 28). Using established protocols, we introduced cysteine residues into the cytoplasmic ends of helices V and VI, the region proposed to rearrange upon activation in GPCRs (11, 12, 14, 18). We then site-specifically labeled these cysteines with the small, non-polar fluorescent probe, bimane, and used the spectral properties of these bimane probes to act as reporter groups for environmental changes around their site of attachment upon formation of the photoactivated state for both rhodopsins.In addition, we measured changes in the relative proximity of the cytoplasmic ends of helix VI to helix V in both rhodopsin and parapinopsin using the tryptophan-induced-quenching of bimane (TrIQ-bimane) fluorescence method (31, 32). TrIQ-bimane measures the efficiency of intramolecular fluorescence quenching of bimane caused by tryptophan (Trp), which occurs in a distance-dependent manner. The goal of this study was to determine whether the helices in both receptors moved in the same way during formation of the active state. Our results show that whereas movement of helix VI relative to helix V occurs during formation of the active state for both parapinopsin and bovine rhodopsin, the “amplitude” of the movement is markedly different between the two rhodopsins.     

Semisynthesis of C-ring modified triptolide analogues and their cytotoxic activities

Several C-ring modified analogues of a potent antileukemic diterpene, triptolide (1), were synthesized and their structure-activity relationships were studied.     

The chromosomal mazEF locus of Streptococcus mutans encodes a functional type II toxin-antitoxin addiction system

Type II chromosomal toxin-antitoxin (TA) modules consist of a pair of genes that encode two components: a stable toxin and a labile antitoxin interfering with the lethal action of the toxin through protein complex formation. Bioinformatic analysis of Streptococcus mutans UA159 genome identified a pair of linked genes encoding a MazEF-like TA. Our results show that S. mutans mazEF genes form a bicistronic operon that is cotranscribed from a σ70-like promoter. Overproduction of S. mutans MazF toxin had a toxic effect on S. mutans which can be neutralized by coexpression of its cognate antitoxin, S. mutans MazE. Although mazF expression inhibited cell growth, no cell lysis of S. mutans cultures was observed under the conditions tested. The MazEF TA is also functional in E. coli, where S. mutans MazF did not kill the cells but rather caused reversible cell growth arrest. Recombinant S. mutans MazE and MazF proteins were purified and were shown to interact with each other in vivo, confirming the nature of this TA as a type II addiction system. Our data indicate that MazF is a toxic nuclease arresting cell growth through the mechanism of RNA cleavage and that MazE inhibits the RNase activity of MazF by forming a complex. Our results suggest that the MazEF TA module might represent a cell growth modulator facilitating the persistence of S. mutans under the harsh conditions of the oral cavity.