Dynamic action of an intrinsically disordered protein in DNA compaction that induces mycobacterial dormancy.

Mycobacteria are the major human pathogens with the capacity to become dormant persisters. Mycobacterial DNA-binding protein 1 (MDP1), an abundant histone-like protein in dormant mycobacteria, induces dormancy phenotypes, e.g. chromosome compaction and growth suppression. For these functions, the polycationic intrinsically disordered region (IDR) is essential. However, the disordered property of IDR stands in the way of clarifying the molecular mechanism. Here we clarified the molecular and structural mechanism of DNA compaction by MDP1. Using high-speed atomic force microscopy, we observed that monomeric MDP1 bundles two adjacent DNA duplexes side-by-side via IDR. Combined with coarse-grained molecular dynamics simulation, we revealed the novel dynamic DNA cross-linking model of MDP1 in which a stretched IDR cross-links two DNA duplexes like double-sided tape. IDR is able to hijack HU function, resulting in the induction of strong mycobacterial growth arrest. This IDR-mediated reversible DNA cross-linking is a reasonable model for MDP1 suppression of the genomic function in the resuscitable non-replicating dormant mycobacteria.

Conversion map from quantitative parameter mapping to myelin water fraction: comparison with R1·R2* and myelin water fraction in white matter.

OBJECTIVE: To clarify the relationship between myelin water fraction (MWF) and R1⋅R2* and to develop a method to calculate MWF directly from parameters derived from QPM, i.e., MWF converted from QPM (MWFQPM). MATERIALS AND METHODS: Subjects were 12 healthy volunteers. On a 3 T MR scanner, dataset was acquired using spoiled gradient-echo sequence for QPM. MWF and R1⋅R2* maps were derived from the multi-gradient-echo (mGRE) dataset. Volume-of-interest (VOI) analysis using the JHU-white matter (WM) atlas was performed. All the data in the 48 WM regions measured VOI were plotted, and quadratic polynomial approximations of each region were derived from the relationship between R1·R2* and the two-pool model-MWF. The R1·R2* map was converted to MWFQPM map. MWF atlas template was generated using converted to MWF from R1·R2* per WM region. RESULTS: The mean MWF and R1·R2* values for the 48 WM regions were 11.96 ± 6.63%, and 19.94 ± 4.59 s-2, respectively. A non-linear relationship in 48 regions of the WM between MWF and R1·R2* values was observed by quadratic polynomial approximation (R2 ≥ 0.963, P < 0.0001). DISCUSSION: MWFQPM map improved image quality compared to the mGRE-MWF map. Myelin water atlas template derived from MWFQPM may be generated with combined multiple WM regions.

The flexible N-terminal motif of uL11 unique to eukaryotic ribosomes interacts with P-complex and facilitates protein translation.

Eukaryotic uL11 contains a conserved MPPKFDP motif at the N-terminus that is not found in archaeal and bacterial homologs. Here, we determined the solution structure of human uL11 by NMR spectroscopy and characterized its backbone dynamics by 15N-1H relaxation experiments. We showed that these N-terminal residues are unstructured and flexible. Structural comparison with ribosome-bound uL11 suggests that the linker region between the N-terminal domain and C-terminal domain of human uL11 is intrinsically disordered and only becomes structured when bound to the ribosomes. Mutagenesis studies show that the N-terminal conserved MPPKFDP motif is involved in interacting with the P-complex and its extended protuberant domain of uL10 in vitro. Truncation of the MPPKFDP motif also reduced the poly-phenylalanine synthesis in both hybrid ribosome and yeast mutagenesis studies. In addition, G→A/P substitutions to the conserved GPLG motif of helix-1 reduced poly-phenylalanine synthesis to 9-32% in yeast ribosomes. We propose that the flexible N-terminal residues of uL11, which could extend up to ∼25 Šfrom the N-terminal domain of uL11, can form transient interactions with the uL10 that help to fetch and fix it into a position ready for recruiting the incoming translation factors and facilitate protein synthesis.

Arabidopsis ROOT PHOTOTROPISM2 Is a Light-Dependent Dynamic Modulator of Phototropin1.

The Arabidopsis (Arabidopsis thaliana) blue light photoreceptor phototropin1 (phot1) is a blue light-activated Ser/Thr protein kinase that mediates various light responses, including phototropism. The function of phot1 in hypocotyl phototropism is dependent on the light induction of ROOT PHOTOTROPISM2 (RPT2) proteins within a broad range of blue light intensities. It is not yet known however how RPT2 contributes to the photosensory adaptation of phot1 to high intensity blue light and the phototropic responses under bright light conditions. We show that RPT2 suppresses the activity of phot1 and demonstrate that RPT2 binds to the PHOT1 light, oxygen or voltage sensing1 (LOV1) domain that is required for its high photosensitivity. Our biochemical analyses revealed that RPT2 inhibits autophosphorylation of phot1, suggesting that it suppresses the photosensitivity and/or kinase activity of phot1 through the inhibition of LOV1 function. We found that RPT2 proteins are degraded via a ubiquitin-proteasome pathway when phot1 is inactive and are stabilized under blue light in a phot1-dependent manner. We propose that RPT2 is a molecular rheostat that maintains a moderate activation level of phot1 under any light intensity conditions.

A novel ribosome-dimerization protein found in the hyperthermophilic archaeon Pyrococcus furiosus using ribosome-associated proteomics.

Ribosome dimerization is one of the bacterial events that suppresses protein synthesis in the stationary phase. Protein factors responsible for ribosome dimerization in bacteria are well characterized, whereas no information is available for the corresponding factors in archaeal and eukaryotic cells. Here we describe a protein found among the ribosome-associated proteins which dimerizes the 30S ribosomal subunit of the archaeon Pyrococcus furiosus. The ribosome-associated proteins were prepared by high-salt wash of crude ribosomes, and analyzed by nanoflow liquid chromatography-tandem mass spectrometry (nano LC-MS/MS). Of the detected proteins we focused on a protein (PF0560) whose Protein Score was the highest of all of the function-unknown proteins. PF0560 protein had a pronounced effect on the sedimentation pattern of the 30S ribosomal subunit; addition of this protein to isolated 30S subunit reduced the 30S fraction and increased the amount of the 50S fraction. This increase presumably corresponds to the dimer of the 30S subunit. The PF0560-dependent 30S-dimerization, was also observed by gel electrophoretic analysis. This effect was not observed in EDTA-treated 30S subunit, with protein-free 16S rRNA or with bacterial/eukaryotic ribosomal small subunits. Furthermore, PF0560 protein suppressed the formation of functional 70S ribosomes. These results suggest that PF0560 is a novel 30S dimerization factor, which might participate in regulation of archaeal translation.

Well-Controlled Living Polymerization of N-Propargylamides and Their Derivatives by Rhodium Catalysis.

A substantially improved method for living polymerization of N-propargylamides and their derivatives has been developed. Rhodium(I) complexes bearing an aryl-substituted 1,3,5-hexatriene chain can work as excellent initiators of the polymerization of such non-conjugated terminal alkynes to give the corresponding cis-stereoregular polymers having a narrow molecular weight distribution. The typical living nature has been confirmed by investigating the effects of initial feed ratios of the monomer to the initiator on the molecular weight of the resulting polymers as well as multistage polymerization. Moreover, we demonstrated that the present method enables functionalization of both polymer chain ends and synthesis of novel block copolymers consisting of poly(N-propargylamide) and poly(phenylacetylene) blocks with a narrow molecular weight distribution.

Deletion of PIKfyve alters alveolar macrophage populations and exacerbates allergic inflammation in mice.

Alveolar macrophages (AMs) are specialized tissue-resident macrophages that orchestrate the immune responses to inhaled pathogens and maintain organ homeostasis of the lung. Dysregulation of AMs is associated with allergic inflammation and asthma. Here, we examined the role of a phosphoinositide kinase PIKfyve in AM development and function. Mice with conditionally deleted PIKfyve in macrophages have altered AM populations. PIKfyve deficiency results in a loss of AKT activation in response to GM-CSF, a cytokine critical for AM development. Upon exposure to house dust mite extract, mutant mice display severe lung inflammation and allergic asthma accompanied by infiltration of eosinophils and lymphoid cells. Moreover, they have defects in production of retinoic acid and fail to support incorporation of Foxp3+ Treg cells in the lung, resulting in exacerbation of lung inflammation. Thus, PIKfyve plays a role in preventing excessive lung inflammation through regulating AM function.

Impact of sinus surgery on type 2 airway and systemic inflammation in asthma.

Objective: Asthma is frequently associated with chronic rhinosinusitis with nasal polyps (CRSwNP). Although endoscopic sinus surgery (ESS) improves asthma control in CRSwNP patients with asthma, the mechanism that underlies the response to surgical treatment is still unclear. We evaluated the relevance of changes in asthma control and changes in airway/systemic inflammation in eosinophilic CRSwNP patients with not well controlled asthma who underwent ESS.Methods: We prospectively assessed changes in the asthma control questionnaire (ACQ) score, blood eosinophil counts (B-Eos), forced expiratory volume in 1 s (FEV1), and fraction of exhaled nitric oxide (FeNO) levels at 1-week before and 8 and 52 weeks after ESS.Results: Twenty-five subjects were analyzed. The ACQ score, B-Eos, and FeNO decreased, and FEV1 increased significantly after ESS. In the period from baseline to 52 weeks after ESS, changes in ACQ were significantly correlated with the changes in blood eosinophil counts (r = 0.58, p<.01) and FeNO (r = 0.45, p<.05). Ten subjects (40%) showed consistently improved asthma control at 52-weeks after ESS. In the remaining subjects, although the ACQ score temporarily improved at 8-weeks after ESS, but eventually deteriorated at 52-weeks. Higher levels of total immunoglobulin E were associated with long-term improved asthma control after ESS.Conclusions: In eosinophilic CRSwNP patients with asthma, sinus surgery impacts asthma control through the suppression of airway/systemic type 2 inflammation. The present study reinforced the common pathophysiology of type 2 inflammation between the upper and lower airways.

Leptin stimulates gonadotropin release and ovarian development in marine teleost chub mackerel.

Leptin transmits information about energy stored in the periphery to the reproductive axis and is an essential signal for puberty initiation in mammals; however, to date, few studies have focused on the direct effects of leptin stimulation on reproductive factors in fish. This study demonstrated the effect of leptin stimulation on important reproductive factors and ovarian development in the marine teleost chub mackerel (Scomber japonicus). We prepared recombinant leptin and conducted functional analyses through in vitro bioassays using primary pituitary cells, long-term leptin treatment administered to pre-pubertal females, and intracerebroventricular (ICV) administration. The results showed that leptin stimulation strongly induced gonadotropin (follicle-stimulating hormone: FSH and luteinizing hormone: LH) secretion from pituitary cells collected from pre-pubertal females, and that long-term leptin treatment significantly promoted ovarian development and triggered pubertal onset. Furthermore, ICV administration of leptin did not affect kisspeptin gene expression but significantly upregulated gonadotropin-releasing hormone 1 (gnrh1), fshb and lhb gene expression in sexually immature females. These results strongly suggest leptin as an important signal for reproductive-axis activation in chub mackerel.

The ribosomal stalk protein is crucial for the action of the conserved ATPase ABCE1.

The ATP-binding cassette (ABC) protein ABCE1 is an essential factor in ribosome recycling during translation. However, the detailed mechanochemistry of its recruitment to the ribosome, ATPase activation and subunit dissociation remain to be elucidated. Here, we show that the ribosomal stalk protein, which is known to participate in the actions of translational GTPase factors, plays an important role in these events. Biochemical and crystal structural data indicate that the conserved hydrophobic amino acid residues at the C-terminus of the archaeal stalk protein aP1 binds to the nucleotide-binding domain 1 (NBD1) of aABCE1, and that this binding is crucial for ATPase activation of aABCE1 on the ribosome. The functional role of the stalk•ABCE1 interaction in ATPase activation and the subunit dissociation is also investigated using mutagenesis in a yeast system. The data demonstrate that the ribosomal stalk protein likely participates in efficient actions of both archaeal and eukaryotic ABCE1 in ribosome recycling. The results also show that the stalk protein has a role in the function of ATPase as well as GTPase factors in translation.

Structural and Mutagenesis Studies Evince the Role of the Extended Protuberant Domain of Ribosomal Protein uL10 in Protein Translation.

The lateral stalk of ribosomes constitutes the GTPase-associated center and is responsible for recruiting translation factors to the ribosomes. The eukaryotic stalk contains a P-complex, in which one molecule of uL10 (formerly known as P0) protein binds two copies of P1/P2 heterodimers. Unlike bacterial uL10, eukaryotic uL10 has an extended protuberant (uL10ext) domain inserted into the N-terminal RNA-binding domain. Here, we determined the solution structure of the extended protuberant domain of Bombyx mori uL10 by nuclear magnetic resonance spectroscopy. Comparison of the structures of the B. mori uL10ext domain with eRF1-bound and eEF2-bound ribosomes revealed significant structural rearrangement in a "hinge" region surrounding Phe183, a residue conserved in eukaryotic but not in archaeal uL10. 15N relaxation analyses showed that residues in the hinge region have significantly large values of transverse relaxation rates. To test the role of the conserved phenylalanine residue, we created a yeast mutant strain expressing an F181A variant of uL10. An in vitro translation assay showed that the alanine substitution increased the level of polyphenylalanine synthesis by ∼33%. Taken together, our results suggest that the hinge motion of the uL10ext domain facilitates the binding of different translation factors to the GTPase-associated center during protein synthesis.

High-resolution crystal structure of peptidyl-tRNA hydrolase from Thermus thermophilus.

Peptidyl-tRNA hydrolase (Pth) cleaves the ester bond between the peptide and the tRNA of peptidyl-tRNA molecules, which are the products of defective translation, to recycle the tRNA for further rounds of protein synthesis. Pth is ubiquitous in nature, and its activity is essential for bacterial viability. Here, we have determined the crystal structure of Pth from Thermus thermophilus (TtPth) at 1.00 Å resolution. This is the first structure of a Pth from a thermophilic bacterium and the highest resolution Pth structure reported so far. The present atomic resolution data enabled the calculation of anisotropic displacement parameters for all atoms, which revealed the directionality of the fluctuations of key regions for the substrate recognition. Comparisons between TtPth and mesophilic bacterial Pths revealed that their structures are similar overall. However, the structures of the N- and C-terminal, loop-helix α4, and helix α6 regions are different. In addition, the helix α1 to strand ß4 region of TtPth is remarkably different from those of the mesophilic bacterial Pths, because this region is 9 or 10 amino acid residues shorter than those of the mesophilic bacterial Pths. This shortening seems to contribute to the thermostability of TtPth. To further understand the determinants for the thermostability of TtPth, we compared various structural factors of TtPth with those of mesophilic bacterial Pths. The data suggest that the decreases in accessible surface area and thermolabile amino acid residues, and the increases in ion pairs, hydrogen bonds, and proline residues cooperatively contribute to the thermostability of TtPth.

Planar Antiaromatic Core-Modified 24π Hexaphyrin(1.0.1.0.1.0) and 32π Octaphyrin(1.0.1.0.1.0.1.0) Bearing Alternate Hybrid Diheterole Units.

The Lewis acid catalyzed self-condensation of hybrid diheterole (furan-pyrrole and thiophene-pyrrole) precursors has afforded novel Hückel antiaromatic 24π hexaphyrin(1.0.1.0.1.0) and 32π octaphyrin(1.0.1.0.1.0.1.0) structures without ß-annulated bridges. Single-crystal X-ray diffraction analysis of the hybrid porphyrinoids (S3 N3 -ox and O4 N4 -ox) revealed a nearly planar conformation and the 1 H NMR spectra suggest the presence of paratropic ring currents. These antiaromatic macrocycles show characteristic optical features and underwent reversible two-electron reduction to Hückel aromatic 26π- and 34π-electron species, respectively, as is evident from the results of spectroscopic and theoretical studies (nucleus-independent chemical shift (NICS) and anisotropy of the current-induced density (ACID) calculations). The incorporation of hybrid diheteroles alternately into expanded porphyrin skeletons provides a novel approach to the fine-tuning of the electronic structures of planar antiaromatic macrocycles.

Hybridization and Mismatch Discrimination Abilities of 2',4'-Bridged Nucleic Acids Bearing 2-Thiothymine or 2-Selenothymine Nucleobase.

Oligonucleotides modified with 2'- O,4'- C-spirocyclopropylene-bridged nucleic acid (scpBNA) exhibit excellent duplex-forming ability with their complementary single-stranded RNA (ssRNA). Here, we demonstrate that scpBNA bearing a 2-thiothymine (scpBNA-S2T) or 2-selenothymine (scpBNA-Se2T) nucleobase provides robust mismatch discrimination capabilities to oligonucleotides without compromising their high binding affinities toward the full complementary ssRNA. X-ray crystallographic analysis of a self-assembling oligonucleotide featuring 2',4'-BNA/LNA-2-thiothymine (2',4'-BNA/LNA-S2T, where 2',4'-BNA and LNA stand for "2'- O,4'- C-methylene-bridged nucleic acid" and "locked nucleic acid", respectively), a prototype of scpBNA-S2T, revealed that the 2-thiocarbonyl moiety plays a crucial role in the destabilization of thymine-guanine mismatched wobble base pairs.

Synthesis and biophysical properties of 5'-thio-2',4'-BNA/LNA oligonucleotide.

Phosphorothioate modification of oligonucleotides is one of the most promising chemical modifications in nucleic acid therapeutics. Structurally similar 5'-thio or phosphorothiolate-modified nucleotides, in which the 5'-bridging oxygen atom is replaced with a sulfur atom, are attracting attention and gaining importance in oligonucleotide-based research. In our present study, we synthesized 5'-thio-2',4'-BNA/LNA monomers bearing thymine or 5-methylcytosine nucleobase. The 5'-thio-2',4'-BNA/LNA monomers were successfully incorporated into target oligonucleotides, and their nuclease stability and binding affinity with complementary strands were evaluated.

Binding of translation elongation factors to individual copies of the archaeal ribosomal stalk protein aP1 assembled onto aP0.

Ribosomes in all organisms contain oligomeric and flexible proteins called stalks, which are responsible for the recruitment of translational GTPase factors to the ribosome. Archaeal ribosomes have three stalk homodimers (aP1)2 that constitute a heptameric complex with the anchor protein aP0. We investigated the factor binding ability of aP1 proteins assembled onto aP0, by gel-retardation assays. The isolated aP0(aP1)2(aP1)2(aP1)2 complex, as well as the form bound to the Escherichia coli 50S core, as a hybrid 50S particle, interacted strongly with elongation factor aEF2, but weakly with aEF1A. These interactions were disrupted by a point mutation, F107S, at the C-terminus of aP1. To examine the ability of each copy of aP0-associated aP1 to bind to elongation factors, we constructed aP0·aP1 variant trimers, composed of an aP0 mutant and a single (aP1)2 dimer. Biochemical and quantitative analyses revealed that the resultant three trimers, aP0(aP1)2I, aP0(aP1)2II, and aP0(aP1)2III, individually bound two molecules of aEF2, suggesting that each copy of the aP1 C-terminal region in the aP0·aP1 trimers can bind tightly to aEF2. Interestingly, the unstable binding of aEF1A to each of the three aP0·aP1 trimers was remarkably stabilized in the presence of aEF2. The stability of the aEF1A binding to the stalk complex may be affected by the presence of aEF2 bound to the complex, by an unknown mechanism.

Synthesis and Properties of 7-Deazapurine- and 8-Aza-7-deazapurine-Locked Nucleic Acid Analogues: Effect of the Glycosidic Torsion Angle.

Conformationally restricted nucleoside analogues 2',4'-BNA/LNA-7-deazaguanine (LNA-7cG) and 2',4'-BNA/LNA-8-aza-7-deazaguanine (LNA-8n7cG), which avoid extra hydrogen bond formation at the 7-position of the guanine nucleobase, were successfully synthesized and incorporated into oligonucleotides. While the LNA-7cG-containing oligonucleotides show high duplex-forming ability with complementary DNA and RNA similar to LNA-G, the LNA-8n7cG-containing oligonucleotide has lower binding affinity than that of natural 2'-deoxyguanosine. This disparity in thermostability is also observed in 7-deazaadenosine analogues (LNA-7cA, LNA-8n7cA). Thermodynamic parameters and computational chemistry revealed that an inappropriate glycosidic torsion angle χ of 2',4'-BNA/LNA-8-aza-7-deazapurine analogues destabilizes duplex formation in contrast to 2',4'-BNA/LNA-7-deazapurine analogues. This result indicates that the nucleobase rotation angle plays an important role in duplex binding affinity. In addition, LNA-7cG-modified oligonucleotide effectively suppresses aggregation even in a guanine-rich sequence.

Crystal structure of translation initiation factor 5B from the crenarchaeon Aeropyrum pernix.

Initiation factor 5B (IF5B) is a universally conserved translational GTPase that catalyzes ribosomal subunit joining. In eukaryotes, IF5B directly interacts via a groove in its domain IV with initiation factor 1A (IF1A), another universally conserved initiation factor, to accomplish efficient subunit joining. Here, we have determined the first structure of a crenarchaeal IF5B, which revealed that the archaea-specific region of IF5B (helix α15) binds and occludes the groove of domain IV. Therefore, archaeal IF5B cannot access IF1A in the same manner as eukaryotic IF5B. This fact suggests that different relationships between IF5B and IF1A exist in archaea and eukaryotes.

Functional role of the C-terminal tail of the archaeal ribosomal stalk in recruitment of two elongation factors to the sarcin/ricin loop of 23S rRNA.

Two types of elongation factors alternate in their binding to the factor-binding center of the ribosome. Both binding events are accompanied by GTP hydrolysis and drive the translation elongation cycle. The multicopy ribosomal protein family, termed the stalk, contributes actively to the elongation process. Recent evidence indicates that the mobile C-terminal tail of archaeal stalk aP1 directly interacts with both the elongation factors aEF1A and aEF2. To investigate the functional significance of these interactions in recruitment of elongation factors to the factor-binding center of the ribosome, we substituted the archaeal stalk complex aL10•aP1 for the bL10•bL12 stalk complex in the Escherichia coli 50S subunit. The resultant hybrid ribosome accessed archaeal aEF1A and aEF2 in a manner dependent on the C-terminal tail containing the hydrophobic residues Leu103, Leu106 and Phe107. Bases G2659 and A2660 in the sarcin/ricin loop (SRL) of 23S rRNA were protected against DMS modification by both factors as was A1067 by aEF2. Mutagenesis indicated that this protection was dependent on the intact C-terminal tail of aP1. The results suggest a crucial role for the interactions between the stalk C-terminal tail and elongation factors in their recruitment to the SRL of 23S rRNA within the ribosome.