Quantum spin nematic phase in a square-lattice iridate.

Spin nematic is a magnetic analogue of classical liquid crystals, a fourth state of matter exhibiting characteristics of both liquid and solid1,2. Particularly intriguing is a valence-bond spin nematic3-5, in which spins are quantum entangled to form a multipolar order without breaking time-reversal symmetry, but its unambiguous experimental realization remains elusive. Here we establish a spin nematic phase in the square-lattice iridate Sr2IrO4, which approximately realizes a pseudospin one-half Heisenberg antiferromagnet in the strong spin-orbit coupling limit6-9. Upon cooling, the transition into the spin nematic phase at TC ≈ 263 K is marked by a divergence in the static spin quadrupole susceptibility extracted from our Raman spectra and concomitant emergence of a collective mode associated with the spontaneous breaking of rotational symmetries. The quadrupolar order persists in the antiferromagnetic phase below TN ≈ 230 K and becomes directly observable through its interference with the antiferromagnetic order in resonant X-ray diffraction, which allows us to uniquely determine its spatial structure. Further, we find using resonant inelastic X-ray scattering a complete breakdown of coherent magnon excitations at short-wavelength scales, suggesting a many-body quantum entanglement in the antiferromagnetic state10,11. Taken together, our results reveal a quantum order underlying the Néel antiferromagnet that is widely believed to be intimately connected to the mechanism of high-temperature superconductivity12,13.

Resonant inelastic X-ray scattering endstation at the 1C beamline of Pohang Light Source II.

An endstation for resonant inelastic X-ray scattering (RIXS), dedicated to operations in the hard X-ray regime, has been constructed at the 1C beamline of Pohang Light Source II. At the Ir L3-edge, a total energy resolution of 34.2 meV was achieved, close to the theoretical estimation of 34.0 meV, which considers factors such as the incident energy bandpass, intrinsic analyzer resolution, geometrical broadening of the spectrometer, finite beam-size effect and Johann aberration. The performance of the RIXS instrument is demonstrated by measuring the RIXS spectra of Sr2IrO4. The endstation can be easily reconfigured to measure energy-integrated intensities with very low background for diffuse scattering and diffraction experiments.

Discovery of compounds that reactivate p53 mutants in vitro and in vivo.

The tumor suppressor p53 is the most frequently mutated protein in human cancer. The majority of these mutations are missense mutations in the DNA binding domain of p53. Restoring p53 tumor suppressor function could have a major impact on the therapy for a wide range of cancers. Here we report a virtual screening approach that identified several small molecules with p53 reactivation activities. The UCI-LC0023 compound series was studied in detail and was shown to bind p53, induce a conformational change in mutant p53, restore the ability of p53 hotspot mutants to associate with chromatin, reestablish sequence-specific DNA binding of a p53 mutant in a reconstituted in vitro system, induce p53-dependent transcription programs, and prevent progression of tumors carrying mutant p53, but not p53null or p53WT alleles. Our study demonstrates feasibility of a computation-guided approach to identify small molecule corrector drugs for p53 hotspot mutations.

Ketogenesis impact on liver metabolism revealed by proteomics of lysine ß-hydroxybutyrylation.

Ketone bodies are bioactive metabolites that function as energy substrates, signaling molecules, and regulators of histone modifications. ß-hydroxybutyrate (ß-OHB) is utilized in lysine ß-hydroxybutyrylation (Kbhb) of histones, and associates with starvation-responsive genes, effectively coupling ketogenic metabolism with gene expression. The emerging diversity of the lysine acylation landscape prompted us to investigate the full proteomic impact of Kbhb. Global protein Kbhb is induced in a tissue-specific manner by a variety of interventions that evoke ß-OHB. Mass spectrometry analysis of the ß-hydroxybutyrylome in mouse liver revealed 891 sites of Kbhb within 267 proteins enriched for fatty acid, amino acid, detoxification, and one-carbon metabolic pathways. Kbhb inhibits S-adenosyl-L-homocysteine hydrolase (AHCY), a rate-limiting enzyme of the methionine cycle, in parallel with altered metabolite levels. Our results illuminate the role of Kbhb in hepatic metabolism under ketogenic conditions and demonstrate a functional consequence of this modification on a central metabolic enzyme.

The cooperative assembly of shelterin bridge provides a kinetic gateway that controls telomere length homeostasis.

Shelterin is a six-protein complex that coats chromosome ends to ensure their proper protection and maintenance. Similar to the human shelterin, fission yeast shelterin is composed of telomeric double- and single-stranded DNA-binding proteins, Taz1 and Pot1, respectively, bridged by Rap1, Poz1 and Tpz1. The assembly of the proteinaceous Tpz1-Poz1-Rap1 complex occurs cooperatively and disruption of this shelterin bridge leads to unregulated telomere elongation. However, how this biophysical property of bridge assembly is integrated into shelterin function is not known. Here, utilizing synthetic bridges with a range of binding properties, we find that synthetic shelterin bridge lacking cooperativity requires a linker pair that matches the native bridge in complex lifespan but has dramatically higher affinity. We find that cooperative assembly confers kinetic properties on the shelterin bridge allowing disassembly to function as a molecular timer, regulating the duration of the telomere open state, and consequently telomere lengthening to achieve a defined species-specific length range.

The histone H3K9M mutation synergizes with H3K14 ubiquitylation to selectively sequester histone H3K9 methyltransferase Clr4 at heterochromatin.

Oncogenic histone lysine-to-methionine mutations block the methylation of their corresponding lysine residues on wild-type histones. One attractive model is that these mutations sequester histone methyltransferases, but genome-wide studies show that mutant histones and histone methyltransferases often do not colocalize. Using chromatin immunoprecipitation sequencing (ChIP-seq), here, we show that, in fission yeast, even though H3K9M-containing nucleosomes are broadly distributed across the genome, the histone H3K9 methyltransferase Clr4 is mainly sequestered at pericentric repeats. This selective sequestration of Clr4 depends not only on H3K9M but also on H3K14 ubiquitylation (H3K14ub), a modification deposited by a Clr4-associated E3 ubiquitin ligase complex. In vitro, H3K14ub synergizes with H3K9M to interact with Clr4 and potentiates the inhibitory effects of H3K9M on Clr4 enzymatic activity. Moreover, binding kinetics show that H3K14ub overcomes the Clr4 aversion to H3K9M and reduces its dissociation. The selective sequestration model reconciles previous discrepancies and demonstrates the importance of protein-interaction kinetics in regulating biological processes.

Quality-Control Mechanism for Telomerase RNA Folding in the Cell.

Long non-coding RNAs can often fold into different conformations. Telomerase RNA, an essential component of the telomerase ribonucleoprotein (RNP) enzyme, must fold into a defined structure to fulfill its function with the protein catalytic subunit (TERT) and other accessory factors. However, the mechanism by which the correct folding of telomerase RNA is warranted in a cell is still unknown. Here we show that La-related protein Pof8 specifically recognizes the conserved pseudoknot region of telomerase RNA and instructs the binding of the Lsm2-8 complex to its mature 3' end, thus selectively protecting the correctly folded RNA from exonucleolytic degradation. In the absence of Pof8, TERT assembles with misfolded RNA and produces little telomerase activity. Therefore, Pof8 plays a key role in telomerase RNA folding quality control, ensuring that TERT only assembles with functional telomerase RNA to form active telomerase. Our finding reveals a mechanism for non-coding RNA folding quality control.

Structural Basis for Shelterin Bridge Assembly.

Telomere elongation through telomerase enables chromosome survival during cellular proliferation. The conserved multifunctional shelterin complex associates with telomeres to coordinate multiple telomere activities, including telomere elongation by telomerase. Similar to the human shelterin, fission yeast shelterin is composed of telomeric sequence-specific double- and single-stranded DNA-binding proteins, Taz1 and Pot1, respectively, bridged by Rap1, Poz1, and Tpz1. Here, we report the crystal structure of the fission yeast Tpz1475-508-Poz1-Rap1467-496 complex that provides the structural basis for shelterin bridge assembly. Biochemical analyses reveal that shelterin bridge assembly is a hierarchical process in which Tpz1 binding to Poz1 elicits structural changes in Poz1, allosterically promoting Rap1 binding to Poz1. Perturbation of the cooperative Tpz1-Poz1-Rap1 assembly through mutation of the "conformational trigger" in Poz1 leads to unregulated telomere lengthening. Furthermore, we find that the human shelterin counterparts TPP1-TIN2-TRF2 also assemble hierarchically, indicating cooperativity as a conserved driving force for shelterin assembly.

Spatial Organization and Molecular Interactions of the Schizosaccharomyces pombe Ccq1-Tpz1-Poz1 Shelterin Complex.

The shelterin complex is a macromolecular assembly of proteins that binds to and protects telomeric DNA, which composes the ends of all linear chromosomes. Shelterin proteins prevent chromosome ends from fusing together and from eliciting erroneous induction of DNA damage response pathways. In addition, shelterin proteins play key roles in regulating the recruitment and activation of telomerase, an enzyme that extends telomeric DNA. In fission yeast, Schizosaccharomyces pombe, interactions between the shelterin proteins Ccq1, Tpz1, and Poz1 are important for regulating telomerase-mediated telomere synthesis and thus telomere length homeostasis. Here, we used electron microscopy combined with genetic labeling to define the three-dimensional arrangement of the S. pombe Ccq1-Tpz1-Poz1 (CTP) complex. Crosslinking mass spectrometry was used to identify individual residues that are in proximity to the protein-protein interfaces of the assembled CTP complex. Together, our data provide a first glimpse into the architectural design of the CTP complex and reveals unique interactions that are important in maintaining the S. pombe telomere in a non-extendible state.

Multi-step coordination of telomerase recruitment in fission yeast through two coupled telomere-telomerase interfaces.

Tightly controlled recruitment of telomerase, a low-abundance enzyme, to telomeres is essential for regulated telomere synthesis. Recent studies in human cells revealed that a patch of amino acids in the shelterin component TPP1, called the TEL-patch, is essential for recruiting telomerase to telomeres. However, how TEL-patch-telomerase interaction integrates into the overall orchestration of telomerase regulation at telomeres is unclear. In fission yeast, Tel1(ATM)/Rad3(ATR)-mediated phosphorylation of shelterin component Ccq1 during late S phase is involved in telomerase recruitment through promoting the binding of Ccq1 to a telomerase accessory protein Est1. Here, we identify the TEL-patch in Tpz1(TPP1), mutations of which lead to decreased telomeric association of telomerase, similar to the phosphorylation-defective Ccq1. Furthermore, we find that telomerase action at telomeres requires formation and resolution of an intermediate state, in which the cell cycle-dependent Ccq1-Est1 interaction is coupled to the TEL-patch-Trt1 interaction, to achieve temporally regulated telomerase elongation of telomeres.

Dissecting Fission Yeast Shelterin Interactions via MICro-MS Links Disruption of Shelterin Bridge to Tumorigenesis.

Shelterin, a six-member complex, protects telomeres from nucleolytic attack and regulates their elongation by telomerase. Here, we have developed a strategy, called MICro-MS (Mapping Interfaces via Crosslinking-Mass Spectrometry), that combines crosslinking-mass spectrometry and phylogenetic analysis to identify contact sites within the complex. This strategy allowed identification of separation-of-function mutants of fission yeast Ccq1, Poz1, and Pot1 that selectively disrupt their respective interactions with Tpz1. The various telomere dysregulation phenotypes observed in these mutants further emphasize the critical regulatory roles of Tpz1-centered shelterin interactions in telomere homeostasis. Furthermore, the conservation between fission yeast Tpz1-Pot1 and human TPP1-POT1 interactions led us to map a human melanoma-associated POT1 mutation (A532P) to the TPP1-POT1 interface. Diminished TPP1-POT1 interaction caused by hPOT1-A532P may enable unregulated telomere extension, which, in turn, helps cancer cells to achieve replicative immortality. Therefore, our study reveals a connection between shelterin connectivity and tumorigenicity.

Sphingosinicella ginsenosidimutans sp. nov., with ginsenoside converting activity.

The Gram-reaction-negative, strictly aerobic, non-motile, nonspore-forming, and rod-shaped bacterial strain designated BS11(T) was isolated from the compost and its taxonomic position was investigated by using a polyphasic approach. Strain BS11(T) grew optimally at 30-37°C and at pH 7.0 in the absence of NaCl on nutrient agar. Strain BS11(T) displayed ß-glucosidase activity that was responsible for its ability to transform ginsenoside Rb1 (one of the dominant active components of ginseng) to Rd. On the basis of 16S rRNA gene sequence similarity, strain BS11(T) was shown to belong to the family Sphingomonadaceae and was related to Sphingosinicella vermicomposti YC7378(T) (96.3% sequence similarity), S. xenopeptidilytica 3-2W4(T) (96.2%), S. microcystinivorans Y2(T) (96.1%), and S. soli KSL-125(T) (95.9%). The G+C content of the genomic DNA was 64.9%. The major menaquinone was Q-10 and the major fatty acids were summed feature 7 (comprising C18:1 ω7c/ω9t/ω12t; 40.6%), C16:0 (22.5%), C17:1 ω6c (13.7%) and C17:0 (9.1%). DNA and chemotaxonomic data supported the affiliation of strain BS11(T) to the genus Sphingosinicella. Strain BS11(T) could be differentiated genotypically and phenotypically from the recognized species of the genus Sphingosinicella. The novel isolate therefore represents a novel species, for which the name Sphingosinicella ginsenosidimutans sp. nov. is proposed, with the type strain BS11(T) (=KACC 16619T =JCM 18201(T)).

Acute dietary nitrate supplementation does not augment submaximal forearm exercise hyperemia in healthy young men.

Despite the popularity of dietary nitrate supplementation and the growing evidence base of its potential ergogenic and vascular health benefits, there is no direct information about its effects on exercising limb blood flow in humans. We hypothesized that acute dietary nitrate supplementation from beetroot juice would augment the increases in forearm blood flow, as well as the progressive dilation of the brachial artery, during graded handgrip exercise in healthy young men. In a randomized, double-blind, placebo-controlled crossover study, 12 young (22 ± 2 years) healthy men consumed a beetroot juice (140 mL Beet-It Sport, James White Juice Company) that provided 12.9 mmol (0.8 g) of nitrate or placebo (nitrate-depleted Beet-It Sport) on 2 study visits. At 3 h postconsumption, brachial artery diameter, flow, and blood velocity were measured (Doppler ultrasound) at rest and during 6 exercise intensities. Nitrate supplementation raised plasma nitrate (19.5-fold) and nitrite (1.6-fold) concentrations, and lowered resting arterial pulse wave velocity (PWV) versus placebo (all p < 0.05), indicating absorption, conversion, and a biological effect of this supplement. The supplement-associated lowering of PWV was also negatively correlated with plasma nitrite (r = -0.72, p = 0.0127). Despite these systemic effects, nitrate supplementation had no effect on brachial artery diameter, flow, or shear rates at rest (all p ≥ 0.28) or during any exercise workload (all p ≥ 0.18). These findings suggest that acute dietary nitrate supplementation favorably modifies arterial PWV, but does not augment blood flow or brachial artery vasodilation during nonfatiguing forearm exercise in healthy young men.

Identification and characterization of a ginsenoside-transforming ß-glucosidase from Pseudonocardia sp. Gsoil 1536 and its application for enhanced production of minor ginsenoside Rg2(S).

The ginsenoside Rg2(S), which is one of the pharmaceutical components of ginseng, is known to have neuroprotective, anti-inflammation, and anti-diabetic effects. However, the usage of ginsenoside Rg2(S) is restricted owing to the small amounts found in white and red ginseng. To enhance the production of ginsenoside Rg2(S) as a 100 gram unit with high specificity, yield, and purity, an enzymatic bioconversion method was developed to adopt the recombinant glycoside hydrolase (BglPC28), which is a ginsenoside-transforming recombinant ß-glucosidase from Pseudonocardia sp. strain Gsoil 1536. The gene, termed bglPC28, encoding ß-glucosidase (BglPC28) belonging to the glycoside hydrolase family 3 was cloned. bglPC28 consists of 2,232 bp (743 amino acid residues) with a predicted molecular mass of 78,975 Da. This enzyme was overexpressed in Escherichia coli BL21(DE3) using a GST-fused pGEX 4T-1 vector system. The optimum conditions of the recombinant BglPC28 were pH 7.0 and 37 °C. BglPC28 can effectively transform the ginsenoside Re to Rg2(S); the Km values of PNPG and Re were 6.36 ± 1.10 and 1.42 ± 0.13 mM, respectively, and the Vmax values were 40.0 ± 2.55 and 5.62 ± 0.21 µmol min-1 mg-1 of protein, respectively. A scaled-up biotransformation reaction was performed in a 10 L jar fermenter at pH 7.0 and 30°C for 12 hours with a concentration of 20 mg/ml of ginsenoside Re from American ginseng roots. Finally, 113 g of Rg2(S) was produced from 150 g of Re with 84.0 ± 1.1% chromatographic purity. These results suggest that this enzymatic method could be usefully exploited in the preparation of ginsenoside Rg2(S) in the cosmetics, functional food, and pharmaceutical industries.

Characterization of recombinant ß-glucosidase from Arthrobacter chlorophenolicus and biotransformation of ginsenosides Rb1, Rb 2, Rc, and Rd.

The focus of this study was the cloning, expression, and characterization of recombinant ginsenoside hydrolyzing ß-glucosidase from Arthrobacter chlorophenolicus with an ultimate objective to more efficiently bio-transform ginsenosides. The gene bglAch, consisting of 1,260 bp (419 amino acid residues) was cloned and the recombinant enzyme, overexpressed in Escherichia coli BL21 (DE3), was characterized. The GST-fused BglAch was purified using GST·Bind agarose resin and characterized. Under optimal conditions (pH 6.0 and 37°C) BglAch hydrolyzed the outer glucose and arabinopyranose moieties of ginsenosides Rb1 and Rb2 at the C20 position of the aglycone into ginsenoside Rd. This was followed by hydrolysis into F2 of the outer glucose moiety of ginsenoside Rd at the C3 position of the aglycone. Additionally, BglAch more slowly transformed Rc to F2 via C-Mc1 (compared to hydrolysis of Rb1 or Rb2). These results indicate that the recombinant BglAch could be useful for the production of ginsenoside F2 for use in the pharmaceutical and cosmetic industries.

Divalent metal ion-based catalytic mechanism of the Nudix hydrolase Orf153 (YmfB) from Escherichia coli.

YmfB from Escherichia coli is the Nudix hydrolase involved in the metabolism of thiamine pyrophosphate, an important compound in primary metabolism and a cofactor of many enzymes. In addition, it hydrolyzes (d)NTPs to (d)NMPs and inorganic orthophosphates in a stepwise manner. The structures of YmfB alone and in complex with three sulfates and two manganese ions determined by X-ray crystallography, when compared with the structures of other Nudix hydrolases such as MutT, Ap4Aase and DR1025, provide insight into the unique hydrolysis mechanism of YmfB. Mass-spectrometric analysis confirmed that water attacks the terminal phosphates of GTP and GDP sequentially. Kinetic analysis of binding-site mutants showed that no individual residue is absolutely required for catalytic activity, suggesting that protein residues do not participate in the deprotonation of the attacking water. Thermodynamic integration calculations show that a hydroxyl ion bound to two divalent metal ions attacks the phosphate directly without the help of a nearby catalytic base.

PLP undergoes conformational changes during the course of an enzymatic reaction.

Numerous enzymes, such as the pyridoxal 5'-phosphate (PLP)-dependent enzymes, require cofactors for their activities. Using X-ray crystallography, structural snapshots of the L-serine dehydratase catalytic reaction of a bacterial PLP-dependent enzyme were determined. In the structures, the dihedral angle between the pyridine ring and the Schiff-base linkage of PLP varied from 18° to 52°. It is proposed that the organic cofactor PLP directly catalyzes reactions by active conformational changes, and the novel catalytic mechanism involving the PLP cofactor was confirmed by high-level quantum-mechanical calculations. The conformational change was essential for nucleophilic attack of the substrate on PLP, for concerted proton transfer from the substrate to the protein and for directing carbanion formation of the substrate. Over the whole catalytic cycle, the organic cofactor catalyzes a series of reactions, like the enzyme. The conformational change of the PLP cofactor in catalysis serves as a starting point for identifying the previously unknown catalytic roles of organic cofactors.

Crystal structures of d-alanine-d-alanine ligase from Xanthomonas oryzae pv. oryzae alone and in complex with nucleotides.

D-Alanine-D-alanine ligase (DDL) catalyzes the biosynthesis of d-alanyl-d-alanine, an essential bacterial peptidoglycan precursor, and is an important drug target for the development of antibacterials. We determined four different crystal structures of DDL from Xanthomonas oryzae pv. oryzae (Xoo) causing Bacteria Blight (BB), which include apo, ADP-bound, ATP-bound, and AMPPNP-bound structures at the resolution between 2.3 and 2.0 Å. Similarly with other DDLs, the active site of XoDDL is formed by three loops from three domains at the center of enzyme. Compared with d-alanyl-d-alanine and ATP-bound TtDDL structure, the γ-phosphate of ATP in XoDDL structure was shifted outside toward solution. We swapped the ω-loop (loop3) of XoDDL with those of Escherichia coli and Helicobacter pylori DDLs, and measured the enzymatic kinetics of wild-type XoDDL and two mutant XoDDLs with the swapped ω-loops. Results showed that the direct interactions between ω-loop and other two loops are essential for the active ATP conformation for D-ala-phosphate formation.

Characterization of a ginsenoside-transforming ß-glucosidase from Paenibacillus mucilaginosus and its application for enhanced production of minor ginsenoside F2.

A novel ß-glucosidase (BglPm) was identified from Paenibacillus mucilaginosus KCTC 3870(T) which has ginsenoside converting activity. The gene, termed bglPm, consists of 1,260 bp and belongs to glycoside hydrolase family 1 (GH1). After being overexpressed and purified from Escherichia coli, the enzymatic properties of BglPm were investigated. The enzyme exhibited an optimal activity at 45°C and pH 7.5 and showed high bioconversion ability for major ginsenoside Rb1 and Rd into ginsenoside F2. Thus, it was used for mass production of relatively high pure F2 from relatively abundant protopanaxadiol type ginsenosides mixture (PPDGM) with combined usage of ginsenoside Rc-hydrolyzing enzyme. Scale-up of production using 250 g of the PPDGM resulted in 152 g of F2 with 80.1% chromatography purity and 95.7% recovery. These results suggest that this enzyme would be useful in the preparation of pharmacologically active ginsenoside F2 in the functional food and pharmaceutical industries.

Roseomonas sediminicola sp. nov., isolated from fresh water.

A Gram-stain negative, strictly aerobic, non-motile, non-spore-forming, and rod-shaped bacterial strain designated FW-3(T) was isolated from fresh water and its taxonomic position was investigated by using a polyphasic approach. Strain FW-3(T) was found to grow at 10-37 °C and at pH 7.0 in the absence of NaCl on nutrient agar. On the basis of 16S rRNA gene sequence similarity, strain FW-3(T) was shown to belong to the family Acetobacteraceae and to be related to Roseomonas lacus TH-G33(T) (97.2 % sequence similarity) and Roseomonas terrae DS-48(T) (96.4 %). The G+C content of the genomic DNA was determined to be 68.0 %. The major menaquinone was determined to be Q-10 and the major fatty acids were identified as summed feature 7 (comprising C18:1 ω9c/ω12t/ω7c as defined by the MIDI system; 55.4 %), and C18:1 2OH (29.8 %). DNA and chemotaxonomic data supported the affiliation of strain FW-3(T) to the genus Roseomonas. Strain FW-3(T) could be differentiated genotypically and phenotypically from the recognized species of the genus Roseomonas. The novel isolate therefore represents a novel species, for which the name Roseomonas sediminicola sp. nov. is proposed, with the type strain FW-3(T) (=KACC 16616(T) = JCM 18210(T)).