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SummaryProtein Phosphatase 2A (PP2A) plays an essential role in many aspects of cellular physiology. The PP2A holoenzyme consists of a heterodimeric core enzyme, which comprises a scaffolding subunit and a catalytic subunit, and a variable regulatory subunit. Here we report the crystal structure of the heterotrimeric PP2A holoenzyme involving the regulatory subunit B′/B56/PR61. Surprisingly, the B′/PR61 subunit has a HEAT-like (huntingtin-elongation-A subunit-TOR-like) repeat structure, similar to that of the scaffolding subunit. The regulatory B′/B56/PR61 subunit simultaneously interacts with the catalytic subunit as well as the conserved ridge of the scaffolding subunit. The carboxyterminus of the catalytic subunit recognizes a surface groove at the interface between the B′/B56/PR61 subunit and the scaffolding subunit. Compared to the scaffolding subunit in the PP2A core enzyme, formation of the holoenzyme forces the scaffolding subunit to undergo pronounced conformational rearrangements. This structure reveals significant ramifications for understanding the function and regulation of PP2A.

SummaryThe serine/threonine phosphatase protein phosphatase 2A (PP2A) plays an essential role in many aspects of cellular functions and has been shown to be an important tumor suppressor. The core enzyme of PP2A comprises a 65 kDa scaffolding subunit and a 36 kDa catalytic subunit. Here we report the crystal structures of the PP2A core enzyme bound to two of its inhibitors, the tumor-inducing agents okadaic acid and microcystin-LR, at 2.6 and 2.8 Å resolution, respectively. The catalytic subunit recognizes one end of the elongated scaffolding subunit by interacting with the conserved ridges of HEAT repeats 11–15. Formation of the core enzyme forces the scaffolding subunit to undergo pronounced structural rearrangement. The scaffolding subunit exhibits considerable conformational flexibility, which is proposed to play an essential role in PP2A function. These structures, together with biochemical analyses, reveal significant insights into PP2A function and serve as a framework for deciphering the diverse roles of PP2A in cellular physiology.

Phosphotyrosyl phosphatase activator (PTPA), also known as PP2A phosphatase activator, is a conserved protein from yeast to human. Here we report the 1.9 A crystal structure of human PTPA, which reveals a previously unreported fold consisting of three subdomains: core, lid, and linker. Structural analysis uncovers a highly conserved surface patch, which borders the three subdomains, and an associated deep pocket located between the core and the linker subdomains. The conserved surface patch and the deep pocket are responsible for binding to PP2A and ATP, respectively. PTPA and PP2A A-C dimer together constitute a composite ATPase. PTPA binding to PP2A results in a dramatic alteration of substrate specificity, with enhanced phosphotyrosine phosphatase activity and decreased phosphoserine phosphatase activity. This function of PTPA strictly depends on the composite ATPase activity. These observations reveal significant insights into the function and mechanism of PTPA and have important ramifications for understanding PP2A function.

LIGHT is an important costimulatory molecule for T cell immunity. Recent studies have further implicated its role in innate immunity and inflammatory diseases, but its cellular and molecular mechanisms remain elusive. We report here that LIGHT is upregulated and functions as a proinflammatory cytokine in 2 independent experimental hepatitis models, induced by concanavalin A and Listeria monocytogenes. Molecular mutagenesis studies suggest that soluble LIGHT protein produced by cleavage from the cell membrane plays an important role in this effect through the interaction with the lymphotoxin-beta receptor (LTbetaR) but not herpes virus entry mediator. NK1.1+ T cells contribute to the production, but not the cleavage or effector functions, of soluble LIGHT. Importantly, treatment with a mAb that specifically interferes with the LIGHT-LTbetaR interaction protects mice from lethal hepatitis. Our studies thus identify a what we believe to be a novel function of soluble LIGHT in vivo and offer a potential target for therapeutic interventions in hepatic inflammatory diseases.

Membrane fusion between virus and host cells is the key step for enveloped virus entry and is mediated by the viral envelope fusion protein. In murine coronavirus, mouse hepatitis virus (MHV), the spike (S) protein mediates this process. Recently, the formation of anti-parallel 6-helix bundle of the MHV S protein heptad repeat (HR) regions (HR1 and HR2) has been confirmed, implying coronavirus has a class I fusion protein. This bundle is also called fusion core. To facilitate the solution of the crystal structure of this fusion core, we deployed an Escherichia coli in vitro expression system to express the HR1 and HR2 regions linked together by a flexible linker as a single chain (named 2-helix). This 2-helix polypeptide subsequently assembled into a typical 6-helix bundle. This bundle has been analyzed by a series of biophysical and biochemical techniques and confirmed that the design technique can be used for coronavirus as we successfully used for members of paramyxoviruses.

It is emerging that enveloped viruses may adopt a unique entry/fusion mechanism; in paramyxoviruses, including Sendai virus (SeV), the attachment protein HN (or its homologue H or G) binds a cellular receptor which triggers conformational changes of its fusion protein, F. There are at least three conformations of the F protein in the current fusion model: the pre-fusion native conformation, the pre-hairpin intermediate conformation and the post-fusion coiled-coil conformation. The fusion mechanism of SeV, a member of the Paramyxoviridae family, has been well established and several structural and functional domains or modules have been proposed from studies of its F protein. However, biochemical and biophysical studies of the heptad-repeat (HR) regions (HR1 and HR2) have not been systematically carried out. HR1 and HR2 strongly interact with each other to form a stable six-helix coiled-coil bundle as the post-fusion conformation. In this study, a single-chain HR1-linker-HR2 protein of SeV was prepared in an Escherichia coli expression system and biochemical and biophysical analyses showed it to form a typical six-helix coiled-coil bundle; its trigonal crystals diffracted X-rays to 2.5 A resolution. The crystal structure will help to reveal the structural requirements of the post-fusion coiled-coil conformation of SeV F protein.

In this paper Co-doped Al-substituted α-Ni(OH)2 was prepared by a homogeneous precipitation method and its electrochemical behavior, in two kinds of electrolyte (7 M KOH solution and 7 M KOH saturated with Al(OH)3), was investigated by galvanostatic charge-discharge method, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The electrochemical properties of the commercial β-Ni(OH)2 in both electrolyte was also studied in order to compare with that of α-Ni(OH)2. The results show that Co-doped Al-substituted α-Ni(OH)2 has the largest capacity in 7 M KOH solution (300 mAh g−1). The addition of Al to KOH solution is harmful to the reversible capacity and cycle life of both α-Ni(OH)2 and β-Ni(OH)2. X-ray diffraction (XRD) patterns show that, for Co-doped Al-substituted α-Ni(OH)2, its turbostratic structure maintains after cycles in both alkaline electrolyte, despite the interlayer distance becoming smaller. For β-Ni(OH)2, however, γ-NiOOH species appeared after cycling in both electrolyte and its content is higher after cycling in 7 M KOH saturated with Al(OH)3. And adding Al into KOH solution caused the decrease of solution conductivity and increased the apparent diffusion coefficient which is evidenced by electrochemical impedance spectroscopy.

The surface transmembrane glycoprotein is responsible for mediating virion attachment to cell and subsequent virus-cell membrane fusion. However, the molecular mechanisms for the viral entry of coronaviruses remain poorly understood. The crystal structure of the fusion core of mouse hepatitis virus S protein, which represents the first fusion core structure of any coronavirus, reveals a central hydrophobic coiled coil trimer surrounded by three helices in an oblique, antiparallel manner. This structure shares significant similarity with both the low pH-induced conformation of influenza hemagglutinin and fusion core of HIV gp41, indicating that the structure represents a fusion-active state formed after several conformational changes. Our results also indicate that the mechanisms for the viral fusion of coronaviruses are similar to those of influenza virus and HIV. The coiled coil structure has unique features, which are different from other viral fusion cores. Highly conserved heptad repeat 1 (HR1) and HR2 regions in coronavirus spike proteins indicate a similar three-dimensional structure among these fusion cores and common mechanisms for the viral fusion. We have proposed the binding regions of HR1 and HR2 of other coronaviruses and a structure model of their fusion core based on our mouse hepatitis virus fusion core structure and sequence alignment. Drug discovery strategies aimed at inhibiting viral entry by blocking hairpin formation may be applied to the inhibition of a number of emerging infectious diseases, including severe acute respiratory syndrome.

Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) is a newly identified member of Family Coronaviridae. Coronavirus envelope spike protein S is a class I viral fusion protein which is characterized by the existence of two heptad repeat regions (HR1 and HR2) (forming a complex called fusion core). Here we report that by using in vitro bio-engineering techniques, SARS-CoV HR1 and HR2 bind to each other and form a typical 6-helix bundle. The HR2, either as a synthetic peptide or as a GST-fusion polypeptide, is a potent inhibitor of virus entry. The results do show that SARS-CoV follows the general fusion mechanism of class I viruses and this lays the ground for identification of virus fusion/entry inhibitors for this devastating emerging virus.

Crystal structure of the YTH domain of YTHDF2 reveals mechanism for recognition of N6-methyladenosine

Interconversion of white and brown adipocytes occurs between anabolic and catabolic states. The molecular mechanism regulating this phenotypic switch remains largely unknown. This study explores the role of tuberous sclerosis complex 1 (TSC1)–mechanistic target of rapamycin (mTOR) signaling in the conversion of brown to white adipose tissue (WAT). A colony of Fabp4-Tsc1−/− mice, in which the Tsc1 gene was specifically deleted by the fatty acid binding protein 4 (FABP4)-Cre, was established. Western blotting and immunostaining demonstrated the absence of TSC1 and activation of ribosomal protein S6 kinase 1, the downstream target of mTOR complex 1 (mTORC1) signaling, in the brown adipose tissues (BATs) of Fabp4-Tsc1−/− mice. Accumulation of lipid droplets in BAT was significantly increased. Levels of brown adipocyte markers were markedly downregulated, while white adipocyte markers were upregulated. Rapamycin reversed the conversion from BAT to WAT in Fabp4-Tsc1−/− mice. Deletion of the Tsc1 gene in cultured brown preadipocytes significantly increased the conversion to white adipocytes. FoxC2 mRNA, the transcriptional factor for brown adipocyte determination, was significantly decreased, while mRNAs for retinoblastoma protein, p107 and RIP140, the transcriptional factors for white adipocyte determination, increased in the BAT of Fabp4-Tsc1−/− mice. Our study demonstrates that TSC1-mTORC1 signaling contributes to the brown-to-white adipocyte phenotypic switch.

Enhanced activity of interleukin 17 (IL-17) producing T helper 17 (Th17) cells plays an important role in autoimmune and inflammatory diseases. Significant loss of body weight and appetite is associated with chronic inflammation and immune activation, suggesting the cross talk between immune and neuroendocrine systems. Ghrelin has been shown to regulate the organism immune function. However, the effects of ghrelin on the differentiation of Th17 cells remain elusive. In the present study, we observed the enhanced differentiation of Th17 cells in spleens of growth hormone secretagogue receptor 1a (GHSR1a)-/- mice. Treatment of ghrelin repressed Th17 cell differentiation in a time- and concentration-dependent manner. Phosphorylation of mammalian target of rapamycin (mTOR) and signal transducer and activator of transcription 3 (STAT3) was increased in the spleens of GHSR1a-/- mice. Activation of mTOR signaling by injection of Cre-expressiong adenovirus into tuberous sclerosis complex 1 (TSC1) loxp/loxp mice increased the differentiation of Th17 cells in spleen, which was associated with an increment in the phosphorylation of STAT3. Activation of mTOR signaling by leucine or overexpression of p70 ribosome protein subunit 6 kinase 1 (S6K1) activated mTOR signaling in isolated T cells, while reversed the ghrelin-induced inhibition of iTh17 cell differentiation. In conclusion, mTOR mediates the inhibitory effect of ghrelin on the differentiation of Th17 cells by interacting with STAT3.

Several Gram-negative pathogens of plants and animals and some eukaryotic associated bacteria use type III protein-secretion systems (T3SSs) to deliver bacterial virulence-associated ‘effector’ proteins directly into host cells. HopU1 is a type III effector protein from the plant pathogen Pseudomonas syringae, which causes plant bacterial speck disease. HopU1 quells host immunity through ADP-ribosylation of GRP7 as a substrate. HopU1 has been reported as the first ADP-ribosyltransferase virulence protein to be identified in a plant pathogen. Although several structures of ADP-ribosyltransferases have been determined to date, no structure of an ADP-ribosyltransferase from a plant pathogen has been determined. Here, the protein expression, purification, crystallization and preliminary crystallographic analysis of HopU1 are reported. Diffracting crystals were grown by hanging-drop vapour diffusion using polyethylene glycol 10 000 as a precipitant. Native and SAD data sets were collected using native and selenomethionine-derivative HopU1 crystals. The diffraction pattern of the crystal extended to 2.7 A resolution using synchrotron radiation. The crystals belonged to space group P43, with unit-cell parameters a = 92.6, b = 92.6, c = 101.6 A.

Fusion of members of the Paramyxoviridae family involves two glycoproteins: the attachment protein and the fusion protein. Changes in the fusion-protein conformation were caused by binding of the attachment protein to the cellular receptor. In the membrane-fusion process, two highly conserved heptad-repeat (HR) regions, HR1 and HR2, are believed to form a stable six-helix coiled-coil bundle. However, no crystal structure has yet been determined for this state in the mumps virus (MuV, a member of the Paramyxoviridae family). In this study, a single-chain protein consisting of two HR regions connected by a flexible amino-acid linker (named 2-Helix) was expressed, purified and crystallized by the hanging-drop vapour-diffusion method. A complete X-ray data set was obtained in-house to 2.2 A resolution from a single crystal. The crystal belongs to space group C2, with unit-cell parameters a = 161.2, b = 60.8, c = 40.1 A, beta = 98.4 degrees. The crystal structure will help in understanding the molecular mechanism of Paramyxoviridae family membrane fusion.

The fusion core of bovine immunodeficiency virus (BIV) gp40 is proposed to be involved in membrane fusion. However, no crystal structures are yet available. A predicted protein module BIV2-Helix of BIVgp40 has been expressed in Escherichia coli and purified by chromatography. Recombinant BIV2-Helix was crystallized using the hanging-drop vapour-diffusion technique at 291 K. The crystals were grown in MPD and belonged to the primitive rhombohedral space group R3, with unit-cell parameters a = 39.17, b = 39.17, c = 295.05 A and two molecules per asymmetric unit. X-ray diffraction data were collected to 1.76 A in the home laboratory from a single crystal.

Many concepts of programmed cell death (PCD), also known as apoptosis, were derived from genetic investigation of cell deaths in the nematode Caenorhabditis elegans (Horvitz et al. 1994). Initiation of PCD in C. elegans is controlled by four genes—ced-3, ced-4, ced-9, and egl-1—that act in a linear pathway (Fig. 1A; Horvitz 2003). The cell-killing protease CED-3 belongs to the caspase family (Yuan et al. 1993; Xue et al. 1996) and is synthesized as an inactive zymogen in cells. Activation of CED-3 occurs in three sequential steps (Fig. 1B). First, in response to cell death stimuli, the proapoptotic, BH3-only protein EGL-1 is activated and negatively regulates the anti-apoptotic protein CED-9 through direct interaction (Conradt and Horvitz 1998; del Peso et al. 1998, 2000; Parrish et al. 2000). Second, binding of EGL-1 to CED-9 leads to allosteric disruption of the interactions between CED-9 and CED-4, resulting in the release of CED-4 from the inhibitory CED-9–CED-4 complex (Hengartner and Horvitz 1994; Chinnaiyan et al. 1997; James et al. 1997; Spector et al. 1997; Wu et al. 1997; Chen et al. 2000). Third and finally, the released proapoptotic protein CED-4 facilitates autocataltytic activation of the CED-3 zymogen (Yuan and Horvitz 1992; Chinnaiyan et al. 1997; Irmler et al. 1997; Seshagiri and Miller 1997; Wu et al. 1997; Yang et al. 1998). Elucidating the molecular mechanisms by which CED-3, CED-4, CED-9, and EGL-1 control the onset of PCD is central to understanding the regulation of PCD. Figure 1. CED-4-mediated CED-3 activation is a central step to initiate PCD in C. elegans. (A) Four genes—egl-1, ced-9, ced-4, and ced-3—act in a linear fashion to control the onset of PCD in C. elegans. (B) Current model of the linear PCD pathway. ... Rigorous investigations in the past decade have revealed mechanistic insights into the first two steps of the linear PCD pathway in C. elegans (Fig. 1B). First, EGL-1 uses an extended BH3 helix to recognize a hydrophobic groove on the surface of CED-9 (Yan et al. 2004). Second, binding of EGL-1 to CED-9 induces pronounced conformational changes in CED-9, which, through allostery, destabilize the interactions between CED-9 and CED-4, ultimately leading to the dissociation of CED-9 from an asymmetric dimer of CED-4 (Yan et al. 2005; Yan et al. 2006b). The freed CED-4 dimer further oligomerizes to form the CED-4 apoptosome, which is responsible for the activation of CED-3. In sharp contrast to the mechanistic elucidation of the first two steps in the linear PCD pathway, the third and arguably most important step—how CED-4 mediates the activation of CED-3—remains largely enigmatic. At present, there is little information on how the CED-4 apoptosome specifically recognizes the CED-3 zymogen. There is no experimental identification of any specific sequence or structural motifs that are responsible for the interactions between CED-4 and CED-3. The unorthodox experimental revelation that the octameric CED-4 apoptosome only binds two molecules of CED-3 has engendered much excitement as well as doubt in the field, both of which ask for mechanistic explanation. In this study, we address these questions by deciphering a central piece of the puzzle: how CED-3 is specifically recognized by CED-4. The crystal structure unambiguously shows that CED-3 is bound within the hutch of the funnel-shaped CED-4 apoptosome, whereas our structural analysis indicates that only two CED-3 molecules can be accommodated within the hutch. These structural findings have important ramifications for understanding initiator caspase activation.

Recently, there exists a discrepancy on the lithium ion de-intercalation mechanism for LiCoPO4 electrode. In the present work, the study was focused upon exploring the origin of this discrepancy by studying the dependence of the impedance spectrum on the state of charge and the carbon content. For the pure LiCoPO4 electrode, the two plateaus in the charge curve are at 4.82 and 4.92 V. We have also studied the variation of electrochemical impedance spectroscopies (EISs) with the state of charge. The EIS measurement has shown that the total interfacial resistance increases as the state of charge increases for the pure LiCoPO4 electrode. If higher content of sucrose was added in the precursor (this implies higher carbon content in the synthesized sample), only one potential plateau can be found in the charge curve. For this electrode, the total interfacial resistance decreases with the state of charge. Especially, the total interfacial resistance has a dramatic decrease when the state of charge increases from 20% to 40%. It is believed that the influence of carbon impurity on the variation tendency of the EIS pattern may reflect the change of the fine structure. For the pure LiCoPO4 electrode, the intermediate phase is Li0.20~0.45CoPO4.

Osteoclast-stimulating factor increases osteoclast formation and bone resorption through a cellular signal transduction cascade, possibly by its interaction with c-Src or related family members. Crystals of human osteoclast-stimulating factor were obtained by the hanging-drop vapour-diffusion method using ammonium sulfate as a precipitant. The crystals are primitive orthorhombic and belong to P222 or a related space group, with unit-cell parameters a = 38.1, b = 54.9, c = 64.7 A.

The aetiological agent of an emergent outbreak of atypical pneumonia, severe acute respiratory syndrome (SARS), is a positive-stranded RNA virus (SARS-CoV) belonging to the Coronaviridae family with a genome that differs substantially from those of other known coronaviruses. Highly conserved heptad-repeat (HR1 and HR2) regions in class I viral fusion proteins, including spike protein from SARS coronavirus, interact with each other to form a six-helix bundle, which is called a fusion core. The crystal structure of the fusion core is expected to greatly facilitate drug design. Crystals of the fusion core of SARS-CoV spike protein have been grown at 291 K using PEG 4000 as precipitant. The diffraction pattern of the crystal extends to 2.8 A resolution at 100 K in-house. The crystals have unit-cell parameters a = 121.2, b = 66.3, c = 70.0 A, alpha = gamma = 90, beta = 107.4 degrees and belong to space group C2. Assuming the presence of six molecules per asymmetric unit, the solvent content is estimated to be about 28%.

Crystals of a 2-Helix fusion-core construct of MHV spike protein (commonly referred to as E2) have been grown at 291 K using PEG 4000 as precipitant. The diffraction pattern of the crystal extends to 2.8 A resolution at 100 K in-house. Furthermore, a selenomethionine (SeMet) derivative of MHV spike protein fusion core has been overexpressed and purified. The derivative crystals were obtained under similar conditions and three different wavelength data sets were collected to 2.4 A resolution from a single derivative crystal at BSRF (Beijing Synchrotron Radiation Facility). The crystals have unit-cell parameters a = b = 48.3, c = 199.6 A, alpha = beta = 90, gamma = 120 degrees and belong to space group R3. Assuming the presence of two molecules in the asymmetric unit, the solvent content is calculated to be about 46%.

For modern and future circular accelerators, especially high-intensity proton synchrotrons or colliders, the electron cloud effect is a key issue. So, in order to reduce the electron cloud effect, exploring very low secondary electron yield (SEY) material or coating used in vacuum tubes becomes necessary. In this article, we studied the SEY characteristics of graphene films with different thicknesses which were deposited on copper substrates using chemical vapor deposition. The SEY tests were done at temperatures of 25 °C and vacuum pressure of (2 − 6) × 10−9 torr. The properties of the deposited graphene films were investigated by X-ray photoelectron spectroscopy (XPS) and Raman spectroscopy. The SEY curves show that the number of graphene layers has a great effect on the SEY of graphene films. The maximum SEY of graphene films decreases with the increase of the number of layers. The maximum SEY of 6–8 layers of graphene film is 1.25. These results have a great significance for next-generation particle accelerators.

Precise knowledge of the resistance in each phase of a permanent magnet synchronous machine renders possible the performance optimization of a PMSM drive. It can be applied to torque ripple minimization, fault diagnosis for inverter, and high quality monitoring of e.g. temperature or the aging condition of electrical contacts. This paper provides a method to accurately estimate each individual stator phase resistance of a three-phase PMSM. The resistances are identified using recursive least squares minimization in real time. The estimation algorithm is based on a PMSM model in rotational reference frame. To achieve a high precision estimation in all points of operation the motor model takes account of magnetic saturation and cross-magnetization that are captured by offline estimation. The reliability of the proposed scheme is verified in simulations and test-bench experiments.

The tolerances and limitations in design and manufacturing of both motors and inverters lead to parasitic resistances in the phases. Therefore, the phase resistances are asymmetric, which causes an asymmetry among the phase currents which in turn leads to torque ripples. This paper proposes a novel scheme for torque ripple minimization using online estimation of unbalanced stator resistances. The stator resistances of a permanent magnet synchronous motor are estimated by recursive least squares in rotational reference frame in real time. Torque ripple reduction is achieved by a compensation scheme in stationary reference frame. Because the proposed estimation method requires relatively little additional computing time in a field-oriented control, it could also be applied in online fault diagnosis. The effectiveness of the proposed scheme is proven in simulations and test-bench experiments.

Simple mass-spring model is often used as the model of complex turbine-generator shaft when simulating subsynchronous oscillation (SSO). However, this model cannot precisely describe torsional vibration characteristics of turbine-generator shaft. For estimating the effect of shaft model on subsynchronous oscillation, simple mass-spring model of uncertain parameters was used in this paper. The AC-DC hybrid power system model was put up by using PSCAD/EMTDC. Subsynchronous damping controller (SSDC) was designed to damp subsynchronous oscillation and the effect of shaft parameters variation on SSDC was analyzed. The probabilistic collocation method was applied to research the validity of SSDC when considering shaft parameter uncertainty.

The An-Bao line low frequency oscillation occurred in Hebei province power grid of China was analyzed in this paper. The oscillation modes of Hebei power grid were obtained by eigenvalue analysis. The An-Bao line oscillation frequency was obtained though applying Prony method to analyze field data. Simulation results indicate that the An-Bao line low frequency oscillation cannot be interpreted by negative damping theory. The resonance mechanism of low frequency oscillation was put up in this paper, which can interpret the An-Bao line low frequency oscillation. The probability of power disturbance of prime mover is researched in this paper to support the resonance mechanism. The coupling model between thermodynamic system of plant and power system was set up using MATLAB. The simulation results indicate that power system low frequency oscillations of resonance mechanism might be caused by the disturbance of control valve, when its frequency is close to the power system natural oscillations frequency. If the speed-droop of governing system is too small in some running point, control valve disturbance with natural frequency of power system can be induced by speed deviation.

Dynamic regulation of histone methylation represents a fundamental epigenetic mechanism underlying eukaryotic gene regulation, yet little is known about how the catalytic activities of histone demethylases are regulated. Here, we identify and characterize NPAC/GLYR1 as an LSD2/KDM1b-specific cofactor that stimulates H3K4me1 and H3K4me2 demethylation. We determine the crystal structures of LSD2 alone and LSD2 in complex with the NPAC linker region in the absence or presence of histone H3 peptide, at resolutions of 2.9, 2.0, and 2.25 A, respectively. These crystal structures and further biochemical characterization define a dodecapeptide of NPAC (residues 214-225) as the minimal functional unit for its cofactor activity and provide structural determinants and a molecular mechanism underlying the intrinsic cofactor activity of NPAC in stimulating LSD2-catalyzed H3K4 demethylation. Thus, these findings establish a model for how a cofactor directly regulates histone demethylation and will have a significant impact on our understanding of catalytic-activity-based epigenetic regulation.

Inflammation has been implicated as a contributing factor in the development of abdominal aortic aneurysms (AAA). Quercetin, a natural flavonoid with anti-inflammatory properties, is known for its beneficial effects on vascular disease. In this study, we examined the effects of quercetin to inflammatory cell infiltration, subsequent expression of cytokines and activation of proteases on the expansion of experimental AAA. Aneurysms were induced by abluminal application of calcium chloride in C57/BL6 mice. Quercetin (60 mg/kg) was administered once daily by gavage beginning 2 weeks before AAA induction and continuing for 8 weeks. Mice treated with quercetin exhibited a 32.7% reduction in aortic size compared with vehicle-treated controls. Prevention of AAA was associated with preservation of medial structure, as well as a relative reduction in macrophage and CD3(+) T cell infiltration in aortic tissue, inflammatory cytokines release and nuclear factor κB activation. Quercetin also reduced the expression of matrix metalloproteinase (MMP)-2, MMP-9, cathepsin B, and cathepsin K in aortic tissue. In addition, quercetin treatment increased tissue inhibitors of metalloproteinases (TIMP)-1 gene expression. These data indicate that quercetin may be useful for the prevention and treatment of AAA via blocking the inflammatory response and inhibiting the proteases involved in the pathogenesis of this disease.

UHRF1 (Ubiquitin-like, with PHD and RING finger domains 1) plays an important role in DNA CpG methylation, heterochromatin function and gene expression. Overexpression of UHRF1 has been suggested to contribute to tumorigenesis. However, regulation of UHRF1 is largely unknown. Here we show that the deubiquitylase USP7 interacts with UHRF1. Using interaction-defective and catalytic mutants of USP7 for complementation experiments, we demonstrate that both physical interaction and catalytic activity of USP7 are necessary for UHRF1 ubiquitylation and stability regulation. Mass spectrometry analysis identified phosphorylation of serine (S) 652 within the USP7-interacting domain of UHRF1, which was further confirmed by a UHRF1 S652 phosphor (S652ph)-specific antibody. Importantly, the S652ph antibody identifies phosphorylated UHRF1 in mitotic cells and consistently S652 can be phosphorylated by the M phase-specific kinase CDK1-cyclin B in vitro. UHRF1 S652 phosphorylation significantly reduces UHRF1 interaction with USP7 in vitro and in vivo, which is correlated with a decreased UHRF1 stability in the M phase of the cell cycle. In contrast, UHRF1 carrying the S652A mutation, which renders UHRF1 resistant to phosphorylation at S652, is more stable. Importantly, cells carrying the S652A mutant grow more slowly suggesting that maintaining an appropriate level of UHRF1 is important for cell proliferation regulation. Taken together, our findings uncovered a cell cycle-specific signaling event that relieves UHRF1 from its interaction with USP7, thus exposing UHRF1 to proteasome-mediated degradation. These findings identify a molecular mechanism by which cellular UHRF1 level is regulated, which may impact cell proliferation.

Acute renal injury is one of the most frequent complications after cardiopulmonary bypass (CPB). This study was designed to evaluate the potential protective effect of erythropoietin (EPO) on CPB-induced renal injury in a rat model. Male Sprague-Dawley rats were randomly divided into three groups, sham-operated group (sham), control CPB group (control), erythropoietin CPB group (EPO). Blood samples were collected at the beginning, at the end of CPB, and at 0.5, 1, 2 and 24 h post-operation, and the kidneys were harvested 24 h postoperatively and observed by optical microscopy. Levels of serum creatinine (Cr) and blood urea nitrogen (BUN) were assayed. Tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6(IL-6) levels in the renal tissues were evaluated by the method of enzyme linked immunosorbent assay (ELISA). Protein and mRNA levels of nuclear factor kappa B p65 (NF-κB p65), intercellular adhesion molecule-1 (ICAM-1) were also determined using western blot and real-time PCR respectively. Serum Cr and BUN levels as well as TNF-α, IL-1β and IL-6 levels in renal tissues in control group were significantly higher than those in the sham group. However, the levels of above biomarkers were markedly decreased in EPO group when comparing with control group. Furthermore, NF-κB p65, ICAM-1 protein and mRNA expression were significantly down-regulated in EPO group comparing with control group. In addition, microscopic examinations revealed that histological injury was alleviated when treated with EPO. The results indicated that EPO potently protected against CPB-induced acute renal injury and inhibited expression of NF-κB p65 and inflammatory response.

According to the management rules about spinning reserve ancillary services in CSG(China Southern Power Grid Co., Ltd.), the date of spinning reserve ancillary services in Yunnan power grid is counted, it's found that the current compensation capacity algorithm is not well reflect the spinning reserve demand of the grid side. This paper proposes the concept of the validity coefficient of spinning reserve to improve the compensation algorithm, which have been tested and verified by the actual operation date. It can be used for reference on the better use of Yunnan power grid spinning reserve resources and the development of the spinning reserve ancillary service market.

Crystal structure of PHD domain of UHRF1 and insights into recognition of unmodified histone H3 arginine residue 2

By now wide-area measurement system (WAMS) has been widely used in many power systems. The load characteristics data are essential for measurement-based load modeling. So the feasibility of load modeling based on WAMS need be researched. In this paper, the uploading data from phasor measurement unit (PMU) and local fault recording data of PMU were used to identify the parameters of composite load model separately. Simulation results indicate that WAMS based composite load model has higher veracity than traditional static load model. For real-time load modeling, the trajectory sensitivity method was used to reducing the identified parameters of composite load model. The validity of reduction method was illustrated by a case of measurement-based composite load modeling.

Load model has great uncertainty characteristic due to the time-variation of load. As a consequent, it is essential to analyze the influence of load model on power system stability. The effect of load model structure and parameter on Yunnan power grid transient stability is researched in this paper. Simulation results indicate that load model has great influence on Yunnan power great transient stability. Under some conditions, opposite conclusion about stability may be arrived at using different load model structure or parameter. By calculating critical cut time of different fault type, it is found that there is no absolute conservative load model. So it is necessary to establish actual load model of Yunnan power grid.

SummaryIDH1 and IDH2 mutations occur frequently in gliomas and acute myeloid leukemia, leading to simultaneous loss and gain of activities in the production of α-ketoglutarate (α-KG) and 2-hydroxyglutarate (2-HG), respectively. Here we demonstrate that 2-HG is a competitive inhibitor of multiple α-KG-dependent dioxygenases, including histone demethylases and the TET family of 5-methlycytosine (5mC) hydroxylases. 2-HG occupies the same space as α-KG does in the active site of histone demethylases. Ectopic expression of tumor-derived IDH1 and IDH2 mutants inhibits histone demethylation and 5mC hydroxylation. In glioma, IDH1 mutations are associated with increased histone methylation and decreased 5-hydroxylmethylcytosine (5hmC). Hence, tumor-derived IDH1 and IDH2 mutations reduce α-KG and accumulate an α-KG antagonist, 2-HG, leading to genome-wide histone and DNA methylation alterations.

The derivation and proposal of major electrochemical techniques used to determine and calculate the electrochemical kinetic parameters is basically based on the electrochemical reaction taking place at liquid/solid or liquid/liquid interface in which all the reactants and products are soluble in liquid aqueous solution or liquid mercury electrode, or are volatile gas. Such electrochemical reaction system is classical and traditional (ERS1). Recently, the electrochemical behavior of some materials used as the active electrode materials in chemical power sources has attracted much attention. In chemical power source systems, either reactant or product, or both are insoluble. This kind of electrochemical reaction system (ERS2) is slightly different from ERS1. The application of these electrochemical techniques/equations to chemical power sources’ system requires carefulness. The misuse of these electrochemical techniques can be easily found in the literatures and some of them even lead to a wrong conclusion. In this review, almost all the electrochemical techniques to measure the exchange current and diffusion coefficient were compiled for reference to the readers, including pulse step, electrochemical impedance, alternating cyclic voltammetry, etc. The necessary requirements/conditions to apply these techniques have been briefly discussed and some simple examples were also discussed for a better understanding.

H3K9me2 and H3K27me2 are important epigenetic marks associated with transcription repression, while H3K4me3 is associated with transcription activation. It has been shown that active and repressive histone methylations distribute in a mutually exclusive manner, but the underlying mechanism was poorly understood. Here we identified ceKDM7A, a PHD (plant homeodomain)- and JmjC domain-containing protein, as a histone demethylase specific for H3K9me2 and H3K27me2. We further demonstrated that the PHD domain of ceKDM7A bound H3K4me3 and H3K4me3 co-localized with ceKDM7A at the genome-wide level. Disruption of the PHD domain binding to H3K4me3 reduced the demethylase activity in vivo, and loss of ceKDM7A reduced the expression of its associated target genes. These results indicate that ceKDM7A is recruited to the promoter to demethylate H3K9me2 and H3K27me2 and activate gene expression through the binding of the PHD domain to H3K4me3. Thus, our study identifies a dual-specificity histone demethylase and provides novel insights into the regulation of histone methylation.

Nosiheptide-resistance methyltransferase (NSR) methylates 23S rRNA at the nucleotide adenosine 1067 in Escherichia coli and thus contributes to resistance against nosiheptide, a sulfur-containing peptide antibiotic. Here, the expression, purification and crystallization of NSR from Streptomyces actuosus are reported. Diffracting crystals were grown by the hanging-drop vapour-diffusion method in reservoir solution consisting of 0.35 M ammonium chloride, 24%(w/v) PEG 3350, 0.1 M MES pH 5.7 at 293 K. Native data have been collected from the apo enzyme and a SAM complex, as well as apo SeMet SAD data. The diffraction patterns of the apo form of NSR, of NSR complexed with SAM and of SeMet-labelled NSR crystals extended to 1.90, 1.95 and 2.25 A resolution, respectively, using synchrotron radiation. All crystals belonged to space group P2(1), with approximate unit-cell parameters a = 64.6, b = 69.6, c = 64.9 A, beta = 117.8 degrees .

Protein phosphatase 2A (PP2A) regulates many essential aspects of cellular physiology. Members of the regulatory B/B55/PR55 family are thought to play a key role in the dephosphorylation of Tau, whose hyperphosphorylation contributes to Alzheimer's disease. The underlying mechanisms of the PP2A-Tau connection remain largely enigmatic. Here, we report the complete reconstitution of a Tau dephosphorylation assay and the crystal structure of a heterotrimeric PP2A holoenzyme involving the regulatory subunit Balpha. We show that Balpha specifically and markedly facilitates dephosphorylation of the phosphorylated Tau in our reconstituted assay. The Balpha subunit comprises a seven-bladed beta propeller, with an acidic, substrate-binding groove located in the center of the propeller. The beta propeller latches onto the ridge of the PP2A scaffold subunit with the help of a protruding beta hairpin arm. Structure-guided mutagenesis studies revealed the underpinnings of PP2A-mediated dephosphorylation of Tau.

The Nipah and Hendra viruses are highly pathogenic paramyxoviruses that recently emerged from flying foxes to cause serious disease outbreaks in humans and livestock in Australia, Malaysia, Singapore and Bangladesh. Their unique genetic constitution, high virulence and wide host range set them apart from other paramyxoviruses. These characteristics have led to their classification into the new genus Henpavirus within the family Paramyxoviridae and to their designation as Biosafety Level 4 pathogens. The fusion protein, an enveloped glycoprotein essential for viral entry, belongs to the family of class I fusion proteins and is characterized by the presence of two heptad repeat (HR) regions, HR1 and HR2. These two regions associate to form a fusion-active hairpin conformation that juxtaposes the viral and cellular membranes to facilitate membrane fusion and enable subsequent viral entry. The Hendra and Nipah virus fusion core proteins were crystallized and their structures determined to 2.2 A resolution. The Nipah and Hendra fusion core structures are six-helix bundles with three HR2 helices packed against the hydrophobic grooves on the surface of a central coiled coil formed by three parallel HR1 helices in an oblique antiparallel manner. Because of the high level of conservation in core regions, it is proposed that the Nipah and Hendra virus fusion cores can provide a model for membrane fusion in all paramyxoviruses. The relatively deep grooves on the surface of the central coiled coil represent a good target site for drug discovery strategies aimed at inhibiting viral entry by blocking hairpin formation.

The fusion proteins of enveloped viruses mediating the fusion between the viral and cellular membranes comprise two discontinuous heptad repeat (HR) domains located at the ectodomain of the enveloped glycoproteins. The crystal structure of the fusion protein core of Mumps virus (MuV) was determined at 2.2 A resolution. The complex is a six-helix bundle in which three HR1 peptides form a central highly hydrophobic coiled-coil and three HR2 peptides pack against the hydrophobic grooves on the surface of central coiled-coil in an oblique antiparallel manner. Fusion core of MuV, like those of simian virus 5 and human respiratory syncytium virus, forms typical 3-4-4-4-3 spacing. The similar charecterization in HR1 regions, as well as the existence of O-X-O motif in extended regions of HR2 helix, suggests a basic rule for the formation of the fusion core of viral fusion proteins.

Four evolutionally conserved proteins -EGL-1, CED-9, CED-4 and CED-3- collectively control the initiation of programmed cell death (PCD) in Caenorhabditis elegans. Activation of CED-3, the cell killing caspase, requires CED-4. The pro-death function of CED-4 is inhibited by the mitochondria-bound CED-9. Crystal structure of the 150-kDa CED-4-CED-9 complex at 2.6 A resolution reveals a 2:1 stoichiometry between CED-4 and CED-9. EGL-1 binding to CED-9 results in the dissociation of CED-4 from the CED-4-CED-9 complex. The freed CED-4 dimer further dimerizes to form a tetramer. Only the CED-4 tetramer, but not dimer or monomer, is capable of activating CED-3. Thus, CED-9 inhibits CED-4-mediated activation of CED-3 by sequestering CED-4 dimer from further dimerization. On the basis of structural and biochemical analyses, working models are proposed to explain the mechanism by which CED-4 facilitates CED-3 activation.

Severe acute respiratory syndrome coronavirus (SARS-CoV) is a newly emergent virus responsible for a worldwide epidemic in 2003. The coronavirus spike proteins belong to class I fusion proteins, and are characterized by the existence of two heptad repeat (HR) regions, HR1 and HR2. The HR1 region in coronaviruses is predicted to be considerably longer than that in other type I virus fusion proteins. Therefore the exact binding sequence to HR2 from the HR1 is not clear. In this study, we defined the region of HR1 that binds to HR2 by a series of biochemical and biophysical measures. Subsequently the defined HR1 (902−952) and HR2 (1145−1184) chains, which are different from previously defined binding regions, were linked together by a flexible linker to form a single-chain construct, 2-Helix. This protein was expressed in Escherichia coli and forms a typical six-helix coiled coil bundle. Highly conserved HR regions between mouse hepatitis virus (MHV) and SARS-CoV spike proteins suggest a similar three-dimensional structure for the two fusion cores. Here, we constructed a homology model for SARS coronavirus fusion core based on our biochemical analysis and determined the MHV fusion core structure. We also propose an important target site for fusion inhibitor design and several strategies, which have been successfully used in fusion inhibitor design for human immunodeficiency virus (HIV), for the treatment of SARS infection.

Severe acute respiratory syndrome coronavirus is a newly emergent virus responsible for a recent outbreak of an atypical pneumonia. The coronavirus spike protein, an enveloped glycoprotein essential for viral entry, belongs to the class I fusion proteins and is characterized by the presence of two heptad repeat (HR) regions, HR1 and HR2. These two regions are understood to form a fusion-active conformation similar to those of other typical viral fusion proteins. This hairpin structure likely juxtaposes the viral and cellular membranes, thus facilitating membrane fusion and subsequent viral entry. The fusion core protein of severe acute respiratory syndrome coronavirus spike protein was crystallized, and the structure was determined at 2.8 A of resolution. The fusion core is a six-helix bundle with three HR2 helices packed against the hydrophobic grooves on the surface of central coiled coil formed by three parallel HR1 helices in an oblique antiparallel manner. This structure shares significant similarity with the fusion core structure of mouse hepatitis virus spike protein and other viral fusion proteins, suggesting a conserved mechanism of membrane fusion. Drug discovery strategies aimed at inhibiting viral entry by blocking hairpin formation, which have been successfully used in human immunodeficiency virus 1 inhibitor development, may be applicable to the inhibition of severe acute respiratory syndrome coronavirus on the basis of structural information provided here. The relatively deep grooves on the surface of the central coiled coil will be a good target site for the design of viral fusion inhibitors.