A Pivotal Role for Tryptophan 447 in Enzymatic Coupling of Human Endothelial Nitric Oxide Synthase (eNOS): EFFECTS ON TETRAHYDROBIOPTERIN-DEPENDENT CATALYSIS AND eNOS DIMERIZATION*

Tetrahydrobiopterin (BH4) is a required cofactor for the synthesis of NO by NOS. Bioavailability of BH4 is a critical factor in regulating the balance between NO and superoxide production by endothelial NOS (eNOS coupling). Crystal structures of the mouse inducible NOS oxygenase domain reveal a homologous BH4-binding site located in the dimer interface and a conserved tryptophan residue that engages in hydrogen bonding or aromatic stacking interactions with the BH4 ring. The role of this residue in eNOS coupling remains unexplored. We overexpressed human eNOS W447A and W447F mutants in novel cell lines with tetracycline-regulated expression of human GTP cyclohydrolase I, the rate-limiting enzyme in BH4 synthesis, to determine the importance of BH4 and Trp-447 in eNOS uncoupling. NO production was abolished in eNOS-W447A cells and diminished in cells expressing W447F, despite high BH4 levels. eNOS-derived superoxide production was significantly elevated in W447A and W447F versus wild-type eNOS, and this was sufficient to oxidize BH4 to 7,8-dihydrobiopterin. In uncoupled, BH4-deficient cells, the deleterious effects of W447A mutation were greatly exacerbated, resulting in further attenuation of NO and greatly increased superoxide production. eNOS dimerization was attenuated in W447A eNOS cells and further reduced in BH4-deficient cells, as demonstrated using a novel split Renilla luciferase biosensor. Reduction of cellular BH4 levels resulted in a switch from an eNOS dimer to an eNOS monomer. These data reveal a key role for Trp-447 in determining NO versus superoxide production by eNOS, by effects on BH4-dependent catalysis, and by modulating eNOS dimer formation.     

Regulation of a Dynamic Interaction between Two Microtubule-binding Proteins,EB1 and TIP150, by the Mitotic p300/CBP-associated Factor (PCAF) Orchestrates Kinetochore Microtubule Plasticity and Chromosome Stability during Mitosis

The microtubule cytoskeleton network orchestrates cellular dynamics and chromosome stability in mitosis. Although tubulin acetylation is essential for cellular plasticity, it has remained elusive how kinetochore microtubule plus-end dynamics are regulated by p300/CBP-associated factor (PCAF) acetylation in mitosis. Here, we demonstrate that the plus-end tracking protein, TIP150, regulates dynamic kinetochore-microtubule attachments by promoting the stability of spindle microtubule plus-ends. Suppression of TIP150 by siRNA results in metaphase alignment delays and perturbations in chromosome biorientation. TIP150 is a tetramer that binds an end-binding protein (EB1) dimer through the C-terminal domains, and overexpression of the C-terminal TIP150 or disruption of the TIP150-EB1 interface by a membrane-permeable peptide perturbs chromosome segregation. Acetylation of EB1-PCAF regulates the TIP150 interaction, and persistent acetylation perturbs EB1-TIP150 interaction and accurate metaphase alignment, resulting in spindle checkpoint activation. Suppression of the mitotic checkpoint serine/threonine protein kinase, BubR1, overrides mitotic arrest induced by impaired EB1-TIP150 interaction, but cells exhibit whole chromosome aneuploidy. Thus, the results identify a mechanism by which the TIP150-EB1 interaction governs kinetochore microtubule plus-end plasticity and establish that the temporal control of the TIP150-EB1 interaction by PCAF acetylation ensures chromosome stability in mitosis.     

Meclizine Inhibits Mitochondrial Respiration through Direct Targeting of Cytosolic Phosphoethanolamine Metabolism

We recently identified meclizine, an over-the-counter drug, as an inhibitor of mitochondrial respiration. Curiously, meclizine blunted respiration in intact cells but not in isolated mitochondria, suggesting an unorthodox mechanism. Using a metabolic profiling approach, we now show that treatment with meclizine leads to a sharp elevation of cellular phosphoethanolamine, an intermediate in the ethanolamine branch of the Kennedy pathway of phosphatidylethanolamine biosynthesis. Metabolic labeling and in vitro enzyme assays confirmed direct inhibition of the cytosolic enzyme CTP:phosphoethanolamine cytidylyltransferase (PCYT2). Inhibition of PCYT2 by meclizine led to rapid accumulation of its substrate, phosphoethanolamine, which is itself an inhibitor of mitochondrial respiration. Our work identifies the first pharmacologic inhibitor of the Kennedy pathway, demonstrates that its biosynthetic intermediate is an endogenous inhibitor of respiration, and provides key mechanistic insights that may facilitate repurposing meclizine for disorders of energy metabolism.     

Individual N-Glycans Added at Intervals along the Stalk of the Nipah Virus G Protein Prevent Fusion but Do Not Block the Interaction with the Homologous F Protein

The promotion of membrane fusion by most paramyxoviruses requires an interaction between the viral attachment and fusion (F) proteins to enable receptor binding by the former to trigger the activation of the latter for fusion. Numerous studies demonstrate that the F-interactive sites on the Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) and measles virus (MV) hemagglutinin (H) proteins reside entirely within the stalk regions of those proteins. Indeed, stalk residues of NDV HN and MV H that likely mediate the F interaction have been identified. However, despite extensive efforts, the F-interactive site(s) on the Nipah virus (NiV) G attachment glycoprotein has not been identified. In this study, we have introduced individual N-linked glycosylation sites at several positions spaced at intervals along the stalk of the NiV G protein. Five of the seven introduced sites are utilized as established by a retardation of electrophoretic mobility. Despite surface expression, ephrinB2 binding, and oligomerization comparable to those of the wild-type protein, four of the five added N-glycans completely eliminate the ability of the G protein to complement the homologous F protein in the promotion of fusion. The most membrane-proximal added N-glycan reduces fusion by 80%. However, unlike similar NDV HN and MV H mutants, the NiV G glycosylation stalk mutants retain the ability to bind F, indicating that the fusion deficiency of these mutants is not due to prevention of the G-F interaction. These findings suggest that the G-F interaction is not mediated entirely by the stalk domain of G and may be more complex than that of HN/H-F.     

The PA and HA Gene-Mediated High Viral Load and Intense Innate Immune Response in the Brain Contribute to the High Pathogenicity of H5N1 Avian Influenza Virus in Mallard Ducks

Most highly pathogenic avian influenza A viruses cause only mild clinical signs in ducks, serving as an important natural reservoir of influenza A viruses. However, we isolated two H5N1 viruses that are genetically similar but differ greatly in virulence in ducks. A/Chicken/Jiangsu/k0402/2010 (CK10) is highly pathogenic, whereas A/Goose/Jiangsu/k0403/2010 (GS10) is low pathogenic. To determine the genetic basis for the high virulence of CK10 in ducks, we generated a series of single-gene reassortants between CK10 and GS10 and tested their virulence in ducks. Expression of the CK10 PA or hemagglutinin (HA) gene in the GS10 context resulted in increased virulence and virus replication. Conversely, inclusion of the GS10 PA or HA gene in the CK10 background attenuated the virulence and virus replication. Moreover, the PA gene had a greater contribution. We further determined that residues 101G and 237E in the PA gene contribute to the high virulence of CK10. Mutations at these two positions produced changes in virulence, virus replication, and polymerase activity of CK10 or GS10. Position 237 plays a greater role in determining these phenotypes. Moreover, the K237E mutation in the GS10 PA gene increased PA nuclear accumulation. Mutant GS10 viruses carrying the CK10 HA gene or the PA101G or PA237E mutation induced an enhanced innate immune response. A sustained innate response was detected in the brain rather than in the lung and spleen. Our results suggest that the PA and HA gene-mediated high virus replication and the intense innate immune response in the brain contribute to the high virulence of H5N1 virus in ducks.     

Dysbiosis Signature of Fecal Microbiota in Colorectal Cancer Patients

The human gut microbiota is a complex system that is essential to the health of the host. Increasing evidence suggests that the gut microbiota may play an important role in the pathogenesis of colorectal cancer (CRC). In this study, we used pyrosequencing of the 16S rRNA gene V3 region to characterize the fecal microbiota of 19 patients with CRC and 20 healthy control subjects. The results revealed striking differences in fecal microbial population patterns between these two groups. Partial least-squares discriminant analysis showed that 17 phylotypes closely related to Bacteroides were enriched in the gut microbiota of CRC patients, whereas nine operational taxonomic units, represented by the butyrate-producing genera Faecalibacterium and Roseburia, were significantly less abundant. A positive correlation was observed between the abundance of Bacteroides species and CRC disease status (R?=?0.462, P?=?0.046?<?0.5). In addition, 16 genera were significantly more abundant in CRC samples than in controls, including potentially pathogenic Fusobacterium and Campylobacter species at genus level. The dysbiosis of fecal microbiota, characterized by the enrichment of potential pathogens and the decrease in butyrate-producing members, may therefore represent a specific microbial signature of CRC. A greater understanding of the dynamics of the fecal microbiota may assist in the development of novel fecal microbiome-related diagnostic tools for CRC.     

A Microbial Link between Elevated CO2 and Methane Emissions that is Plant Species-Specific

Rising atmospheric CO₂ levels alter the physiology of many plant species, but little is known of changes to root dynamics that may impact soil microbial mediation of greenhouse gas emissions from wetlands. We grew co-occurring wetland plant species that included an invasive reed canary grass (Phalaris arundinacea L.) and a native woolgrass (Scirpus cyperinus L.) in a controlled greenhouse facility under ambient (380 ppm) and elevated atmospheric CO₂ (700 ppm). We hypothesized that elevated atmospheric CO₂ would increase the abundance of both archaeal methanogen and bacterial methanotroph populations through stimulation of plant root and shoot biomass. We found that methane levels emitted from S. cyperinus shoots increased 1.5-fold under elevated CO₂, while no changes in methane levels were detected from P. arundincea. The increase in methane emissions was not explained by enhanced root or shoot growth of S. cyperinus. Principal components analysis of the total phospholipid fatty acid (PLFA) recovered from microbial cell membranes revealed that elevated CO₂ levels shifted the composition of the microbial community under S. cyperinus, while no changes were detected under P. arundinacea. More detailed analysis of microbial abundance showed no impact of elevated CO₂ on a fatty acid indicative of methanotrophic bacteria (18:2ω6c), and no changes were detected in the terminal restriction fragment length polymorphism (T-RFLP) relative abundance profiles of acetate-utilizing archaeal methanogens. Plant carbon depleted in ¹³C was traced into the PLFAs of soil microorganisms as a measure of the plant contribution to microbial PLFA. The relative contribution of plant-derived carbon to PLFA carbon was larger in S. cyperinus compared with P. arundinacea in four PLFAs (i14:0, i15:0, a15:0, and 18:1ω9t). The δ¹³C isotopic values indicate that the contribution of plant-derived carbon to microbial lipids could differ in rhizospheres of CO₂-responsive plant species, such as S. cyperinus in this study. The results from this study show that the CO₂–methane link found in S. cyperinus can occur without a corresponding change in methanogen and methanotroph relative abundances, but PLFA analysis indicated shifts in the community profile of bacteria and fungi that were unique to rhizospheres under elevated CO₂.     

A Method for Molecular Analysis of Catalase Gene Diversity in Seawater

Catalase plays an important role in the metabolism of marine bacteria and has potential impact on the marine environment. Four PCR primers were designed to amplify the catalase gene fragments in marine bacteria by applying metagenomic DNA from Yellow Sea surface water as the template. Of the four reproducible target PCR products, the longest one with 900 bp were chosen for catalase gene library construction by the T-vector and the white Escherichia coli colonies in the library was screened through restriction-digesting the reamplified insert fragments by the selected restriction endonuclease MboI, and then the bands of the resulting products were displayed in the agarose gel by electrophoresis. The unique restriction fragment length polymorphism (RFLP) pattern was selected and the corresponding catalase gene fragments were sequenced, which verified that every unique RFLP pattern represented one type of catalase. This PCR–RFLP method above was established to investigate the bacterial catalase diversity in seawater.