Xiaofeng Zheng

Publications (42) View all

• Article: Crystallization and X-ray crystallographic analysis of the cap-binding domain of influenza A virus H1N1 polymerase subunit PB2.

  Yong Liu, Geng Meng, Ming Luo, Xiaofeng Zheng
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  ABSTRACT: PB2 is one of the subunits of the influenza virus heterotrimeric polymerase. By its cap-binding domain (PB2cap), PB2 captures the 5' cap of the host pre-mRNA to generate a capped 5' oligonucleotide primer for virus transcription. The crystal structure of influenza A virus H3N2 PB2cap with bound cap analogue m(7)GTP has been reported previously. To show the substrate-free structural details of PB2cap and clarify whether obvious conformational changes exist between the substrate-free and substrate-bound cap-binding domain, we have successfully obtained the crystal of substrate-free H1N1 PB2cap. The crystal of H1N1 PB2cap diffracted to a high resolution of 1.32 Å. The crystal symmetry belongs to space group P1 with unit-cell parameters a = 29.49, b = 37.04, c = 38.33 Å, α = 71.10, β = 69.84, γ = 75.85°. There is one molecule in the asymmetric unit.
  Acta Crystallographica Section F Structural Biology and Crystallization Communications 03/2013; 69(Pt 3):280-3. · 0.51 Impact Factor
• Article: Structural and Functional Characterization of K339T Substitution Identified in the PB2 Cap-binding Pocket of Influenza A Virus.

  Yong Liu, Kun Qin, Geng Meng, Jinfang Zhang, Jianfang Zhou, Guangyu Zhao, Ming Luo, Xiaofeng Zheng
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  ABSTRACT: Influenza virus RNA-dependent RNA polymerase (RdRp) is a heterotrimer composed of PA, PB1 and PB2 subunits. RdRp is required for both transcription and replication of influenza viral RNA taking place in the nucleus of infected cells. A 'cap-snatching' mechanism is employed to generate a 5'-capped primer for transcription, in which the cap-binding domain of PB2 (PB2cap) captures the 5' cap of the host pre-mRNA. Our statistical analysis of PB2 sequences showed that residue Lys339 located in the cap-binding pocket of H5N1 PB2cap was gradually replaced by Thr339 over the past decade. To understand the role of this amino acid polymorphism, we solved the crystal structures of PB2cap with or without a pre-mRNA cap analog m7GTP in the presence of Lys339 or Thr339. The structures showed that Lys339 contributes to binding the γ-phosphate group of m7GTP, and the replacement of Lys339 by Thr eliminates this interaction. Isothermal titration calorimetry analysis showed that Thr339 attenuated the PB2cap cap-binding activity in vitro compared to Lys339. Further functional studies confirmed that Thr339-PB2-containing RNP has a reduced influenza polymerase activity and RNA synthesis activity, and a reconstituted H5N1 virus containing the Thr339 substitution exhibited a lower virulence to mice while more active replication in MDCK cells. The K339T substitution in the cap-binding pocket of PB2 modulates the polymerase activity and virulence by regulating the cap-binding activity. It is informative to track variations in the cap-binding pocket of PB2 in surveillance of the evolution and spread of influenza virus.
  Journal of Biological Chemistry 02/2013; · 4.77 Impact Factor
• Article: HSCARG Inhibits NADPH Oxidase Activity through Regulation of the Expression of p47phox.

  Weichun Xiao, Yanyan Peng, Yong Liu, Zhi Li, Senlin Li, Xiaofeng Zheng
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  ABSTRACT: Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase catalyzes the transfer of electrons from NADPH to O2, which is the main source of reactive oxygen species (ROS) in nonphagocytic cells. Excess ROS are toxic; therefore, keeping ROS in homeostasis in cells can protect cells from oxidative damage. It is meaningful to further understand the molecular mechanism by which ROS homeostasis is mediated. Human protein HSCARG is a newly identified oxidative sensor and a negative regulator of NF-κB. Here, we find that HSCARG represses the cellular ROS generation through inhibiting mRNA and protein expression of p47phox, a subunit of NADPH oxidase. In contrast, shRNA-mediated HSCARG knockdown increases endogenous p47phox expression level. And HSCARG has no obvious effect on ROS production in p47phox-depleted cells. Furthermore, HSCARG regulates p47phox through inhibition of NF-κB activity. Our findings identify HSCARG as a novel regulator in regulation of the activity of NADPH oxidase and ROS homeostasis.
  PLoS ONE 01/2013; 8(3):e59301. · 4.09 Impact Factor
• Article: Identification of a novel nuclear-localized adenylate kinase 6 from Arabidopsis thaliana as an essential stem growth factor.

  Xue Feng, Ruonan Yang, Xiaofeng Zheng, Feiyun Zhang
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  ABSTRACT: Adenylate kinase (AK; EC 2.7.4.3) is highly conserved across a wide range of organisms, including Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana, and Homo sapiens. While AK6 orthologs play important roles in the growth of yeast and worms, the physiological function of AK6 in A. thaliana is still unknown. In this study, we first cloned and expressed Arabidopsis adenylate kinase 6 (AAK6). Enzyme assays revealed that AAK6 has characteristic adenylate kinase properties, with ATP as the preferred phosphate donor and AMP as the best phosphate acceptor. A subcellular localization assay demonstrated that AAK6 had a predominant nuclear localization. Through biochemical purification and mass spectrometry analysis, a putative homolog of the S. cerevisiae Rps14 protein was identified as a partner of AAK6. Most importantly, we observed that aak6 T-DNA insertion mutants had decreased stem growth compared with wild-type plants. These data indicate that AAK6 exhibits adenylate kinase activity and is an essential growth factor in Arabidopsis.
  Plant Physiology and Biochemistry 10/2012; · 2.84 Impact Factor
• Article: Rigidity of wedge loop in PACSIN 3 protein is a key factor in dictating diameters of tubules.

  Xiaoyun Bai, Geng Meng, Ming Luo, Xiaofeng Zheng
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  ABSTRACT: BAR (Bin/amphiphysin/Rvs) domain-containing proteins participate in cellular membrane remodeling. The F-BAR proteins normally generate low curvature tubules. However, in the PACSIN subfamily, the F-BAR domain from PACSIN 1 and 2 can induce both high and low curvature tubules. We found that unlike PACSIN 1 and 2, PACSIN 3 could only induce low curvature tubules. To elucidate the key factors that dictate the tubule curvature, crystal structures of all three PACSIN F-BAR domains were determined. A novel type of lateral interaction mediated by a wedge loop is observed between the F-BAR neighboring dimers. Comparisons of the structures of PACSIN 3 with PACSIN 1 and 2 indicate that the wedge loop of PACSIN 3 is more rigid, which influences the lateral interactions between assembled dimers. We further identified the residues that affect the rigidity of the loop by mutagenesis and determined the structures of two PACSIN 3 wedge loop mutants. Our results suggest that the rigidity-mediated conformations of the wedge loop correlate well with the various crystal packing modes and membrane tubulations. Thus, the rigidity of the wedge loop is a key factor in dictating tubule diameters.
  Journal of Biological Chemistry 05/2012; 287(26):22387-96. · 4.77 Impact Factor