Ye Sun Han

Structural basis for cold adaptation. Sequence, biochemical properties, and crystal structure of malate dehydrogenase from a psychrophile Aquaspirillium arcticum.

Aquaspillium arcticum is a psychrophilic bacterium that was isolated from arctic sediment and grows optimally at 4 °C. We have cloned, purified, and characterized malate dehydrogenase from A. arcticum (Aa MDH). We also have determined the crystal structures of apo-Aa MDH, Aa MDH·NADH binary complex, and Aa MDH·NAD·oxaloacetate ternary complex at 1.9-, 2.1-, and 2.5-Å resolutions, respectively. The Aa MDH sequence is most closely related to the sequence of a thermophilic MDH fromThermus flavus (Tf MDH), showing 61% sequence identity and over 90% sequence similarity. Stability studies show that Aa MDH has a half-life of 10 min at 55 °C, whereas Tf MDH is fully active at 90 °C for 1 h. Aa MDH shows 2–3-fold higher catalytic efficiency compared with a mesophilic or a thermophilic MDH at the temperature range 4–10 °C. Structural comparison of Aa MDH and Tf MDH suggests that the increased relative flexibility of active site residues, favorable surface charge distribution for substrate and cofactor, and the reduced intersubunit ion pair interactions may be the major factors for the efficient catalytic activity of Aa MDH at low temperatures.

Structure-based identification of a novel NTPase from Methanococcus jannaschii.

Almost half of the entire set of predicted genomic products from Methanococcus jannaschii are classified as functionally unknown hypothetical proteins. We present a structure-based identification of the biochemical function of a protein with an as yet unknown function from a M. jannaschii gene, Mj0226. The crystal structure of Mj0226 protein determined at 2.2 Å resolution reveals that the protein is a homodimer and each monomer folds into an elongated α/β structure of a new fold family. Comparisons of Mj0226 protein with protein structures in the database, however, indicate that one part of the protein is homologous to some of the nucleotide-binding proteins. Biochemical analysis shows that Mj0226 protein is a novel nucleotide triphosphatase that can efficiently hydrolyze nonstandard nucleotides such as XTP to XMP or ITP to IMP, but not the standard nucleotides, in the presence of Mg2+ or Mn2+ ions.

Genistein-induced apoptosis of human breast cancer MCF-7 cells involves calpain-caspase and apoptosis signaling kinase 1-p38 mitogen-activated protein kinase activation cascades.

The molecular mechanisms of genistein-induced apoptosis of human breast cancer MCF-7 cells were investigated. Genistein showed 50% cell growth inhibition at IC50=27.5±0.8 μmol/l in 24 h incubation under 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay conditions. Genistein is known to express both cell growth activity at nanomolar concentrations and anti-cell growth activity at micromolar concentrations. It was found that genistein at 100 μmol/l concentration effectively induced apoptosis of MCF-7 cells in 24 h. Genistein-induced apoptosis involved activation of calpain, caspase 7 and poly(ADP ribose) polymerase. Dantrolene, an inhibitor of Ca2+ release from the endoplasmic reticulum, inhibited genistein-induced activation of calpain and caspase 7, in addition to effectively negating genistein-induced apoptosis. MCF-7 cells treated with genistein also showed increased phosphorylation of p38 mitogen-activated protein kinase, whereas no effect was observed for extracellular signal-regulating kinase 1/2. Phosphorylation of apoptosis signaling kinase 1, an upstream regulator of p38 mitogen-activated protein kinase, was also increased by genistein treatment. Genistein-induced phosphorylation of apoptosis signaling kinase 1 and p38 mitogen-activated protein kinase was diminished by the presence of dantrolene. These results suggest that genistein-induced apoptosis in MCF-7 cells is mediated through calpain–caspase 7 and apoptosis signaling kinase 1–p38 mitogen-activated protein kinase activation cascades that involve Ca2+ release from the endoplasmic reticulum.

Ubiquitin ligase CHIP induces TRAF2 proteasomal degradation and NF‐κB inactivation to regulate breast cancer cell invasion

Transcriptional factor nuclear factor‐kappaB (NF‐κB) plays a crucial role in human breast cancer cell invasion and metastasis. The carboxyl terminus of Hsc70‐interacting protein (CHIP) is a U‐box‐type ubiquitin ligase that induces ubiquitination and proteasomal degradation of its substrate proteins. In this study, we investigated the role of CHIP in the NF‐κB pathway in the invasion of MDA‐MB‐231 cells, a highly aggressive breast cancer cell line. We showed that overexpression of CHIP significantly inhibits the invasion of the MDA‐MB‐231 cells. The overexpression of CHIP suppressed expression of urokinase plasminogen activator (uPA) and matrix metalloproteinase‐9 (MMP‐9) in MDA‐MB‐231 cells. Moreover, CHIP strongly inhibited the nuclear localization and the transcriptional activity of NF‐κB. The activation of the IkappaB kinase complex (IKK) was also blocked by CHIP overexpression. Importantly, CHIP overexpression resulted in a significant decrease in the level of TNF receptor‐associated factor 2 (TRAF2), an upstream key player in the NF‐κB pathway. However, the level of TRAF2 was restored after treatment with a proteasome inhibitor, MG‐132. Moreover, CHIP overexpression promoted the ubiquitination of TRAF2. We also found cell invasion significantly decreased in cells transfected with TRAF2 small interfering RNA (siRNA). In contrast, when CHIP expression was suppressed by siRNA in poorly invasive MCF‐7 cells, cell invasion significantly increased in conjunction with enhanced NF‐κB activation and TRAF2 levels. Taken together, these results suggest that CHIP regulates NF‐κB‐mediated cell invasion via the down‐regulation of TRAF2. J. Cell. Biochem. 112: 3612–3620, 2011.

Expression and characterization of a novel enantioselective lipase from Acinetobacter species SY-01

A novel lipase gene, lipase A, of Acinetobacter species SY-01 (A. species SY-01) was cloned, sequenced, and expressed in Bacillus subtilis 168. The deduced amino acid (aa) sequences for the lipase A and its chaperone, lipase-specific chaperone, were found to encode mature proteins of 339 aa (37.2 kDa) and 347 aa (38.1 kDa), respectively. The aa sequence of lipase A and lipase-specific chaperone shared high homology 82 and 67% identity with the lipase A and the lipase B of A. species RAG-1. This new lipase was defined as a group I Proteobacterial lipase family. The expressed lipase A was purified through sequential treatment with Q-Sepharose, Resource Q, and Superdex-S75 columns. The maximal activity was observed at 50 degrees C for hydrolysis of p-nitrophenyl monoesters and found to be stable at pH 9-11, with optimal activity at pH 10. Lipase A hydrolyzed wide range of fatty acid esters of p-nitrophenyl, but preferentially hydrolyzed short length acyl chains (C2 and C4). Moreover, lipase A from A. species SY-01 catalyzed hydrolysis of the two acetate isomers of cis-(+/-)-2-(bromomethyl)-2-(2,4-dichloro phenyl)-1,3-dioxolane-4-methyl acetate, an intermediate required for the synthesis of Itraconazole which was an anti-fungal drug, at different rate and yielded cis-(-)-isomer in 81.5% conversion with 91.9% enantiomeric excess.

Cloning and expression of superoxide dismutase from Aquifex pyrophilus, a hyperthermophilic bacterium

A superoxide dismutase (SOD) gene of Aquifex pyrophilus, a marine hyperthermophilic bacterium, was cloned, sequenced, expressed in Escherichia coli, and its gene product characterized. This is the first SOD from a hyperthermophilic bacterium that has been cloned. It is an iron‐containing homo‐oligomeric protein with a monomeric molecular mass of 24.2 kDa. The DNA‐derived amino acid sequence is more similar to those of known Mn‐ and Fe‐SODs from thermophilic archaea than of Cu, Zn‐SODs. The metal binding residues found in all SOD sequences from different species are also conserved in A. pyrophilus SOD. The protein is biochemically active only as an oligomer and is resistant to thermal denaturation.

Repair activities of human 8-oxoguanine DNA glycosylase are stimulated by the interaction with human checkpoint sensor Rad9-Rad1-Hus1 complex

Rad9-Rad1-Hus1 (9-1-1) is a checkpoint protein complex playing roles in DNA damage sensing, cell cycle arrest, DNA repair or apoptosis. Human 8-oxoguanine DNA glycosylase (hOGG1) is the major DNA glycosylase responsible for repairing a specific aberrantly oxidized nucleotide, 7,8-dihydro-8-oxoguanine (8-oxoG). In this study, we identified a novel interaction between hOGG1 and human 9-1-1, and investigated the functional consequences of this interaction. Co-immunoprecipitation assays using transiently transfected HEK293 cells demonstrated an interaction between hOGG1 and the 9-1-1 proteins. Subsequently, GST pull-down assays using bacterially expressed and purified hOGG1-His and GST-fused 9-1-1 subunits (GST-hRad9, GST-hRad1, and GST-hHus1) demonstrated that hOGG1 interacted directly with the individual subunits of the human 9-1-1 complex. In vitro excision assay, which employed a DNA duplex containing an 8-oxoG/C mismatch, showed that hRad9, hRad1, and hHus1 enhanced the 8-oxoG excision and beta-elimination activities of hOGG1. In addition, the presence of hRad9, hRad1, and hHus1 enhanced the formation of covalently cross-linked hOGG1-8-oxoG/C duplex complexes, as determined by a trapping assay using NaBH(4). A trimeric human 9-1-1 complex was purified from Escherichia coli cell transformed with hRad9, His-fused hRad1, or His-fused hHus1 expressing vectors. It also showed the similar activity to enhance in vitro hOGG1 glycosylase activity, compared with individual human 9-1-1 subunits. Detection of 8-oxoG in HEK293 cells using flow cytometric and spectrofluorometric analysis revealed that over-expression of hOGG1 or human 9-1-1 reduced the formation of 8-oxoG residues following the H(2)O(2) treatment. The highest 8-oxoG reduction was observed in HEK293 cells over-expressing hOGG1 and all the three subunits of human 9-1-1. These indicate that individual human 9-1-1 subunits and human 9-1-1 complex showed almost the same abilities to enhance the in vitro 8-oxoG excision activity of hOGG1, but that the greatest effect to remove 8-oxoG residues in H(2)O(2)-treated cells was derived from the 9-1-1 complex as a whole.

Association of hepatitis B virus polymerase with promyelocytic leukemia nuclear bodies mediated by the S100 family protein p11

Hepatitis B virus (HBV) polymerase (Pol) interacts with cellular chaperone proteins and thereby performs multiple functions necessary for viral replication. Yeast two-hybrid analysis was applied to identify additional cellular targets required for HBV Pol function. HBV Pol interacted with S100A10 (p11), a Ca(2+)-modulated protein previously shown to bind to annexin II. The interaction between HBV Pol and p11 was confirmed by co-immunoprecipitation of the two proteins synthesized either in vitro or in transfected cells and by inhibition of the DNA polymerase activity of HBV Pol by p11. Immunofluorescence analysis of transfected human cell lines revealed that, although most HBV Pol and p11 was restricted to the cytoplasm, a small proportion of each protein colocalized as nuclear speckles; HBV Pol was not detected in the nucleus in the absence of p11. The HBV Pol-p11 nuclear speckles coincided with nuclear bodies containing the promyelocytic leukemia protein PML. Furthermore, the association of HBV Pol-p11 with PML was increased by exposure of cells to EGTA and inhibited by valinomycin. These results suggest a role for p11 in modulation of HBV Pol function and implicate PML nuclear bodies and intracellular Ca(2+) in viral replication.

Expression of the active human and duck hepatitis B virus polymerases in heterologous system of Pichia methanolica

We expressed the Hepatitis B virus polymerase (HBV P protein) using a recently introduced yeast system, Pichia methanolica. HBV (1-680 amino acids) and Duck Hepatitis B virus (DHBV, 1-780 amino acids) polymerase were expressed and showed DNA dependent DNA polymerase (DDDP). The DHBV polymerase had RNA dependent DNA polymerase (RDDP) and RNase H activities. We present a new simplified way of obtaining active viral P protein using the yeast expression system. The viral P proteins proved to be stable and were not aggregated in the yeast system.

Stereochemical control in diastereoselective reduction of α-substituted-β-ketoesters using a reductase purified from Kluyveromyces marxianus

An NADPH-dependent carbonyl reductase that shows (3R)-selective reducing activity for α-substituted-β-ketoesters was purified from Kluyveromyces marxianus and racemic alkyl 2-substituted-3-oxobutanoates were reduced to the corresponding (2S,3R)- or (2R,3R)-2-substituted-3-hydroxybutanoates with enantiomeric purity (> 99%) and diastereoselectivity (24 ∼ 98%).