Jenq-Kuen Huang

Functional expression of Francisella tularensis FabH and FabI, potential antibacterial targets

Francisella tularensis is an extremely infectious airborne pathogen that has long been considered as a potential biological weapon. Enzymes of fatty acid synthesis (FAS) pathway are attractive targets for the development of new antibacterial agents because of differences between the biosynthesis pathways of bacteria and mammals. We report here the first expression of three functional enzymes in F. tularensis FAS-II pathway: FabH (3-oxoacyl-acyl carrier protein synthase III) which initiates elongation in FAS-II; FabD (Malonyl-CoA-acyl carrier protein transacylase) which catalyzes the transfer of a malonyl moiety from malonyl-CoA to ACP generating malonyl-ACP, and FabI (enoyl-ACP reductase) which catalyzes the reduction of enoyl-acyl-ACP derivatives. The genes encoding the FabD, FabH, and FabI were custom synthesized and cloned in pET15b expression vector. Each recombinant His-tagged fusion protein was overexpressed by IPTG induction, and then purified by affinity chromatography on a Ni-NTA column. The purified FabH and FabI have been used as targets for new drug development. Screening of a class of indole-2-carboxylic acid compounds has led to the discovery of several new compounds with promising activity against F. tularensis FabH or FabI enzymes. For example, indole derivative WIUAKP-001 inhibited 80% the FabH enzyme at 40 microM with IC(50) value of 2 microM whereas WIUAKP-031 inhibited 98% the FabI enzyme at 37.5 microM with IC(50) value of 6 microM. These compounds hold great promise for future development of new indole derivatives as inhibitors of type II FAS enzymes, and as potential new treatment for tularemia.

Modulation of Nitrosative Stress via Glutathione-Dependent Formaldehyde Dehydrogenase and S-Nitrosoglutathione Reductase

Glutathione-dependent formaldehyde dehydrogenase (GFD) from Taiwanofungus camphorata plays important roles in formaldehyde detoxification and antioxidation. The enzyme is bifunctional. In addition to the GFD activity, it also functions as an effective S-nitrosoglutathione reductase (GSNOR) against nitrosative stress. We investigated the modulation of HEK (human embryonic kidney) 293T cells under nitrosative stress by transfecting a codon optimized GFD cDNA from Taiwanofungus camphorata (Tc-GFD-O) to these cells. The parental and transfected HEK 293T cells were then subjected to S-nitrosoglutathione treatment to induce nitrosative stress. The results showed that in Tc-GFD-O-transfected 293T cells, the expression and activity of GFD increased. Additionally, these cells under the nitrosative stress induced by S-nitrosoglutathione showed both higher viability and less apoptosis than the parental 293T cells. This finding suggests that the Tc-GFD-O in HEK 293T cells may provide a protective function under nitrosative stress.

Molecular Cloning of Bovine eIF5A and Deoxyhypusine Synthase cDNA

Deoxyhypusine synthase is the first of the two enzymes that catalyzes the maturation of eukaryotic initiation factor 5A (eIF5A). The mature eIF5A is the only known protein in eukaryotic cells that contains the unusual amino acid hypusine (Nϵ-(4-amino-2(R)-hydroxybutyl)lysine). Synthesis of hypusine is essential for the function of eIF5A in eukaryotic cell proliferation and survival. Here we describe the cloning and characterization of bovine eIF5A and bovine deoxyhypusine synthase. The deduced bovine eIF5A protein is 100% identical to human eIF5A-1, and the deduced bovine deoxyhypusine synthase protein showed a 93% identity to the human protein.

Molecular Cloning and Biochemical Characterization of Recombinant Laccase from ＂Rigidoporus Vinctus＂

Laccases are multi-copper oxidases that widely distributed in plants and fungi. These enzymes catalyze oxidation of various compounds including phenolics and non-phenolics, and have been used in many industrial processes such as paper industry, biobleaching and bioremediation. A full length cDNA (1767 bp) encoding a putative laccase (Lac) from ＂Rigidoporus vinctus＂ was cloned by polymerase chain reaction (PCR). The coding region of RvLac encodes 517 amino acids which contained four conserved putative copper-binding regions. To further characterize the RvLac, the coding region was subcloned into an expression vector pET-20b(+) and transformed into ＂E. coli＂ BL21 (DE3) pLysS. Expression of the Lac was induced by IPTG and the recombinant His-tagged Lac was purified by Ni_2+ -nitrilotriacetic acid Sepharose superflow column. The purified enzyme was revealed as a single band on SDS-PAGE with molecular mass of ~57 kDa. Furthermore, the enzyme activity and kinetics were determined by ABTS assay. The Michaelis constant value for ABTS is 0.07 mM. The enzyme has a half-life of 4.6 min at 75℃. The enzyme is active under a pH 2 treatment for 30 min.

Subcloning and Expression of Functional Human Cathepsin B and K in E. coli: Characterization and Inhibition by Flavonoids

1.1 Cathepsins Cathepsins, originally identified as lysosomal proteases, play a fundamental role in intracellular protein turnover in lysosomes. However, several cathepsins and variants of cathepsins can also be found on the cell membrane, in the cytosol, nucleus, mitochondria, and extracellular space. These cathepsins are involved in a variety of important physiological and pathological processes [reviewed in: (Brix et al., 2008; Frlan and Gobec, 2006; Lutgens et al., 2007; Mohamed and Sloane, 2006; Nomura and Katunuma, 2005; Obermajer et al., 2008; Reiser et al., 2010; Stoka et al., 2005; Turk et al., 2001; Vasiljeva et al., 2007; Victor and Sloane, 2007)]. Cathepsins are classified mechanistically into groups which include serine (cathepsins A and G), aspartic (cathepsins D and E), and cysteine cathepsins (cathepsins B, C, F, H, L, K, O, S, V, W, and X). This classification is based on the nucleophilic residues present on their active sites responsible for proteolytic cleavage (Rawlings et al., 2006; Turk et al., 2001). Cathepsins are synthesized as zymogens composed of a signal peptide, a propeptide, and mature protein of distinct length and substrate specificity for individual cathepsins (Rawlings et al., 2006). The signal peptide is cleaved in the Endoplasmic Reticulum and the pro-protein is activated by proteolytic removal of the N-terminal pro-peptide either by autocatalysis in acidic environments, or by other proteases. The pro-peptide region of the cathepsin plays multiple roles. It can act as an inhibitor to block access to the active site that regulates cathepsin activity. In addition the propeptide can act as an intramolecular chaperone that assists in protein folding, or as a trafficking signal that targets the protein to its destination (Turk et al., 2002). Cathepsins exhibit a broad range of functions and tissue expression (Brix et al., 2008; Turk et al., 2001). Some of the cathepsins are ubiquitously expressed and others are tissue or cell-type specific. Cathepsins have been shown to be involved in the process of tumor invasion and metastasis (Bialas and Kafarski, 2009; Lindeman et al., 2004; Nomura and Katunuma, 2005; Obermajer