Role of cytochrome P450 monooxygenase in the bioactivation of aflatoxin B1

In a previous study, the gene EgP450 that encodes the proteins of 505 amino acids was isolated from oil palm. The recombinant protein EgP450 is bound to phenylurea-like herbicides which detoxify the substance. Aflatoxin B1 (AFB1), a mycotoxin produced by Aspergillus sp., is another toxic compound that is known to cause acute toxic effects and act as a hepatocarcinogenic agent. This study aimed to examine the role of EgP450 enzyme in mycotoxin bioactivation in human mesenchymal stem cells (hMSCs). Docking analysis showed that EgP450 is bound to the group of carcinogens, which includes AFB1, n-(2-fluorenyl) acetamide, n-n-butyl-n-butan-4-ol-nitrosamine, n-nitrosodiethylamine, n-nitrosodiethylamine and n-nitrosodimethylamine. An in vivo aflatoxin toxicity test on hMSCs and AFB1 induces the expression of Bmi-1 which is one of the markers for the development of cancer. The presence of EgP450 at 0.15 μg/mL could reduced the Bmi-1 expression in AFB1 induced cells. Moreover, this protein also showed some antioxidant activity. These results exhibited the enormous potential of EgP450 in the detoxification processes

Characterization of a Novel Binding Protein for Fortilin/TCTP — Component of a Defense Mechanism against Viral Infection in Penaeus monodon

The Fortilin (also known as TCTP) in Penaeus monodon (PmFortilin) and Fortilin Binding Protein 1 (FBP1) have recently been shown to interact and to offer protection against the widespread White Spot Syndrome Virus infection. However, the mechanism is yet unknown. We investigated this interaction in detail by a number of in silico and in vitro analyses, including prediction of a binding site between PmFortilin/FBP1 and docking simulations. The basis of the modeling analyses was well-conserved PmFortilin orthologs, containing a Ca2+-binding domain at residues 76–110 representing a section of the helical domain, the translationally controlled tumor protein signature 1 and 2 (TCTP_1, TCTP_2) at residues 45–55 and 123–145, respectively. We found the pairs Cys59 and Cys76 formed a disulfide bond in the C-terminus of FBP1, which is a common structural feature in many exported proteins and the “x–G–K–K” pattern of the amidation site at the end of the C-terminus. This coincided with our previous work, where we found the “x–P–P–x” patterns of an antiviral peptide also to be located in the C-terminus of FBP1. The combined bioinformatics and in vitro results indicate that FBP1 is a transmembrane protein and FBP1 interact with N-terminal region of PmFortilin

Isolation, expression analysis and characterization of EgNDL, a NDR-like protein in oil palm (Elaeis guineensis Jacq.)

A novel cDNA of a SF21-like protein or NDR-like protein (EgNDL) from Elaeis guineensis Jacq. is 1,044 bp in length and encodes a putative protein with a 347-amino-acid open reading frame. The EgNDL showed 93% identity to the pollenspecific SF21-like protein of Phoenix dactylifera and also showed 79% identity to the NDL protein of Theobroma cacao. Expression analysis of the EgNDL gene in various tissues showed that EgNDL was expressed in the anthers, pistils, mesocarp and leaves. The high expression of EgNDL among three oil palm varieties was significantly expressed in Pisifera (P<0.05), which is commonly used as a male parent in crosses. Computational tools were used to predict the protein and concluded that EgNDL is a putative membrane protein that may function in a signal transduction pathway during pollen development. In this context, knowledge regarding EgNDL and its potential role in plant developmental processes will benefit oil palm breeding programs

Amino acid sequence alignment of the HbPR-1 from <i>H</i>. <i>brasiliensis</i> with its homologous proteins of different plant species.

<p>The sequences of the plant PR-1 proteins were obtained from the GenBank database: <i>Citrus sinensis</i> (XP_006486822.1), <i>Vitis vinifera</i> (XP_ 002273416.1), <i>Prunus mum</i> (XP_008236225.1), <i>Glycine max</i> (XP_003545771.1), <i>Malus domstica</i> (XP_008370577.1), <i>Ficus pumila var</i>. <i>awkeotsang</i> (AFK93500.1). The sequences were aligned by Clustal-X (Thompson et al., 2001). The conserved amino acid sequences were highlighted in red which indicates 100% conserved sequences. The arrowhead indicated the cleavage site between the signal peptide and the mature protein. The positions of the cysteine residues forming disulfide linkages were shown as C. CRISP family signature 1 (CRISP_1) were highlighted in green and CRISP family signature 2 (CRISP_2) were highlighted in blue.</p

Molecular Cloning of <i>HbPR-1</i> Gene from Rubber Tree, Expression of <i>HbPR-1</i> Gene in <i>Nicotiana benthamiana</i> and Its Inhibition of <i>Phytophthora palmivora</i>

<div><p>This is the first report to present a full-length cDNA (designated <i>HbPR-1</i>) encoding a putative basic HbPR-1 protein from rubber tree (<i>Hevea brasiliensis</i>) treated with salicylic acid. It was characterized and also expressed in <i>Nicotiana benthamiana</i> using <i>Agrobacterium</i>-mediated transient gene expression system in order to investigate the role of <i>HbPR-1</i> gene in rubber tree against its oomycete pathogen <i>Phytopthora palmivora</i> and to produce recombinant HbPR-1 protein for microbial inhibition test. The <i>HbPR-1</i> cDNA was 647 bp long and contained an open reading frame of 492 nucleotides encoding 163 amino acid residues with a predicted molecular mass of 17,681 Da and an isoelectric point (pI) of 8.56, demonstrating that HbPR-1 protein belongs to the basic PR-1 type. The predicted 3D structure of HbPR-1 was composed of four α-helices, three β-sheets, seven strands, and one junction loop. Expression and purification of recombinant HbPR-1 protein were successful using <i>Agrobacterium</i>-mediated transient expression and one-step of affinity chromatography. Heterologous expression of <i>HbPR-1</i> in <i>N</i>. <i>benthamiana</i> reduced necrosis areas which were inoculated with <i>P</i>. <i>palmivora</i> zoospores, indicating that the expressed HbPR-1 protein played an important role in plant resistance to pathogens. The purified recombinant HbPR-1 protein was found to inhibit 64% of <i>P</i>. <i>palmivora</i> zoospore germination on a water agar plate compared with control, suggesting that it was an antimicrobial protein against <i>P</i>. <i>palmivora</i>.</p></div

The putative binding sites of HbPR-1 protein.

<p>(A) The putative binding sites of HbPR-1 protein were predicted by the I-TASSER. The result suggested the possible locations of HbPR-1 protein interacting with glycerol (red-yellow color), Zn²⁺ (navy blue color) and EAH (pink-red color). (B-D) The putative residues of HbPR-1 protein that might be bound to the glycerol molecule (B), Zn²⁺ ion (C) and EAH molecule (D), respectively.</p

Transient expression of HbPR-1 protein in <i>N</i>. <i>benthamiana</i>.

<p>Total proteins and intercellular fluids were isolated from agroinfiltrated <i>N</i>. <i>benthamiana</i> plants and were visualized by Western blot analysis. Lane M represents protein standard and lanes 1–4 represent total proteins isolated from infiltrated <i>N</i>. <i>benthamiana</i> leaves with infiltration buffer (Mock, lane 1), <i>A</i>. <i>tumefaciens</i> GV3101 expressing pJL3-p19 (lane 2), <i>A</i>. <i>tumefaciens</i> C58C1 expressing pGD_HbPR-1 (lane 3) and a mixture of <i>A</i>. <i>tumefaciens</i> strains expressing pJL3-p19 and pGD_HbPR-1 (lane 4). Lane 5 and 6 represent intercellular fluids isolated from infiltrated <i>N</i>. <i>benthamiana</i> leaves with <i>A</i>. <i>tumefaciens</i> GV3101 expressing pJL3-p19 (lane 5) and a mixture of <i>A</i>. <i>tumefaciens</i> strains expressing pJL3-p19 and pGD_HbPR-1 (lane 6), respectively. The numbers on the left represent the size of molecular weight markers.</p

Purification of HbPR-1 protein expressed in the extracellular space of <i>N</i>. <i>benthamiana</i> leaves.

<p>(A) SDS-PAGE of the intercellular fluids isolated from <i>N</i>. <i>benthamiana</i> leaves. Lane 1 indicates intercellular fluid isolated from <i>N</i>. <i>benthamiana</i> leaves infiltrated with <i>A</i>. <i>tumefaciens strain</i> GV3101 carrying the pJL3-p19 and lane 2 indicates intercellular fluid isolated from <i>N</i>. <i>benthamiana</i> leaves co-infiltrated with <i>A</i>. <i>tumefaciens</i> C58-C1 carrying the pGD_HbPR-1 and <i>A</i>. <i>tumefaciens</i> GV3101 carrying the pJL3-P19. (B) SDS-PAGE of the purified HbPR-1 protein. The protein was purified from intercellular fluid shown in A (lane 2) by affinity chromatography with complete his-tag resin. Lane M indicates protein standard and the numbers on the left represent the size of molecular weight markers.</p

Molecular interaction models of <i>Pm</i>Fortilin/FBP1.

<p>(A top) A space-filling model representing the combination of two possible interactions of FBP1 (grey), at the opposite sides of <i>Pm</i>Fortilin (blue). (A bottom) A cartoon of the space-filling model showing the two conformations, A and B and the binding of <i>Pm</i>Fortilin to the C-terminus of FBP1. The predictions were performed with four separate modes: Balance, Electrostatic, Hydrophobic and VdW+Elec mode. (B–E) The lowest energy conformations of each of the four docking modes. The <i>Pm</i>Fortilin molecule (blue) and FBP1 (pink, yellow, magenta and green).</p