• Journal of Microbiology and Biotechnology
• /
• v.24 no.3
• /
• pp.379-385
• /
• 2014

The purpose of this study was to compare the ability of an engineered Escherichia coli to degrade chlorpyrifos (Cp) using an organophosphorus hydrolase enzyme, encoded in both Flavobacterium sp. ATCC 27551 or Pseudomonas diminuta, by employing the Lpp-OmpA chimera and the N-terminal domain of the ice nucleation protein as anchoring motifs. Tracing of the expression location of the recombinant protein using SDS-PAGE showed the presentation of OPH by both anchors on the outer membrane. This is the first report on the presentation of OPH on the cell surface by Lpp-OmpA under the control of the T7 promoter. The results showed cell growth in the presence of Cp as the sole source of energy, without growth inhibition, and with higher whole-cell activity for both cells harboring plasmids pENVO and pELMO, at approximately 10,342.85 and 10,857.14 U/mg, respectively. Noticeably, the protein displayed by pELMO was lower than the protein displayed by pENVO. It can be concluded that Lpp-OmpA can display less protein, but more functional OPH protein. These results highlight the high potential, of both engineered bacteria, for use in the bioremediation of pesticide-contaminated sources in the environment.

• Molecules and Cells
• /
• v.26 no.4
• /
• pp.329-337
• /
• 2008

Src-family kinases are critically involved in the control of cytoskeleton organization and in the generation of integrin-dependent signaling responses, inducing tyrosine phosphorylation of many signaling and cytoskeletal proteins. Activity of the Src family of tyrosine kinases is tightly controlled by inhibitory phosphorylation of a carboxy-terminal tyrosine residue, inducing an inactive conformation through binding with its SH2 domain. Dephosphorylation of C-ter tyrosine, as well as its deletion of substitution with phenylalanine in oncogenic Src kinases, leads to autophosphorylation at a tyrosine in the activation loop, thereby leading to enhanced Src activity. Beside this phophorylation/dephosphorylation circuitry, cysteine oxidation has been recently reported as a further mechanism of enzyme activation. Mounting evidence describes Src activation via its redox regulation as a key outcome in several circumstances, including growth factor and cytokines signaling, integrin-mediated cell adhesion and motility, membrane receptor cross-talk as well in cell transformation and tumor progression. Among the plethora of data involving Src kinase in physiological and pathophysiological processes, this review will give emphasis to the redox component of the regulation of this master kinase.

• Korean Journal of Agricultural Science
• /
• v.45 no.4
• /
• pp.561-573
• /
• 2018

Oomycetes are known to secrete a vast arsenal of effectors that modulate the host defense system as well as facilitate establishing a parasitic infection in plants. In recent years, tremendous progress has been made in the field of effectromics based on studies of oomycetes, especially the cytoplasmic family of RXLR effectors. Yet, the biology of the RXLR effector family is still poorly understood. There has been a consensus regarding the structure of the RXLR motif in the mycologist community. However, the function of the RXLR motif is still unclear. First, different models have suggested that the role of the RXLR motif is either in translocation to a target destination inside a host cell or in the cleavage of itself followed by secretion. Second, recent studies have suggested different functional models for the RXLR motif. According to a widely accepted model, the RXLR motif is directly involved in the translocation of effectors to target sites. In contrast, a new study has proposed that the RXLR motif is involved in secretion rather than translocation. Thus, this review is an attempt to summarize the recent advances made in the functional analysis of the N-terminal domain of RXLR effectors.

• Journal of Microbiology and Biotechnology
• /
• v.32 no.8
• /
• pp.1034-1040
• /
• 2022

Fas-associated death domain (FADD) is an adapter molecule that bridges the interaction between receptor-interacting protein 1 (RIP1) and aspartate-specific cysteine protease-8 (caspase-8). As the primary mediator of apoptotic cell death, caspase-8 has two N-terminal death-effector domains (DEDs) and it interacts with other proteins in the DED subfamily through several conserved residues. In the tumor necrosis receptor-1 (TNFR-1)-dependent signaling pathway, apoptosis is triggered by the caspase-8/FADD complex by stimulating receptor internalization. However, the molecular mechanism of complex formation by the DED proteins remains poorly understood. Here, we found that direct DED-DED interaction between FADD and caspase-8 and the structure-based mutations (Y8D/I128A, E12A/I128A, E12R/I128A, K39A/I128A, K39D/I128A, F122A/I128A, and L123A/I128A) of caspase-8 disrupted formation of the stable DED complex with FADD. Moreover, the monomeric crystal structure of the caspase-8 DEDs (F122A/I128A) was solved at 1.7 Å. This study will provide new insight into the interaction mechanism and structural characteristics between FADD and caspase-8 DED subfamily proteins.

• Parasites, Hosts and Diseases
• /
• v.48 no.4
• /
• pp.319-324
• /
• 2010

A family of calcium-dependent protein kinases (CDPKs) is a unique enzyme which plays crucial roles in intracellular calcium signaling in plants, algae, and protozoa. CDPKs of malaria parasites are known to be key regulators for stage-specific cellular responses to calcium, a widespread secondary messenger that controls the progression of the parasite. In our study, we identified a gene encoding Plasmodium vivax CDPK4 (PvCDPK4) and characterized its molecular property and cellular localization. PvCDPK4 was a typical CDPK which had well-conserved N-terminal kinase domain and C-terminal calmodulin-like structure with 4-EF hand motifs for calcium-binding. The recombinant protein of EF hand domain of PvCDPK4 was expressed in Echerichia coli and a 34 kDa product was obtained. Immunofluorescence assay by confocal laser microscopy revealed that the protein was expressed at the mature schizont of P. vivax. The expression of PvCDPK4-EF in schizont suggests that it may participate in the proliferation or egress process in the life cycle of this parasite.

• BMB Reports
• /
• v.37 no.5
• /
• pp.538-545
• /
• 2004

A new CRT binding factor (CBF) gene designated Cbcbf25 was cloned from Capsella bursa-pastoris, a wild grass, by the rapid amplification of cDNA ends (RACE). The full-length cDNA of Cbcbf25 was 898 bp with a 669 bp open reading frame (ORF) encoding a putative DRE/CRT (LTRE)-binding protein of 223 amino acids. The predicted CbCBF25 protein contained a potential nuclear localization signal (NLS) in its N-terminal region followed by an AP2 DNA-binding motif and a possible acidic activation domain in the C-terminal region. Bioinformatic analysis revealed that Cbcbf25 has a high level of similarity with other CBF genes like cbf1, cbf2, and cbf3 from Arabidopsis thaliana, and Bncbf5, Bncbf7, Bncbf16, and Bncbf17 from Brassica napus. A cold acclimation assay showed that Cbcbf25 was expressed immediately after cold triggering, but this expression was transient, suggesting that it concerns cold acclimation. Our study implies that Cbcbf25 is an analogue of other CBF genes and may participate in cold-response, by for example, controlling the expression of cold-regulated genes or increasing the freezing tolerance of plants.

• Biomedical Science Letters
• /
• v.13 no.4
• /
• pp.263-272
• /
• 2007

Nebulin is a giant actin binding protein (600-900 kDa) which is specific to skeletal muscle. This protein is known to regulate thin filaments length in sarcomere as a molecular template. The C-terminus of nebulin is located in the Z-disc of muscle sarcomere and is bound to other proteins such like myopalladin, titin, archvillin, and desmin. The N-terminus of nebulin binds to tropomodulin at the pointed ends of the thin filaments. In recent research, nebulin not only found in brain but also expressed in heart, stomach, and liver. So, the roles of nebulin in non-muscle tissue have been studied. However, lack of information or studies on nebulin binding proteins and nebulin function in brain are available so far. Therefore, the current study have investigated a novel binding partner of Nebulin C-terminus by using yeast two-hybrid screening with human brain cDNA library. Nebulin C-terminus, containing simple repeats, serine rich and SH3 domain, interacts with osteonectin C-terminal region. The specific interaction of nebulin and osteonectin were confirmed in vitro by using GST pull-down assay and reconfirmed in vivo by using transfected COS-7 cells with EGFP-tagged nebulin and DsRed-tagged osteonectin. Consequently, this study identified SH3 domain in nebulin C-terminus specifically binds to extracellular Ca-binding (EeC domain in osteonectin. Also, nebulin C-terminus fusion protein colocalized with osteonectin EC domain fusion protein in transfected COS-7 cells. The current study found the interaction between nebulin and osteonectin in human brain for the first time and suggested the nebulin in brain may be associated with osteonectin, as a regulator of cell cycle progression and mitosis.

• Molecules and Cells
• /
• v.21 no.2
• /
• pp.261-268
• /
• 2006

The Drosophila methuselah (mth) mutant has an approximately 35 percent increase in average lifespan, and enhanced resistance to various forms of stress, including starvation, high temperature, and dietary paraquat. To examine the transcriptional regulation of mth, we used luciferase assays employing Drosophila S2 cells. Two positive control elements were found at -542 ~ -272 (PE1) and +28 ~ +217 (PE2), where putative binding sites for transcription factors including Dorsal (Dl) were identified. Cotransfection of a Dl expression plasmid with a mth-luciferase reporter plasmid resulted in decreased reporter activity. PE1 and PE2, the minimal elements for strong promoter activity, were required for maximal repression by Dl protein. The N-terminal Rel homology domain (RHD) of Dl was not sufficient for repression of mth. We demonstrated by chromatin affinity precipitation (ChAP) assays in S2 cells that Dl bound to the putative PE1 binding site. Unexpectedly, semi-quantitative RT-PCR analysis revealed that the level of mth transcripts was reduced in dl flies. However, the in vivo result support the view that mth expression is regulated by dl, since it is well known that Dl functions as both a transcriptional activator and repressor depending on what other transcription factors are present. These findings suggest that both innate immunity and resistance to stress are controlled by Dl protein.

• Parasites, Hosts and Diseases
• /
• v.54 no.4
• /
• pp.385-391
• /
• 2016

The discovery and understanding of antigenic proteins are essential for development of a vaccine against malaria. In Plasmodium falciparum, Pf92 have been characterized as a merozoite surface protein, and this protein is expressed at the late schizont stage, but no study of Pv92, the orthologue of Pf92 in P. vivax, has been reported. Thus, the protein structure of Pv92 was analyzed, and the gene sequence was aligned with that of other Plasmodium spp. using bioinformatics tools. The recombinant Pv92 protein was expressed and purified using bacterial expression system and used for immunization of mice to gain the polyclonal antibody and for evaluation of antigenicity by protein array. Also, the antibody against Pv92 was used for subcellular analysis by immunofluorescence assay. The Pv92 protein has a signal peptide and a sexual stage s48/45 domain, and the cysteine residues at the N-terminal of Pv92 were completely conserved. The N-terminal of Pv92 was successfully expressed as soluble form using a bacterial expression system. The antibody raised against Pv92 recognized the parasites and completely merged with PvMSP1-19, indicating that Pv92 was localized on the merozoite surface. Evaluation of the human humoral immune response to Pv92 indicated moderate antigenicity, with 65% sensitivity and 95% specificity by protein array. Taken together, the merozoite surface localization and antigenicity of Pv92 implicate that it might be involved in attachment and invasion of a merozoite to a new host cell or immune evasion during invasion process.

• Journal of Life Science
• /
• v.32 no.3
• /
• pp.241-248
• /
• 2022

NF-κB acts as a critical transcription factor in inflammation and innate immunity, and it is also closely involved in cell survival and tumorigenesis via induction of anti-apoptotic genes. In these processes, NF-κB cooperates with multiple other signaling molecules and pathways, and although many studies have demonstrated that Hsp90 regulates NF-κB activity, the exact mechanism is unclear. In this study, we investigated the relationship between Hsp90 and IKKγ in the regulation of NF-κB using expression plasmids of IKK complex components. Wild-type and deletion mutants of IKKγ were expressed together with Hsp90, and the combined regulatory effect of Hsp90 and IKKγ on NF-κB activation was assayed. The results show that Hsp90 activates NF-κB by promoting the phosphorylation and degradation of IκBα and that activation of NF-κB by NIK and LPS was increased by Hsp90. IKKγ elevated the effect of Hsp90 on NF-κB activation by increasing phosphorylation and degradation of IκBα. The positive regulation on NF-κB by Hsp90 and IKKγ was also proved in analysis with IKKβ-EE, the constitutively active form of IKKβ. In experiments with the deletion mutants of IKKγ, the N-terminal IKKβ binding domain, C-terminal leucine zipper, and zinc finger domains of IKKγ were found not necessary for the positive regulation of NF-κB activity. Additionally, the expression of pro-inflammatory cytokines was synergistically elevated by Hsp90 and IKKγ. These results indicate that inhibiting the interaction between Hsp90 and IKKγ is a possible strategic method for controlling NF-κB and related diseases.