Biomedical subjects

Jeong Dong Bahk

Publications and source records attributed to Jeong Dong Bahk.

11 recordsLinked to original sources

PubMed2005

Phosphorylation and concomitant structural changes in human 2-Cys peroxiredoxin isotype I differentially regulate its peroxidase and molecular chaperone functions.

The H2O2-catabolizing peroxidase activity of human peroxiredoxin I (hPrxI) was previously shown to be regulated by phosphorylation of Thr90. Here, we show that hPrxI forms multiple oligomers with distinct secondary structures. HPrxI is a dual function protein, since it can behave either as a peroxidase or as a molecular chaperone. The effects of phosphorylation of hPrxI on its protein structure and dual functions were determined using site-directed mutagenesis, in which the phosphorylation site was substituted with aspartate to mimic the phosphorylated status of the protein (T90D-hPrxI). Phosphorylation of the protein induces significant changes in its protein structure from low molecular weight (MW) protein species to high MW protein complexes as well as its dual functions. In contrast to the wild type (WT)- and T90A-hPrxI, the T90D-hPrxI exhibited a markedly reduced peroxidase activity, but showed about sixfold higher chaperone activity than WT-hPrxI.

Amino Acid Substitution↗

PubMed2005

OsGAP1 functions as a positive regulator of OsRab11-mediated TGN to PM or vacuole trafficking.

The Ypt/Rab family of small G-proteins is important in regulating vesicular transport. Rabs hydrolyze GTP very slowly on their own and require GTPase-activating proteins (GAPs). Here we report the identification and characterization of OsGAP1, a Rab-specific rice GAP. OsGAP1 strongly stimulated OsRab8a and OsRab11, which are homologs of the mammalian Rab8 and Rab11 proteins that are essential for Golgi to plasma membrane (PM) and trans-Golgi network (TGN) to PM trafficking, respectively. Substitution of two invariant arginines within the catalytic domain of Oryza sativa GTPase-activating protein 1 (OsGAP1) with alanines significantly inhibited its GAP activity. In vivo targeting experiments revealed that OsGAP1 localizes to the TGN or pre-vacuolar compartment (PVC). A yeast expression system demonstrated that wild-type OsGAP1 facilitates O. sativa dissociation inhibitor 3 (OsGDI3)-catalyzed OsRab11 recycling at an early stage, but the OsGAP1(R385A) and (R450A) mutants do not. Thus, GTP hydrolysis is essential for Rab recycling. Moreover, expression of the OsGAP1 mutants in Arabidopsis protoplasts inhibited the trafficking of some cargo proteins, including the PM-localizing H+-ATPase-green fluorescent protein (GFP) and Ca2+-ATPase8-GFP and the central vacuole-localizing Arabidopsis aleurain-like protein (AALP)-GFP. The OsGAP1 mutants caused these proteins to accumulate at the Golgi apparatus. Surprisingly, OsRab11 overproduction relieved the inhibitory effect of the OsGAP1 mutants on vesicular trafficking. OsRab8a had no such effect. Thus, the OsGAP1 mutants may inhibit TGN to PM or central vacuole trafficking because they induce the sequestration of endogenous Rab11. We propose that OsGAP1 facilitates vesicular trafficking from the TGN to the PM or central vacuole by both stimulating the GTPase activity of OsRab11 and increasing the recycling of inactive OsRab11.

Alanine↗

PubMed2005

Role of RepB in the replication of plasmid pJB01 isolated from Enterococcus faecium JC1.

The plasmid pJB01 (GenBank Accession No. AY425961) isolated from the pathogenic bacterium, Enterococcus faecium JC1, is 2235 base pairs in length and consists of a putative double-strand origin (dso), a single-strand origin, a counter-transcribed RNA, and three open reading frames. A comparison of a few replication factors and motifs, bind and nic regions, for replication initiation on the nucleotide sequence level revealed that it belongs to the pMV158 family among RC-replicating plasmids. A runoff DNA synthesis assay demonstrated that nicking occurred between G525 and A526, which is located on the internal loop of a putative secondary structure in the dso. Unlike all the other plasmids of the pMV158 family having two or three direct repeats, pJB01 has three non-tandem direct repeats of 5'-CAACAAA-3' separated by four nucleotides, as the RepB-binding site in the dso. Moreover, the nick site on the internal loop is located at 77 nucleotides upstream from the RepB-binding region. Irrespective of the structural difference of direct repeats from other members of the pMV158 family, we think, it is still a new member of this plasmid family. The introduction of mutations in conserved regions of RepB confirmed that RepB N-moiety is important for nicking/nick-closing activity. Within N-moiety, especially all of the motif R-III, the Y100 in R-IV and Y116 in R-V residues, played particularly critical roles in this activity, however, for its binding, both of the N- and C-moieties of RepB were needed.

Amino Acid Sequence↗

PubMed2004

Arabidopsis C-terminal domain phosphatase-like 1 and 2 are essential Ser-5-specific C-terminal domain phosphatases.

Transcription and mRNA processing are regulated by phosphorylation and dephosphorylation of the C-terminal domain (CTD) of RNA polymerase II, which consists of tandem repeats of a Y(1)S(2)P(3)T(4)S(5)P(6)S(7) heptapeptide. Previous studies showed that members of the plant CTD phosphatase-like (CPL) protein family differentially regulate osmotic stress-responsive and abscisic acid-responsive transcription in Arabidopsis thaliana. Here we report that AtCPL1 and AtCPL2 specifically dephosphorylate Ser-5 of the CTD heptad in Arabidopsis RNA polymerase II, but not Ser-2. An N-terminal catalytic domain of CPL1, which suffices for CTD Ser-5 phosphatase activity in vitro, includes a signature DXDXT acylphosphatase motif, but lacks a breast cancer 1 CTD, which is an essential component of the fungal and metazoan Fcp1 CTD phosphatase enzymes. The CTD of CPL1, which contains two putative double-stranded RNA binding motifs, is essential for the in vivo function of CPL1 and includes a C-terminal 23-aa signal responsible for its nuclear targeting. CPL2 has a similar domain structure but contains only one double-stranded RNA binding motif. Combining mutant alleles of CPL1 and CPL2 causes synthetic lethality of the male but not the female gametes. These results indicate that CPL1 and CPL2 exemplify a unique family of CTD Ser-5-specific phosphatases with an essential role in plant growth and development.

Arabidopsis↗

PubMed2004

Bax-induced cell death of Arabidopsis is meditated through reactive oxygen-dependent and -independent processes.

An Arabidopsis protoplast system was developed for dissecting plant cell death in individual cells. Bax, a mammalian pro-apoptotic member of the Bcl-2 family, induces apoptotic-like cell death in Arabidopsis. Bax accumulation in Arabidopsis mesophyll protoplasts expressing murine Bax cDNA from a glucocorticoid-inducible promoter results in cytological characteristics of apoptosis, namely DNA fragmentation, increased vacuolation, and loss of plasma membrane integrity. In vivo targeting analysis monitored using jellyfish green fluorescent protein (GFP) reporter indicated full-length Bax was localized to the mitochondria, as it does in animal cells. Deletion of the carboxyl-terminal transmembrane domain of Bax completely abolished targeting to mitochondria. Bax expression was followed by reactive oxygen species (ROS) accumulation. Treatment of protoplasts with the antioxidant N -acetyl- -cysteine (NAC) during induction of Bax expression strongly suppressed Bax-mediated ROS production and the cell death phenotype. However, some population of the ROS depleted cells still induced cell death, indicating that there is a process that Bax-mediated plant cell death is independent of ROS accumulation. Accordingly, suppression of Bax-mediated plant cell death also takes place in two different processes. Over-expression of a key redox-regulator, Arabidopsis nucleoside diphosphate kinase 2 (AtNDPK2) down-regulated ROS accumulation and suppressed Bax-mediated cell death and transient expression of Arabidopsis Bax inhibitor-1 (AtBI-1) substantially suppressed Bax-induced cell death without altering cellular ROS level. Taken together, our results collectively suggest that the Bax-mediated cell death and its suppression in plants is mediated by ROS-dependent and -independent processes.

Animals↗

PubMed2003

Rice C2-domain proteins are induced and translocated to the plasma membrane in response to a fungal elicitor.

Hundreds of proteins involved in signaling pathways contain a Ca(2+)-dependent membrane-binding motif called the C2-domain. However, no small C2-domain proteins consisting of a single C2-domain have been reported in animal cells. We have isolated two cDNA clones, OsERG1a and OsERG1b, that encode two small C2-domain proteins of 156 and 159 amino acids, respectively, from a fungal elicitor-treated rice cDNA library. The clones are believed to have originated from a single gene by alternative splicing. Transcript levels of the OsERG1 gene are dramatically elevated by a fungal elicitor prepared from Magnaporthe grisea or by Ca(2+) ions. The OsERG1 protein produced in Escherichia coli binds to phospholipid vesicles in a Ca(2+)-dependent manner and is translocated to the plasma membrane of plant cells by treatment with either a fungal elicitor or a Ca(2+) ionophore. These results suggest that OsERG1 proteins containing a single C2-domain are involved in plant defense signaling systems.

Amino Acid Motifs↗

PubMed2003

The effect of pH and various cations on the GTP hydrolysis of rice heterotrimeric G-protein alpha subunit expressed in Escherichia coli.

Previously, we reported the biochemical properties of RGA1 that is expressed in Escherichia coli (Seo et al., 1997). The activities of RGA1 that hydrolyzes and binds guanine nucleotide were dependent on the MgCl(2) concentration. The steady state rate constant (k(cat) ) for GTP hydrolysis of RGA1 at 2 mM MgCl(2) was 0.0075 +/- 0.0001 min(-1). Here, we examined the effects of pH and cations on the GTPase activity. The optimum pH at 2 mM MgCl(2) was approximately 6.0; whereas, the pH at 2 mM NH(4)Cl was approximately 4.0. The result from the cation dependence on the GTPase (guanosine 5'-triphosphatase) activity of RGA1 under the same condition showed that the GTP hydrolysis rate (k(cat)= 0.0353 min(-1)) under the condition of 2 mM NH(4)Cl at pH 4.0 was the highest. It corresponded to about 3.24-fold of the k(cat) value of 0.0109 min(-1) in the presence of 2 mM MgCl(2) at pH 6.0.

Cations↗

PubMed2003

Molecular and biochemical analyses of OsRab7, a rice Rab7 homolog.

Rab7 is a small GTP-binding protein important in early to late endosome/lysosome vesicular transport in mammalian cells. We have isolated a Rab7 cDNA clone, OsRab7, from a cold-treated rice cDNA library by the subtraction screening method. The cDNA encodes a polypeptide of 206 amino acids with a calculated molecular mass of about 23 kDa. Its predicted amino acid sequence shows significantly high identity with the sequences of other Rab7 proteins. His-tagged OsRab7 bound to radiolabeled GTPgammaS in a specific and stoichiometric manner. Biochemical and structural properties of the Rab7 wild type (WT) protein were compared to those of Q67L and T22N mutants. The detergent 3-([3-cholamidopropyl]dimethylammonio)-1-propane sulfonate (CHAPS) increased the guanine nucleotide binding and hydrolysis activities of Rab7WT. The OsRab7Q67L mutant showed much lower GTPase activity compared to the WT protein untreated with CHAPS, and the T22N mutant showed no GTP binding activity at all. The OsRab7Q67L mutant was constitutively active for guanine nucleotide binding while the T22N mutant (dominant negative) showed no guanine nucleotide binding activity. When bound to GTP, the Rab7WT and the Q67L mutants were protected from tryptic proteolysis. The cleavage pattern of the Rab7T22N mutant, however, was not affected by GTP addition. Northern and Western blot analyses suggested that OsRab7 is distributed in various tissues of rice. Furthermore, expression of a rice Rab7 gene was differentially regulated by various environmental stimuli such as cold, NaCl, dehydration, and ABA. In addition, subcellular localization of OsRab7 was investigated in the Arabidopsis protoplasts by a double-labeling experiment using GFP-fused OsRab7 and FM4-64. GFP-OsRab7 is localized to the vacuolar membrane, suggesting that OsRab7 is implicated in a vesicular transport to the vacuole in plant cells.

Abscisic Acid↗

PubMed2002

Partial purification and properties of a phosphatidylinositol 4,5-bisphosphate hydrolyzing phospholipase C from the soluble fraction of soybean sprouts.

Three soluble enzyme fractions (F-I, F-II, and F-III) that hydrolyze phophoinositides were separated from soybean sprouts by using Matrex green gel column chromatography. Among the three phosphatidylinositol (PI)-specific phopholipsase C (PLC) enzymes, only the third fraction (F-III) was able to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) as well as phosphatidylinositol (PI) and phosphatidylinositol phosphate (PIP) as substrates. The F-I and F-II fractions only showed enzymatic activities for PI and PIP. The PIP2-hydrolyzing PLC protein, F-III, was partially purified using the chromatographic steps of the Matrex green gel, phenyl Toyopearl, Matrex orange gel, Mono S cation exchange, and superose 6 gel filtration columns. The molecular weight of the F-III protein was estimated to be about 64 kDa on SDS-PAGE. The protein showed immunocross-reactivity with a polyclonal antibody that was prepared against the X and Y motifs of animal PLC enzymes, the conserved catalytic domains. Ca2+ ion critically affected the PIP2-hydrolyzing PLC activity of the F-III protein, representing maximal activity at 10 microM Ca2+ concentration. The PIP2-hydrolyzing PLC activity of the protein was also significantly increased by sodium deoxycholate (SDC) from 0.05 to 0.08%. However, the activity was greatly reduced above the concentration, and no activity was detected at 0.3% SDC. In addition, the protein exhibited maximal PIP2-hydrolyzing PLC activity at pH, in the range of 6.5-7.5.

Antibodies↗

PubMed2002

Carnitine uptake by AGP2 in yeast Saccharomyces cerevisiae is dependent on Hog1 MAP kinase pathway.

The AGP2 gene encodes a plasma membrane carnitine transporter in S. cerevisiae. Here, we report the identification of AGP2 as an osmotic stress response gene. AGP2 was isolated from mTn3 tagged mutants that contained in-frame fusions with lacZ. The expression of AGP2 was down-regulated by osmotic stresses, including NaCl, sorbitol, and KCI. We also found that carnitine uptake was inhibited by NaCl. In the ssk1delta stelldelta double-mutant strain, the expression of AGP2 and the uptake of carnitine were greatly reduced compared to the wild-type strain. Furthermore, carnitine uptake was inhibited by the constitutive expression of PBS2, which encodes a MAPKK that activates Hog1. We concluded, therefore, that the HOG pathway plays an important role in the regulation of carnitine uptake in S. cerevisiae.

Amino Acid Transport Systems↗

PubMed2002

Over-expression of a seed specific hevein-like antimicrobial peptide from Pharbitis nil enhances resistance to a fungal pathogen in transgenic tobacco plants.

Two hevein-like peptides from the seed of Pharbitis nil, designated Pharbitis nil antimicrobial peptide 1 (Pn-AMP1) and Pn-AMP2, had been purified previously. Both exhibit potent in vitro antifungal activity against a broad spectrum of phytopathogenic fungi. We now report the isolation of two cDNA clones, designated pnAMP-h1 and pnAMP-h2, and the corresponding genomic clones encoding these proteins from mature seeds of P. nil. Comparison of the deduced amino acid sequence to that of the mature protein suggests that the peptides are produced as a prepropeptide consisting of an N-terminal signal peptide, the mature protein and C-terminal domains. The transcripts of the two genes are accumulated seed--specifically, and the maximum transcripts are observed in the mid-to-late stage of seed development. Constitutive over-expression of the pnAMP-h2 cDNA in transgenic tobacco under the control of the cauliflower mosaic virus 35S promoter conferred enhanced resistance against the oomycete Phytophthora parasitica, the causal agent of black shank disease. Thus the Pn-AMPs may play a role in the protection of seeds and may be useful as a novel gene source to engineer plants resistant to fungal pathogens.

Amino Acid Sequence↗