Biomedical subjects

Hyo-Il Jung

Publications and source records attributed to Hyo-Il Jung.

11 recordsLinked to original sources

PubMed2006

[Ca(2+)]-dependent generation of intracellular reactive oxygen species mediates maitotoxin-induced cellular responses in human umbilical vein endothelial cells.

Maitotoxin (MTX) is known as one of the most potent marine toxins involved in Ciguatera poisoning, but intracellular signaling pathways caused by MTX was not fully understood. Thus, we have investigated whether intracellular reactive oxygen species (ROS) are involved in MTX-induced cellular responses in human umbilical vein endothelial cells. MTX induced a dose-dependent increase of intracellular [Ca(2+)]. MTX stimulated the production of intracellular ROS in a dose- and time-dependent manner, which was suppressed by BAPTA-AM, an intracellular Ca(2+) che-lator. Ionomycin also elevated the ROS production in a dose-dependent manner. MTX elevated transamidation activity in a time-dependent manner and the activation was largely inhibited by transfection of tissue transglutaminase siRNA. The activation of tissue transglutaminase and ERK1/2 by MTX was sup-pressed by BAPTA-AM or ROS scavengers. In addition, MTX-induced cell death was significantly de-layed by BAPTA-AM or a ROS scavenger. These results suggest that [Ca(2+)]-dependent generation of in-tracellular ROS, at least in part, play an important role in MTX-stimulated cellular responses, such as activation of tTGase, ERK phosphorylation, and in-duction of cell death, in human umbilical vein endothelial cells.

Calcium↗

PubMed2006

Direct force measurement of the interaction between liposome and the C2A domain of synaptotagmin I using atomic force microscopy.

The binding force between a liposome and the C2A domain of synaptotagmin I was determined by an atomic force microscopy (AFM). Liposomes were immobilized on the surface of the L1 sensor chip and the C2A domains, which recognize phosphatidylserine, were chemically conjugated onto a gold-coated cantilever tip. The average interaction force between the C2A domain and the liposome was 306 (+/-57) pN while the force between untreated cantilever and the liposome was 58 (+/-16) pN. This work helps understand the physicochemical interactions between proteins and lipid vesicles for the design of high affinity protein probes against the apoptotic cell surface.

Binding Sites↗

PubMed2004

Arachidonic acid activates tissue transglutaminase and stress fiber formation via intracellular reactive oxygen species.

We have investigated whether arachidonic acid could regulate tissue transglutaminase (tTGase) via intracellular reactive oxygen species (ROS) in NIH3T3 cells. tTGase was identified in NIH3T3 cells by Western blot and confocal microscopy. Arachidonic acid elevated in situ tTGase activity in dose- and time-dependent manners with a maximal level at 1h, and ROS scavengers, N-(2-mercaptopropionyl)glycine and catalase, blocked the tTGase activation by arachidonic acid. The activation of tTGase by arachidonic acid was largely inhibited by transfection of tTGase siRNA. The role of intracellular ROS in the activation of in situ tTGase was supported by the activation of in situ tTGase by exogenous H(2)O(2). Arachidonic acid stimulated the formation of stress fibers in a dose- and time-dependent manner, and the ROS scavengers suppressed the arachidonic acid-induced formation of stress fibers. These results suggested that the activation of in situ tTGase and stress fiber formation by arachidonic acid was mediated by intracellular ROS in NIH3T3 cells.

Animals↗

PubMed2004

DNA replication licensing in somatic and germ cells.

The DNA replication (or origin) licensing system ensures precise duplication of the genome in each cell cycle and is a powerful regulator of cell proliferation in metazoa. Studies in yeast, Drosophila melanogaster and Xenopus laevis have characterised the molecular machinery that constitutes the licensing system, but it remains to be determined how this important evolutionary conserved pathway is regulated in Homo sapiens. We have investigated regulation of the origin licensing factors Cdc6, Cdt1, Mcm2 and Geminin in human somatic and germ cells. Cdc6 and Cdt1 play an essential role in DNA replication initiation by loading the Mcm2-7 complex, which is required for unwinding the DNA helix, onto chromosomal origins. Geminin is a repressor of origin licensing that blocks Mcm2-7 loading onto origins. Our studies demonstrate that Cdc6, Cdt1 and Mcm2 play a central role in coordinating growth during the proliferation-differentiation switch in somatic self-renewing systems and that Cdc6 expression is rate-limiting for acquisition of replication competence in primary oocytes. In striking contrast, we show that proliferation control during male gametogenesis is not linked to Cdc6 or Mcm2, but appears to be coordinated by the negative regulator Geminin with Cdt1 becoming rate-limiting in late prophase. Our data demonstrate a striking sexual dimorphism in the mechanisms repressing origin licensing and preventing untimely DNA synthesis during meiosis I, implicating a pivotal role for Geminin in maintaining integrity of the male germline genome.

Alternative Splicing↗

PubMed2003

Prediction of the binding site on E1 in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus.

The beta-subunit (E1beta) of the pyruvate decarboxylase (E1, alpha(2)beta(2)) component of the Bacillus stearothermophilus pyruvate dehydrogenase complex was comparatively modelled based on the crystal structures of the homologous 2-oxoisovalerate decarboxylase of Pseudomonas putida and Homo sapiens. Based on this homology modelling, alanine-scanning mutagenesis studies revealed that the negatively charged side chain of Glu285 and the hydrophobic side chain of Phe324 are of particular importance in the interaction with the peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase component of the complex. These results help to identify the site of interaction on the E1beta subunit and are consistent with thermodynamic evidence of a mixture of electrostatic and hydrophobic interactions being involved.

Alanine↗

PubMed2003

Reaction mechanism of the heterotetrameric (alpha2beta2) E1 component of 2-oxo acid dehydrogenase multienzyme complexes.

Pyruvate decarboxylase (E1) catalyzes the first two reactions of the four involved in oxidative decarboxylation of pyruvate by the pyruvate dehydrogenase (PDH) multienzyme complex. It requires thiamin diphosphate to bring about the decarboxylation of pyruvate, which is followed by the reductive acetylation of a lipoyl group covalently bound to the N(6) amino group of a lysine residue in the second catalytic component, a dihydrolipoyl acetyltransferase (E2). Replacement of two histidine residues in the E1alpha and E1beta chains of the heterotetrameric E1 (alpha(2)beta(2)) component of the PDH complex of Bacillus stearothermophilus, considered possible proton donors at the active site, was carried out. Subsequent characterization of the mutants permitted different roles to be assigned to these two particular residues in the reaction catalyzed by E1: E1alpha His271 to stabilize the dianion formed during decarboxylation of the 2-oxo acid and E1beta His128 to provide the proton required to protonate the incoming dithiolane ring in the subsequent reductive acetylation of the lipoyl goup. On the basis of these and other results from a separate investigation into the roles of individual residues in a loop region in the E1alpha chain close to the active site of E1 [Fries, M., Chauhan, H. J., Domingo, G. J., Jung, H., and Perham, R. N. (2002) Eur. J. Biochem. 270, 861-870] together with work from other laboratories, a detailed mechanism for the E1 reaction can be formulated.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

PubMed2003

Site-directed mutagenesis of a loop at the active site of E1 (alpha2beta2) of the pyruvate dehydrogenase complex. A possible common sequence motif.

Limited proteolysis of the pyruvate decarboxylase (E1, alpha2beta2) component of the pyruvate dehydrogenase (PDH) multienzyme complex of Bacillus stearothermophilus has indicated the importance for catalysis of a site (Tyr281-Arg282) in the E1alpha subunit (Chauhan, H.J., Domingo, G.J., Jung, H.-I. & Perham, R.N. (2000) Eur. J. Biochem. 267, 7158-7169). This site appears to be conserved in the alpha-subunit of heterotetrameric E1s and multiple sequence alignments suggest that there are additional conserved amino-acid residues in this region, part of a common pattern with the consensus sequence -YR-H-D-YR-DE-. This region lies about 50 amino acids on the C-terminal side of a 30-residue motif previously recognized as involved in binding thiamin diphosphate (ThDP) in all ThDP-dependent enzymes. The role of individual residues in this set of conserved amino acids in the E1alpha chain was investigated by means of site-directed mutagenesis. We propose that particular residues are involved in: (a) binding the 2-oxo acid substrate, (b) decarboxylation of the 2-oxo acid and reductive acetylation of the tethered lipoyl domain in the PDH complex, (c) an "open-close" mechanism of the active site, and (d) phosphorylation by the E1-specific kinase (in eukaryotic PDH and branched chain 2-oxo acid dehydrogenase complexes).

Amino Acid Sequence↗

PubMed2003

Interactions of the peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase component in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus.

The enzymes pyruvate decarboxylase (E1) and dihydrolipoyl dehydrogenase (E3) bind tightly but in a mutually exclusive manner to the peripheral subunit-binding domain (PSBD) of dihydrolipoyl acetyltransferase in the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. The use of directed mutagenesis, surface plasmon resonance detection and isothermal titration microcalorimetry revealed that several positively charged residues of the PSBD, most notably Arg135, play an important part in the interaction with both E1 and E3, whereas Met131 makes a significant contribution to the binding of E1 only. This indicates that the binding sites for E1 and E3 on the PSBD are overlapping but probably significantly different, and that additional hydrophobic interactions may be involved in binding E1 compared with E3. Arg135 of the PSBD was also replaced with cysteine (R135C), which was then modified chemically by alkylation with increasingly large aliphatic groups (R135C -methyl, -ethyl, -propyl and -butyl). The pattern of changes in the values of DeltaG degrees, DeltaH degrees and TDeltaS degrees that were found to accompany the interaction with the variant PSBDs differed between E1 and E3 despite the similarities in the free energies of their binding to the wild-type. The importance of a positive charge on the side-chain at position 135 for the interaction of the PSBD with E3 and E1 was apparent, although lysine was found to be an imperfect substitute for arginine. The results offer further evidence of entropy-enthalpy compensation ('thermodynamic homeostasis') - a feature of systems involving a multiplicity of weak interactions.

Acetyltransferases↗

PubMed2002

Identification of key amino acid residues in the assembly of enzymes into the pyruvate dehydrogenase complex of Bacillus stearothermophilus: a kinetic and thermodynamic analysis.

Structural studies have shown that electrostatic interactions play a major part in the binding of dihydrolipoyl dehydrogenase (E3) to the peripheral subunit-binding domain (PSBD) of the dihydrolipoyl acyltransferase (E2) in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. The binding is characterized by a small, unfavorable enthalpy change (deltaH degrees = +2.2 kcal/mol) and a large, positive entropy change (TdeltaS degrees = +14.8 kcal/mol). The contributions of individual surface residues of the PSBD of E2 to its interaction with E3 have been assessed by alanine-scanning mutagenesis, surface plasmon resonance detection, and isothermal titration calorimetry. The mutation R135A in the PSBD gave rise to a significant decrease (120-fold) in the binding affinity; two other mutations (R139A and R156A) were associated with smaller effects. The binding of the R135A mutant to E3 was accompanied by a favorable enthalpy (deltaH degrees = -2.6 kcal/mol) and a less positive entropy change (TdeltaS degrees = +7.2 kcal/mol). The midpoint melting temperature (T(m)) of E3-PSBD complexes was determined by differential scanning calorimetry. The R135A mutation caused a significant decrease (5 degrees C) in the T(m), compared with the wild-type complex. The results reveal the importance of Arg135 of the PSBD as a key residue in the molecular recognition of E3 by E2, and as a major participant in the overall entropy-driven binding process. Further, the effects of mutagenesis on the deltaCp of subunit association illustrate the difficulties in attributing changes in heat capacity to specific classes of interactions.

Acetyltransferases↗

PubMed2002

Thermodynamic analysis of the binding of component enzymes in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus.

The peripheral subunit-binding domain (PSBD) of the dihydrolipoyl acetyltransferase (E2, EC 2.3.1.12) binds tightly but mutually exclusively to dihydrolipoyl dehydrogenase (E3, EC 1.8.1.4) and pyruvate decarboxylase (E1, EC 1.2.4.1) in the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. Isothermal titration calorimetry (ITC) experiments demonstrated that the enthalpies of binding (DeltaH degrees ) of both E3 and E1 with the PSBD varied with salt concentration, temperature, pH, and buffer composition. There is little significant difference in the free energies of binding (DeltaG degrees = -12.6 kcal/mol for E3 and = -12.9 kcal/mol for E1 at pH 7.4 and 25 degrees C). However, the association with E3 was characterized by a small, unfavorable enthalpy change (DeltaH degrees = +2.2 kcal/mol) and a large, positive entropy change (TDeltaS degrees = +14.8 kcal/mol), whereas that with E1 was accompanied by a favorable enthalpy change (DeltaH degrees = -8.4 kcal/mol) and a less positive entropy change (TDeltaS degrees = +4.5 kcal/mol). Values of DeltaC(p) of -316 cal/molK and -470 cal/molK were obtained for the binding of E3 and E1, respectively. The value for E3 was not compatible with the DeltaC(p) calculated from the nonpolar surface area buried in the crystal structure of the E3-PSBD complex. In this instance, a large negative DeltaC(p) is not indicative of a classical hydrophobic interaction. In differential scanning calorimetry experiments, the midpoint melting temperature (T(m)) of E3 increased from 91 degrees C to 97.1 degrees C when it was bound to PSBD, and that of E1 increased from 65.2 degrees C to 70.0 degrees C. These high T(m) values eliminate unfolding as a major source of the anomalous DeltaC(p) effects at the temperatures (10-37 degrees C) used for the ITC experiments.

Calorimetry, Differential Scanning↗

PubMedDate not supplied

Detection of apoptosis using the C2A domain of synaptotagmin I.

Binding of annexin V or the C2A domain of synaptotagmin I to phosphatidylserine expressed on the surface of apoptotic cells can, when labeled with appropriate probe molecules, be used to detect the presence of apoptosis using radionuclide, magnetic resonance, and optical imaging techniques. The preparation of a biotinylated C2A-GST fusion protein is described, and its capability, when used in conjunction with fluorescein-labeled streptavidin, of detecting apoptotic cells by flow cytometry is compared directly with the performance of a commercial preparation of fluorescein-labeled annexin V. Biotinylated C2A-GST, when used in conjunction with streptavidin-conjugated superparamagnetic iron oxide nanoparticles or Gd-chelate-avidin conjugates, was shown to be capable of detecting apoptotic cells using T(2)-weighted or T(1)-weighted magnetic resonance imaging experiments, respectively.

Animals↗