Biomedical subjects

Eun-Woo Lee

Publications and source records attributed to Eun-Woo Lee.

11 recordsLinked to original sources

PubMed2026

A refined MASH-HCC model identifies macrophage Gadd45b as a key orchestrator of inflammation-driven neoplastic progression.

Metabolic dysfunction-associated steatohepatitis (MASH) is emerging as a leading driver of hepatocellular carcinoma (HCC), yet the molecular mechanisms linking metabolic stress, chronic inflammation and tumorigenesis remain poorly understood. Here we established a metabolically relevant, time-efficient MASH-to-HCC model in C57BL/6N mice by combining a MASH diet with controlled CCl4 administration, enabling stepwise recapitulation of MASH-associated neoplastic progression. Using this model, we identified growth arrest and DNA damage 45b (Gadd45b) as a novel MASH-derived protumorigenic regulator selectively activated under metabolic stress. Integrated analyses of human bulk and single-cell transcriptomic datasets and mouse transcriptomic deconvolution revealed concordant macrophage remodeling and GADD45B/Gadd45b expression dynamics during MASH-to-HCC progression. Mechanistically, fatty acids and TNFα preferentially induced Gadd45b in macrophages, where it amplified TNFα-NF-κB signaling. Macrophage-derived inflammatory signals subsequently induced Gadd45b and NF-κB activation in hepatocytes, establishing a feed-forward inflammatory loop that promoted fibrogenic and partial EMT-like programs and tumor spheroid formation. Importantly, temporal profiling during spheroid formation and progression revealed transient induction of Gadd45b during early spheroid establishment, but not during later progression, indicating that Gadd45b-mediated inflammatory signaling primarily promotes tumor initiation rather than subsequent growth. Consistent with human data, Gadd45b expression increased with disease severity and positively correlated with inflammatory factors in the MASH-HCC model, whereas pharmacological inhibition attenuated the Gadd45b-inflammation signaling axis. Collectively, our findings establish macrophage Gadd45b as a key orchestrator linking metabolic stress, chronic inflammation, and neoplastic transformation during MASH-to-HCC progression. Our refined MASH-HCC model provides a robust platform for mechanistic studies and preclinical evaluation of inflammation-targeted therapies.

Journal Article↗

PubMed2006

Jab1 as a mediator of nuclear export and cytoplasmic degradation of p53.

Jun activation domain-binding protein 1 (Jab1) is involved in various cellular mechanisms including development in Drosophila and mouse, cell cycle control and signal transduction pathways. Recent studies also determined that Jab1 functions as a nuclear exporter and inducer of cytoplasmic degradation for several proteins including p53, p27, capsid of West Nile virus, and Smad4/7 proteins. In particular, p53 is shown to bind to and to be exported into the cytoplasm by Jab1, which helps to maintain low levels of p53 under normal conditions. This review was undertaken in an effort to understand the biological significance of the homeostasis of p53 as maintained in the presence of Jab1. Based on our observations, we have provided potential mechanistic hypotheses for the nuclear export of p53 in coordination with Jab1 and the role of other factors in these processes.

Active Transport, Cell Nucleus↗

PubMed2006

Jab1 mediates cytoplasmic localization and degradation of West Nile virus capsid protein.

The clinical manifestations of West Nile virus (WNV), a member of the Flavivirus family, include febrile illness, sporadic encephalitis, and paralysis. The capsid (Cp) of WNV is thought to participate in these processes by inducing apoptosis through mitochondrial dysfunction and activation of caspase-9 and caspase-3. To further identify the molecular mechanism of the WNV capsid protein (WNVCp), yeast two-hybrid assays were employed using WNV-Cp as bait. Jab1, the fifth subunit of the COP9 signalosome, was subsequently identified as a molecule that interacts with WNVCp. Immunoprecipitation and glutathione S-transferase pulldown assays confirmed that direct interaction could occur between WNVCp and Jab1. Immunofluorescence microscopy demonstrated that the overexpressed WNVCp, which localized to the nucleolus, was translocated to the cytoplasm upon its co-expression with Jab1. When treated with leptomycin B, Jab1-facilitated nuclear exclusion of WNVCp was prevented, which indicated that the CRM1 complex is required for Jab1-facilitated nuclear export of WNVCp. Moreover, Jab1 promoted the degradation of WNVCp in a proteasome-dependent way. Consistent with this, WNVCp-mediated cell cycle arrest at the G(2) phase in H1299 was prevented by exogenous Jab1. Finally, an analysis of WNVCp deletion mutants indicated that the first 15 amino acids were required for interaction with Jab1. Furthermore, the double-point mutant of the WNVCp, P5A/P8A, was incapable of binding to Jab1. These results indicate that Jab1 has a potential protective effect against pathogenic WNVCp and might provide a novel target site for the treatment of disease caused by WNV.

Active Transport, Cell Nucleus↗

PubMed2006

Jab1 induces the cytoplasmic localization and degradation of p53 in coordination with Hdm2.

The biological mechanisms for maintaining the basal level of p53 in normal cells require nuclear exclusion and cytoplasmic degradation. Here, we showed that Jab1 facilitates p53 nuclear exclusion and its subsequent degradation in coordination with Hdm2. p53 was excluded from the nucleus in the presence of Jab1; this exclusion was prevented by leptomycin B treatment. Nuclear export of p53 was accompanied by a decrease in the levels of p53, as well as of its target proteins, which include p21 and Bax. Domain analyses of Jab1 showed that the N-terminal domain, 1-110, was capable of inducing cytoplasmic translocation of p53. Furthermore, 110-191 was required to facilitate the degradation of p53. Neither of these mutants incorporated into the CSN complex, indicating that Jab1 could affect the levels of p53 independent of intact CSN complex. Conversely, Jab1 was incapable of translocating and degrading two p53 mutants, W23S and 6KR, neither of which could be modified by Hdm2. Moreover, Jab1 did not affect the cellular localization or protein levels of p53 in p53 and Hdm2 double-null mouse embryo fibroblasts. We further observed that the ablation of endogenous Jab1 by small interfering RNA prevented Hdm2-mediated p53 nuclear exclusion. Under stressed conditions, which could sequester Hdm2 in its inactive state, Jab1 did not affect p53. Our studies implicate that Jab1 is required to remove post-translationally modified p53 and provide a novel target for p53-related cancer therapies.

Animals↗

PubMed2006

Identification of the mitochondrial targeting signal of the human equilibrative nucleoside transporter 1 (hENT1): implications for interspecies differences in mitochondrial toxicity of fialuridine.

We have previously shown that the human equilibrative nucleoside transporter 1 (hENT1) is expressed and functional in the mitochondrial membrane and that this expression enhances the mitochondrial toxicity of the nucleoside drug, fialuridine (FIAU) (Lai, Y., Tse, C. M., and Unadkat, J. D. (2004) J. Biol. Chem. 279, 4490-4497). Here we report on identification of the mitochondrial targeting sequence of hENT1. Using confocal microscopy and different truncated and point mutants of hENT1-YFP (yellow fluorescent protein) expressed in Madin-Darby canine kidney cells, we identified amino acid residues Pro(71),Glu(72), and Asn(74) (the PEXN motif) of hENT1 as important in mitochondrial targeting of hENT1. Identification of this mitochondrial targeting sequence provides a possible explanation for the dramatic difference in mitochondrial toxicity of FIAU between humans and rodents. Although the mouse ENT1 (mENT1), expressed in Madin-Darby canine kidney cells, can transport FIAU, confocal microscopy showed that mENT1-GFP (green fluorescent protein) was not localized to the mitochondria. Consistent with this observation, mitochondria isolated from mouse livers did not transport FIAU. Sequence alignment of hENT1, mENT1, and rat ENT1 (rENT1) showed that the PEXN motif of hENT1 was substituted with a PAXS motif in both mENT1 and rENT1. Substitution of PAXS in mENT1 with PEXN (to create mENT1-PEXN-GFP) and of PEXN in hENT1 with PAXS (to create hENT1-PAXS-YFP) resulted in partial mitochondrial localization of mENT1-PEXN-GFP and loss of mitochondrial localization of hENT1-PAXS-YFP. This is the first time that the mitochondrial targeting signal of hENT1 has been identified. Our data suggest that the lack of mitochondrial toxicity of FIAU in mice is due to the lack of mENT1 targeting to and expression in the mitochondria.

Amino Acid Sequence↗

PubMed2006

Electrophysiological characterization and modeling of the structure activity relationship of the human concentrative nucleoside transporter 3 (hCNT3).

We characterized the electrophysiology, kinetics, and quantitative structure-activity relationship (QSAR) of the human concentrative nucleoside transporter 3 (hCNT3) expressed in Xenopus laevis oocytes by measuring substrate-induced inward currents using a two-microelectrode voltage-clamp system. At membrane potentials between -30 and -150 mV, sodium activation of gemcitabine transport was sigmoidal, with a K0.5 of 8.5+/-0.3 mM for Na+ and a Hill coefficient of 2.2+/-0.25 independent of membrane potential. We measured the Imax and K0.5 for substrate at -50 mV for the nucleoside analog drugs gemcitabine (638+/-58 nA, 59.7+/-17.5 microM), ribavirin (546+/-37 nA, 61.0+/-13.2 microM), AZT (420+/-4 nA, 310+/-9 microM), and 3-deazauridine (506+/-30 nA, 50.8+/-9.90 microM). K0.5 and Imax for substrate were dependent on membrane potential (both increasing as the membrane became more hyperpolarized) for all four drugs. hCNT3 also exhibited pre-steady-state currents. The quantitative structure-activity relationship (QSAR) was examined using comparative molecular field analysis and comparative molecular similarity indices analysis of the inward currents induced by 27 nucleoside analogs with substitutions at both the ribose and the nucleobase. Two statistically significant QSAR models identified electrostatic interaction as the major force in hCNT3 transport and attributed a critical role to the 3'-hydroxyl position of hCNT3 substrates. Steric hindrance at the 3-position and positive charge at the 5-position of the pyrimidine ring were favorable for transport. Two hCNT3 pharmacophore models revealed the minimal features required for hCNT3 transport as two hydrogen bond acceptors at 3'-OH and 5'-O and the hydrophobic center occupied by the base ring.

Algorithms↗

PubMed2004

Conserved residues F316 and G476 in the concentrative nucleoside transporter 1 (hCNT1) affect guanosine sensitivity and membrane expression, respectively.

The functional significance of two highly conserved amino acid residues, F316 [putative transmembrane domain (TM)7] and G476 (putative TM11), in the concentrative nucleoside transporter hCNT1 (SLC28A1) was examined by performing site-directed mutagenesis. Conservative mutations at these positions (F316A, F316Y, G476A, and G476L) were generated and expressed in Madin-Darby canine kidney (MDCK) cells as fusion polypeptides with green fluorescent protein (GFP). Unlike wild-type hCNT1, G476A-GFP and G476L-GFP were not expressed in the plasma membrane in undifferentiated or differentiated MDCK cells and had no functional activity. Like wild-type hCNT1, F316A-GFP and F316Y-GFP were expressed in the plasma membrane of undifferentiated MDCK cells and in the apical membrane of differentiated MDCK cells. Remarkably, transport of [(3)H]uridine by F316Y-GFP or F316A-GFP was highly sensitive to inhibition by guanosine. Furthermore, genotyping of exon 11 of hCNT1 (TM7) in a panel of 260 anonymous human DNA samples revealed a novel F316H variant (TT>CA; 1/260). When expressed in MDCK cells, [(3)H]uridine transport by F316H was also found to be sensitive to inhibition by guanosine (IC(50) = 148 microM). The effect of the F316H mutation resembles the N4 type nucleoside transporter phenotype previously reported to be present in human kidneys. We suggest that the N4 transport system is a naturally occurring variant of hCNT1, perhaps at the F316 position. Collectively, our data show that G476 is important for correct membrane targeting, folding, and/or intracellular processing of hCNT1. In addition, we have discovered that hCNT1 displays natural variation at position F316 and that the variant F316H confers on the transporter an unusual sensitivity to inhibition by guanosine.

Amino Acid Sequence↗

PubMed2003

EfrAB, an ABC multidrug efflux pump in Enterococcus faecalis.

A DNA fragment responsible for resistance to antimicrobial agents was cloned from the chromosomal DNA of Enterococcus faecalis ATCC 29212 by using drug-hypersensitive mutant Escherichia coli KAM32 as a host cell. Cells of E. coli KAM32 harboring a recombinant plasmid (pAEF82) carrying the DNA fragment became resistant to many structurally unrelated antimicrobial agents, such as norfloxacin, ciprofloxacin, doxycycline, acriflavine, 4',6-diamidino-2-phenylindole, tetraphenylphosphonium chloride, daunorubicin, and doxorubicin. Since the sequence of the whole genome of E. faecalis is known, we sequenced several portions of the DNA insert in plasmid pAEF82 and identified two open reading frames within the insert. We designated the genes efrA and efrB. A search of the deduced amino acid sequences of EfrA and EfrB revealed that they are similar to each other and that they belong to the ATP-binding cassette (ABC) family of multidrug efflux transporters. Transformed E. coli KAM32 cells harboring efrAB showed energy-dependent efflux of acriflavine. The efflux activity was inhibited by reserpine, verapamil, and sodium-o-vanadate, known inhibitors of ABC efflux pumps.

ATP-Binding Cassette Transporters↗

PubMed2003

Molecular cloning and characterization of an ABC multidrug efflux pump, VcaM, in Non-O1 Vibrio cholerae.

A gene responsible for multidrug resistance was cloned from the chromosomal DNA of non-O1 Vibrio cholerae NCTC 4716 by using as a host drug-hypersensitive Escherichia coli strain KAM32, which lacks major multidrug efflux pumps. E. coli cells transformed with the gene showed elevated levels of resistance to a number of structurally dissimilar drugs, such as tetracycline, norfloxacin, ciprofloxacin, doxorubicin, daunomycin, 4',6-diamidino-2-phenylindole, and Hoechst 33342. We determined the nucleotide sequence and found one open reading frame. We designated the gene vcaM. The deduced product, VcaM, seems to be a polypeptide with 619 amino acid residues (69 kDa) that has a putative topology of six transmembrane segments in the N-terminal hydrophobic domain, followed by an ATP binding domain in the C-terminal hydrophilic region. The sequence of VcaM was shown to be similar to those of human multidrug resistance proteins P-glycoprotein MDR1 and lactococcal LmrA, which are driven by ATP. The efflux of Hoechst 33342 and doxorubicin from cells possessing VcaM was detected. The efflux activity was inhibited by reserpine and sodium o-vanadate, which are potent inhibitors of MDR1 and LmrA. Thus, we conclude that VcaM is a member of the family of multidrug efflux pumps of the ATP binding cassette type and the first experimentally proven example of a multidrug efflux pump of this family in gram-negative bacteria.

ATP-Binding Cassette Transporters↗

PubMed2003

Functional cloning and expression of emeA, and characterization of EmeA, a multidrug efflux pump from Enterococcus faecalis.

A fragment of chromosomal DNA from Enterococcus faecalis ATCC 29212 was cloned using Escherichia coli KAM32 host cells lacking major multidrug efflux pumps. E. coli KAM32 cells were sensitive to many antimicrobial agents, and the transformed cells harboring a recombinant plasmid became resistant to several structurally unrelated antimicrobial agents such as tetraphenylphosphonium chloride, 4',6-diamidino-2-phenylindole (DAPI), Hoechst 33342, acriflavine, benzalkonium chloride, norfloxacin and ethidium bromide. This suggests that the cloned DNA fragment carries a gene(s) encoding a multidrug efflux pump. Determination of the nucleotide sequence of the cloned DNA revealed a gene designated as emeA. The transformed E. coli cells showed efflux activity of several antimicrobial agents such as DAPI, Hoechst 33342 and acriflavine. Efflux of DAPI via EmeA was strongly inhibited by reserpine.

Bacterial Proteins↗

PubMed2003

Multidrug resistance in Serratia marcescens and cloning of genes responsible for the resistance.

Six clinically isolated strains of Serratia marcescens were tested for their drug resistance. All showed fairly high resistance to many antimicrobial agents tested including norfloxacin, streptomycin, ampicillin, erythromycin, tetracycline, chloramphenicol, and antimicrobial dyes. Using the drug-hypersensitive strain of Escherichia coli KAM32 as the host, we cloned the genes responsible for multidrug resistance from chromosomal DNA of one of the strains of S. marcescens, NUSM8906. We obtained 28 hybrid plasmids that made host cells resistant to several antimicrobial agents. Many of the transformants harboring each of the plasmids showed multidrug resistance, and some showed resistance to specific drugs. The hybrid plasmids were classified into several groups based on their drug specificity. It appears that each class of plasmid carries different types of drug resistance genes. Analysis of such genes will reveal the multiple mechanisms involved in multidrug resistance in S. marcescens.

Anti-Bacterial Agents↗