Hyun-Woo Rhee
Ulsan National Institute of Science and Technology
Network
Latest external collaboration on country level. Dive into details by clicking on the dots.
Publication
Featured researches published by Hyun-Woo Rhee.
PLOS Pathogens | 2010
Wei Gao; Kyra Chua; John K. Davies; Hayley J. Newton; Torsten Seemann; Paul F. Harrison; Natasha E. Holmes; Hyun-Woo Rhee; Jong-In Hong; Elizabeth L. Hartland; Timothy P. Stinear; Benjamin P. Howden
Staphylococcus aureus frequently invades the human bloodstream, leading to life threatening bacteremia and often secondary foci of infection. Failure of antibiotic therapy to eradicate infection is frequently described; in some cases associated with altered S. aureus antimicrobial resistance or the small colony variant (SCV) phenotype. Newer antimicrobials, such as linezolid, remain the last available therapy for some patients with multi-resistant S. aureus infections. Using comparative and functional genomics we investigated the molecular determinants of resistance and SCV formation in sequential S. aureus isolates from a patient who had a persistent and recurrent S. aureus infection, after failed therapy with multiple antimicrobials, including linezolid. Two point mutations in key staphylococcal genes dramatically affected clinical behaviour of the bacterium, altering virulence and antimicrobial resistance. Most strikingly, a single nucleotide substitution in relA (SACOL1689) reduced RelA hydrolase activity and caused accumulation of the intracellular signalling molecule guanosine 3′, 5′-bis(diphosphate) (ppGpp) and permanent activation of the stringent response, which has not previously been reported in S. aureus. Using the clinical isolate and a defined mutant with an identical relA mutation, we demonstrate for the first time the impact of an active stringent response in S. aureus, which was associated with reduced growth, and attenuated virulence in the Galleria mellonella model. In addition, a mutation in rlmN (SACOL1230), encoding a ribosomal methyltransferase that methylates 23S rRNA at position A2503, caused a reduction in linezolid susceptibility. These results reinforce the exquisite adaptability of S. aureus and show how subtle molecular changes cause major alterations in bacterial behaviour, as well as highlighting potential weaknesses of current antibiotic treatment regimens.
Journal of the American Chemical Society | 2008
Hyun-Woo Rhee; Chang-Ro Lee; Seung-Hyon Cho; Miryung Song; Michael Cashel; Hyon E. Choy; Young-Jae Seok; Jong-In Hong
We have developed the first selective fluorescent chemosensor (PyDPA) for (p)ppGpp, a bacterial and plant alarmone. By using pyrene-excimer fluorescence, PyDPA shows very good selectivity for (p)ppGpp from among other nucleotides in water. PyDPA was used for the real-time detection of in vitro ppGpp synthesis by bacterial ribosomal complexes.
Journal of the American Chemical Society | 2016
Jung Seung Nam; Myeong-Gyun Kang; Juhye Kang; Sunyoung Park; Shin Jung C. Lee; Hyun-Tak Kim; Jeong Kon Seo; Oh Hoon Kwon; Mi Hee Lim; Hyun-Woo Rhee; Tae-Hyuk Kwon
Protein inactivation by reactive oxygen species (ROS) such as singlet oxygen ((1)O2) and superoxide radical (O2(•-)) is considered to trigger cell death pathways associated with protein dysfunction; however, the detailed mechanisms and direct involvement in photodynamic therapy (PDT) have not been revealed. Herein, we report Ir(III) complexes designed for ROS generation through a rational strategy to investigate protein modifications by ROS. The Ir(III) complexes are effective as PDT agents at low concentrations with low-energy irradiation (≤ 1 J cm(-2)) because of the relatively high (1)O2 quantum yield (> 0.78), even with two-photon activation. Furthermore, two types of protein modifications (protein oxidation and photo-cross-linking) involved in PDT were characterized by mass spectrometry. These modifications were generated primarily in the endoplasmic reticulum and mitochondria, producing a significant effect for cancer cell death. Consequently, we present a plausible biologically applicable PDT modality that utilizes rationally designed photoactivatable Ir(III) complexes.
Angewandte Chemie | 2010
Hyun-Woo Rhee; Seung Hwan Lee; Ik-Soo Shin; So Jung Choi; Hun Hee Park; Kyungja Han; Tai Hyun Park; Jong-In Hong
Protein phosphorylation is the most universal form of posttranslational modification of cell-signal transduction in living organisms. The human kinome comprises 518 protein kinases that control protein phosphorylation; irregular control of protein phosphorylation is a major cause of diseases such as cancer. Therefore, accurate probing of the kinase activity of a target protein is crucial for cancer diagnosis and high-throughput screening of anticancer drugs. 3] For the high-throughput analysis of kinase activity, several research groups have developed various types of protein or peptide chips using radioactive labeling with [Pg]-adenosine 5’-triphosphate (ATP) or using antibody hybridization. However, a crucial problem involved in the use of these onchip detection methods is that some kinases show decreased activities on the surfaces of chips because of reduced enzyme accessibility to the substrate. In recent years, several pharmaceutical and biotechnology companies have developed homogeneous kinase assay systems based on fluorescence polarization (FP) for developing anticancer drugs. These platforms (kinase assay systems) utilize peptide substrates with an N-terminal fluorophore and phospho-specific antibodies or phosphopeptide (or phosphoprotein)-binding nanoparticles (IMAP). However, FPbased detection has been reported to be very sensitive to fluorescence interference, and it is liable to produce false positives when used to screen a large number of compounds. Furthermore, there are no reports on the real-time monitoring of kinase activity in cell lysates through FP-based kinase detection; this is because many cellular components can bind to the fluorescent peptides and produce false positives for FP. Recently, peptideor protein-linked synthetic fluorescent probes that are sensitive to certain protein kinases have been reported by the research groups of Lawrence, Imperiali, Sames, and Hamachi. Ting, Tsien, and co-workers used fluorescent proteins to develop an in vivo probe system to detect kinase. These synthetic probes enabled real-time fluorescence monitoring of the specific activity of kinases in cellular lysates, and exhibited immense potential for use in the development of kinase activity inhibitors for certain kinases. However, it is still difficult to predict and determine the optimal sites for attaching fluorophores near the phosphorylated sites on the substrate peptides or proteins; the attachment of these fluorophores is necessary to induce significant changes in the fluorescence signal after phosphorylation of the substrate peptides or proteins by a specific kinase. Therefore, a general strategy for developing a synthetic fluorescent kinase probe is desired. We designed chemosensors Dab-DPA and PTZ-DPA (Scheme 1) to develop a simple but powerful kinase assay tool based on fluorescence intensity changes (ON/OFF). Using these chemosensors, we show for the first time the diagnosis of chronic myelogenous leukemia (CML) through real-time fluorescence monitoring of Abelson (Abl) tyrosine kinase activity and the development of a fluorescence-based homogeneous kinase assay system on a microfluidic chip. As shown in Scheme 1, Dab-DPA consists of a bis(Zndipicolylamine) complex and a dabcyl (Dab) fluorescence quencher, and PTZ-DPA consists of the dipicolylamine complex and a phenothiazine (PTZ) fluorescence quencher. Dab and PTZ quench fluorescence by F rster resonance energy transfer (FRET) and photoinduced electron transfer (PET), 11g] respectively. The bis(Zn-dipicolylamine) complex is a well-known synthetic receptor that strongly and selectively binds to phosphate in aqueous solution. DabDPA and PTZ-DPA are synthesized in a few steps (see the Supporting Information). PTZ is a good fluorescence quencher but there are very few reports on its use as such, except for the isoalloxazine ring of flavins. 11g] To prove that PTZ can be used as a general fluorescence quencher for other fluorophores, such as carboxyfluorescein (FAM) or tetramethyrhodamine (TMR), we performed electrochemical analyses of PTZ, FAM, and TMR. Figure 1a shows the cyclic voltammograms of 1 mm PTZ, TMR, and FAM; the Pt-disk working electrode is immersed in acetonitrile with 0.1m tetrabutylammonium hexafluorophosphate (TBAPF6) as supporting electrolyte. The observed waves are assigned to the oxidation of PTZ and the fluorophores (TMR and FAM). PTZ undergoes nearly Nernstian oxidation at E1/2,ox = 0.63 V with a peak separation [*] Dr. H.-W. Rhee, S. H. Lee, Dr. I.-S. Shin, S. J. Choi, Prof. Dr. T. H. Park, Prof. Dr. J.-I. Hong Department of Chemistry School of Chemical & Biological Engineering Seoul National University, Seoul 151-747 (Korea) Fax: (+ 82)2-889-1568 E-mail: thpark@snu.ac.kr jihong@snu.ac.kr
Cell Reports | 2016
Song-Yi Lee; Myeong-Gyun Kang; Jong-Seok Park; Geunsik Lee; Alice Y. Ting; Hyun-Woo Rhee
Deciphering the sub-compartmental location of a given protein of interest may help explain its physiological function, but it can be challenging to do using optical or biochemical methods. Imaging with electron microscopy (EM) can provide highly resolved mapping of proteins; however, EM requires complex sample preparation and a specialized facility. Here, we use engineered ascorbate peroxidase (APEX)-generated molecular labeling patterns to provide information regarding intracellular microenvironments in living cells. Using APEX labeling of specific proteins, we uncovered subcellular localization at sub-compartmental resolution and successfully elucidated the membrane protein topology of HMOX1 and sub-mitochondrial localization of recently identified mitochondrial proteins. This method can be expanded to confirm sub-mitochondrial localization and membrane topologies of previously identified mitochondrial proteins.
Chemistry: A European Journal | 2013
Sang Wook Lee; Hyun-Woo Rhee; Young-Tae Chang; Jong-In Hong
Hydrogen peroxide (H2O2), the simplest peroxide, is a precursor molecule of other reactive oxygen species (ROS), such as the hydroxyl radical (COH). In living cells, H2O2 is generated as a byproduct from various cellular aerobic oxidation reactions such as glucose oxidation by glucose oxidase (GOx). H2O2 widely causes oxidative damage on proteins and nucleic acids, and it is assumed that the accumulation of this damage is responsible for the aging process. H2O2 also has physiological functions as a cellular signaling molecule in many cases. Therefore, precise detection of H2O2 is crucial for understanding H2O2-related biochemical mechanisms. The most widely accepted optical method for measuring H2O2 is the horseradish peroxidase (HRP)-catalyzed oxidation of fluorogenic substrates. This method is considered sensitive and precise, but rather expensive because it requires purified enzymes. Recently, Chang and his co-workers developed boronate-based fluorescent probes that enabled the direct detection of H2O2. [6] Usually, these sensors are designed to quench fluorescence due to the presence of an electron-deficient boronic acid ester group. When this boronate group reacts with H2O2 to yield a hydroxyl moiety, the fluorescence of the probe is drastically increased by regeneration of a “push–pull” pair in the fluorophore. However, quantitative assay of H2O2 is still challenging because of this “night and day” property of the sensors in many applications. For practical needs, the simultaneous recording of two measurable fluorescence intensities that are of seesaw-type ratiometric change would be a better alternative for the quantitative detection of H2O2. Boronate-based ratiometric sensors for H2O2, which utilize the Fçrster resonance energy-transfer (FRET)-based mechanism, and an intramolecular charge-transfer (ICT)based strategy have been recently reported. However, the emission ratio change based on FRET is limited (less than 8-fold). An ICT-based sensor using H2O2-mediated deprotection of carbobenzyloxy (Cbz) boronate exhibits 75-fold emission ratio changes. However, the probe s fluorescence change rate is much slower (t1/2>45 min) than a directly conjugated aryl boronate sensor without the Cbz moiety (t1/2<30 min), [9] because the fluorescence change should occur upon the H2O2-mediated oxidation of boronate followed by the benzyl deprotection reaction. Therefore, we wanted to develop directly conjugated aryl boronate-based probes (Figure 1) that would enable dramatic ratiometric sensing of H2O2 in a short time. Although the electronic state change of boronate-based H2O2 probes can be rationally designed from the electron push–pull strategy, it is hard to predict whether the resulting H2O2 probes would generate fluorescence upon reaction with H2O2. Therefore, we aimed to find ratiometric fluorescent probes for H2O2 through a focused, combinatorial approach. For the best chance of finding a hit molecule to sense H2O2, we used formylphenyl boronate ester (1–3) as one combinatorial block for the styryl dye synthesis. For the structural diversity of fluorophores, we used five different N-methylpicolinium and N-methylquinolinium blocks (A–E) (see Figure S1 in the Supporting Information). The Knoevenagel condensation reaction between 3-formylphenyl boronate esters and 5-N-methylpicolinium and N-methylquinolinium iodides provided 15 fluorescent styryl probes. As shown in Figure 1a, each condensation reaction produced a fully conjugated fluorophore. Each product was purified and separated through flash silica gel column chromatography and preparative HPLC (prep-HPLC). It turned out that 12 of 15 dyes have a quantum yield considerably higher than 0.010. Each probe s observed mass, absorption wavelength, emission wavelength, and quantum yield are reported in Table S1 in the Supporting Information. To find ratiometric fluorescent H2O2 probes, we investigated the spectral fluorescence changes of styryl dyes (each 50 mm) upon addition of H2O2. As expected, most of these dyes showed a distinct fluorescence change with H2O2, as shown in Figure 1. Among them, 1 A, 1 B, 1 C, and 3 D underwent redshifts within 30 min of incubation with H2O2. Probes 1 A and 1 C, especially, showed a redshift of over [a] S. W. Lee, Prof. J.-I. Hong Department of Chemistry, Seoul National University Seoul 151-747 (Korea) Fax: (+82) 2-889-1568 E-mail : jihong@snu.ac.kr [b] Prof. H.-W. Rhee School of Nano-Bioscience and Chemical Engineering Ulsan National Institute of Science and Technology (UNIST) Ulsan 689-798 (Korea) [c] Prof. Y.-T. Chang Department of Chemistry National University of Singapore, 3 Science Drive Singapore, 117543 Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201302523.
Analytical Chemistry | 2014
Ik-Soo Shin; Rohit Chand; Sang Wook Lee; Hyun-Woo Rhee; Yong-Sang Kim; Jong-In Hong
Herein, we report a homogeneous assay for protein kinase activity using an electrochemistry-based probe. The approach involves a peptide substrate conjugated with a redox tag and the phosphate-specific receptor immobilized on an electrode surface. The peptide substrate phosphorylated by a protein kinase binds to the receptor site of the probe, which results in a redox current under voltammetric measurement. Our method was successfully applied even in the presence of citrated human blood and modified to enable a single-use, chip-based electrochemical assay for kinase activity.
Biosensors and Bioelectronics | 2014
Seung Hwan Lee; Hyun-Woo Rhee; Danny van Noort; Hong Jai Lee; Hee Ho Park; Ik-Soo Shin; Jong-In Hong; Tai Hyun Park
Protein kinases control cellular functions by regulating protein phosphorylation. Monitoring protein kinase activity is essential for medical diagnosis and drug screening. Here, we present a novel microfluidic device for performing simple and versatile protein kinase assays, which utilizes a microbead-based chemosensor. An automatic mix-and-measure technique was achieved using integrated pneumatic valves. After mixing each reagent for the kinase assay, the mixture was transferred to the sensing chamber. Then, phosphorylated and fluorescence-labeled peptides were captured and detected by the chemosensor. A fluorescence signal was observed depending on the presence of the kinase. Furthermore, activities of various kinases in the cell lysate and the inhibitory effect of specific chemicals on the kinases were monitored. These results indicate that chemosensor-based microfluidic chips can be developed as a versatile kinase assay system.
Bioorganic & Medicinal Chemistry Letters | 2013
Sung-Jean Kim; Hyun-Woo Rhee; Hyun-Joo Park; Hye-Yeon Kim; Hyo-Soo Kim; Jong-In Hong
We developed activity-based fluorescent probes for detecting human serum albumin (HSA) on the basis of its pseudo-esterase activity. These probes could also detect HSA in blood-contaminated tissue samples.
Journal of the American Chemical Society | 2017
Song-Yi Lee; Myeong-Gyun Kang; Sanghee Shin; Chulhwan Kwak; Taejoon Kwon; Jeong Kon Seo; Jong-Seo Kim; Hyun-Woo Rhee
The inner mitochondrial membrane (IMM) proteome plays a central role in maintaining mitochondrial physiology and cellular metabolism. Various important biochemical reactions such as oxidative phosphorylation, metabolite production, and mitochondrial biogenesis are conducted by the IMM proteome, and mitochondria-targeted therapeutics have been developed for IMM proteins, which is deeply related for various human metabolic diseases including cancer and neurodegenerative diseases. However, the membrane topology of the IMM proteome remains largely unclear because of the lack of methods to evaluate it in live cells in a high-throughput manner. In this article, we reveal the in vivo topological direction of 135 IMM proteins, using an in situ-generated radical probe with genetically targeted peroxidase (APEX). Owing to the short lifetime of phenoxyl radicals generated in situ by submitochondrial targeted APEX and the impermeability of the IMM to small molecules, the solvent-exposed tyrosine residues of both the matrix and intermembrane space (IMS) sides of IMM proteins were exclusively labeled with the radical probe in live cells by Matrix-APEX and IMS-APEX, respectively and identified by mass spectrometry. From this analysis, we confirmed 58 IMM protein topologies and we could determine the topological direction of 77 IMM proteins whose topology at the IMM has not been fully characterized. We also found several IMM proteins (e.g., LETM1 and OXA1) whose topological information should be revised on the basis of our results. Overall, our identification of structural information on the mitochondrial inner-membrane proteome can provide valuable insights for the architecture and connectome of the IMM proteome in live cells.