Publications by authors named "Kyeong Kyu Kim"

161 Publications

An Antibacterial Nanorobotic Approach for the Specific Targeting and Removal of Multiple Drug-Resistant Staphylococcus aureus.

Small 2021 Apr 10:e2100257. Epub 2021 Apr 10.

Department of Precision Medicine, Institute for Antimicrobial Resistance Research and Therapeutics, Graduate School of Basic Medical Sciences (GSBMS), Sungkyunkwan University School of Medicine, Suwon, 16419, South Korea.

Methicillin-resistant Staphylococcus aureus (MRSA) causes diseases ranging from skin infections to lethal sepsis and has become a serious threat to human health due to multiple-drug resistance (MDR). Therefore, a resistance-free antibacterial therapy is necessary to overcome MDR MRSA infections. In this study, an antibacterial nanorobot (Ab-nanobot) is developed wherein a cell wall-binding domain (CBD)-endolysin, acting as a sensor, is covalently conjugated with an actuator consisting of an iron oxide/silica core-shell. The CBD-endolysin sensor shows an excellent specificity to detect, bind, and accumulate on the S. aureus USA300 cell surface even in a bacterial consortium, and in host cell infections. Ab-nanobot specifically captures and kills MRSA in response to medically approved radiofrequency (RF) electromagnetic stimulation (EMS) signal. When Ab-nanobot receives the RF-EMS signal on the cell surface, actuator induces cell death in MRSA with 99.999% removal within 20 min by cell-wall damage via generation of localized heat and reactive oxygen species. The in vivo efficacy of Ab-nanobot is proven using a mice subcutaneous skin infection model. Collectively, this study offers a nanomedical resistance-free strategy to overcome MDR MRSA infections by providing a highly specific nanorobot for S. aureus.
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http://dx.doi.org/10.1002/smll.202100257DOI Listing
April 2021

Genome-Wide Analysis of Sequence Type 72 Isolates Provides Insights Into Resistance Against Antimicrobial Agents and Virulence Potential.

Front Microbiol 2020 20;11:613800. Epub 2021 Jan 20.

Department of Precision Medicine, Sungkyunkwan University School of Medicine, Suwon, South Korea.

sequence type 72 (ST72) is a major community-associated (CA) methicillin-resistant (MRSA) that has rapidly entered the hospital setting in Korea, causing mild superficial skin wounds to severe bloodstream infections. In this study, we sequenced and analyzed the genomes of one methicillin-resistant human isolate and one methicillin-sensitive human isolate of ST72 from Korea, K07-204 and K07-561, respectively. We used a subtractive genomics approach to compare these two isolates to other 27 ST72 isolates to investigate antimicrobial resistance (AMR) and virulence potential. Furthermore, we validated genotypic differences by phenotypic characteristics analysis. Comparative and subtractive genomics analysis revealed that K07-204 contains methicillin (), ampicillin (), erythromycin (), aminoglycoside (), and tetracycline (, tetracycline efflux pump) resistance genes while K07-561 has ampicillin () and tetracycline () resistance genes. In addition to antibiotics, K07-204 was reported to show resistance to lysostaphin treatment. K07-204 also has additional virulence genes (, , , , , , and ) compared to K07-561, which may explain the differential virulence potential of these human isolates of ST72. Unexpectedly, the virulence potential of K07-561 was higher in an wax-worm infection model than that of K07-204, putatively due to the presence of a 20-fold higher staphyloxanthin concentration than K07-204. Comprehensive genomic analysis of these two human isolates, with 27 ST72 isolates, and USA300 (ST8) suggested that acquisition of both virulence and antibiotics resistance genes by ST72 isolates might have facilitated their adaptation from a community to a hospital setting where the selective pressure imposed by antibiotics selects for more resistant and virulent isolates. Taken together, the results of the current study provide insight into the genotypic and phenotypic features of various ST72 clones across the globe, delivering more options for developing therapeutics and rapid molecular diagnostic tools to detect resistant bacteria.
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http://dx.doi.org/10.3389/fmicb.2020.613800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854921PMC
January 2021

MD-TSPC4: Computational Method for Predicting the Thermal Stability of I-Motif.

Int J Mol Sci 2020 Dec 23;22(1). Epub 2020 Dec 23.

Department of Precision Medicine, Institute for Antimicrobial Resistance Research and Therapeutics, Sungkyunkwan University School of Medicine, Suwon 16419, Korea.

I-Motif is a tetrameric cytosine-rich DNA structure with hemi-protonated cytosine: cytosine base pairs. Recent evidence showed that i-motif structures in human cells play regulatory roles in the genome. Therefore, characterization of novel i-motifs and investigation of their functional implication are urgently needed for comprehensive understanding of their roles in gene regulation. However, considering the complications of experimental investigation of i-motifs and the large number of putative i-motifs in the genome, development of an in silico tool for the characterization of i-motifs in the high throughput scale is necessary. We developed a novel computation method, MD-TSPC4, to predict the thermal stability of i-motifs based on molecular modeling and molecular dynamic simulation. By assuming that the flexibility of loops in i-motifs correlated with thermal stability within certain temperature ranges, we evaluated the correlation between the root mean square deviations (RMSDs) of model structures and the thermal stability as the experimentally obtained melting temperature (Tm). Based on this correlation, we propose an equation for Tm prediction from RMSD. We expect this method can be useful for estimating the overall structure and stability of putative i-motifs in the genome, which can be a starting point of further structural and functional studies of i-motifs.
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http://dx.doi.org/10.3390/ijms22010061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7793491PMC
December 2020

Pontin arginine methylation by CARM1 is crucial for epigenetic regulation of autophagy.

Nat Commun 2020 12 8;11(1):6297. Epub 2020 Dec 8.

Creative Research Initiatives Center for Epigenetic Code and Diseases, Department of Biological Sciences, Seoul National University, Seoul, 08826, South Korea.

Autophagy is a catabolic process through which cytoplasmic components are degraded and recycled in response to various stresses including starvation. Recently, transcriptional and epigenetic regulations of autophagy have emerged as essential mechanisms for maintaining homeostasis. Here, we identify that coactivator-associated arginine methyltransferase 1 (CARM1) methylates Pontin chromatin-remodeling factor under glucose starvation, and methylated Pontin binds Forkhead Box O 3a (FOXO3a). Genome-wide analyses and biochemical studies reveal that methylated Pontin functions as a platform for recruiting Tip60 histone acetyltransferase with increased H4 acetylation and subsequent activation of autophagy genes regulated by FOXO3a. Surprisingly, CARM1-Pontin-FOXO3a signaling axis can work in the distal regions and activate autophagy genes through enhancer activation. Together, our findings provide a signaling axis of CARM1-Pontin-FOXO3a and further expand the role of CARM1 in nuclear regulation of autophagy.
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http://dx.doi.org/10.1038/s41467-020-20080-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7722926PMC
December 2020

Draft Genome Sequences of Lysostaphin-Resistant (K07-204) and Lysostaphin-Susceptible (K07-561) Staphylococcus aureus Sequence Type 72 Strains Isolated from Patients in South Korea.

Microbiol Resour Announc 2020 Dec 3;9(49). Epub 2020 Dec 3.

Department of Precision Medicine, School of Medicine, Sungkyunkwan University, Suwon, South Korea

Methicillin-resistant sequence type 72 (ST72) is prevalent in South Korea and has shown resistance to multiple antimicrobials. ST72 isolates display different levels of resistance to the antistaphylococcal lysostaphin. Draft genome sequencing of ST72 human isolates exhibiting lysostaphin resistance or susceptibility was performed to better understand the mechanism of lysostaphin resistance using subtractive genomics.
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http://dx.doi.org/10.1128/MRA.01057-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7714846PMC
December 2020

Functional Identification of Serine Hydroxymethyltransferase as a Key Gene Involved in Lysostaphin Resistance and Virulence Potential of Strains.

Int J Mol Sci 2020 Nov 30;21(23). Epub 2020 Nov 30.

Department of Precision Medicine, Institute for Antimicrobial Resistance Research and Therapeutics, Sungkyunkwan University School of Medicine, Suwon 16419, Korea.

Gaining an insight into the mechanism underlying antimicrobial-resistance development in is crucial for identifying effective antimicrobials. We isolated sequence type 72 from a patient in whom the infection was highly resistant to various antibiotics and lysostaphin, but no known resistance mechanisms could explain the mechanism of lysostaphin resistance. Genome-sequencing followed by subtractive and functional genomics revealed that serine hydroxymethyltransferase ( or gene) plays a key role in lysostaphin resistance. Serine hydroxymethyltransferase (SHMT) is indispensable for the one-carbon metabolism of serine/glycine interconversion and is linked to folate metabolism. Functional studies revealed the involvement of SHMT in lysostaphin resistance, as Δ was susceptible to the lysostaphin, while complementation of the knockout expressing restored resistance against lysostaphin. In addition, the Δ showed reduced virulence under (mammalian cell lines infection) and (wax-worm infection) models. The SHMT inhibitor, serine hydroxymethyltransferase inhibitor 1 (SHIN1), protected the 50% of the wax-worm infected with wild type . These results suggest SHMT is relevant to the extreme susceptibility to lysostaphin and the host immune system. Thus, the current study established that SHMT plays a key role in lysostaphin resistance development and in determining the virulence potential of multiple drug-resistant .
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http://dx.doi.org/10.3390/ijms21239135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7731198PMC
November 2020

Sphingosylphosphorylcholine blocks ovariectomy-induced bone loss by suppressing Ca /calmodulin-mediated osteoclast differentiation.

J Cell Mol Med 2021 Jan 23;25(1):473-483. Epub 2020 Nov 23.

Department of Biological Sciences, Sungkyunkwan University, Suwon, Korea.

Osteoporosis is a disease in which bone mineral density decreases due to abnormal activity of osteoclasts, and is commonly found in post-menopausal women who have decreased levels of female hormones. Sphingosylphosphorylcholine (SPC) is an important biological lipid that can be converted to sphingosine-1-phosphate (S1P) by autotaxin. S1P is known to be involved in osteoclast activation by stimulating osteoblasts, but bone regulation by SPC is not well understood. In this study, we found that SPC strongly inhibits RANKL-induced osteoclast differentiation. SPC-induced inhibitory effects on osteoclast differentiation were not affected by several antagonists of S1P receptors or pertussis toxin, suggesting cell surface receptor independency. However, SPC inhibited RANKL-induced calcineurin activation and subsequent NFATc1 activity, leading to decrease of the expression of Trap and Ctsk. Moreover, we found that bone loss in an experimental osteoporosis mouse model was recovered by SPC injection. SPC also blocked ovariectomy-induced body weight increase and Nfatc1 gene expression in mice. We also found that SPC inhibits RANKL-induced osteoclast differentiation in human macrophages. Since currently available treatments for osteoporosis, such as administration of female hormones or hormone receptor modulators, show serious side effects, SPC has potential as a new agent for osteoporosis treatment.
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http://dx.doi.org/10.1111/jcmm.16101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7810965PMC
January 2021

Proline Hinged Amphipathic α-Helical Peptide Sensitizes Gram-Negative Bacteria to Various Gram-Positive Antibiotics.

J Med Chem 2020 12 18;63(23):14937-14950. Epub 2020 Nov 18.

Department of Chemistry & Education, Seoul National University, Seoul 08826, Korea.

Gram-negative bacteria are becoming resistant to almost all currently available antibiotics. Systemically designed antimicrobial peptides (AMPs) are attractive agents to enhance the activities of antibiotics. We constructed a small Pro-scanning library using amphipathic model peptides. Measurements of minimum inhibitory concentration (MIC) against and hemolytic activities showed that one of the Pro-hinged peptides, KL-L9P, displays the highest specificity toward . Moreover, KL-L9P sensitizes to be responsive to most antibiotics that are not active against Gram-negative bacteria. The results of biochemical experiments show that KL-L9P promotes the rearrangement of the bacterial membrane that enables hydrophobic antibiotics to permeate. Finally, the results of animal tests demonstrate that KL-L9P strongly sensitizes Gram-negative bacteria to linezolid (Lzd), rifampicin (Rif), or clarithromycin (Clr). Thus, KL-L9P operates as a sensitizer to extend the antibacterial activity of most antibiotics to Gram-negative bacteria.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01506DOI Listing
December 2020

Exosome-Mediated Differentiation of Mouse Embryonic Fibroblasts and Exocrine Cells into β-Like Cells and the Identification of Key miRNAs for Differentiation.

Biomedicines 2020 Nov 9;8(11). Epub 2020 Nov 9.

Department of Precision Medicine, Sungkyunkwan University School of Medicine, Suwon 16419, Korea.

Diabetes is a concerning health malady worldwide. Islet or pancreas transplantation is the only long-term treatment available; however, the scarcity of transplantable tissues hampers this approach. Therefore, new cell sources and differentiation approaches are required. Apart from the genetic- and small molecule-based approaches, exosomes could induce cellular differentiation by means of their cargo, including miRNA. We developed a chemical-based protocol to differentiate mouse embryonic fibroblasts (MEFs) into β-like cells and employed mouse insulinoma (MIN6)-derived exosomes in the presence or absence of specific small molecules to encourage their differentiation into β-like cells. The differentiated β-like cells were functional and expressed pancreatic genes such as Pdx1, Nkx6.1, and insulin 1 and 2. We found that the exosome plus small molecule combination differentiated the MEFs most efficiently. Using miRNA-sequencing, we identified miR-127 and miR-709, and found that individually and in combination, the miRNAs differentiated MEFs into β-like cells similar to the exosome treatment. We also confirmed that exocrine cells can be differentiated into β-like cells by exosomes and the exosome-identified miRNAs. A new differentiation approach based on the use of exosome-identified miRNAs could help people afflicted with diabetes.
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http://dx.doi.org/10.3390/biomedicines8110485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7695333PMC
November 2020

Targeted epigenetic modulation using a DNA-based histone deacetylase inhibitor enhances cardiomyogenesis in mouse embryonic stem cells.

J Cell Physiol 2021 May 8;236(5):3946-3962. Epub 2020 Nov 8.

Department of Precision Medicine, Sungkyunkwan University School of Medicine, Suwon, Republic of Korea.

The epigenome has an essential role in orchestrating transcriptional activation and modulating key developmental processes. Previously, we developed a library of pyrrole-imidazole polyamides (PIPs) conjugated with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor, for the purpose of sequence-specific modification of epigenetics. Based on the gene expression profile of SAHA-PIPs and screening studies using the α-myosin heavy chain promoter-driven reporter and SAHA-PIP library, we identified that SAHA-PIP G activates cardiac-related genes. Studies in mouse ES cells showed that SAHA-PIP G could enhance the generation of spontaneous beating cells, which is consistent with upregulation of several cardiac-related genes. Moreover, ChIP-seq results confirmed that the upregulation of cardiac-related genes is highly correlated with epigenetic activation, relevant to the sequence-specific binding of SAHA-PIP G. This proof-of-concept study demonstrating the applicability of SAHA-PIP not only improves our understanding of epigenetic alterations involved in cardiomyogenesis but also provides a novel chemical-based strategy for stem cell differentiation.
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http://dx.doi.org/10.1002/jcp.30140DOI Listing
May 2021

Identification, characterization, and immobilization of a novel YbfF esterase from Halomonas elongata.

Int J Biol Macromol 2020 Dec 6;165(Pt A):1139-1148. Epub 2020 Oct 6.

Department of Chemistry, Graduate School of Natural Science, Sookmyung Women's University, Seoul 04310, Republic of Korea. Electronic address:

The YbfF esterase family, which has a bifurcated binding pocket for diverse ligands, could serve as excellent biocatalysts in industrial and biotechnological applications. Here, the identification, characterization, and immobilization of a novel YbfF esterase (YbfF) from Halomonas elongata DSM 2581 is reported. Biochemical characterization of YbfF was carried out using activity staining, chromatographic analysis, kinetic analysis, activity assay, acetic acid release, and pH-indicator-based hydrolysis. YbfF displayed broad substrate specificity, including that for p-nitrophenyl esters, glucose pentaacetate, tert-butyl acetate, and β-lactam-containing compounds, with high efficiency. Based on a homology model of YbfF, Trp in the substrate-binding pocket, a critical residue for catalytic activity and substrate specificity was identified and characterized. Furthermore, crosslinked enzyme aggregates and nanoflower formation were explored to enhance the chemical stability and recyclability of YbfF. The present study is the first report of a YbfF esterase from extremophiles, and explains its protein stability, catalytic activity, substrate specificities and diversities, kinetics, functional residues, amyloid formation, and immobilization.
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http://dx.doi.org/10.1016/j.ijbiomac.2020.09.247DOI Listing
December 2020

Improved differentiation of human adipose stem cells to insulin-producing β-like cells using PDFGR kinase inhibitor Tyrphostin9.

Biochem Biophys Res Commun 2020 11 12;533(1):132-138. Epub 2020 Sep 12.

Department of Precision Medicine, Sungkyunkwan University School of Medicine, Suwon, 16419, Republic of Korea. Electronic address:

Diabetes mellitus (DM) is a metabolic syndrome where insulin secretion or the response to insulin produced by the body is compromised. The only available long-term treatment is the transplantation of pancreas or islet for procuring β-cells. However, due to the shortage of β-cell sources from the tissues, differentiation of pluripotent stem cells or terminally differentiated cells into β-cell is proposed as an alternative strategy. Previously, human adipose-derived stem cells (ADSCs) were reported to be converted into β-like cells by a stepwise treatment of chemicals and growth factors. However, due to the low conversion efficiency, the clinical application was not feasible. In this study, we developed a modified conversion protocol with improved yield and functionality, which is achieved by changing the culture method and addition of Tyrphostin9, a platelet-derived growth factor receptor (PDGFR) kinase inhibitor. Tyrphostin9 was identified from a cell-based chemical screening using the mCherry reporter under the control of the Pdx1 promoter. The β-like cells differentiated under the new protocol showed a 3.6-fold increase in the expression of Pdx1, a marker for pancreatic differentiation, as compared to the previous protocol. We propose that Tyrphostin9 contributes to the β-like cell differentiation by playing a dual role; enhancing the definitive endoderm generation by inhibiting the PI3K signaling and suppressing the taurine-mediated proliferation of definitive endoderm. Importantly, these differentiated cells responded well to low and high glucose stimulations compared to cells differentiated by the previous protocol, as confirmed by the 2.0-fold increase in the C-peptide release. As ADSCs are abundant, easily isolated, and autologous in nature, improved differentiation approaches to generate β-like cells from ADSCs would provide a better opportunity for treating diabetes.
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http://dx.doi.org/10.1016/j.bbrc.2020.08.090DOI Listing
November 2020

New screening system using Twist1 promoter activity identifies dihydrorotenone as a potent drug targeting cancer-associated fibroblasts.

Sci Rep 2020 04 27;10(1):7058. Epub 2020 Apr 27.

Department of Health Science and Technology, Samsung Advanced Institute for Health Science and Technology, Sungkyunkwan University, Seoul, Republic of Korea.

Cancer-associated fibroblasts (CAFs) are the most abundant stromal cells in tumor microenvironments. These cells strongly support tumor progression and are considered to be potent therapeutic targets. Therefore, drugs targeting CAFs have been developed, but most of them have failed in clinical trials. The discovery of additional drugs to inactivate or eliminate CAFs is thus essential. In this study, we developed a high-throughput screening system to find anti-CAF drugs using reporter cells that express Twist1 promoter-GFP. This screening system uses the activity of the Twist1 promoter as an indicator of CAF activation because Twist1 is known to be a central player in CAF activation. Using this screening system, we found that dihydrorotenone (DHR), an inhibitor of electron transfer chain complex 1 in mitochondria, can effectively deactivate CAFs. DHR-treated CAFs exhibited reduced expression of CAF-enriched markers, decreased capability of collagen gel contraction, and impaired ability to engage in tumor-promoting activities, such as facilitating the proliferation and colonization of cancer cells. Furthermore, conditioned media from DHR-treated CAFs attenuated tumor progression in mice grafted with MNK28 cells. In conclusion, DHR can be considered as a candidate drug targeting CAFs.
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http://dx.doi.org/10.1038/s41598-020-63996-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7184745PMC
April 2020

Biodiesel and flavor compound production using a novel promiscuous cold-adapted SGNH-type lipase (SGNH1) from the psychrophilic bacterium .

Biotechnol Biofuels 2020 16;13:55. Epub 2020 Mar 16.

1Department of Chemistry, College of Natural Science, Sookmyung Women's University, Seoul, 04310 South Korea.

Background: Biodiesel and flavor compound production using enzymatic transesterification by microbial lipases provides mild reaction conditions and low energy cost compared to the chemical process. SGNH-type lipases are very effective catalysts for enzymatic transesterification due to their high reaction rate, great stability, relatively small size for convenient genetic manipulations, and ease of immobilization. Hence, it is highly important to identify novel SGNH-type lipases with high catalytic efficiencies and good stabilities.

Results: A promiscuous cold-adapted SGNH-type lipase (SGNH1) from was catalytically characterized and functionally explored. SGNH1 displayed broad substrate specificity that included -butyl acetate, glucose pentaacetate, and -nitrophenyl esters with excellent stability and high efficiency. Important amino acids (N83, M86, R87, F131, and I173F) around the substrate-binding pocket were shown to be responsible for catalytic activity, substrate specificity, and reaction kinetics. Moreover, immobilized SGNH1 was used to produce high yields of butyl and oleic esters.

Conclusions: This work provides a molecular understanding of substrate specificities, catalytic regulation, immobilization, and industrial applications of a promiscuous cold-adapted SGNH-type lipase (SGNH1) from . This is the first analysis on biodiesel and flavor synthesis using a cold-adapted halophilic SGNH-type lipase from a species.
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http://dx.doi.org/10.1186/s13068-020-01696-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7074997PMC
March 2020

Ubiquitin-Specific Protease 21 Promotes Colorectal Cancer Metastasis by Acting as a Fra-1 Deubiquitinase.

Cancers (Basel) 2020 Jan 14;12(1). Epub 2020 Jan 14.

Department of Precision Medicine, Sungkyunkwan University School of Medicine, Suwon 16419, Korea.

Fos-related-antigen-1 (Fra-1), a member of the activator protein-1 (AP-1) transcription factor superfamily, has an essential role in cancer progress and metastasis and Fra-1 is considered a therapeutic target in metastatic cancer including metastatic colorectal cancer (mCRC). However, its regulation at protein level has not yet been clearly elucidated. We found that ubiquitin-specific protease 21 (USP21) increases Fra-1 stability by deubiquitinating Fra-1 and enhances the expression of Fra-1 target genes in colon cancer cells. We also showed that USP21 controlled Fra-1-dependent migration and invasion activities. The oncogenic property of USP21 was confirmed by a significant reduction in liver metastasis when USP21-knockdown cancer cells were injected intrasplenically into mice. Consistently, clinicopathological analysis of colorectal cancer patients revealed a correlation of USP21 expression with high-grade carcinoma and life span. These results demonstrate that USP21 enhances Fra-1 stability and AP-1 target gene expression by deubiquitinating Fra-1. Therefore, USP21 is considered an attractive therapeutic target in mCRC with high Fra-1 expression.
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http://dx.doi.org/10.3390/cancers12010207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7017141PMC
January 2020

Characterization and Immobilization of a Novel SGNH Family Esterase (SGNH1) from NCFM.

Int J Mol Sci 2019 Dec 21;21(1). Epub 2019 Dec 21.

Department of Chemistry, College of Natural Science, Sookmyung Women's University, Seoul 04310, Korea.

The SGNH family esterases are highly effective biocatalysts due to their strong catalytic efficiencies, great stabilities, relatively small sizes, and ease of immobilization. Here, a novel SGNH family esterase (SGNH1) from NCFM, which has homologues in many species, was identified, characterized, and immobilized. SGNH1 is highly active towards acetate- or butyrate-containing compounds, such as -nitrophenyl acetate or 1-naphthyl acetate. Enzymatic properties of SGNH1, including thermal stability, optimum pH, chemical stability, and urea stability, were investigated. Interestingly, SGNH1 displayed a wide range of substrate specificity that included glyceryl tributyrate, -butyl acetate, and glucose pentaacetate. Furthermore, immobilization of SGNH1 by crosslinked enzyme aggregates (CLEAs) showed enhanced thermal stability and efficient recycling property. In summary, this work paves the way for molecular understandings and industrial applications of a novel SGNH family esterase (SGNH1) from .
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http://dx.doi.org/10.3390/ijms21010091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6981805PMC
December 2019

Molecular Characterization of a Novel Family VIII Esterase with β-Lactamase Activity (EstA) from sp.

Biomolecules 2019 11 26;9(12). Epub 2019 Nov 26.

Department of Chemistry, College of Natural Science, Sookmyung Women's University, Seoul 04310, Korea.

Molecular information about family VIII esterases, which have similarities with class C β-lactamases and penicillin-binding proteins, remains largely unknown. In this study, a novel family VIII esterase with β-lactamase activity (EstA) from sp. was characterized using several biochemical and biophysical methods. EstA was effective on a broad range of substrates including tertiary butyl acetate, glyceryl tributyrate, glucose pentaacetate, olive oil, and -nitrophenyl esters. Additionally, EstA hydrolyzed nitrocefin, cefotaxime, and 7-aminocephalosporanic acid. Interestingly, two forms of immobilized EstA (CLEAs-EstA and mCLEAs-EstA) showed high recycling property and enhanced stability, but hybrid nanoflowers (hNFs) of EstA require improvement. This study provides a molecular understanding of substrate specificities, catalytic regulation, and immobilization of EstA, which can be efficiently used in biotechnological applications.
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http://dx.doi.org/10.3390/biom9120786DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6995599PMC
November 2019

Molecular Characterization of a Novel Cold-Active Hormone-Sensitive Lipase (HSL) from .

Biomolecules 2019 11 5;9(11). Epub 2019 Nov 5.

Department of Chemistry, College of Natural Science, Sookmyung Women's University, Seoul 04310, Korea.

Bacterial hormone-sensitive lipases (bHSLs), which are homologous to the catalytic domains of human HSLs, have received great interest due to their uses in the preparation of highly valuable biochemicals, such as drug intermediates or chiral building blocks. Here, a novel cold-active HSL from (HSL) was examined and its enzymatic properties were investigated using several biochemical and biophysical methods. Interestingly, HSL acted on a large variety of substrates including tertiary alcohol esters and fish oils. Additionally, this enzyme was highly tolerant to high concentrations of salt, detergents, and glycerol. Furthermore, immobilized HSL retained its activity for up to six cycles of use. Homology modeling suggested that aromatic amino acids (Trp, Tyr, Phe, Trp, and Phe) in close proximity to the substrate-binding pocket were important for enzyme activity. Mutational analysis revealed that Tyr played an important role in substrate specificity, thermostability, and enantioselectivity. In summary, the current study provides an invaluable insight into the novel cold-active HSL from , which can be efficiently and sustainably used in a wide range of biotechnological applications.
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http://dx.doi.org/10.3390/biom9110704DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6921082PMC
November 2019

Unraveling the Regulatory G-Quadruplex Puzzle: Lessons From Genome and Transcriptome-Wide Studies.

Front Genet 2019 18;10:1002. Epub 2019 Oct 18.

Department of Molecular Cell Biology, Institute for Antimicrobial Resistance Research and Therapeutics, Sungkyunkwan University School of Medicine, Suwon, South Korea.

G-quadruplexes (G4s) are among the best-characterized DNA secondary structures and are enriched in regulatory regions, especially promoters, of several prokaryote and eukaryote genomes, indicating a possible role in regulation of genes. Many studies have focused on evaluating the impact of specific G4-forming sequences in the promoter regions of genes. However, the lack of correlation between the presence of G4s and the functional impact on gene regulation, evidenced by the variable expression fold change in the presence of G4 stabilizers, shows that not all G4s affect transcription in the same manner. This indicates that the regulatory effect of the G4 is significantly influenced by its position, the surrounding DNA topology, and other environmental factors within the cell. In this review, we compare individual gene studies with high-throughput differential expression studies to highlight the importance of formulating a combined approach that can be applied in humans, bacteria, and viruses to better understand the effect of G4-mediated gene regulation.
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http://dx.doi.org/10.3389/fgene.2019.01002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6813735PMC
October 2019

Pneumococcal VncR Strain-Specifically Regulates Capsule Polysaccharide Synthesis.

Front Microbiol 2019 1;10:2279. Epub 2019 Oct 1.

School of Pharmacy, Sungkyunkwan University, Suwon, South Korea.

Capsular polysaccharides (CPS), a major virulence factor in , become thicker during blood invasion while not during asymptomatic nasopharyngeal colonization. However, the underlying mechanism controlling this differential pneumococcal CPS regulation remain unclear. Here, we show how VncR, the response regulator of the vancomycin resistance locus ( operon), regulates CPS expression in mutants in three serotype (type 2, 3, and 6B) backgrounds upon exposure to serum lactoferrin (LF). Comparative analysis of CPS levels in the wild type (WT) of three strains and their isogenic mutants after LF exposure revealed a strain-specific alteration in CPS production. Consistently, VncR-mediated strain-specific CPS production is correlated with pneumococcal virulence, . Electrophoretic mobility-shift assay and co-immunoprecipitation revealed an interaction between VncR and the promoter () in the presence of serum. In addition, analysis uncovered this protein-DNA interaction, suggesting that VncR binds with the , and recognizes the strain-specific significance of the tandem repeats in . Taken together, the interaction of VncR and after serum exposure plays an essential role in regulating differential strain-specific CPS production, which subsequently determines strain-specific systemic virulence. This study highlights how host protein LF contributes to pneumococcal VncR-mediated CPS production. As CPS plays a significant role in immune evasion, these findings suggest that drugs designed to interrupt the VncR-mediated CPS production could help to combat pneumococcal infections.
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http://dx.doi.org/10.3389/fmicb.2019.02279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6781885PMC
October 2019

Contribution of SLC22A12 on hypouricemia and its clinical significance for screening purposes.

Sci Rep 2019 10 7;9(1):14360. Epub 2019 Oct 7.

Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University, Seoul, Republic of Korea.

Differentiating between inherited renal hypouricemia and transient hypouricemic status is challenging. Here, we aimed to describe the genetic background of hypouricemia patients using whole-exome sequencing (WES) and assess the feasibility for genetic diagnosis using two founder variants in primary screening. We selected all cases (N = 31) with extreme hypouricemia (<1.3 mg/dl) from a Korean urban cohort of 179,381 subjects without underlying conditions. WES and corresponding downstream analyses were performed for the discovery of rare causal variants for hypouricemia. Two known recessive variants within SLC22A12 (p.Trp258*, pArg90His) were identified in 24 out of 31 subjects (77.4%). In an independent cohort, we identified 50 individuals with hypouricemia and genotyped the p.Trp258* and p.Arg90His variants; 47 of the 50 (94%) hypouricemia cases were explained by only two mutations. Four novel coding variants in SLC22A12, p.Asn136Lys, p.Thr225Lys, p.Arg284Gln, and p.Glu429Lys, were additionally identified. In silico studies predict these as pathogenic variants. This is the first study to show the value of genetic diagnostic screening for hypouricemia in the clinical setting. Screening of just two ethnic-specific variants (p.Trp258* and p.Arg90His) identified 87.7% (71/81) of Korean patients with monogenic hypouricemia. Early genetic identification of constitutive hypouricemia may prevent acute kidney injury by avoidance of dehydration and excessive exercise.
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http://dx.doi.org/10.1038/s41598-019-50798-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6779878PMC
October 2019

Structural and functional characterization of a novel cold-active S-formylglutathione hydrolase (SfSFGH) homolog from Shewanella frigidimarina, a psychrophilic bacterium.

Microb Cell Fact 2019 Aug 19;18(1):140. Epub 2019 Aug 19.

Unit of Polar Genomics, Korea Polar Research Institute, Incheon, 21990, Republic of Korea.

Background: S-Formylglutathione is hydrolyzed to glutathione and formate by an S-formylglutathione hydrolase (SFGH) (3.1.2.12). This thiol esterase belongs to the esterase family and is also known as esterase D. SFGHs contain highly conserved active residues of Ser-Asp-His as a catalytic triad at the active site. Characterization and investigation of SFGH from Antarctic organisms at the molecular level is needed for industrial use through protein engineering.

Results: A novel cold-active S-formylglutathione hydrolase (SfSFGH) from Shewanella frigidimarina, composed of 279 amino acids with a molecular mass of ~ 31.0 kDa, was characterized. Sequence analysis of SfSFGH revealed a conserved pentapeptide of G-X-S-X-G found in various lipolytic enzymes along with a putative catalytic triad of Ser148-Asp224-His257. Activity analysis showed that SfSFGH was active towards short-chain esters, such as p-nitrophenyl acetate, butyrate, hexanoate, and octanoate. The optimum pH for enzymatic activity was slightly alkaline (pH 8.0). To investigate the active site configuration of SfSFGH, we determined the crystal structure of SfSFGH at 2.32 Å resolution. Structural analysis shows that a Trp182 residue is located at the active site entrance, allowing it to act as a gatekeeper residue to control substrate binding to SfSFGH. Moreover, SfSFGH displayed more than 50% of its initial activity in the presence of various chemicals, including 30% EtOH, 1% Triton X-100, 1% SDS, and 5 M urea.

Conclusions: Mutation of Trp182 to Ala allowed SfSFGH to accommodate a longer chain of substrates. It is thought that the W182A mutation increases the substrate-binding pocket and decreases the steric effect for larger substrates in SfSFGH. Consequently, the W182A mutant has a broader substrate specificity compared to wild-type SfSFGH. Taken together, this study provides useful structure-function data of a SFGH family member and may inform protein engineering strategies for industrial applications of SfSFGH.
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http://dx.doi.org/10.1186/s12934-019-1190-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6699074PMC
August 2019

A novel enantioselective SGNH family esterase (NmSGNH1) from Neisseria meningitides: Characterization, mutational analysis, and ester synthesis.

Biochim Biophys Acta Mol Cell Biol Lipids 2019 10 17;1864(10):1438-1448. Epub 2019 Jul 17.

Department of Chemistry, College of Natural Science, Sookmyung Women's University, Seoul 04310, Republic of Korea. Electronic address:

In Neisseria sp., SGNH family esterases are involved in bacterial pathogenesis as well as cell wall peptidoglycan maturation. Here, a novel enantioselective SGNH family esterase (NmSGNH1) from Neisseria meningitidis, which has sequence similarity to carbohydrate esterase (CE3) family, was catalytically characterized and functionally explored. NmSGNH1 exhibited a wide range of substrate specificities including naproxol acetate, tert-butyl acetate, glucose pentaacetate as well as p-nitrophenyl esters. Deletion of C-terminal residues (NmSGNH1Δ11) led to the altered substrate specificity, reduced catalytic activity, and increased thermostability. Furthermore, a hydrophobic residue of Leu in the substrate-binding pocket was identified to be critical in catalytic activity, thermostability, kinetics, and enantioselectivity. Interestingly, immobilization of NmSGNH1 by hybrid nanoflowers (hNFs) and crosslinked enzyme aggregates (CLEAs) showed increased level of activity, recycling property, and enhanced stability. Finally, synthesis of butyl acetate, oleic acid esters, and fatty acid methyl esters (FAMEs) were verified. In summary, this work provides a molecular understanding of substrate specificities, catalytic regulation, immobilization, and industrial applications of a novel SGNH family esterase from Neisseria meningitidis.
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http://dx.doi.org/10.1016/j.bbalip.2019.07.007DOI Listing
October 2019

Targeting Mannitol Metabolism as an Alternative Antimicrobial Strategy Based on the Structure-Function Study of Mannitol-1-Phosphate Dehydrogenase in Staphylococcus aureus.

mBio 2019 07 9;10(4). Epub 2019 Jul 9.

Department of Molecular Cell Biology, Institute for Antimicrobial Research and Therapeutics, Sungkyunkwan University School of Medicine, Suwon, South Korea

Mannitol-1-phosphate dehydrogenase (M1PDH) is a key enzyme in mannitol metabolism, but its roles in pathophysiological settings have not been established. We performed comprehensive structure-function analysis of M1PDH from USA300, a strain of community-associated methicillin-resistant , to evaluate its roles in cell viability and virulence under pathophysiological conditions. On the basis of our results, we propose M1PDH as a potential antibacterial target. cell viability assessment of Δ knockout and complemented strains confirmed that M1PDH is essential to endure pH, high-salt, and oxidative stress and thus that M1PDH is required for preventing osmotic burst by regulating pressure potential imposed by mannitol. The mouse infection model also verified that M1PDH is essential for bacterial survival during infection. To further support the use of M1PDH as an antibacterial target, we identified dihydrocelastrol (DHCL) as a competitive inhibitor of M1PDH (M1PDH) and confirmed that DHCL effectively reduces bacterial cell viability during host infection. To explain physiological functions of M1PDH at the atomic level, the crystal structure of M1PDH was determined at 1.7-Å resolution. Structure-based mutation analyses and DHCL molecular docking to the M1PDH active site followed by functional assay identified key residues in the active site and provided the action mechanism of DHCL. Collectively, we propose M1PDH as a target for antibiotic development based on its physiological roles with the goals of expanding the repertory of antibiotic targets to fight antimicrobial resistance and providing essential knowledge for developing potent inhibitors of M1PDH based on structure-function studies. Due to the shortage of effective antibiotics against drug-resistant , new targets are urgently required to develop next-generation antibiotics. We investigated mannitol-1-phosphate dehydrogenase of USA300 (M1PDH), a key enzyme regulating intracellular mannitol levels, and explored the possibility of using M1PDH as a target for developing antibiotic. Since mannitol is necessary for maintaining the cellular redox and osmotic potential, the homeostatic imbalance caused by treatment with a M1PDH inhibitor or knockout of the gene encoding M1PDH results in bacterial cell death through oxidative and/or mannitol-dependent cytolysis. We elucidated the molecular mechanism of M1PDH and the structural basis of substrate and inhibitor recognition by enzymatic and structural analyses of M1PDH. Our results strongly support the concept that targeting of M1PDH represents an alternative strategy for developing a new class of antibiotics that cause bacterial cell death not by blocking key cellular machinery but by inducing cytolysis and reducing stress tolerance through inhibition of the mannitol pathway.
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http://dx.doi.org/10.1128/mBio.02660-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6623548PMC
July 2019

Identification of a new Z-DNA inducer using SYBR green 1 as a DNA conformation sensor.

FEBS Lett 2019 09 10;593(18):2628-2636. Epub 2019 Jul 10.

Department of Molecular Cell Biology, Institute for Antimicrobial Resistance Research and Therapeutics, Sungkyunkwan University School of Medicine, Suwon, Korea.

Z-DNA, which is left-handed double-stranded DNA, is involved in various cellular processes. However, its biological roles have not been fully evaluated due to the lack of tools available that can control the precise conformational change to Z-DNA in vitro and in vivo. Therefore, the need for identifying new Z-DNA inducers is high. We developed an assay system to monitor the conformational change in DNA utilizing the fluorescence of SYBR green I integrated into a double-stranded oligonucleotide. By applying this assay to screen for compounds that induce the B-DNA to Z-DNA transition, we identified the natural compound aklavin as a novel Z-DNA inducer.
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http://dx.doi.org/10.1002/1873-3468.13513DOI Listing
September 2019

A Selective Inhibitor of Ubiquitin-Specific Protease 4 Suppresses Colorectal Cancer Progression by Regulating β-Catenin Signaling.

Cell Physiol Biochem 2019 ;53(1):157-171

Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon, Republic of Korea.

Background/aims: Dysregulation of deubiquitinating enzymes (DUBs), which regulate the stability of key proteins, has been implicated in many human diseases, including cancers. Thus, DUBs can be considered as potential therapeutic targets for many diseases. Among them, USP4 has been proposed as a promising target for colon cancer drugs since USP4 controls the stability of β-catenin, a key factor in the Wnt signaling involved in the tumorigenesis of colorectal cancer. However, developing potential DUB inhibitors has been hindered because many DUBs harbor similar active site structures and show broad substrate specificities.

Methods: By performing in vitro deubiquitinating activity assays using a chemical library, we identified several potential DUB inhibitors. Among them, only neutral red (NR) showed selective inhibitory activity on USP4 in a cell-based assay system. In colon cancer cells, NR affected the protein stability of β-catenin, as shown by immunoblotting, and it affected the target gene expression of β-catenin, as shown by quantitative real-time PCR. NR's potential as an anticancer drug was further estimated by colony formation and cell migration assays and by using a mouse xenograft model.

Results: We identified NR as an uncompetitive inhibitor of USP4 and validated its effects in colorectal cancer. NR-treated cells showed decreased β-catenin stability and reduced expression of β-catenin target genes. Additionally, treating colon cancer cells with NR significantly reduced colony formation and cell migration, and injecting NR into a mouse xenograft model reduced the tumor volume.

Conclusion: The current results suggest that NR could be developed as an anticancer drug targeting USP4, and they support the possibility of developing specific DUB inhibitors as therapeutic agents.
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http://dx.doi.org/10.33594/000000127DOI Listing
July 2019

Characterization, immobilization, and mutagenesis of a novel cold-active acetylesterase (EaAcE) from Exiguobacterium antarcticum B7.

Int J Biol Macromol 2019 Sep 21;136:1042-1051. Epub 2019 Jun 21.

Department of Chemistry, College of Natural Science, Sookmyung Women's University, Seoul 04310, Republic of Korea. Electronic address:

Cold-active enzymes with distinctive properties from a psychrophilic Exiguobacterium antarcticum B7 could be excellent biocatalysts in industrial and biotechnological processes. Here, the characterization, immobilization, and site-directed mutagenesis of a novel cold-active acetylesterase (EaAcE) from E. antarcticum B7 is reported. EaAcE does not belong to any currently known lipase/esterase family, although there are some sequence similarities with family III and V members. Biochemical characterization of EaAcE was carried out using activity staining, mass spectrometry analysis, circular dichroism spectra, freeze-thaw experiments, kinetic analysis, acetic acid release assays, and enantioselectivity determination. Furthermore, immobilization of EaAcE using four different approaches was explored to enhance its thermal stability and recyclability. Based on a homology model of EaAcE, four mutations (F45A, S118A, S141A, and T216A) within the substrate-binding pocket were investigated to elucidate their roles in EaAcE catalysis and substrate specificity. This work has provided invaluable information on the properties of EaAcE, which can now be used to understand the acetylesterase enzyme family.
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http://dx.doi.org/10.1016/j.ijbiomac.2019.06.108DOI Listing
September 2019

Z-DNA in the genome: from structure to disease.

Biophys Rev 2019 Jun 22;11(3):383-387. Epub 2019 May 22.

Department of Molecular Cell Biology, Institute for Antimicrobial Resistance Research and Therapeutics, Sungkyunkwan University School of Medicine, Suwon, 16419, South Korea.

The scope of studies investigating the architecture of genomic DNA has progressed steadily since the elucidation of the structure of B-DNA. In recent years, several non-canonical DNA structures including Z-DNA, G-quadruplexes, H-DNA, cruciform DNA, and i-motifs have been reported to form in genomic DNA and are closely related to the evolution and development of disease. The ability of these structures to form in genomic DNA indicates that they might have important cellular roles and are therefore retained during evolution. Understanding the impact of the formation of these secondary structures on cellular processes can enable identification of new targets for therapeutics. In this review, we report the state of understanding of Z-DNA structure and formation and their implication in disease. Finally, we state our perspective on the potential of Z-DNA as a therapeutic target.
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http://dx.doi.org/10.1007/s12551-019-00534-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6557933PMC
June 2019

Introduction to the Korean Biophysical Society (KBPS).

Biophys Rev 2019 Jun 11;11(3):267-268. Epub 2019 May 11.

School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Republic of Korea.

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http://dx.doi.org/10.1007/s12551-019-00547-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6557951PMC
June 2019
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