Publications by authors named "Jae-Sung Woo"

30 Publications

  • Page 1 of 1

Identification of a Direct-Acting Antiviral Agent Targeting RNA Helicase via a Graphene Oxide Nanobiosensor.

ACS Appl Mater Interfaces 2021 Jun 26;13(22):25715-25726. Epub 2021 May 26.

Department of Chemistry, Seoul National University, Seoul 08826, Republic of Korea.

Dengue virus (DENV), an arbovirus transmitted by mosquitoes, causes infectious diseases such as dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. Despite the dangers posed by DENV, there are no approved antiviral drugs for treatment of DENV infection. Considering the potential for a global dengue outbreak, rapid development of antiviral agents against DENV infections is crucial as a preemptive measure; thus, the selection of apparent drug targets, such as the viral enzymes involved in the viral life cycle, is recommended. Helicase, a potential drug target in DENV, is a crucial viral enzyme that unwinds double-stranded viral RNA, releasing single-stranded RNA genomes during viral replication. Therefore, an inhibitor of helicase activity could serve as a direct-acting antiviral agent. Here, we introduce an RNA helicase assay based on graphene oxide, which enables fluorescence-based analysis of RNA substrate-specific helicase enzyme activity. This assay demonstrated high reliability and ability for high-throughput screening, identifying a new helicase inhibitor candidate, micafungin (MCFG), from an FDA-approved drug library. As a direct-acting antiviral agent targeting RNA helicase, MCFG inhibits DENV proliferation in cells and an animal model. Notably, , MCFG treatment reduced viremia, inflammatory cytokine levels, and viral loads in several tissues and improved survival rates by up to 40% in a lethal mouse model. Therefore, we suggest MCFG as a potential direct-acting antiviral drug candidate.
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http://dx.doi.org/10.1021/acsami.1c04641DOI Listing
June 2021

Adenine base editing and prime editing of chemically derived hepatic progenitors rescue genetic liver disease.

Cell Stem Cell 2021 Apr 26. Epub 2021 Apr 26.

Department of Surgery, Hanyang University College of Medicine, Seoul 04763, Republic of Korea; HY Indang Center of Regenerative Medicine and Stem Cell Research, Hanyang University, Seoul 04763, Republic of Korea; Department of HY-KIST Bio-convergence, Hanyang University, Seoul 04763, Republic of Korea. Electronic address:

DNA base editors and prime editing technology enable therapeutic in situ correction of disease-causing alleles. These techniques could have broad applications for ex vivo editing of cells prior to transplantation in a range of diseases, but it is critical that the target population is efficiently modified and engrafts into the host. Chemically derived hepatic progenitors (CdHs) are a multipotent population capable of robust engraftment and hepatocyte differentiation. Here we reprogrammed hepatocytes from a mouse model of hereditary tyrosinemia type 1 (HT1) into expandable CdHs and successfully corrected the disease-causing mutation using both adenine base editors (ABEs) and prime editors (PEs). ABE- and PE-corrected CdHs repopulated the liver with fumarylacetoacetate hydrolase-positive cells and dramatically increased survival of mutant HT1 mice. These results demonstrate the feasibility of precise gene editing in transplantable cell populations for potential treatment of genetic liver disease.
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http://dx.doi.org/10.1016/j.stem.2021.04.010DOI Listing
April 2021

Characterization of alfalfa mosaic virus capsid protein using Cryo-EM.

Biochem Biophys Res Commun 2021 Jun 1;559:161-167. Epub 2021 May 1.

BioApplications Inc., Pohang Techno Park Complex, 394 Jigok-ro Nam-gu, Pohang, 37668, South Korea. Electronic address:

VLPs are virus-like particles that comprise viral capsid proteins that can self-assemble and mimic the shape and size of real viral particles; however, because they do not contain genetic material they cannot infect host cells. VLPs have great potential as safe drug/vehicle candidates; therefore, they are gaining popularity in the field of preventive medicine and therapeutics. Indeed, extensive studies are underway to examine their role as carriers for immunization and as vehicles for delivery of therapeutic agents. Here, we examined the possibility of developing VLP-utilizing technology based on an efficient VLP production process and high-resolution structural analysis. Nicotiana benthamiana was used as an expression platform to produce the coat protein of the alfalfa mosaic virus (AMV-CP). About 250 mg/kg of rAMV-CP was produced from Nicotiana benthamiana leaves. Structural analysis revealed that the oligomeric status of rAMV-CP changed according to the composition and pH of the buffer. Size exclusion chromatography and electron microscopy analysis confirmed the optimal conditions for rAMV-CP VLP formation, and a 2.4 Å resolution structure was confirmed by cryo-EM analysis. Based on the efficient protein production, VLP manufacturing technology, and high-resolution structure presented herein, we suggest that rAMV-CP VLP is a useful platform for development of various new drugs.
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http://dx.doi.org/10.1016/j.bbrc.2021.04.060DOI Listing
June 2021

Efficient Human Cell Coexpression System and Its Application to the Production of Multiple Coronavirus Antigens.

Adv Biol (Weinh) 2021 04 11;5(4):e2000154. Epub 2021 Feb 11.

Department of Chemistry, Hanyang University, Seoul, 04763, South Korea.

Coproduction of multiple proteins at high levels in a single human cell line would be extremely useful for basic research and medical applications. Here, a novel strategy for the stable expression of multiple proteins by integrating the genes into defined transcriptional hotspots in the human genome is presented. As a proof-of-concept, it is shown that EYFP is expressed at similar levels from hotspots and that the EYFP expression increases proportionally with the copy number. It is confirmed that three different fluorescent proteins, encoded by genes integrated at different loci, can be coexpressed at high levels. Further, a stable cell line is generated, producing antigens from different human coronaviruses: MERS-CoV and HCoV-OC43. Antibodies raised against these antigens, which contain human N-glycosylation, show neutralizing activities against both viruses, suggesting that the coexpression system provides a quick and predictable way to produce multiple coronavirus antigens, such as the recent 2019 novel human coronavirus.
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http://dx.doi.org/10.1002/adbi.202000154DOI Listing
April 2021

TRIM28 functions as a negative regulator of aggresome formation.

Autophagy 2021 Apr 12:1-17. Epub 2021 Apr 12.

Creative Research Initiatives Center for Molecular Biology of Translation, Korea University, Seoul, Republic of Korea.

Selective recognition and elimination of misfolded polypeptides are crucial for protein homeostasis. When the ubiquitin-proteasome system is impaired, misfolded polypeptides tend to form small cytosolic aggregates and are transported to the aggresome and eventually eliminated by the autophagy pathway. Despite the importance of this process, the regulation of aggresome formation remains poorly understood. Here, we identify TRIM28/TIF1β/KAP1 (tripartite motif containing 28) as a negative regulator of aggresome formation. Direct interaction between TRIM28 and CTIF (cap binding complex dependent translation initiation factor) leads to inefficient aggresomal targeting of misfolded polypeptides. We also find that either treatment of cells with poly I:C or infection of the cells by influenza A viruses triggers the phosphorylation of TRIM28 at S473 in a way that depends on double-stranded RNA-activated protein kinase. The phosphorylation promotes association of TRIM28 with CTIF, inhibits aggresome formation, and consequently suppresses viral proliferation. Collectively, our data provide compelling evidence that TRIM28 is a negative regulator of aggresome formation.BAG3: BCL2-associated athanogene 3; CTIF: CBC-dependent translation initiation factor; CED: CTIF-EEF1A1-DCTN1; DCTN1: dynactin subunit 1; EEF1A1: eukaryotic translation elongation factor 1 alpha 1; EIF2AK2: eukaryotic translation initiation factor 2 alpha kinase 2; HDAC6: histone deacetylase 6; IAV: influenza A virus; IP: immunoprecipitation; PLA: proximity ligation assay; polypeptidyl-puro: polypeptidyl-puromycin; qRT-PCR: quantitative reverse-transcription PCR; siRNA: small interfering RNA.
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http://dx.doi.org/10.1080/15548627.2021.1909835DOI Listing
April 2021

UXT chaperone prevents proteotoxicity by acting as an autophagy adaptor for p62-dependent aggrephagy.

Nat Commun 2021 03 29;12(1):1955. Epub 2021 Mar 29.

Department of Life Sciences, Korea University, Seoul, Republic of Korea.

p62/SQSTM1 is known to act as a key mediator in the selective autophagy of protein aggregates, or aggrephagy, by steering ubiquitinated protein aggregates towards the autophagy pathway. Here, we use a yeast two-hybrid screen to identify the prefoldin-like chaperone UXT as an interacting protein of p62. We show that UXT can bind to protein aggregates as well as the LB domain of p62, and, possibly by forming an oligomer, increase p62 clustering for its efficient targeting to protein aggregates, thereby promoting the formation of the p62 body and clearance of its cargo via autophagy. We also find that ectopic expression of human UXT delays SOD1(A4V)-induced degeneration of motor neurons in a Xenopus model system, and that specific disruption of the interaction between UXT and p62 suppresses UXT-mediated protection. Together, these results indicate that UXT functions as an autophagy adaptor of p62-dependent aggrephagy. Furthermore, our study illustrates a cooperative relationship between molecular chaperones and the aggrephagy machinery that efficiently removes misfolded protein aggregates.
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http://dx.doi.org/10.1038/s41467-021-22252-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8007730PMC
March 2021

Cryo-EM structure of human Cx31.3/GJC3 connexin hemichannel.

Sci Adv 2020 Aug 28;6(35):eaba4996. Epub 2020 Aug 28.

Department of Life Sciences, Korea University, Seoul 02841, Republic of Korea.

Connexin family proteins assemble into hexameric channels called hemichannels/connexons, which function as transmembrane channels or dock together to form gap junction intercellular channels (GJIChs). We determined the cryo-electron microscopy structures of human connexin 31.3 (Cx31.3)/GJC3 hemichannels in the presence and absence of calcium ions and with a hearing-loss mutation R15G at 2.3-, 2.5-, and 2.6-Å resolutions, respectively. Compared with available structures of GJICh in open conformation, Cx31.3 hemichannel shows substantial structural changes of highly conserved regions in the connexin family, including opening of calcium ion-binding tunnels, reorganization of salt-bridge networks, exposure of lipid-binding sites, and collocation of amino-terminal helices at the cytoplasmic entrance. We also found that the hemichannel has a pore with a diameter of ~8 Å and selectively transports chloride ions. Our study provides structural insights into the permeant selectivity of Cx31.3 hemichannel.
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http://dx.doi.org/10.1126/sciadv.aba4996DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7455182PMC
August 2020

Purification of an Intact Human Protein Overexpressed from Its Endogenous Locus Direct Genome Engineering.

ACS Synth Biol 2020 07 2;9(7):1591-1598. Epub 2020 Jul 2.

Department of Chemistry, Hanyang University, Seoul 04763, South Korea.

The overproduction and purification of human proteins is a requisite of both basic and medical research. Although many recombinant human proteins have been purified, current protein production methods have several limitations; recombinant proteins are frequently truncated, fail to fold properly, and/or lack appropriate post-translational modifications. In addition, such methods require subcloning of the target gene into relevant plasmids, which can be difficult for long proteins with repeated domains. Here we devised a novel method for target protein production by introduction of a strong promoter for overexpression and an epitope tag for purification in front of the endogenous human gene, in a sense performing molecular cloning directly in the human genome, which does not require cloning of the target gene. As a proof of concept, we successfully purified intact human Reelin protein, which is lengthy (3460 amino acids) and contains repeating domains, and confirmed that it was biologically functional.
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http://dx.doi.org/10.1021/acssynbio.0c00090DOI Listing
July 2020

Discovery of direct-acting antiviral agents with a graphene-based fluorescent nanosensor.

Sci Adv 2020 May 29;6(22):eaaz8201. Epub 2020 May 29.

Department of Chemistry, Seoul National University, Seoul 08826, Republic of Korea.

Direct-acting agents against viral components are considered as the most promising candidates for the successful antiviral therapeutics. To date, no direct-acting drugs exist for the treatment against dengue virus (DV) infection, which can develop into life-threatening diseases. RNA-dependent RNA polymerase (RdRp), an RNA virus-specific enzyme highly conserved among various viral families, has been known as the broad-range antiviral drug target. Here, we developed an RNA-based graphene biosensor system [RNA nano-graphene oxide system (RANGO)] to enable the fluorescence-based quantitative analysis of the RdRp enzyme activity. We used the RANGO system to a high-throughput chemical screening to identify novel direct-acting antiviral drug candidates targeting DV RdRp from the FDA-approved small-molecule library. RANGO accelerated the massive selection of drug candidates. We found that one of the selected hit compounds, montelukast, showed antiviral activity in vitro and in vivo by directly inhibiting replication of DV and thus relieved related symptoms.
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http://dx.doi.org/10.1126/sciadv.aaz8201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7259931PMC
May 2020

A fluorescent nanobiosensor for the facile analysis of mA RNA demethylase activity.

Chem Commun (Camb) 2020 Apr 26;56(34):4716-4719. Epub 2020 Mar 26.

Department of Chemistry, Seoul National University, Seoul 08826, Republic of Korea.

RNA demethylase has recently been known to be associated with cancer development but its selective inhibitors as anti-cancer agents have rarely been investigated to date. Herein, we have developed a fluorescent nanobiosensor which enables efficient quantitative analysis of RNA demethylase ALKBH5 activity and shows a high potential for robust inhibitor screening.
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http://dx.doi.org/10.1039/c9cc10054gDOI Listing
April 2020

Real-Time Measurement of the Liquid Amount in Cryo-Electron Microscopy Grids Using Laser Diffraction of Regular 2-D Holes of the Grids.

Mol Cells 2020 Mar;43(3):298-303

Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, Center for Food and Bioconvergence, and Research Institute for Agriculture and Life Sciences, CALS, Seoul National University, Seoul 08826, Korea.

Cryo-electron microscopy (cryo-EM) is now the first choice to determine the high-resolution structures of huge protein complexes. Grids with two-dimensional arrays of holes covered with a carbon film are typically used in cryo-EM. Although semi-automatic plungers are available, notable trial-and-error is still required to obtain a suitable grid specimen. Herein, we introduce a new method to obtain thin ice specimens using real-time measurement of the liquid amounts in cryo-EM grids. The grids for cryo-EM strongly diffracted laser light, and the diffraction intensity of each spot was measurable in real-time. The measured diffraction patterns represented the states of the liquid in the holes due to the curvature of the liquid around them. Using the diffraction patterns, the optimal time point for freezing the grids for cryo-EM was obtained in real-time. This development will help researchers rapidly determine highresolution protein structures using the limited resource of cryo-EM instrument access.
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http://dx.doi.org/10.14348/molcells.2020.2238DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7103880PMC
March 2020

Structural insight into glucose repression of the mannitol operon.

Sci Rep 2019 09 26;9(1):13930. Epub 2019 Sep 26.

School of Biological Sciences and Institute of Microbiology, Seoul National University, Seoul, 00826, Korea.

Carbon catabolite repression is a regulatory mechanism to ensure sequential utilization of carbohydrates and is usually accomplished by repression of genes for the transport and metabolism of less preferred carbon compounds by a more preferred one. Although glucose and mannitol share the general components, enzyme I and HPr, of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) for their transport, glucose represses the transport and metabolism of mannitol in a manner dependent on the mannitol operon repressor MtlR in Escherichia coli. In a recent study, we identified the dephosphorylated form of HPr as a regulator determining the glucose preference over mannitol by interacting with and augmenting the repressor activity of MtlR in E. coli. Here, we determined the X-ray structure of the MtlR-HPr complex at 3.5 Å resolution to understand how phosphorylation of HPr impedes its interaction with MtlR. The phosphorylation site (His15) of HPr is located close to Glu108 and Glu140 of MtlR and phosphorylation at His15 causes electrostatic repulsion between the two proteins. Based on this structural insight and comparative sequence analyses, we suggest that the determination of the glucose preference over mannitol solely by the MtlR-HPr interaction is conserved within  the Enterobacteriaceae family.
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http://dx.doi.org/10.1038/s41598-019-50249-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6763467PMC
September 2019

Molecular Basis for the Single-Nucleotide Precision of Primary microRNA Processing.

Mol Cell 2019 02 13;73(3):505-518.e5. Epub 2018 Dec 13.

Center for RNA Research, Institute for Basic Science, Seoul 08826, Korea; School of Biological Sciences, Seoul National University, Seoul 08826, Korea. Electronic address:

Microprocessor, composed of DROSHA and its cofactor DGCR8, initiates microRNA (miRNA) biogenesis by processing the primary transcripts of miRNA (pri-miRNAs). Here we investigate the mechanism by which Microprocessor selects the cleavage site with single-nucleotide precision, which is crucial for the specificity and functionality of miRNAs. By testing ∼40,000 pri-miRNA variants, we find that for some pri-miRNAs the cleavage site is dictated mainly by the mGHG motif embedded in the lower stem region of pri-miRNA. Structural modeling and deep-sequencing-based complementation experiments show that the double-stranded RNA-binding domain (dsRBD) of DROSHA recognizes mGHG to place the catalytic center in the appropriate position. The mGHG motif as well as the mGHG-recognizing residues in DROSHA dsRBD are conserved across eumetazoans, suggesting that this mechanism emerged in an early ancestor of the animal lineage. Our findings provide a basis for the understanding of miRNA biogenesis and rational design of accurate small-RNA-based gene silencing.
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http://dx.doi.org/10.1016/j.molcel.2018.11.005DOI Listing
February 2019

Structural basis of transcobalamin recognition by human CD320 receptor.

Nat Commun 2016 07 14;7:12100. Epub 2016 Jul 14.

Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zurich, 8093 Zurich, Switzerland.

Cellular uptake of vitamin B12 (cobalamin) requires capture of transcobalamin (TC) from the plasma by CD320, a ubiquitous cell surface receptor of the LDLR family. Here we present the crystal structure of human holo-TC in complex with the extracellular domain of CD320, visualizing the structural basis of the TC-CD320 interaction. The observed interaction chemistry can rationalize the high affinity of CD320 for TC and lack of haptocorrin binding. The in vitro affinity and complex stability of TC-CD320 were quantitated using a solid-phase binding assay and thermostability analysis. Stable complexes with TC were also observed for the disease-causing CD320ΔE88 mutant and for the isolated LDLR-A2 domain. We also determined the structure of the TC-CD320ΔE88 complex, which revealed only minor changes compared with the wild-type complex. Finally, we demonstrate significantly reduced in vitro affinity of TC for CD320 at low pH, recapitulating the proposed ligand release during the endocytic pathway.
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http://dx.doi.org/10.1038/ncomms12100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4947154PMC
July 2016

Structure of Human DROSHA.

Cell 2016 Jan 31;164(1-2):81-90. Epub 2015 Dec 31.

Center for RNA Research, Institute for Basic Science, Seoul 08826, Korea; School of Biological Sciences, Seoul National University, Seoul 08826, Korea. Electronic address:

MicroRNA maturation is initiated by RNase III DROSHA that cleaves the stem loop of primary microRNA. DROSHA functions together with its cofactor DGCR8 in a heterotrimeric complex known as Microprocessor. Here, we report the X-ray structure of DROSHA in complex with the C-terminal helix of DGCR8. We find that DROSHA contains two DGCR8-binding sites, one on each RNase III domain (RIIID), which mediate the assembly of Microprocessor. The overall structure of DROSHA is surprisingly similar to that of Dicer despite no sequence homology apart from the C-terminal part, suggesting that DROSHA may have evolved from a Dicer homolog. DROSHA exhibits unique features, including non-canonical zinc-finger motifs, a long insertion in the first RIIID, and the kinked link between Connector helix and RIIID, which explains the 11-bp-measuring "ruler" activity of DROSHA. Our study implicates the evolutionary origin of DROSHA and elucidates the molecular basis of Microprocessor assembly and primary microRNA processing.
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http://dx.doi.org/10.1016/j.cell.2015.12.019DOI Listing
January 2016

Functional Anatomy of the Human Microprocessor.

Cell 2015 Jun 28;161(6):1374-87. Epub 2015 May 28.

Center for RNA Research, Institute for Basic Science, Seoul 151-742, Korea; School of Biological Sciences, Seoul National University, Seoul 151-742, Korea. Electronic address:

MicroRNA (miRNA) maturation is initiated by Microprocessor composed of RNase III DROSHA and its cofactor DGCR8, whose fidelity is critical for generation of functional miRNAs. To understand how Microprocessor recognizes pri-miRNAs, we here reconstitute human Microprocessor with purified recombinant proteins. We find that Microprocessor is an ∼364 kDa heterotrimeric complex of one DROSHA and two DGCR8 molecules. Together with a 23-amino acid peptide from DGCR8, DROSHA constitutes a minimal functional core. DROSHA serves as a "ruler" by measuring 11 bp from the basal ssRNA-dsRNA junction. DGCR8 interacts with the stem and apical elements through its dsRNA-binding domains and RNA-binding heme domain, respectively, allowing efficient and accurate processing. DROSHA and DGCR8, respectively, recognize the basal UG and apical UGU motifs, which ensure proper orientation of the complex. These findings clarify controversies over the action mechanism of DROSHA and allow us to build a general model for pri-miRNA processing.
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http://dx.doi.org/10.1016/j.cell.2015.05.010DOI Listing
June 2015

BSA as additive: A simple strategy for practical applications of PNA in bioanalysis.

Biosens Bioelectron 2015 Jul 20;69:167-73. Epub 2015 Feb 20.

Department of Chemistry, Seoul National University, Seoul 151-747, Republic of Korea; Center for RNA Research, Institute for Basic Science, Seoul National University, Seoul 151-747, Republic of Korea; Lemonex Inc., Seoul 151-742, Republic of Korea. Electronic address:

Application of peptide nucleic acid (PNA) in bioanalysis has been limited due to its nonspecific adsorption onto hydrophobic surface in spite of favorable properties such as higher chemical/biological stability, specificity and binding affinity towards target nucleic acids compared to natural nucleic acid probes. Herein, we employed BSA in PNA application to enhance the stability of PNA in hydrophobic containers and improve the sensing performance of the DNA sensor based on graphene oxide (GO) and PNA. Addition of 0.01% BSA in a PNA solution effectively prevented the adsorption of PNA on hydrophobic surface and increased the portion of the effective PNA strands for target binding without interfering duplex formation with a complementary target sequence. In the GO based biosensor using PNA, BSA interrupted the unfavorable adsorption of PNA/DNA duplex on GO surface, while allowing the adsorption of ssPNA, resulting in improvement of the performance of the DNA sensor system by reducing the detection limit by 90-folds.
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http://dx.doi.org/10.1016/j.bios.2015.02.030DOI Listing
July 2015

MeCP2 caught moonlighting as a suppressor of MicroRNA processing.

Dev Cell 2014 Mar;28(5):477-8

Center for RNA Research, Institute for Basic Science, Seoul 151-742, Korea; School of Biological Sciences, Seoul National University, Seoul 151-742, Korea. Electronic address:

MeCP2 is a transcriptional regulator important for neurodevelopment and is involved in Rett syndrome and autism. In this issue of Developmental Cell, Cheng and colleagues (2014) report that MeCP2 also regulates microRNA biogenesis. MeCP2 phosphorylation induces a direct interaction with DGCR8, leading to reduced microRNA processing and retardation of dendritic growth.
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http://dx.doi.org/10.1016/j.devcel.2014.02.015DOI Listing
March 2014

Rescue of deleterious mutations by the compensatory Y30F mutation in ketosteroid isomerase.

Mol Cells 2013 Jul 3;36(1):39-46. Epub 2013 Jun 3.

Department of Life Science, WCU Program, Pohang University of Science and Technology, Pohang, 790-784, Korea.

Proteins have evolved to compensate for detrimental mutations. However, compensatory mechanisms for protein defects are not well understood. Using ketosteroid isomerase (KSI), we investigated how second-site mutations could recover defective mutant function and stability. Previous results revealed that the Y30F mutation rescued the Y14F, Y55F and Y14F/Y55F mutants by increasing the catalytic activity by 23-, 3- and 1.3-fold, respectively, and the Y55F mutant by increasing the stability by 3.3 kcal/mol. To better understand these observations, we systematically investigated detailed structural and thermodynamic effects of the Y30F mutation on these mutants. Crystal structures of the Y14F/Y30F and Y14F/Y55F mutants were solved at 2.0 and 1.8 previoulsy solved structures of wild-type and other mutant KSIs. Structural analyses revealed that the Y30F mutation partially restored the active-site cleft of these mutant KSIs. The Y30F mutation also increased Y14F and Y14F/Y55F mutant stability by 3.2 and 4.3 kcal/mol, respectively, and the melting temperatures of the Y14F, Y55F and Y14F/Y55F mutants by 6.4°C, 5.1°C and 10.0°C, respectively. Compensatory effects of the Y30F mutation on stability might be due to improved hydrophobic interactions because removal of a hydroxyl group from Tyr30 induced local compaction by neighboring residue movement and enhanced interactions with surrounding hydrophobic residues in the active site. Taken together, our results suggest that perturbed active-site geometry recovery and favorable hydrophobic interactions mediate the role of Y30F as a secondsite suppressor.
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http://dx.doi.org/10.1007/s10059-013-0013-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3887930PMC
July 2013

X-ray structure of the Yersinia pestis heme transporter HmuUV.

Nat Struct Mol Biol 2012 Dec 11;19(12):1310-5. Epub 2012 Nov 11.

Institute of Molecular Biology and Biophysics, Eidgenössische Technische Hochschule (ETH) Zürich, CH-8093 Zürich, Switzerland.

HmuUV is a bacterial ATP-binding cassette (ABC) transporter that catalyzes heme uptake into the cytoplasm of the gram-negative pathogen Yersinia pestis. We report the crystal structure of HmuUV at 3.0 Å resolution in a nucleotide-free state, which features a heme translocation pathway in an outward-facing conformation, poised to accept a heme from the cognate periplasmic binding protein HmuT. A new assay allowed us to determine in vitro rates of HmuUV-catalyzed heme transport into proteoliposomes and to establish the role of conserved residues in the translocation pathway of HmuUV and at the interface with HmuT. Differences in architecture relative to the related vitamin B(12) transporter BtuCD suggest an adaptation of HmuUV for its smaller substrate. Our study also suggests that type II ABC importers, which include bacterial iron-siderophore, heme and cobalamin transporters, have a coupling mechanism distinct from that of other ABC transporters.
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http://dx.doi.org/10.1038/nsmb.2417DOI Listing
December 2012

Crystal structure of DeSI-1, a novel deSUMOylase belonging to a putative isopeptidase superfamily.

Proteins 2012 Aug 22;80(8):2099-104. Epub 2012 May 22.

Department of Biological Sciences, KAIST Institute for the Biocentury, Korea Advanced Institute of Science and Technology, Daejeon, Korea.

Post-translational modification by small ubiquitin-like modifier (SUMO) can be reversed by sentrin/SUMO-specific proteases (SENPs), the first known class of deSUMOylase. Recently, we identified a new deSUMOylating enzyme DeSI-1, which is distinct from SENPs and belongs to the putative deubiquitinating isopeptidase PPPDE superfamily. Herein, we report the crystal structure of DeSI-1, revealing that this enzyme forms a homodimer and that the groove between the two subunits is the active site harboring two absolutely conserved cysteine and histidine residues that form a catalytic dyad. We also show that DeSI-1 exhibits an extremely low endopeptidase activity toward precursor forms of SUMO-1 and SUMO-2, unlike SENPs.
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http://dx.doi.org/10.1002/prot.24093DOI Listing
August 2012

Two stacked heme molecules in the binding pocket of the periplasmic heme-binding protein HmuT from Yersinia pestis.

J Mol Biol 2010 Nov 1;404(2):220-31. Epub 2010 Oct 1.

Institute ofMolecular Biology and Biophysics, ETH Zürich, HPK D17, Schafmattstrasse 20, CH-8093 Zürich, Switzerland.

The periplasmic binding protein HmuT from Yersinia pestis (YpHmuT) is a component of the heme uptake locus hmu and delivers bound hemin to the inner-membrane-localized, ATP-binding cassette (ABC) transporter HmuUV for translocation into the cytoplasm. The mechanism of this process, heme transport across the inner membrane of pathogenic bacteria, is currently insufficiently understood at the molecular level. Here we describe the crystal structures of the substrate-free and heme-bound states of YpHmuT, revealing two lobes with a central binding cleft. Superposition of the apo and holo states reveals a minor tilting motion of the lobes surrounding concomitant with heme binding. Unexpectedly, YpHmuT binds two stacked hemes in a central binding cleft that is larger than those of the homologous periplasmic heme-binding proteins ShuT and PhuT, both of which bind only one heme. The hemes bound to YpHmuT are coordinated via a tyrosine side chain that contacts the Fe atom of one heme and a histidine that contacts the Fe atom of the other heme. The coordinating histidine is only conserved in a subset of periplasmic heme binding proteins suggesting that its presence predicts the ability to bind two heme molecules simultaneously. The structural data are supported by spectroscopic binding studies performed in solution, where up to two hemes can bind to YpHmuT. Isothermal titration calorimetry suggests that the two hemes are bound in discrete, sequential steps and with dissociation constants (K(D)) of ∼0.29  and ∼29 nM, which is similar to the affinities observed in other bacterial substrate binding proteins. Our findings suggest that the cognate ABC transporter HmuUV may simultaneously translocate two hemes per reaction cycle.
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http://dx.doi.org/10.1016/j.jmb.2010.09.005DOI Listing
November 2010

Regulation of Drosophila vasa in vivo through paralogous cullin-RING E3 ligase specificity receptors.

Mol Cell Biol 2010 Apr 1;30(7):1769-82. Epub 2010 Feb 1.

Department of Biology, McGill University, Montreal, Quebec H3A 1B1, Canada.

In Drosophila species, molecular asymmetries guiding embryonic development are established maternally. Vasa, a DEAD-box RNA helicase, accumulates in the posterior pole plasm, where it is required for embryonic germ cell specification. Maintenance of Vasa at the posterior pole requires the deubiquitinating enzyme Fat facets, which protects Vasa from degradation. Here, we found that Gustavus (Gus) and Fsn, two ubiquitin Cullin-RING E3 ligase specificity receptors, bind to the same motif on Vasa through their paralogous B30.2/SPRY domains. Both Gus and Fsn accumulate in the pole plasm in a Vasa-dependent manner. Posterior Vasa accumulation is precocious in Fsn mutant oocytes; Fsn overexpression reduces ovarian Vasa levels, and embryos from Fsn-overexpressing females form fewer primordial germ cells (PGCs); thus, Fsn destabilizes Vasa. In contrast, endogenous Gus may promote Vasa activity in the pole plasm, as gus females produce embryos with fewer PGCs, and posterior accumulation of Vas is delayed in gus mutant oocytes that also lack one copy of cullin-5. We propose that Fsn- and Gus-containing E3 ligase complexes contribute to establishing a fine-tuned steady state of Vasa ubiquitination that influences the kinetics of posterior Vasa deployment.
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http://dx.doi.org/10.1128/MCB.01100-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2838069PMC
April 2010

Structural insights into the dual nucleotide exchange and GDI displacement activity of SidM/DrrA.

EMBO J 2010 Jan 26;29(2):496-504. Epub 2009 Nov 26.

Department of Life Sciences and Center for Biomolecular Recognition, Pohang University of Science and Technology, Pohang, Kyungbuk, Korea.

GDP-bound prenylated Rabs, sequestered by GDI (GDP dissociation inhibitor) in the cytosol, are delivered to destined sub-cellular compartment and subsequently activated by GEFs (guanine nucleotide exchange factors) catalysing GDP-to-GTP exchange. The dissociation of GDI from Rabs is believed to require a GDF (GDI displacement factor). Only two RabGDFs, human PRA-1 and Legionella pneumophila SidM/DrrA, have been identified so far and the molecular mechanism of GDF is elusive. Here, we present the structure of a SidM/DrrA fragment possessing dual GEF and GDF activity in complex with Rab1. SidM/DrrA reconfigures the Switch regions of the GTPase domain of Rab1, as eukaryotic GEFs do toward cognate Rabs. Structure-based mutational analyses show that the surface of SidM/DrrA, catalysing nucleotide exchange, is involved in GDI1 displacement from prenylated Rab1:GDP. In comparison with an eukaryotic GEF TRAPP I, this bacterial GEF/GDF exhibits high binding affinity for Rab1 with GDP retained at the active site, which appears as the key feature for the GDF activity of the protein.
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http://dx.doi.org/10.1038/emboj.2009.347DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2824451PMC
January 2010

Focal localization of MukBEF condensin on the chromosome requires the flexible linker region of MukF.

FEBS J 2009 Sep 7;276(18):5101-10. Epub 2009 Aug 7.

Center for Biomolecular Recognition and Division of Molecular and Life Science, Pohang University of Science and Technology, Kyungbuk, Korea.

Condensin complexes are the key mediators of chromosome condensation. The MukB-MukE-MukF complex is a bacterial condensin, in which the MukB subunit forms a V-shaped dimeric structure with two ATPase head domains. MukE and MukF together form a tight complex, which binds to the MukB head via the C-terminal winged-helix domain (C-WHD) of MukF. One of the two bound C-WHDs of MukF is forced to detach from two ATP-bound, engaged MukB heads, and this detachment reaction depends on the MukF flexible linker preceding the C-WHD. Whereas MukB is known to focally localize at particular positions in cells by an unknown mechanism, mukE- or mukF-null mutation causes MukB to become dispersed in cells. Here, we report that mutations in MukF causing a defect in the detachment reaction interfere with the focal localization of MukB, and that the dispersed distribution of MukB in cells correlates directly with defects in cell growth and division. The data strongly suggest that the MukB-MukE-MukF condensin forms huge clusters through the ATP-dependent detachment reaction, and this cluster formation is critical for chromosome condensation by this machinery. We also show that the MukF flexible linker is involved in the dimerization and ATPase activity of the MukB head.
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http://dx.doi.org/10.1111/j.1742-4658.2009.07206.xDOI Listing
September 2009

Structural studies of a bacterial condensin complex reveal ATP-dependent disruption of intersubunit interactions.

Cell 2009 Jan;136(1):85-96

Center for Biomolecular Recognition and Division of Molecular and Life Science, Department of Life Sciences, Pohang University of Science and Technology, Pohang, Kyungbuk, 790-784, Korea.

Condensins are key mediators of chromosome condensation across organisms. Like other condensins, the bacterial MukBEF condensin complex consists of an SMC family protein dimer containing two ATPase head domains, MukB, and two interacting subunits, MukE and MukF. We report complete structural views of the intersubunit interactions of this condensin along with ensuing studies that reveal a role for the ATPase activity of MukB. MukE and MukF together form an elongated dimeric frame, and MukF's C-terminal winged-helix domains (C-WHDs) bind MukB heads to constitute closed ring-like structures. Surprisingly, one of the two bound C-WHDs is forced to detach upon ATP-mediated engagement of MukB heads. This detachment reaction depends on the linker segment preceding the C-WHD, and mutations on the linker restrict cell growth. Thus ATP-dependent transient disruption of the MukB-MukF interaction, which creates openings in condensin ring structures, is likely to be a critical feature of the functional mechanism of condensins.
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http://dx.doi.org/10.1016/j.cell.2008.10.050DOI Listing
January 2009

An insight into the mechanistic role of Beclin 1 and its inhibition by prosurvival Bcl-2 family proteins.

Autophagy 2008 May 6;4(4):519-20. Epub 2008 Mar 6.

Center for Biomolecular Recognition, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, Kyungbuk, Korea.

A multiprotein complex composed of Beclin 1, PI(3)KC3 and UVRAG promotes autophagosome formation, while this activity is suppressed by a cohort of antiapoptotic Bcl-2 family members. Recently, we showed that a viral Bcl-2 of murine gamma-herpesvirus 68, known as M11, binds to Beclin 1 with markedly high affinity in comparison with cellular Bcl-2 or Bcl-X(L) that interacts with Beclin 1 weakly.(1) Furthermore, the binding affinity directly correlated with the potency of inhibition of autophagosome formation in cells. Herein, we present additional data showing that Beclin 1 forms a large homo-oligomer, and this oligomerization is partly disrupted by the binding of M11. Oligomerized Beclin 1 is proposed to serve as a platform enabling a concerted action of many molecules of the associating proteins, including Bif-1 that could be directly involved in autophagosome biogenesis on membranes owing to its BAR domain.
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http://dx.doi.org/10.4161/auto.5846DOI Listing
May 2008

Structural and biochemical bases for the inhibition of autophagy and apoptosis by viral BCL-2 of murine gamma-herpesvirus 68.

PLoS Pathog 2008 Feb;4(2):e25

Division of Molecular and Life Sciences, Center for Biomolecular Recognition, Pohang University of Science and Technology, Pohang, Kyungbuk, Korea.

All gammaherpesviruses express homologues of antiapoptotic B-cell lymphoma-2 (BCL-2) to counter the clearance of infected cells by host antiviral defense machineries. To gain insights into the action mechanisms of these viral BCL-2 proteins, we carried out structural and biochemical analyses on the interactions of M11, a viral BCL-2 of murine gamma-herpesvirus 68, with a fragment of proautophagic Beclin1 and BCL-2 homology 3 (BH3) domain-containing peptides derived from an array of proapoptotic BCL-2 family proteins. Mainly through hydrophobic interactions, M11 bound the BH3-like domain of Beclin1 with a dissociation constant of 40 nanomole, a markedly tighter affinity compared to the 1.7 micromolar binding affinity between cellular BCL-2 and Beclin1. Consistently, M11 inhibited autophagy more efficiently than BCL-2 in NIH3T3 cells. M11 also interacted tightly with a BH3 domain peptide of BAK and those of the upstream BH3-only proteins BIM, BID, BMF, PUMA, and Noxa, but weakly with that of BAX. These results collectively suggest that M11 potently inhibits Beclin1 in addition to broadly neutralizing the proapoptotic BCL-2 family in a similar but distinctive way from cellular BCL-2, and that the Beclin1-mediated autophagy may be a main target of the virus.
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http://dx.doi.org/10.1371/journal.ppat.0040025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2222952PMC
February 2008

Structural basis for protein recognition by B30.2/SPRY domains.

Mol Cell 2006 Dec;24(6):967-76

Center for Biomolecular Recognition, Department of Life Sciences, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, Kyungbuk, 790-784, Korea.

B30.2/SPRY domains are found in numerous proteins that cover a wide spectrum of biological functions, including regulation of cytokine signaling and innate retroviral restriction. Herein, we report the crystal structure of the B30.2/SPRY domain of a SPRY domain-containing SOCS box (SSB) protein, GUSTAVUS, complexed with a 20 amino acid peptide derived from the RNA helicase VASA, revealing how these domains recognize target proteins. The peptide-binding site is conformationally rigid and has a preformed pocket. The interaction between the pocket and the Asp-Ile-Asn-Asn-Asn-Asn sequence within the peptide accounts for the high-affinity binding between GUSTAVUS and VASA. This observation led to a facile identification of the Glu-Leu-Asn-Asn-Asn-Leu sequence as the recognition motif in a proapoptotic protein Par-4 for its interaction with a GUSTAVUS homolog, SSB-1. Ensuing analyses indicated that many B30.2/SPRY domains have a similar preformed pocket, which would allow them to bind multiple targets.
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http://dx.doi.org/10.1016/j.molcel.2006.11.009DOI Listing
December 2006

Structural and functional insights into the B30.2/SPRY domain.

EMBO J 2006 Mar 23;25(6):1353-63. Epub 2006 Feb 23.

Division of Molecular and Life Sciences, Department of Life Sciences, Center for Biomolecular Recognition, Pohang University of Science and Technology, Pohang, Kyungbuk, Korea.

The B30.2/SPRY domain is present in approximately 700 eukaryotic (approximately 150 human) proteins, including medically important proteins such as TRIM5alpha and Pyrin. Nonetheless, the functional role of this modular domain remained unclear. Here, we report the crystal structure of an SPRY-SOCS box family protein GUSTAVUS in complex with Elongins B and C, revealing a highly distorted two-layered beta-sandwich core structure of its B30.2/SPRY domain. Ensuing studies identified one end of the beta-sandwich as the surface interacting with an RNA helicase VASA with a 40 nM dissociation constant. The sequence variation in TRIM5alpha responsible for HIV-1 restriction and most of the mutations in Pyrin causing familial Mediterranean fever map on this surface, implicating the corresponding region in many B30.2/SPRY domains as the ligand-binding site. The amino acids lining the binding surface are highly variable among the B30.2/SPRY domains, suggesting that these domains are protein-interacting modules, which recognize a specific individual partner protein rather than a consensus sequence motif.
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http://dx.doi.org/10.1038/sj.emboj.7600994DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1422157PMC
March 2006