Publications by authors named "Ji-Joon Song"

66 Publications

A Novel N-terminal Region to Chromodomain in CHD7 is Required for the Efficient Remodeling Activity.

J Mol Biol 2021 Jun 21;433(18):167114. Epub 2021 Jun 21.

Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), KAIST Institute of BioCentury, Daejeon 34141, Korea. Electronic address:

Chromodomain-Helicase DNA binding protein 7 (CHD7) is an ATP dependent chromatin remodeler involved in maintaining open chromatin structure. Mutations of CHD7 gene causes multiple developmental disorders, notably CHARGE syndrome. However, there is not much known about the molecular mechanism by which CHD7 remodels nucleosomes. Here, we performed biochemical and biophysical analysis on CHD7 chromatin remodeler and uncover that N-terminal to the Chromodomain (N-CRD) interacts with nucleosome and contains a high conserved arginine stretch, which is reminiscent of arginine anchor. Importantly, this region is required for efficient ATPase stimulation and nucleosome remodeling activity of CHD7. Furthermore, smFRET analysis shows the mutations in the N-CRD causes the defects in remodeling activity. Collectively, our results uncover the functional importance of a previously unidentified N-terminal region in CHD7 and implicate that the multiple domains in chromatin remodelers are involved in regulating their activities.
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http://dx.doi.org/10.1016/j.jmb.2021.167114DOI Listing
June 2021

Antigen-Presenting, Self-Assembled Protein Nanobarrels as an Adjuvant-Free Vaccine Platform against Influenza Virus.

ACS Nano 2021 06 11;15(6):10722-10732. Epub 2021 Jun 11.

Department of Biological Sciences, KAIST Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Daejeon 34141, Republic of Korea.

Although naturally occurring, self-assembled protein nanoarchitectures have been utilized as antigen-delivery carriers, and the inability of such carriers to elicit immunogenicity requires additional use of strong adjuvants. Here, we report an immunogenic outer membrane protein BP26-derived nanoarchitecture displaying the influenza extracellular domain of matrix protein-2 (M2e) as a vaccine platform against influenza virus. Genetic engineering of a monomeric BP26 containing four or eight tandem repeats of M2e resulted in a hollow barrel-shaped nanoarchitecture (BP26-M2e nanobarrel). Immunization with BP26-M2e nanobarrels induced a strong M2e-specific humoral immune response that was much greater than that of a physical mixture of soluble M2e and BP26, with or without the use of an alum adjuvant. An anti-M2e antibody generated by BP26-M2e nanobarrel-immunized mice specifically bound to influenza virus-infected cells. Furthermore, in viral challenge tests, BP26-M2e nanobarrels effectively protected mice from influenza virus infection-associated death, even without the use of a conventional adjuvant. A mechanism study revealed that both M2e-specific antibody-dependent cellular cytotoxicity and T cell responses are involved in the vaccine efficacy of BP26-M2e nanobarrels. These findings suggest that the BP26-based nanobarrel developed here represents a versatile vaccine platform that can be used against various viral infections.
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http://dx.doi.org/10.1021/acsnano.1c04078DOI Listing
June 2021

Single-Molecule Imaging Reveals the Mechanism Underlying Histone Loading of AAA+ ATPase Abo1.

Mol Cells 2021 Feb;44(2):79-87

Department of Biological Sciences, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Korea.

Chromatin dynamics is essential for maintaining genomic integrity and regulating gene expression. Conserved bromodomain-containing AAA+ ATPases play important roles in nucleosome organization as histone chaperones. Recently, the high-resolution cryo-electron microscopy structures of Abo1 revealed that it forms a hexameric ring and undergoes a conformational change upon ATP hydrolysis. In addition, single-molecule imaging demonstrated that Abo1 loads H3-H4 histones onto DNA in an ATP hydrolysis-dependent manner. However, the molecular mechanism by which Abo1 loads histones remains unknown. Here, we investigated the details concerning Abo1-mediated histone loading onto DNA and the Abo1- DNA interaction using single-molecule imaging techniques and biochemical assays. We show that Abo1 does not load H2A-H2B histones. Interestingly, Abo1 deposits multiple copies of H3-H4 histones as the DNA length increases and requires at least 80 bp DNA. Unexpectedly, Abo1 weakly binds DNA regardless of ATP, and neither histone nor DNA stimulates the ATP hydrolysis activity of Abo1. Based on our results, we propose an allosteric communication model in which the ATP hydrolysis of Abo1 changes the configuration of histones to facilitate their deposition onto DNA.
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http://dx.doi.org/10.14348/molcells.2021.2242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7941004PMC
February 2021

Cryo-EM structure of Vibrio cholerae aldehyde-alcohol dehydrogenase spirosomes.

Biochem Biophys Res Commun 2021 01 23;536:38-44. Epub 2020 Dec 23.

Center for Natural Sciences, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Daejeon, 34141, Republic of Korea. Electronic address:

Aldehyde-alcohol dehydrogenase (AdhE) is a metabolic enzyme and virulence factor in bacteria. E. coli AdhE (eAdhE) multimerizes into spirosomes that are essential for enzymatic activity. However, it is unknown whether AdhE structure is conserved in divergent bacteria. Here, we present the cryo-EM structure of AdhE (vAdhE) from Vibrio cholerae to 4.31 Å resolution. Overall, vAdhE spirosomes are similar to eAdhE with conserved subunit arrangement. However, divergences in key oligomerization residues cause vAdhE to form labile spirosomes with lower enzymatic activity. Mutating the vAdhE oligomerization interface to mimic eAdhE increases spirosome stability and enzymatic activity to levels comparable to eAdhE. These results support the generality of AdhE spirosome structures, and provide a structural basis to target vAdhE to attenuate bacterial virulence.
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http://dx.doi.org/10.1016/j.bbrc.2020.12.040DOI Listing
January 2021

Mutant Huntingtin Is Cleared from the Brain via Active Mechanisms in Huntington Disease.

J Neurosci 2021 01 11;41(4):780-796. Epub 2020 Dec 11.

Burnett School of Biomedical Sciences, University of Central Florida, Orlando, Florida 32828

Huntington disease (HD) is a neurodegenerative disease caused by a CAG trinucleotide repeat expansion in the huntingtin () gene. Therapeutics that lower HTT have shown preclinical promise and are being evaluated in clinical trials. However, clinical assessment of brain HTT lowering presents challenges. We have reported that mutant HTT (mHTT) in the CSF of HD patients correlates with clinical measures, including disease burden as well as motor and cognitive performance. We have also shown that lowering HTT in the brains of HD mice results in correlative reduction of mHTT in the CSF, prompting the use of this measure as an exploratory marker of target engagement in clinical trials. In this study, we investigate the mechanisms of mHTT clearance from the brain in adult mice of both sexes to elucidate the significance of therapy-induced CSF mHTT changes. We demonstrate that, although neurodegeneration increases CSF mHTT concentrations, mHTT is also present in the CSF of mice in the absence of neurodegeneration. Importantly, we show that secretion of mHTT from cells in the CNS followed by glymphatic clearance from the extracellular space contributes to mHTT in the CSF. Furthermore, we observe secretion of wild type HTT from healthy control neurons, suggesting that HTT secretion is a normal process occurring in the absence of pathogenesis. Overall, our data support both passive release and active clearance of mHTT into CSF, suggesting that its treatment-induced changes may represent a combination of target engagement and preservation of neurons. Changes in CSF mutant huntingtin (mHTT) are being used as an exploratory endpoint in HTT lowering clinical trials for the treatment of Huntington disease (HD). Recently, it was demonstrated that intrathecal administration of a HTT lowering agent leads to dose-dependent reduction of CSF mHTT in HD patients. However, little is known about how HTT, an intracellular protein, reaches the extracellular space and ultimately the CSF. Our findings that HTT enters CSF by both passive release and active secretion followed by glymphatic clearance may have significant implications for interpretation of treatment-induced changes of CSF mHTT in clinical trials for HD.
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http://dx.doi.org/10.1523/JNEUROSCI.1865-20.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7842749PMC
January 2021

Quantification of purified endogenous miRNAs with high sensitivity and specificity.

Nat Commun 2020 11 27;11(1):6033. Epub 2020 Nov 27.

Department of Physics and Astronomy, and Institute of Applied Physics, Seoul National University, Seoul, Republic of Korea.

MicroRNAs (miRNAs) are short (19-24 nt) non-coding RNAs that suppress the expression of protein coding genes at the post-transcriptional level. Differential expression profiles of miRNAs across a range of diseases have emerged as powerful biomarkers, making a reliable yet rapid profiling technique for miRNAs potentially essential in clinics. Here, we report an amplification-free multi-color single-molecule imaging technique that can profile purified endogenous miRNAs with high sensitivity, specificity, and reliability. Compared to previously reported techniques, our technique can discriminate single base mismatches and single-nucleotide 3'-tailing with low false positive rates regardless of their positions on miRNA. By preloading probes in Thermus thermophilus Argonaute (TtAgo), miRNAs detection speed is accelerated by more than 20 times. Finally, by utilizing the well-conserved linearity between single-molecule spot numbers and the target miRNA concentrations, the absolute average copy numbers of endogenous miRNA species in a single cell can be estimated. Thus our technique, Ago-FISH (Argonaute-based Fluorescence In Situ Hybridization), provides a reliable way to accurately profile various endogenous miRNAs on a single miRNA sensing chip.
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http://dx.doi.org/10.1038/s41467-020-19865-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7699633PMC
November 2020

Yeast Chd1p Unwraps the Exit Side DNA upon ATP Binding to Facilitate the Nucleosome Translocation Occurring upon ATP Hydrolysis.

Biochemistry 2020 12 11;59(47):4481-4487. Epub 2020 Nov 11.

Department of Physics and Astronomy, Institute of Applied Physics, Seoul National University, Seoul 08826, Republic of Korea.

Chromodomain-helicase-DNA-binding protein 1 (CHD1) remodels chromatin by translocating nucleosomes along DNA, but its mechanism remains poorly understood. We use single-molecule fluorescence experiments to clarify the mechanism by which yeast CHD1 (Chd1p) remodels nucleosomes. We find that binding of ATP to Chd1p induces transient unwrapping of the DNA on the exit side of the nucleosome, facilitating nucleosome translocation. ATP hydrolysis is required to induce nucleosome translocation. The unwrapped DNA after translocation is then rewrapped after the release of the hydrolyzed nucleotide and phosphate, revealing that each step of the ATP hydrolysis cycle is responsible for a distinct step of nucleosome remodeling. These results show that Chd1p remodels nucleosomes via a mechanism that is unique among the other ATP-dependent chromatin remodelers.
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http://dx.doi.org/10.1021/acs.biochem.0c00747DOI Listing
December 2020

RUNX3 methylation drives hypoxia-induced cell proliferation and antiapoptosis in early tumorigenesis.

Cell Death Differ 2021 Apr 28;28(4):1251-1269. Epub 2020 Oct 28.

Vessel-Organ Interaction Research Center, VOICE (MRC), Department of Molecular Pathophysiology, College of Pharmacy, Kyungpook National University, Daegu, 41566, Republic of Korea.

Inactivation of tumor suppressor Runt-related transcription factor 3 (RUNX3) plays an important role during early tumorigenesis. However, posttranslational modifications (PTM)-based mechanism for the inactivation of RUNX3 under hypoxia is still not fully understood. Here, we demonstrate a mechanism that G9a, lysine-specific methyltransferase (KMT), modulates RUNX3 through PTM under hypoxia. Hypoxia significantly increased G9a protein level and G9a interacted with RUNX3 Runt domain, which led to increased methylation of RUNX3 at K129 and K171. This methylation inactivated transactivation activity of RUNX3 by reducing interactions with CBFβ and p300 cofactors, as well as reducing acetylation of RUNX3 by p300, which is involved in nucleocytoplasmic transport by importin-α1. G9a-mediated methylation of RUNX3 under hypoxia promotes cancer cell proliferation by increasing cell cycle or cell division, while suppresses immune response and apoptosis, thereby promoting tumor growth during early tumorigenesis. Our results demonstrate the molecular mechanism of RUNX3 inactivation by G9a-mediated methylation for cell proliferation and antiapoptosis under hypoxia, which can be a therapeutic or preventive target to control tumor growth during early tumorigenesis.
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http://dx.doi.org/10.1038/s41418-020-00647-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8027031PMC
April 2021

EMPAS: Electron Microscopy Screening for Endogenous Protein Architectures.

Mol Cells 2020 Sep;43(9):804-812

Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Korea.

In cells, proteins form macromolecular complexes to execute their own unique roles in biological processes. Conventional structural biology methods adopt a bottom-up approach starting from defined sets of proteins to investigate the structures and interactions of protein complexes. However, this approach does not reflect the diverse and complex landscape of endogenous molecular architectures. Here, we introduce a top-down approach called Electron Microscopy screening for endogenous Protein ArchitectureS (EMPAS) to investigate the diverse and complex landscape of endogenous macromolecular architectures in an unbiased manner. By applying EMPAS, we discovered a spiral architecture and identified it as AdhE. Furthermore, we performed screening to examine endogenous molecular architectures of human embryonic stem cells (hESCs), mouse brains, cyanobacteria and plant leaves, revealing their diverse repertoires of molecular architectures. This study suggests that EMPAS may serve as a tool to investigate the molecular architectures of endogenous macromolecular proteins.
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http://dx.doi.org/10.14348/molcells.2020.0163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7528680PMC
September 2020

The Polyglutamine Expansion at the N-Terminal of Huntingtin Protein Modulates the Dynamic Configuration and Phosphorylation of the C-Terminal HEAT Domain.

Structure 2020 09 14;28(9):1035-1050.e8. Epub 2020 Jul 14.

Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), KI for the BioCentury, Daejeon 34141, Korea. Electronic address:

The polyQ expansion in huntingtin protein (HTT) is the prime cause of Huntington's disease (HD). The recent cryoelectron microscopy (cryo-EM) structure of HTT-HAP40 complex provided the structural information on its HEAT-repeat domains. Here, we present analyses of the impact of polyQ length on the structure and function of HTT via an integrative structural and biochemical approach. The cryo-EM analysis of normal (Q23) and disease (Q78) type HTTs shows that the structures of apo HTTs significantly differ from the structure of HTT in a HAP40 complex and that the polyQ expansion induces global structural changes in the relative movements among the HTT domains. In addition, we show that the polyQ expansion alters the phosphorylation pattern across HTT and that Ser2116 phosphorylation in turn affects the global structure and function of HTT. These results provide a molecular basis for the effect of the polyQ segment on HTT structure and activity, which may be important for HTT pathology.
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http://dx.doi.org/10.1016/j.str.2020.06.008DOI Listing
September 2020

Aldehyde-alcohol dehydrogenase undergoes structural transition to form extended spirosomes for substrate channeling.

Commun Biol 2020 06 10;3(1):298. Epub 2020 Jun 10.

Department of Biological Sciences, KI for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, 34141, Korea.

Aldehyde-alcohol dehydrogenase (AdhE) is an enzyme responsible for converting acetyl-CoA to ethanol via acetaldehyde using NADH. AdhE is composed of two catalytic domains of aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH), and forms a spirosome architecture critical for AdhE activity. Here, we present the atomic resolution (3.43 Å) cryo-EM structure of AdhE spirosomes in an extended conformation. The cryo-EM structure shows that AdhE spirosomes undergo a structural transition from compact to extended forms, which may result from cofactor binding. This transition leads to access to a substrate channel between ALDH and ADH active sites. Furthermore, prevention of this structural transition by crosslinking hampers the activity of AdhE, suggesting that the structural transition is important for AdhE activity. This work provides a mechanistic understanding of the regulation mechanisms of AdhE activity via structural transition, and a platform to modulate AdhE activity for developing antibiotics and for facilitating biofuel production.
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http://dx.doi.org/10.1038/s42003-020-1030-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7286902PMC
June 2020

Identification of the Antidepressant Vilazodone as an Inhibitor of Inositol Polyphosphate Multikinase by Structure-Based Drug Repositioning.

Mol Cells 2020 Mar;43(3):222-227

Department of Biological Sciences, KAIST, Daejeon 34141, Korea.

Inositol polyphosphate multikinase (IPMK) is required for the biosynthesis of inositol phosphates (IPs) through the phosphorylation of multiple IP metabolites such as IP3 and IP4. The biological significance of IPMK's catalytic actions to regulate cellular signaling events such as growth and metabolism has been studied extensively. However, pharmacological reagents that inhibit IPMK have not yet been identified. We employed a structure-based virtual screening of publicly available U.S. Food and Drug Administration-approved drugs and chemicals that identified the antidepressant, vilazodone, as an IPMK inhibitor. Docking simulations and pharmacophore analyses showed that vilazodone has a higher affinity for the ATP-binding catalytic region of IPMK than ATP and we validated that vilazodone inhibits IPMK's IP kinase activities in vitro . The incubation of vilazodone with NIH3T3-L1 fibroblasts reduced cellular levels of IP5 and other highly phosphorylated IPs without influencing IP4 levels. We further found decreased Akt phosphorylation in vilazodone-treated HCT116 cancer cells. These data clearly indicate selective cellular actions of vilazodone against IPMK-dependent catalytic steps in IP metabolism and Akt activation. Collectively, our data demonstrate vilazodone as a method to inhibit cellular IPMK, providing a valuable pharmacological agent to study and target the biological and pathological processes governed by IPMK.
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http://dx.doi.org/10.14348/molcells.2020.0051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7103885PMC
March 2020

Spectral and photochemical diversity of tandem cysteine cyanobacterial phytochromes.

J Biol Chem 2020 05 17;295(19):6754-6766. Epub 2020 Mar 17.

Department of Biological Sciences, Chungnam National University, Daejeon 34134, Korea

The atypical trichromatic cyanobacterial phytochrome TP1 from ATCC 29133 is a linear tetrapyrrole (bilin)-binding photoreceptor protein that possesses tandem-cysteine residues responsible for shifting its light-sensing maximum to the violet spectral region. Using bioinformatics and phylogenetic analyses, here we established that tandem-cysteine cyanobacterial phytochromes (TCCPs) compose a well-supported monophyletic phytochrome lineage distinct from prototypical red/far-red cyanobacterial phytochromes. To investigate the light-sensing diversity of this family, we compared the spectroscopic properties of TP1 (here renamed TCCP) with those of three phylogenetically diverged TCCPs identified in the draft genomes of sp. PCC7910, sp. PCC10023, and sp. PCC7513. Recombinant photosensory core modules of TCCP, TCCP, and TCCP exhibited violet-blue-absorbing dark-states consistent with dual thioether-linked phycocyanobilin (PCB) chromophores. Photoexcitation generated singly-linked photoproduct mixtures with variable ratios of yellow-orange and red-absorbing species. The photoproduct ratio was strongly influenced by pH and by mutagenesis of TCCP- and phytochrome-specific signature residues. Our experiments support the conclusion that both photoproduct species possess protonated 15 bilin chromophores, but differ in the ionization state of the noncanonical "second" cysteine sulfhydryl group. We found that the ionization state of this and other residues influences subsequent conformational change and downstream signal transmission. We also show that tandem-cysteine phytochromes present in eukaryotes possess similar amino acid substitutions within their chromophore-binding pocket, which tune their spectral properties in an analogous fashion. Taken together, our findings provide a roadmap for tailoring the wavelength specificity of plant phytochromes to optimize plant performance in diverse natural and artificial light environments.
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http://dx.doi.org/10.1074/jbc.RA120.012950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7212627PMC
May 2020

Structural basis of nucleosome assembly by the Abo1 AAA+ ATPase histone chaperone.

Nat Commun 2019 12 17;10(1):5764. Epub 2019 Dec 17.

Department of Biological Sciences and KI for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea.

The fundamental unit of chromatin, the nucleosome, is an intricate structure that requires histone chaperones for assembly. ATAD2 AAA+ ATPases are a family of histone chaperones that regulate nucleosome density and chromatin dynamics. Here, we demonstrate that the fission yeast ATAD2 homolog, Abo1, deposits histone H3-H4 onto DNA in an ATP-hydrolysis-dependent manner by in vitro reconstitution and single-tethered DNA curtain assays. We present cryo-EM structures of an ATAD2 family ATPase to atomic resolution in three different nucleotide states, revealing unique structural features required for histone loading on DNA, and directly visualize the transitions of Abo1 from an asymmetric spiral (ATP-state) to a symmetric ring (ADP- and apo-states) using high-speed atomic force microscopy (HS-AFM). Furthermore, we find that the acidic pore of ATP-Abo1 binds a peptide substrate which is suggestive of a histone tail. Based on these results, we propose a model whereby Abo1 facilitates H3-H4 loading by utilizing ATP.
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http://dx.doi.org/10.1038/s41467-019-13743-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6917787PMC
December 2019

Prolonged half-life of small-sized therapeutic protein using serum albumin-specific protein binder.

J Control Release 2019 12 22;315:31-39. Epub 2019 Oct 22.

Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea. Electronic address:

Many small-sized proteins and peptides, such as cytokines and hormones, are clinically used for the treatment of a variety of diseases. However, their short half-life in blood owing to fast renal clearance usually results in a low therapeutic efficacy and frequent dosing. Here we present the development of a human serum albumin (HSA)-specific protein binder with a binding affinity of 4.3nM through a phage display selection and modular evolution approach to extend the blood half-life of a small-sized therapeutic protein. As a proof-of-concept, the protein binder composed of LRR (Leucine-rich repeat) modules was genetically fused to the N-terminus of Glucagon-like Peptide-1 (GLP-1). The fused GLP-1 was shown to have a significantly improved pharmacokinetic property: The terminal half-life of the fused GLP-1 increased to approximately 10h, and the area under the curve was 5-times higher than that of the control. The utility and potential of our approach was demonstrated by the efficient control of the blood glucose level in type-2 diabetes mouse models using the HSA-specific protein binder-fused GLP-1 over a prolonged time period. The present approach can be effectively used in enhancing the efficacy of small-sized therapeutic proteins and peptides through an enhanced blood circulation time.
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http://dx.doi.org/10.1016/j.jconrel.2019.09.017DOI Listing
December 2019

Aldehyde-alcohol dehydrogenase forms a high-order spirosome architecture critical for its activity.

Nat Commun 2019 10 4;10(1):4527. Epub 2019 Oct 4.

Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, 34141, Korea.

Aldehyde-alcohol dehydrogenase (AdhE) is a key enzyme in bacterial fermentation, converting acetyl-CoA to ethanol, via two consecutive catalytic reactions. Here, we present a 3.5 Å resolution cryo-EM structure of full-length AdhE revealing a high-order spirosome architecture. The structure shows that the aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) active sites reside at the outer surface and the inner surface of the spirosome respectively, thus topologically separating these two activities. Furthermore, mutations disrupting the helical structure abrogate enzymatic activity, implying that formation of the spirosome structure is critical for AdhE activity. In addition, we show that this spirosome structure undergoes conformational change in the presence of cofactors. This work presents the atomic resolution structure of AdhE and suggests that the high-order helical structure regulates its enzymatic activity.
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http://dx.doi.org/10.1038/s41467-019-12427-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6778083PMC
October 2019

Editorial overview: The future after the cryo-EM resolution revolution.

Curr Opin Struct Biol 2019 10 31;58:iii-iv. Epub 2019 Aug 31.

Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Republic of Korea. Electronic address:

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http://dx.doi.org/10.1016/j.sbi.2019.07.007DOI Listing
October 2019

The crystal structure of Capicua HMG-box domain complexed with the ETV5-DNA and its implications for Capicua-mediated cancers.

FEBS J 2019 12 1;286(24):4951-4963. Epub 2019 Aug 1.

Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea.

Capicua (CIC) is a transcriptional repressor and functions downstream of the receptor tyrosine kinase (RTK) signaling pathway. Somatic mutations found in the HMG-box DNA binding domain in CIC have been implicated in several cancers such as oligodendroglioma, oligoastrocytoma, and adenocarcinoma. However, the molecular basis of the DNA binding of CIC and the effect of the somatic mutations found in cancers on DNA binding have not been investigated. Here, we report the crystal structure of the HMG-box domain of CIC complexed with its target DNA, the promoter of Ets Translocation Variant 5 (ETV5). The structure shows that the HMG-box domain has an L-shaped structure and recognizes the minor groove leading to DNA bending. Our structure combined with an electrophoretic mobility shift assay (EMSA) revealed that cancer-associated mutations in the HMG-box domain abrogate the interaction with DNA. These results provide the molecular insight into the DNA binding of CIC and reveal the effects of carcinogenic mutations on DNA binding.
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http://dx.doi.org/10.1111/febs.15008DOI Listing
December 2019

The big picture of chromatin biology by cryo-EM.

Curr Opin Struct Biol 2019 10 22;58:76-87. Epub 2019 Jun 22.

Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea. Electronic address:

Modifications of chromatin structure are one of the key mechanisms regulating epigenetic gene expression. Proteins involved in chromatin modification mainly function as large multi-subunit complexes, and each component in the complex contributes to the function and activity of the complex. However, little is known about the structures of whole complexes and the mechanisms by which the chromatin-modifying complexes function, the functional roles of each component in the complexes, and how the complexes recognize the central unit of chromatin, the nucleosome. This lack of information is partially due to the lack of structural information for whole complexes. Recent advances in cryo-EM have begun to reveal the structures of whole chromatin-modifying complexes that enable us to understand the big picture of chromatin biology. In this review, we discuss the recent discoveries related to the mechanisms of chromatin-modifying complexes.
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http://dx.doi.org/10.1016/j.sbi.2019.05.017DOI Listing
October 2019

Targeting protein and peptide therapeutics to the heart via tannic acid modification.

Nat Biomed Eng 2018 05 30;2(5):304-317. Epub 2018 Apr 30.

Department of Chemistry, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea.

Systemic injection into blood vessels is the most common method of drug administration. However, targeting drugs to the heart is challenging, owing to its dynamic mechanical motions and large cardiac output. Here, we show that the modification of protein and peptide therapeutics with tannic acid-a flavonoid found in plants that adheres to extracellular matrices, elastins and collagens-improves their ability to specifically target heart tissue. Tannic-acid-modified (TANNylated) proteins do not adsorb on endothelial glycocalyx layers in blood vessels, yet they penetrate the endothelium to thermodynamically bind to myocardium extracellular matrix before being internalized by myoblasts. In a rat model of myocardial ischaemia-reperfusion injury, TANNylated basic fibroblast growth factor significantly reduced infarct size and increased cardiac function. TANNylation of systemically injected therapeutic proteins, peptides or viruses may enhance the treatment of heart diseases.
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http://dx.doi.org/10.1038/s41551-018-0227-9DOI Listing
May 2018

Structural basis of recognition and destabilization of the histone H2B ubiquitinated nucleosome by the DOT1L histone H3 Lys79 methyltransferase.

Genes Dev 2019 06 28;33(11-12):620-625. Epub 2019 Mar 28.

Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Korea.

DOT1L is a histone H3 Lys79 methyltransferase whose activity is stimulated by histone H2B Lys120 ubiquitination, suggesting cross-talk between histone H3 methylation and H2B ubiquitination. Here, we present cryo-EM structures of DOT1L complexes with unmodified or H2B ubiquitinated nucleosomes, showing that DOT1L recognizes H2B ubiquitin and the H2A/H2B acidic patch through a C-terminal hydrophobic helix and an arginine anchor in DOT1L, respectively. Furthermore, the structures combined with single-molecule FRET experiments show that H2B ubiquitination enhances a noncatalytic function of the DOT1L-destabilizing nucleosome. These results establish the molecular basis of the cross-talk between H2B ubiquitination and H3 Lys79 methylation as well as nucleosome destabilization by DOT1L.
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http://dx.doi.org/10.1101/gad.323790.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546062PMC
June 2019

Structural Basis of MRG15-Mediated Activation of the ASH1L Histone Methyltransferase by Releasing an Autoinhibitory Loop.

Structure 2019 05 28;27(5):846-852.e3. Epub 2019 Feb 28.

Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Korea. Electronic address:

Human ASH1L is the catalytic subunit of the conserved histone methyltransferase (HMTase) complex AMC that dimethylates lysine 36 in histone H3 (H3K36me2) to promote gene transcription in mammals and flies. Unlike AMC, ASH1L alone shows poor catalytic activity, because access to its substrate binding pocket is blocked by an autoinhibitory loop (AI loop) from the postSET domain. We report the crystal structure of the minimal catalytic active AMC complex containing ASH1L and its partner subunit MRG15. The structure reveals how binding of the MRG domain of MRG15 to a conserved FxLP motif in ASH1L results in the displacement of the AI loop to permit substrates to access the catalytic pocket of the ASH1L SET domain. Together, ASH1L activation by MRG15 therefore represents a delicate regulatory mechanism for how a cofactor activates an SET domain HMTase by releasing autoinhibition.
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http://dx.doi.org/10.1016/j.str.2019.01.016DOI Listing
May 2019

ANKRD9 is associated with tumor suppression as a substrate receptor subunit of ubiquitin ligase.

Biochim Biophys Acta Mol Basis Dis 2018 10 3;1864(10):3145-3153. Epub 2018 Jul 3.

Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea. Electronic address:

Background: Human ANKRD9 (ankyrin repeat domain 9) expression is altered in some cancers.

Methods: We tested genetic association of ANKRD9 with gastric cancer susceptibility and examined functional association of ANKRD9 with altered proliferation of MKN45 gastric cancer cells. We then identified ANKRD9-binding partners in HEK 293 embryonic kidney cells using quantitative proteomics, western blotting and complex reconstitution assays. We finally demonstrated ANKRD9's role of recognizing substrates for ubiquitination using in vitro ubiquitylation assay.

Results: ANKRD9 is associated with cancer susceptibility in a comparison of single-nucleotide polymorphisms between 1092 gastric cancer patients and 1206 healthy controls. ANKRD9 depletion accelerates tumor progression by increasing cellular proliferation, piling up, and anchorage-independent growth of MKN45 cells. We discovered that ANKRD9 is a ubiquitin ligase substrate receptor subunit and has an anti-proliferative activity. ANKRD9 associates with CUL5 (not CUL2), ELOB, ELOC, and presumably RNF7 subunits, which together assemble into a cullin-RING superfamily E3 ligase complex. ANKRD9 belongs to the ASB family of proteins, which are characterized by the presence of ankyrin repeats and a SOCS box. In addition to its interactions with the other E3 ligase subunits, ANKRD9 interacts with two isoforms of inosine monophosphate dehydrogenase (IMPDH). These IMPDH isoforms are cognate substrates of the ANKRD9-containing E3 enzyme, which ubiquitinates them for proteasomal degradation. Their ubiquitination and turnover require the presence of ANKRD9.

Conclusion: ANKRD9, a previously unidentified E3 substrate receptor subunit, functions in tumor suppression by recognizing the oncoprotein IMPDH isoforms for E3 ubiquitination and proteasomal degradation.
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http://dx.doi.org/10.1016/j.bbadis.2018.07.001DOI Listing
October 2018

Novel DNA Aptamers that Bind to Mutant Huntingtin and Modify Its Activity.

Mol Ther Nucleic Acids 2018 Jun 16;11:416-428. Epub 2018 Mar 16.

Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Neurology, Harvard Medical School, Boston, MA 02114, USA. Electronic address:

The CAG repeat expansion that elongates the polyglutamine tract in huntingtin is the root genetic cause of Huntington's disease (HD), a debilitating neurodegenerative disorder. This seemingly slight change to the primary amino acid sequence alters the physical structure of the mutant protein and alters its activity. We have identified a set of G-quadruplex-forming DNA aptamers (MS1, MS2, MS3, MS4) that bind mutant huntingtin proximal to lysines K2932/K2934 in the C-terminal CTD-II domain. Aptamer binding to mutant huntingtin abrogated the enhanced polycomb repressive complex 2 (PRC2) stimulatory activity conferred by the expanded polyglutamine tract. In HD, but not normal, neuronal progenitor cells (NPCs), MS3 aptamer co-localized with endogenous mutant huntingtin and was associated with significantly decreased PRC2 activity. Furthermore, MS3 transfection protected HD NPCs against starvation-dependent stress with increased ATP. Therefore, DNA aptamers can preferentially target mutant huntingtin and modulate a gain of function endowed by the elongated polyglutamine segment. These mutant huntingtin binding aptamers provide novel molecular tools for delineating the effects of the HD mutation and encourage mutant huntingtin structure-based approaches to therapeutic development.
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http://dx.doi.org/10.1016/j.omtn.2018.03.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5992459PMC
June 2018

Regulation and function of H3K36 di-methylation by the trithorax-group protein complex AMC.

Development 2018 04 5;145(7). Epub 2018 Apr 5.

Max-Planck Institute of Biochemistry, Laboratory of Chromatin Biology, Am Klopferspitz 18, 82152 Martinsried, Germany

The Ash1 protein is a trithorax-group (trxG) regulator that antagonizes Polycomb repression at HOX genes. Ash1 di-methylates lysine 36 in histone H3 (H3K36me2) but how this activity is controlled and at which genes it functions is not well understood. We show that Ash1 protein purified from exists in a complex with MRG15 and Caf1 that we named AMC. In and human AMC, MRG15 binds a conserved FxLP motif near the Ash1 SET domain and stimulates H3K36 di-methylation on nucleosomes. -null and catalytic mutants show remarkably specific trxG phenotypes: stochastic loss of HOX gene expression and homeotic transformations in adults. In mutants lacking AMC, H3K36me2 bulk levels appear undiminished but H3K36me2 is reduced in the chromatin of HOX and other AMC-regulated genes. AMC therefore appears to act on top of the H3K36me2/me3 landscape generated by the major H3K36 methyltransferases NSD and Set2. Our analyses suggest that H3K36 di-methylation at HOX genes is the crucial physiological function of AMC and the mechanism by which the complex antagonizes Polycomb repression at these genes.
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http://dx.doi.org/10.1242/dev.163808DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5963871PMC
April 2018

Integrative Structural Investigation on the Architecture of Human Importin4_Histone H3/H4_Asf1a Complex and Its Histone H3 Tail Binding.

J Mol Biol 2018 03 31;430(6):822-841. Epub 2018 Jan 31.

Department of Biological Sciences, Cancer Metastasis Control Center, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Korea. Electronic address:

Importin4 transports histone H3/H4 in complex with Asf1a to the nucleus for chromatin assembly. Importin4 recognizes the nuclear localization sequence located at the N-terminal tail of histones. Here, we analyzed the structures and interactions of human Importin4, histones and Asf1a by cross-linking mass spectrometry, X-ray crystallography, negative-stain electron microscopy, small-angle X-ray scattering and integrative modeling. The cross-linking mass spectrometry data showed that the C-terminal region of Importin4 was extensively cross-linked with the histone H3 tail. We determined the crystal structure of the C-terminal region of Importin4 bound to the histone H3 peptide, thus revealing that the acidic patch in Importin4 accommodates the histone H3 tail, and that histone H3 Lys14 contributes to the interaction with Importin4. In addition, we show that Asf1a modulates the binding of histone H3/H4 to Importin4. Furthermore, the molecular architecture of the Importin4_histone H3/H4_Asf1a complex was produced through an integrative modeling approach. Overall, this work provides structural insights into how Importin4 recognizes histones and their chaperone complex.
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http://dx.doi.org/10.1016/j.jmb.2018.01.015DOI Listing
March 2018

Structural insights into the oligomerization of FtsH periplasmic domain from Thermotoga maritima.

Biochem Biophys Res Commun 2018 01 24;495(1):1201-1207. Epub 2017 Nov 24.

School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 61005, Republic of Korea; Steitz Center for Structural Biology, Gwangju Institute of Science and Technology, Gwangju 61005, Republic of Korea. Electronic address:

Prompt removal of misfolded membrane proteins and misassembled membrane protein complexes is essential for membrane homeostasis. However, the elimination of these toxic proteins from the hydrophobic membrane environment has high energetic barriers. The transmembrane protein, FtsH, is the only known ATP-dependent protease responsible for this task. The mechanisms by which FtsH recognizes, unfolds, translocates, and proteolyzes its substrates remain unclear. The structure and function of the ATPase and protease domains of FtsH have been previously characterized while the role of the FtsH periplasmic domain has not clearly identified. Here, we report the 1.5-1.95 Å resolution crystal structures of the Thermotoga maritima FtsH periplasmic domain (tmPD) and describe the dynamic features of tmPD oligomerization.
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http://dx.doi.org/10.1016/j.bbrc.2017.11.158DOI Listing
January 2018

Biophysical characterization of the basic cluster in the transcription repression domain of human MeCP2 with AT-rich DNA.

Biochem Biophys Res Commun 2018 01 31;495(1):145-150. Epub 2017 Oct 31.

College of Pharmacy, Korea University, 2511 Sejong-ro, Sejong 30019, South Korea. Electronic address:

MeCP2 is a chromatin associated protein which is highly expressed in brain and relevant with Rett syndrome (RTT). There are AT-hook motifs in MeCP2 which can bind with AT-rich DNA, suggesting a role in chromatin binding. Here, we report the identification and characterization of another AT-rich DNA binding motif (residues 295 to 313) from the C-terminal transcription repression domain of MeCP2 by nuclear magnetic resonance (NMR) and isothermal calorimetry (ITC). This motif shows a micromolar affinity to AT-rich DNA, and it binds to the minor groove of DNA like AT-hook motifs. Together with the previous studies, our results provide an insight into a critical role of this motif in chromatin structure and function.
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http://dx.doi.org/10.1016/j.bbrc.2017.10.169DOI Listing
January 2018

Structure-based nuclear import mechanism of histones H3 and H4 mediated by Kap123.

Elife 2017 10 16;6. Epub 2017 Oct 16.

Department of Biological Chemistry, University of Michigan Medical School, Michigan, United States.

Kap123, a major karyopherin protein of budding yeast, recognizes the nuclear localization signals (NLSs) of cytoplasmic histones H3 and H4 and translocates them into the nucleus during DNA replication. Mechanistic questions include H3- and H4-NLS redundancy toward Kap123 and the role of the conserved diacetylation of cytoplasmic H4 (K5ac and K12ac) in Kap123-mediated histone nuclear translocation. Here, we report crystal structures of full-length Kap123 alone and in complex with H3- and H4-NLSs. Structures reveal the unique feature of Kap123 that possesses two discrete lysine-binding pockets for NLS recognition. Structural comparison illustrates that H3- and H4-NLSs share at least one of two lysine-binding pockets, suggesting that H3- and H4-NLSs are mutually exclusive. Additionally, acetylation of key lysine residues at NLS, particularly H4-NLS diacetylation, weakens the interaction with Kap123. These data support that cytoplasmic histone H4 diacetylation weakens the Kap123-H4-NLS interaction thereby facilitating histone Kap123-H3-dependent H3:H4/Asf1 complex nuclear translocation.
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http://dx.doi.org/10.7554/eLife.30244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5677370PMC
October 2017

Arabidopsis FRIGIDA stimulates EFS histone H3 Lys36 methyltransferase activity.

Plant Cell Rep 2017 07 5;36(7):1183-1185. Epub 2017 Jun 5.

Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, 34141, Korea.

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http://dx.doi.org/10.1007/s00299-017-2161-9DOI Listing
July 2017
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