Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Stable inheritance of DNA methylation is critical for maintaining differentiated phenotypes in multicellular organisms. We have recently identified dual mono-ubiquitylation of histone H3 (H3Ub2) by UHRF1 as an essential mechanism to recruit DNMT1 to chromatin. Here, we show that PCNA-associated factor 15 (PAF15) undergoes UHRF1-dependent dual mono-ubiquitylation (PAF15Ub2) on chromatin in a DNA replication-coupled manner. This event will, in turn, recruit DNMT1. During early S-phase, UHRF1 preferentially ubiquitylates PAF15, whereas H3Ub2 predominates during late S-phase. H3Ub2 is enhanced under PAF15 compromised conditions, suggesting that H3Ub2 serves as a backup for PAF15Ub2. In mouse ES cells, loss of PAF15Ub2 results in DNA hypomethylation at early replicating domains. Together, our results suggest that there are two distinct mechanisms underlying replication timing-dependent recruitment of DNMT1 through PAF15Ub2 and H3Ub2, both of which are prerequisite for high fidelity DNA methylation inheritance.

DOI： 10.1038/s41467-020-15006-4

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

The protein UHRF1 is crucial for DNA methylation maintenance. The tandem Tudor domain (TTD) of UHRF1 binds histone H3K9me2/3 with micromolar affinity, as well as unmethylated linker regions within UHRF1 itself, causing auto-inhibition. Recently, we showed that a methylated histone-like region of DNA ligase 1 (LIG1K126me2/me3) binds the UHRF1 TTD with nanomolar affinity, permitting UHRF1 recruitment to chromatin. Here we report the crystal structure of the UHRF1 TTD bound to a LIG1K126me3 peptide. The data explain the basis for the high TTD-binding affinity of LIG1K126me3 and reveal that the interaction may be regulated by phosphorylation. Binding of LIG1K126me3 switches the overall structure of UHRF1 from a closed to a flexible conformation, suggesting that auto-inhibition is relieved. Our results provide structural insight into how UHRF1 performs its key function in epigenetic maintenance.

DOI： 10.1016/j.str.2018.11.012

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.molcel.2017.09.037

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DOI： 10.1016/j.molcel.2017.07.012

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DOI： 10.1038/nature12488

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1073/pnas.1203701109

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DOI： 10.1074/jbc.M111.259630

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DOI： 10.1038/nature07249

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DOI： 10.1073/pnas.0509639103

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DOI： 10.1038/nsmb799

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DNA methylation is faithfully inherited during cell division, playing a crucial role in maintaining cellular identity. The process of DNA methylation maintenance relies on DNA methyltransferase DNMT1 and the ubiquitin E3 ligase UHRF1. UHRF1 facilitates the ubiquitination of both the replication factor PAF15 and histone H3, with each ubiquitin signal regulating replication-coupled and uncoupled DNA methylation maintenance, respectively. Over the past decades, advances in structural biology have significantly deepened our understanding of the molecular mechanisms governing DNA methylation maintenance. In particular, the emergence of cryo-electron microscopy (cryo-EM)-often referred to as the "Resolution Revolution"-has transformed many areas of biology, including epigenetics and chromatin biology. This review focuses on the structural mechanisms of DNA methylation maintenance, as revealed by the three-dimensional structures of key biomolecular complexes and discusses the potential development of inhibitors targeting DNA methylation maintenance factors based on structural insights.

DOI： 10.1266/ggs.25-00051

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The heterochromatin protein 1 (HP1) family recognizes lysine 9-methylated histone H3 (H3K9me) and recruits other transacting factors to establish higher order chromatin structures. In the fission yeast Schizosaccharomyces pombe (S. pombe), two HP1 family proteins, Swi6 and Chp2, play distinct roles in recruiting transacting factors: Swi6 primarily recruits Epe1, a Jumonji C domain-containing protein involved in histone H3K9 demethylation, whereas Chp2 recruits Mit1, a component of the Snf2/Hdac Repressive Complex. However, detailed mechanisms of how multiple HP1 family proteins and their respective interactors work cooperatively or exclusively to form higher order chromatin structures remain elusive. In this study, we investigated the interactions between Swi6 and Epe1. We found that Swi6 interacts with Epe1 through its chromoshadow domain, and identified a unique motif, named the FVI motif, in Epe1 involved in this interaction through detailed mapping of the region. Enhanced green fluorescent protein (EGFP) tethering assays showed that the FVI motif is sufficient to recruit ectopically expressed EGFP to heterochromatic regions, and mutational analyses revealed that conserved hydrophobic residues in this motif are essential for proper targeting. Structural simulations further supported the importance of these residues in Swi6 binding. Interestingly, Mit1 containing the Epe1 FVI motif was recruited to the heterochromatic regions by Swi6 but not by Chp2. Cells expressing mutant Mit1 maintained heterochromatic silencing even in chp2∆ cells, suggesting that Chp2 is not required for heterochromatin formation when Mit1 is recruited by Swi6. These findings highlight distinct HP1-binding motifs in interactors, contributing to functional divergence among HP1 family proteins.

DOI： 10.1096/fj.202402264RR

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DNA methylation maintenance is essential for cell fate inheritance. In differentiated cells, this involves orchestrated actions of DNMT1 and UHRF1. In mice, the high-affinity binding of DPPA3 to the UHRF1 PHD finger regulates UHRF1 chromatin dissociation and cytosolic localization, which is required for oocyte maturation and early embryo development. However, the human DPPA3 ortholog functions during these stages remain unclear. Here, we report the structural basis for human DPPA3 binding to the UHRF1 PHD finger. The conserved human DPPA3 85VRT87 motif binds to the acidic surface of UHRF1 PHD finger, whereas mouse DPPA3 binding additionally utilizes two unique α-helices. The binding affinity of human DPPA3 for the UHRF1 PHD finger was weaker than that of mouse DPPA3. Consequently, human DPPA3, unlike mouse DPPA3, failed to inhibit UHRF1 chromatin binding and DNA remethylation in Xenopus egg extracts effectively. Our data provide novel insights into the distinct function and structure of human DPPA3.

DOI： 10.1038/s42003-024-06434-9

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Authorship：Lead author,　Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

The structural biology of nucleosomes and their complexes with chromatin-associated factors contributes to our understanding of fundamental biological processes in the genome. With the advent of cryo-electron microscopy (cryo-EM), several structures are emerging with histone variants, various species, and chromatin-associated proteins that bind to nucleosomes. Cryo-EM enables visualization of the dynamic states of nucleosomes, leading to the accumulation of knowledge on chromatin-templated biology. Cryo-EM structure of nucleosome in Komagataella pastoris solved by Fukushima et al. provided the insights into transcription ability of RNAPII with nucleosome dynamics. In this commentary, we review the recent advances in the structural biology of nucleosomes and their related biomolecules.

DOI： 10.1093/jb/mvae004

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Language：English  

Mutations of the SNF2 family ATPase HELLS and its activator CDCA7 cause immunodeficiency-centromeric instability-facial anomalies (ICF) syndrome, characterized by hypomethylation at heterochromatin. The unique zinc-finger domain, zf-4CXXC_R1, of CDCA7 is widely conserved across eukaryotes but is absent from species that lack HELLS and DNA methyltransferases, implying its specialized relation with methylated DNA. Here we demonstrate that zf-4CXXC_R1 acts as a hemimethylated DNA sensor. The zf-4CXXC_R1 domain of CDCA7 selectively binds to DNA with a hemimethylated CpG, but not unmethylated or fully methylated CpG, and ICF disease mutations eliminated this binding. CDCA7 and HELLS interact via their N-terminal alpha helices, through which HELLS is recruited to hemimethylated DNA. While placement of a hemimethylated CpG within the nucleosome core particle can hinder its recognition by CDCA7, cryo-EM structure analysis of the CDCA7-nucleosome complex suggests that zf-4CXXC_R1 recognizes a hemimethylated CpG in the major groove at linker DNA. Our study provides insights into how the CDCA7-HELLS nucleosome remodeling complex uniquely assists maintenance DNA methylation.

DOI： 10.1101/2023.12.19.572350

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Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

Abstract

Heterochromatin protein 1 (HP1) is an evolutionarily conserved protein that plays a critical role in heterochromatin assembly. HP1 proteins share a basic structure consisting of an N-terminal chromodomain (CD) and a C-terminal chromoshadow domain (CSD) linked by a disordered hinge region. The CD recognizes histone H3 lysine 9 methylation, a hallmark of heterochromatin, while the CSD forms a dimer to recruit other chromosomal proteins. HP1 proteins have been shown to bind DNA or RNA primarily through the hinge region. However, how DNA or RNA binding contributes to their function remains elusive. Here, we focus on Chp2, one of the two HP1 proteins in fission yeast, and investigate how Chp2's DNA-binding ability contributes to its function. Similar to other HP1 proteins, the Chp2 hinge exhibits clear DNA-binding activity. Interestingly, the Chp2 CSD also shows robust DNA-binding activity. Mutational analysis revealed that basic residues in the Chp2 hinge and at the N-terminus of the CSD are essential for DNA binding, and the combined amino acid substitutions of these residues alter Chp2 stability, impair Chp2 heterochromatin localization and lead to a silencing defect. These results demonstrate that the cooperative DNA-binding activities of Chp2 play an important role in heterochromatin assembly in fission yeast.

DOI： 10.1093/jb/mvad050

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Other Link： https://academic.oup.com/jb/article-pdf/174/4/371/51790768/mvad050.pdf

Language：English   Publishing type：Research paper (scientific journal)   Publisher：eLife Sciences Publications, Ltd  

UHRF1-dependent ubiquitin signaling plays an integral role in the regulation of maintenance DNA methylation. UHRF1 catalyzes transient dual mono-ubiquitylation of PAF15 (PAF15Ub2), which regulates the localization and activation of DNMT1 at DNA methylation sites during DNA replication. Although the initiation of UHRF1-mediated PAF15 ubiquitin signaling has been relatively well characterized, the mechanisms underlying its termination and how they are coordinated with the completion of maintenance DNA methylation have not yet been clarified. This study shows that deubiquitylation by USP7 and unloading by ATAD5 (ELG1 in yeast) are pivotal processes for the removal of PAF15 from chromatin. On replicating chromatin, USP7 specifically interacts with PAF15Ub2 in a complex with DNMT1. USP7 depletion or inhibition of the interaction between USP7 and PAF15 results in abnormal accumulation of PAF15Ub2 on chromatin. Furthermore, we also find that the non-ubiquitylated form of PAF15 (PAF15Ub0) is removed from chromatin in an ATAD5-dependent manner. PAF15Ub2 was retained at high levels on chromatin when the catalytic activity of DNMT1 was inhibited, suggesting that the completion of maintenance DNA methylation is essential for the termination of UHRF1-mediated ubiquitin signaling. This finding provides a molecular understanding of how the maintenance DNA methylation machinery is disassembled at the end of the S phase.

DOI： 10.7554/elife.79013

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Other Link： https://cdn.elifesciences.org/articles/79013/elife-79013-v2.xml

Language：English   Publishing type：Research paper (scientific journal)  

Immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is in most cases-caused by mutations in either DNMT3B, ZBTB24, CDCA7, or HELLS. However, the causative genes of a few ICF patients remain unknown. We, herein, identified UHRF1 as a novel causative gene of one such patient with atypical symptoms. This patient is a compound heterozygote for two previously unreported mutations in UHRF1: c.886C > T (p.R296W) and c.1852C > T (p.R618X). The R618X mutation plausibly caused nonsense-mediated decay, while the R296W mutation changed the higher order structure of UHRF1, which is indispensable for the maintenance of CG methylation along with DNMT1. Genome-wide methylation analysis revealed that the patient had a centromeric/pericentromeric hypomethylation, which is the main ICF signature, but also had a distinctive hypomethylation pattern compared to patients with the other ICF syndrome subtypes. Structural and biochemical analyses revealed that the R296W mutation disrupted the protein conformation and strengthened the binding affinity of UHRF1 with its partner LIG1, and reduced ubiquitylation activity of UHRF1 towards its ubiquitylation substrates, histone H3 and PAF15. We confirmed that the R296W mutation causes hypomethylation at pericentromeric repeats by generating the HEK293 cell-lines that mimic the patient's UHRF1 molecular context. Since proper interactions of the UHRF1 with LIG1, PAF15, and histone H3 are essential for the maintenance of CG methylation, the mutation could disturb the maintenance process. Evidence for the importance of the UHRF1 conformation for CG methylation in humans is, herein, provided for the first time, and deepens our understanding of its role in regulation of CG methylation.

DOI： 10.1093/hmg/ddac291

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

<title>Abstract</title>
Trimethylation of histone H3 at K9 by the lysine methyltransferase, SET domain bifurcated histone lysine methyltransferase 1 (SETDB1) plays a pivotal role in silencing tissue-specific genes and retrotransposable elements. In mammalian cells, SETDB1 undergoes monoubiquitination in the insertion region of the SET domain in an E3 ubiquitin ligase-independent manner. This ubiquitination has been shown to enhance the histone H3-K9 methyltransferase activity of SETDB1; however, the molecular mechanism underlying SETDB1 activation by ubiquitination is unknown. In this study, we developed an Escherichia coli ubiquitination plasmid for the preparation of ubiquitinated SETDB1. Western blotting and mutational analyses showed that co-expression of the SET domain of SETDB1 with the proteins encoded by the ubiquitination plasmid led to site-specific monoubiquitination of the SET domain at K867. An in vitro histone H3 methylation assay demonstrated that the ubiquitinated SET domain of SETDB1 acquired enzymatic activity. Taken together, these findings demonstrate successful preparation of the active form of SETDB1 with the E.coli ubiquitination system, which will aid biochemical and structural studies of ubiquitinated SETDB1.

DOI： 10.1093/jb/mvab087

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Other Link： https://academic.oup.com/jb/article-pdf/170/5/655/42437777/mvab087.pdf

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Infection of certain influenza viruses is triggered when its HA is cleaved by host cell proteases such as proprotein convertases and type II transmembrane serine proteases (TTSP). HA with a monobasic motif is cleaved by trypsin-like proteases, including TMPRSS2 and HAT, whereas the multibasic motif found in high pathogenicity avian influenza HA is cleaved by furin, PC5/6, or MSPL. MSPL belongs to the TMPRSS family and preferentially cleaves [R/K]-K-K-R↓ sequences. Here, we solved the crystal structure of the extracellular region of human MSPL in complex with an irreversible substrate-analog inhibitor. The structure revealed three domains clustered around the C-terminal α-helix of the SPD. The inhibitor structure and its putative model show that the P1-Arg inserts into the S1 pocket, whereas the P2-Lys and P4-Arg interacts with the Asp/Glu-rich 99-loop that is unique to MSPL. Based on the structure of MSPL, we also constructed a homology model of TMPRSS2, which is essential for the activation of the SARS-CoV-2 spike protein and infection. The model may provide the structural insight for the drug development for COVID-19.

DOI： 10.26508/lsa.202000849

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Modification of cytosine plays an important role in epigenetic regulation of gene expression and genome stability. Cytosine is converted to 5-methylcytosine (5mC) by DNA methyltransferase; in turn, 5mC may be oxidized to 5-hydroxymethylcytosine (5hmC) by ten-eleven translocation enzyme. The structural flexibility of DNA is known to affect the binding of proteins to methylated DNA. Here, we have carried out a semi-quantitative analysis of the dynamics of double-stranded DNA (dsDNA) containing various epigenetic modifications by combining data from imino 1H exchange and imino 1H R1ρ relaxation dispersion NMR experiments in a complementary way. Using this approach, we characterized the base-opening (kopen) and base-closing (kclose) rates, facilitating a comparison of the base-opening and -closing process of dsDNA containing cytosine in different states of epigenetic modification. A particularly striking result is the increase in the kopen rate of hemi-methylated dsDNA 5mC/C relative to unmodified or fully methylated dsDNA, indicating that the Watson-Crick base pairs undergo selective destabilization in 5mC/C. Collectively, our findings imply that the epigenetic modulation of cytosine dynamics in dsDNA mediates destabilization of the GC Watson-Crick base pair to allow base-flipping in living cells.

DOI： 10.1093/nar/gkaa1210

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DNA methylation controls gene expression, and once established, DNA methylation patterns are faithfully copied during DNA replication by the maintenance DNA methyltransferase Dnmt1. In vivo, Dnmt1 interacts with Uhrf1, which recognizes hemimethylated CpGs. Recently, we reported that Uhrf1-catalyzed K18- and K23-ubiquitinated histone H3 binds to the N-terminal region (the replication focus targeting sequence, RFTS) of Dnmt1 to stimulate its methyltransferase activity. However, it is not yet fully understood how ubiquitinated histone H3 stimulates Dnmt1 activity. Here, we show that monoubiquitinated histone H3 stimulates Dnmt1 activity toward DNA with multiple hemimethylated CpGs but not toward DNA with only a single hemimethylated CpG, suggesting an influence of ubiquitination on the processivity of Dnmt1. The Dnmt1 activity stimulated by monoubiquitinated histone H3 was additively enhanced by the Uhrf1 SRA domain, which also binds to RFTS. Thus, Dnmt1 activity is regulated by catalysis (ubiquitination)-dependent and -independent functions of Uhrf1.

DOI： 10.1111/gtc.12732

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DNMT1 is a C5-DNA methyltransferase that plays a pivotal role in DNA methylation maintenance. During early and mid S-phase, DNMT1 accumulates at DNA replication sites by binding to proliferating cell nuclear antigen (PCNA), an essential factor for DNA replication, through a PIP box motif. However, the molecular mechanism by which the DNMT1 PIP box motif binds to PCNA remains unclear. Here, we report the crystal structure of PCNA bound to DNMT1 PIP box peptide. The structure reveals the detailed interaction between PCNA and DNMT1 PIP box; conserved glutamine and hydrophobic/aromatic residues in the PIP box are recognized by the Q- and hydrophobic pockets of PCNA, respectively. The structure also shows novel intramolecular interactions within the PIP box motif, which stabilize the helix conformation in the PIP box. Our data provide structural insight into the recruitment of DNMT1 to replication sites by PCNA.

DOI： 10.1016/j.bbrc.2019.06.060

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DOI： 10.1111/febs.14205

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Uhrf1-dependent histone H3 ubiquitylation plays a crucial role in the maintenance of DNA methylation via the recruitment of the DNA methyltransferase Dnmt1 to DNA methylation sites. However, the involvement of deubiquitylating enzymes (DUBs) targeting ubiquitylated histone H3 in the maintenance of DNA methylation is largely unknown. With the use of Xenopus egg extracts, we demonstrate here that Usp7, a ubiquitin carboxyl-terminal hydrolase, forms a stable complex with Dnmt1 and is recruited to DNA methylation sites during DNA replication. Usp7 deubiquitylates ubiquitylated histone H3 in vitro. Inhibition of Usp7 activity or its depletion in egg extracts results in enhanced and extended binding of Dnmt1 to chromatin, suppressing DNA methylation. Depletion of Usp7 in HeLa cells causes enhanced histone H3 ubiquitylation and enlargement of Dnmt1 nuclear foci during DNA replication. Our results thus suggest that Usp7 is a key factor that regulates maintenance of DNA methylation.

DOI： 10.1038/s41598-017-00136-5

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DOI： 10.1093/pcp/pcv177

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DOI： 10.1038/ncomms7116

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DOI： 10.1038/ncomms6340

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DOI： 10.1107/S2053230X14016926

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Publishing type：Research paper (scientific journal)   Publisher：International Union of Crystallography (IUCr)  

Interleukin (IL)-18 [1], a proinflammatory cytokine belonging to the IL-1 superfamily, plays important roles in both innate and adaptive immune system, which is involved in not only the function of host defense mechanism but also allergic reactions. IL-18 is secreted by various types of cells and strongly augments the production of interferon-γ. IL-18 is synthesized as a biologically inactive precursor (proIL-18), which is matured by the action of caspase-1 in the cell upon stimulation. The matured IL-18 is subsequently secreted extracellularly and binds to IL-18 receptor α (Rα) and IL-18 receptor β (Rβ) in a stepwise manner, forming the IL-18/Rα/Rβ ternary complex. The complex initiates the signaling that finally activates NF-κB via the MyD88 dependent pathway [2]. Here, we show the crystal structure of IL-18 (Fig a), the IL-18/Rα binary complex (Fig b) and the IL-18/Rα/Rβ signaling ternary complex (Fig c, d) at 2.33, 3.10 and 3.10 Å resolution, respectively. Overall, the recognition manner of IL-18 by the receptors was similar to that of IL-1β [3], although some remarkable differences such as the orientation of Ig-like domains of IL-18Rβ were revealed. We also demonstrate that carbohydrate chains on IL-18Rα contributes to the recognition of IL-18. Biochemical experiments based on the structure identify amino acid residues that are important in forming the ternary complex and the signal transduction. Our results not only show the common extracellular signaling architecture of IL-1 family cytokines but also unveil unique recognition mechanism of IL-18 with the detailed atomic structures. The structure of the signaling ternary complex of IL-18 would contribute to both understanding the pathogeneses of the IL-18 related diseases and designing new efficient therapeutic agents.

DOI： 10.1107/s2053273314097423

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DOI： 10.1074/jbc.M113.523209

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DOI： 10.1021/ja405745v

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DOI： 10.1074/jbc.M112.433284

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DOI： 10.1074/jbc.M112.431098

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DOI： 10.1039/c3cc39205h

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DOI： 10.1002/prot.22667

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DOI： 10.1038/embor.2009.218

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DOI： 10.1074/jbc.M608148200

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DOI： 10.1021/bi061180d

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DOI： 10.1074/jbc.M503390200

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DOI： 10.1107/S1744309105016325

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DOI： 10.1107/S0907444903022741

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Language：Japanese   Publisher：(公社)日本生化学会  

J-GLOBAL

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DOI： 10.5940/jcrsj.57.53

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J-GLOBAL

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DOI： 10.1107/S0108767311094335

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CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/2008377313

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：The Crystallographic Society of Japan  

Peptidylarginine deimianse 4 (PAD4) is a Ca<SUP>2+</SUP>-dependent enzyme that catalyzes the conversion of both arginine and mono-methyl arginine in histones into citrullines, and regulates both histone argininine methylation level and gene activity. Its gene is susceptibility locus for rheumatoid arthritis (RA) . Here we present the crystal structure of Ca<SUP>2+</SUP>-free wild-type PAD4, which shows that the polypeptide chain adopts an elongated fold in which the N-terminal domain forms two immunoglobulin-like subdomains, and the C-terminal domain forms an α/β propeller structure. Five Ca<SUP>2+</SUP>-binding sites, none of which adopts an EF-hand motif, were identified in the structure of a Ca<SUP>2+</SUP>-bound inactive mutant with and without bound substrate. These structural data indicate that Ca<SUP>2+</SUP>binding induces conformational changes that generate the active site cleft. Our findings identify a novel mechanism for enzyme activation by Ca<SUP>2+</SUP>ions, and are important for understanding the mechanism of protein citrullination and for developing PAD-inhibiting drugs for the treatment of RA.

DOI： 10.5940/jcrsj.47.268

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Other Link： https://jlc.jst.go.jp/DN/JALC/00256402078?from=CiNii

Event date： 2022.11 - 2022.12

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Event date： 2021.3

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