• BMB Reports
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• v.42 no.11
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• pp.764-768
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• 2009

The tandem ubiquitin-interacting motif (UIM) domain located at the N-terminus of Receptor Associated Protein 80 (RAP80) plays a crucial role in ionizing radiation (IR)-induced DNA damage response. RAP80 translocates to sites of IR-induced DNA damage through interaction of its UIM domain with ubiquitinated H2A and Lys63-linked polyubiquitin chains. The exact mechanism, however, through which RAP80 associates with Lys63-linked polyubiquitin chains is not clear. Here, we show by in vitro GST-pull down assays that modifying the linker region between the tandem ubiquitin binding domains of RAP80 changes the binding affinity for Lys63-linked polyubiquitin chains and affects translocation to sites of DNA breaks. Based on these findings, we suggest that the length of the linker region between the tandem ubiquitin binding domains of RAP80 may be a key factor in the binding of RAP80 with Lys63-linked polyubiquitin chains as well as in the translocation of RAP80 to DNA break sites.

• Journal of the Korean Magnetic Resonance Society
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• v.15 no.1
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• pp.80-89
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• 2011

3D structures of STAM1 UIM-ubiquitin complex were presented to predict and analyze the interaction between UIM and ubiquitin. To generate the protein-peptide complex structure, the RosettaDock method was used with and without NMR restraints. High resolution complex structure was acquired successfully and evaluated electrostatic interaction in the protein-peptide binding with several charged residues at the binding site. From docking results, the Rosettadock method could be useful to acquire essential information of protein-protein or protein-peptide interaction with minimal biological evidences.

• BMB Reports
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• v.49 no.2
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• pp.73-80
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• 2016

Autophagy is a process tightly regulated by various autophagy-related proteins. It is generally classified into non-selective and selective autophagy. Whereas non-selective autophagy is triggered when the cell is under starvation, selective autophagy is involved in eliminating dysfunctional organelles, misfolded and/or ubiquitylated proteins, and intracellular pathogens. These components are recognized by autophagy receptors and delivered to phagophores. Several selective autophagy receptors have been identified and characterized. They usually have some common domains, such as motif, a specific cargo interacting (ubiquitin-dependent or ubiquitin-independent) domain. Recently, structural data of these autophagy receptors has been described, which provides an insight of their function in the selective autophagic process. In this review, we summarize the most up-to-date findings about the structure-function of autophagy receptors that regulates selective autophagy.

• Journal of the Korean Chemical Society
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• v.68 no.1
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• pp.25-31
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• 2024

The p97 adenosine triphosphatase is a key player in protein homeostasis, responsible for unfolding ubiquitylated substrates. It engages with various adaptor proteins through its N-terminal domain, with the p97-p47 complex attracting particular attention for its involvement in membrane remodeling. Although the structures of p97 in complex with the Ubiquitin regulatory X (UBX) domain from various adaptors have been reported, the stoichiometry is conflicting. Here, we report the crystal structure of the p97 N-D1 hexamer in complex with the p47 UBX domain at a resolution of 2.7 Å. The structure reveals a stoichiometry of 6:6 between the p97 N-D1 and the p47 UBX domain. These findings provide valuable insights into the binding stoichiometry of p97 N-D1 and p47 UBX domain, which are crucial for understanding the role of p97 and adaptor proteins in cellular processes such as the ubiquitin-proteasome pathway, membrane fusion, and cell cycle regulation.

• Parasites, Hosts and Diseases
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• v.53 no.1
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• pp.65-75
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• 2015

Clonorchis sinensis habitating in the bile duct of mammals causes clonorchiasis endemic in East Asian countries. Parkin is a RING-between-RING protein and has E3-ubiquitin ligase activity catalyzing ubiquitination and degradation of substrate proteins. A cDNA clone of C. sinensis was predicted to encode a polypeptide homologous to parkin (CsParkin) including 5 domains (Ubl, RING0, RING1, IBR, and RING2). The cysteine and histidine residues binding to $Zn^{2+}$ were all conserved and participated in formation of tertiary structural RINGs. Conserved residues were also an E2-binding site in RING1 domain and a catalytic cysteine residue in the RING2 domain. Native CsParkin was determined to have an estimated molecular weight of 45.7 kDa from C. sinensis adults by immunoblotting. CsParkin revealed E3-ubiquitin ligase activity and higher expression in metacercariae than in adults. CsParkin was localized in the locomotive and male reproductive organs of C. sinensis adults, and extensively in metacercariae. Parkin has been found to participate in regulating mitochondrial function and energy metabolism in mammalian cells. From these results, it is suggested that CsParkin play roles in energy metabolism of the locomotive organs, and possibly in protein metabolism of the reproductive organs of C. sinensis.

• Journal of Plant Biotechnology
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• v.36 no.1
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• pp.53-58
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• 2009

Calcium signals can be transduced by binding calmodulin (CaM), a $Ca^{2+}$ sensor in eukaryotes, is known to be involved in the regulation of diverse cellular functions. We isolated a CaM-binding protein 63 kD (AtCBP63) from the pathogen-treated Arabidopsis cDNA expression library. Recently, AtCBP63 was identified as a CaM bining protein. The CaM binding domain of AtCBP63 was reported to be located in its N-terminal region, In this study, however, we showed that ACaM2 could specifically bind to second CaM-binding domain (CaMBD) of AtCBP63 at the C-terminal region. The specific binding of CaM to CaM binding domain was confirmed by a gel mobility shift assay, a split ubiquitin assay, site-directed mutagenesis, and a competition assay using a $Ca^{2+}$/CaM-dependent enzyme. The gene expression of AtCBP63 was induced by pathogens and pathogens related second messengers. This result suggests that a CaM binding protein, AtCBP63, may play role in pathogen defense signaling pathway.

• Journal of Plant Biotechnology
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• v.43 no.2
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• pp.204-212
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• 2016

To understand the responses of grapevines in response to cold stress causing the limited growth and development, differentially expressed genes (DEGs) were screened through transcriptome analysis of shoots from 2 grapevine cultivars ('Campbell Early' and 'Muscat Baily A') kept at -$2^{\circ}C$ for 4 days. In gene ontology analysis of DEGs from 'Campbell Early', there were 17,424 clones related with biological process, 28,954 with cellular component, and 6,972 with molecular function genes in response to freezing temperature. The major induced genes included dehydrin xero 1, K-box region and MADS-box transcription factor family protein, and MYB domain protein 36, and inhibited genes included light-harvesting chlorophyll B-binding protein 3, FASCICLIN-like arabinoogalactan 9, and pectin methylesterase 61 in 'Campbell Early' grapevines. In gene ontology analysis of DEGs from 'Muscat Baily A', there were 1,157 clones related with biological process, 1,350 with cellular component, and 431 with molecular function gene. The major induced genes of 'Muscat Baily A' included NB-ARC domain-containing disease resistance protein, fatty acid hydrozylase superfamily, and isopentenyltransferase 3, and inhibited genes included binding, IAP-like protein 1, and pentatricopeptide repeat superfamily protein. All major DEGs were shown to be expressed differentially by freezing temperature in real time-PCR analysis. Protein domain analysis using InterPro Scan revealed that ubiquitin-protein ligase was redundant in both tested grapevines. Transcriptome profile of shoots exposed to cold can provide new insights into the molecular basis of tolerance to low-temperature in grapevines, and can be used as resources for development new grapevines tolerant to coldness.

• Journal of Life Science
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• v.30 no.5
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• pp.483-490
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• 2020

The ubiquitin system uses ligases and deubiquitinases (DUBs) to regulate ubiquitin position on protein substrates and is involved in many biological processes which determine stability, activity, and interaction of the target substrate. DUBs are classified in six groups according to catalytic domain, namely ubiquitin-specific proteases (USPs); ubiquitin C-terminal hydrolases (UCHs); ovarian tumor proteases (OTUs); Machado Joseph Disease proteases (MJDs); motif interacting with Ub (MIU)-containing novel DUB family (MINDY); and Jab1/MPN/MOV34 metalloenzymes (JAMMs). Otubain 1 (OTUB1) is a DUB in the OTU family which possesses both canonical and non-canonical activity and can regulate multiple cellular signaling pathways. In this review, we describe the function of OTUB1 through regulation of its canonical and non-canonical activities in multiple specifically cancer-associated pathways. The canonical activity of OTUB1 inhibits protein ubiquitination by cleaving Lys48 linkages while its non-canonical activity prevents ubiquitin transfer onto target proteins through binding to E2-conjugating enzymes, resulting in the induction of protein deubiquitination. OTUB1 can therefore canonically and non-canonically promote tumor cell proliferation, invasion, and drug resistance through regulating FOXM1, ERα, KRAS, p53, and mTORC1. Moreover, clinical research has demonstrated that OTUB1 overexpresses with high metastasis in many tumor types including breast, ovarian, esophageal squamous, and glioma. Therefore, OTUB1 has been suggested as a diagnosis marker and potential therapeutic target for oncotherapy.

• Molecules and Cells
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• v.44 no.3
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• pp.146-159
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• 2021

DNA methylation, and consequent down-regulation, of tumour suppressor genes occurs in response to epigenetic stimuli during cancer development. Similarly, human oncoviruses, including human papillomavirus (HPV), up-regulate and augment DNA methyltransferase (DNMT) and histone deacetylase (HDAC) activities, thereby decreasing tumour suppressor genes (TSGs) expression. Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1), an epigenetic regulator of DNA methylation, is overexpressed in HPV-induced cervical cancers. Here, we investigated the role of UHRF1 in cervical cancer by knocking down its expression in HeLa cells using lentiviral-encoded short hairpin (sh)RNA and performing cDNA microarrays. We detected significantly elevated expression of thioredoxin-interacting protein (TXNIP), a known TSG, in UHRF1-knockdown cells, and this gene is hypermethylated in cervical cancer tissue and cell lines, as indicated by whole-genome methylation analysis. Up-regulation of UHRF1 and decreased TXNIP were further detected in cervical cancer by western blot and immunohistochemistry and confirmed by Oncomine database analysis. Using chromatin immunoprecipitation, we identified the inverted CCAAT domain-containing UHRF1-binding site in the TXNIP promoter and demonstrated UHRF1 knockdown decreases UHRF1 promoter binding and enhances TXNIP expression through demethylation of this region. TXNIP promoter CpG methylation was further confirmed in cervical cancer tissue by pyrosequencing and methylation-specific polymerase chain reaction. Critically, down-regulation of UHRF1 by siRNA or UHRF1 antagonist (thymoquinone) induces cell cycle arrest and apoptosis, and ubiquitin-specific protease 7 (USP7), which stabilises and promotes UHRF1 function, is increased by HPV viral protein E6/E7 overexpression. These results indicate HPV might induce carcinogenesis through UHRF1-mediated TXNIP promoter methylation, thus suggesting a possible link between CpG methylation and cervical cancer.

• Journal of Photoscience
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• v.12 no.1
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• pp.1-9
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• 2005

The ubiquitin E3 ligase COP1 (Constitutive Photomorphogenesis 1) is a protein repressor of photomorphogenesis in Arabidopsisplants, and it found in various organisms, including animals. The COP1 protein regulates the stability of many of the light-signaling components that are involved in photomorphogenesis and in the developmental processes. To study the effect of COP1 on flowering in a short day plant, we have cloned a full-length of PnCOP1 (Pharbitis nil COP1) cDNA from Pharbitis nil Choisy cv. Violet, and we examined its transcript levels under various conditions. A full-length PnCOP1 cDNA consists of 2,280 bp nucleotidesthat contain 47 bp of 5'-UTR, 232 bp of 3'-UTR including the poly (A) tail, and 1,998 bp of the coding sequence. The deduced amino acid sequence contains 666 amino acids, giving it a theoretical molecular weight of 75 kD and a isolectric point of 6.2. The PnCOP1 contains three distinct domains, an N-terminal $Zn^2+$-binding RING-finger domain, a coiled-coil structure, and WD40 repeats at the C-terminal, implying that the protein plays a role in protein-protein interactions. The PnCOP1 transcript was detected in the cotyledon, hypocotyls and leaves, but not in root. The levels of the PnCOP1 transcript were reduced in leaves that were a farther distance away from the cotyledons. The expression level of the PnCOP1 gene was inhibited by light, while the expression was increased in the dark. During the floral inductive 16 hour-dark period for Pharbitis nil, the expression was increased and it reached its maximum at the 12th hour of the dark period. The levels of PnCOP1 mRNA were dramatically reduced upon light illumination. These results suggest that PnCOP1 may play an important function in the floral induction of Pharbitis nil.