• BMB Reports
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• v.37 no.2
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• pp.144-147
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• 2004

Molecular amplification and sequencing of genomic DNA that encodes camel polyubiquitin (PUBC1) was performed by a polymerase chain reaction (PCR) using various sets of primers. The amplification generated a number of DNA fragments, which were sequenced and compared with the polyubiquitin coding sequences of various species. One DNA fragment that conformed to 325 bp was found to be 95 and 88% homologous to the sequences of human polyubiquitin B and C, respectively. The DNA translated into 108 amino acids that corresponded to two fused units of ubiquitin with no intervening sequence, which indicates that it is a polyubiquitin and contains at least two units of ubiquitin. Although, variations were found in the nucleotide sequence when compared to those of other species, the amino acid sequence was 100% homologous to the polyubiquitin sequences of humans, mice, and rats. This is the first report of the polyubiquitin DNA coding sequence and its corresponding amino acid sequence from camels, amplified using direct genomic DNA preparations.

• Bulletin of the Korean Chemical Society
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• v.33 no.4
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• pp.1285-1289
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• 2012

RAP80 plays a key role in DNA damage responses by recognizing K63-linked polyubiquitin moieties through its two ubiquitin-interacting motif (UIM) domains. The linker between the two UIMs possesses a phosphorylation site, but the relationship between phosphorylation and polyubiquitin recognition remains elusive. We investigated the interaction between a phosphorylation-mimic RAP80 mutant S101E and linear polyubiquitins, structurally equivalent to the K63-linked ones, using isothermal titration calorimetry (ITC). ITC analysis revealed differential binding affinities for linear tetraubiquitin by otherwise equivalent UIMs in S101E. Mutational analysis supported such differential polyubiquitin recognition by S101E. Our results suggest a potential crosstalk between polyubiquitin recognition and phosphorylation in RAP80.

• 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.

• BMB Reports
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• v.41 no.3
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• pp.177-183
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• 2008

Ubiquitin is an essential, highly-conserved small regulatory protein in eukaryotic cells. It covalently modifies a wide variety of targeted proteins in the forms of monomer and polymers, altering the conformation and binding properties of the proteins and thus regulating proteasomal delivery, protein activities and localization. Mass spectrometry has emerged as an indispensable tool for in-depth characterization of protein ubiquitination. Ubiquitinated proteins in cell lysates are usually enriched by affinity chromatography and subsequently analyzed by mass spectrometry for identification and quantification. Ubiquitin-conjugated amino acid residues can be determined by unique mass shift caused by the modification. Moreover, the complex structure of polyubiquitin chains on substrates can be dissected by bottom-up and middle-down mass spectrometric approaches, revealing potential novel functions of polyubiquitin linkages. Here I review the advances and caveats of these strategies, emphasizing caution in the validation of ubiquitinated proteins and in the interpretation of raw data.

• Proceedings of the Zoological Society Korea Conference
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• 1997.10b
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• pp.267.2-267
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• 1997

No Abstract, See Full Text

• BMB Reports
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• v.47 no.9
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• pp.475-482
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• 2014

Ubiquitin (Ub) is a versatile signaling molecule that plays important roles in a variety of cellular processes. Cellular Ub pools, which are composed of free Ub and Ub conjugates, are in dynamic equilibrium inside cells. In particular, increasing evidence suggests that Ub homeostasis, or the maintenance of free Ub above certain threshold levels, is important for cellular function and survival under normal or stress conditions. Accurate determination of various Ub species, including levels of free Ub and specific Ub chain linkages, have become possible in biological specimens as a result of the introduction of the proteomic approach using mass spectrometry. This technology has facilitated research on dynamic properties of cellular Ub pools and has provided tools for in-depth investigation of Ub homeostasis. In this review, we have also discussed the consequences of the disruption of Ub pool dynamics and homeostasis via deletion of polyubiquitin genes or mutations of deubiquitinating enzymes. The common consequence was a reduced availability of free Ub and a significant impact on the function and viability of cells. These observations further indicate that the levels of free Ub are important determinants for cellular protection.

• Journal of Plant Biotechnology
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• v.4 no.1
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• pp.7-15
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• 2002

Eight cDNAs corresponding to fruit-specific genes were isolated from ripened melon through differential screening. Sequence comparison indicated that six of these cDNAs encoded proteins were previously characterized into aminocyclopropane-1-carboxylate (ACC) oxidase, abscisic acid, stress and ripening inducible (ASR) gene, RINC-H2 zinc finger protein, pyruvate decarboxylase, or polyubiquitin. RFS2 and RFS5 were the same clone encoding polyubiquitin. The other cDNAs showed no significant homology with known protein sequences. The ASR homologue (Asr1) gene was further characterized on the cDNA and genomic structure. The deduced amino acid sequence had similar characteristics to other plant ASR. The Asr1 genomic DNA consisted of 2 exons and 1 intron, which is similar to the structure of other plants ASR genes. The promoter region of the Asr1 gene contained several putative functional cis-elements such as an abscisic acid responsive element (ABRE), an ethylene responsive element (ERE), a C-box or DPBf-1 and 2, Myb binding sites, a low temperature responsive element (LTRE) and a metal responsive element (MRE). The findings imply that these elements may play important roles in the response to plant hormones and environmental stresses in the process of fruit development. The results of this study suggest that the expressions of fruit specific and ripening-related cDNAs are closely associated with the stress response.

• Biomedical Science Letters
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• v.27 no.3
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• pp.182-186
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• 2021

Parkinson disease (PD) is becoming one of the most neurodegenerative disorder worldwide. The deposited aggregates have been connected in the pathophysiology of PD, which are degraded either by ubiquitin-proteasomal system (UPS) or autophagy-lysosomal pathway (ALP). Leucin-rich repeat kinase 2 (LRRK2), one of the neurodegenerative proteins of PD is also stringently controlled by both UPS and ALP degradation as well. However, the polyubiquitination pattern of LRRK2 aggregates is largely unknown. Here, we found that K63-linked polyubiquitinations of G2019S mutant, most familial variant for PD, is highly enhanced compared to those of wild type LRRK2 (WT). In addition, in the presence of overexpressed p62/SQSTM-1, ubiquitination of LRRK2 WT or D1994A was reduced, whereas G2019S mutant was not diminished significantly. Therefore, we propose that degradation of G2019S via UPS is more involved with K63-linked ubiquitination than K48-linked ubiquitination, and overexpressed p62/SQSTM-1 does not enhance degradative effect on G2019S variant.

• Animal cells and systems
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• v.5 no.1
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• pp.85-90
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• 2001

Yolk platelets, one of the main food stores in the embryonic development, are composed of proteins. However, little is known about the identity of proteins utilized at certain stages of embryogenesis. In this study, we followed the fates of embryonic storage proteins by using an anti-polyubiquitin monoclonal antibody (mAB) as a probe. The mAb recognized the major storage proteins of Drosophila, Xenopus and chicken eggs. In the Drosophila embryo, the mAb-reactive 45-kDa protein was not used until stage 11 but was used up at stage 16 when the embryo completed segmentation. In the Xenopus embryo, the mAb-reactive 111 kDa protein was mostly utilized between stages 42 and 45 implying that the protein might be an energy source used just prior to feeding on food. By N-terminal sequencing the storage protein of Xenopus embryo was identified as a lipovitellin 1. This study confirms that storage proteins are used almost simultaneously at certain stages of embryogenesis and that vitellogenin 1 is the last storage protein in Xenopus embryogenesis.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.4
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• pp.2173-2179
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• 2013

Ubiquitin carboxyl-terminal hydrolase 37 (UCH37, also called UCHL5), a member of the deubiquitinating enzymes, can suppress protein degradation through disassembling polyubiquitin from the distal subunit of the chain. It has been proved that UCH37 can be activated by proteasome ubiqutin chain receptor Rpn13 and incorporation into the 19S complex. UCH37, which has been reported to assist in the mental development of mice, may play an important role in oncogenesis, tumor invasion and migration. Further studies will allow a better understanding of roles in cell physiology and pathology, embryonic development and tumor formation, hopefully providing support for the idea that UCH37 may constitute a new interesting target for the development of anticancer drugs.