• BMB Reports
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• v.56 no.3
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• pp.172-177
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• 2023

BEST family is a class of Ca²⁺-activated Cl- channels evolutionary well conserved from bacteria to human. The human BEST paralogs (BEST1-BEST4) share significant amino acid sequence homology in the N-terminal region, which forms the transmembrane helicases and contains the direct calcium-binding site, Ca²⁺-clasp. But the cytosolic C-terminal region is less conserved in the paralogs. Interestingly, this domain-specific sequence conservation is also found in the BEST1 orthologs. However, the functional role of the C-terminal region in the BEST channels is still poorly understood. Thus, we aimed to understand the functional role of the C-terminal region in the human and mouse BEST1 channels by using electrophysiological recordings. We found that the calcium-dependent activation of BEST1 channels can be modulated by the C-terminal region. The C-terminal deletion hBEST1 reduced the Ca²⁺-dependent current activation and the hBEST1-mBEST1 chimera showed a significantly reduced calcium sensitivity to hBEST1 in the HEK293 cells. And the C-terminal domain could regulate cellular expression and plasma membrane targeting of BEST1 channels. Our results can provide a basis for understanding the C-terminal roles in the structure-function of BEST family proteins.

• BMB Reports
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• v.49 no.6
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• pp.337-343
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• 2016

Understanding of trafficking, processing, and degradation mechanisms of amyloid precursor protein (APP) is important because APP can be processed to produce β-amyloid (Aβ), a key pathogenic molecule in Alzheimer's disease (AD). Here, we found that APP contains KFERQ motif at its C-terminus, a consensus sequence for chaperone-mediated autophagy (CMA) or microautophagy which are another types of autophagy for degradation of pathogenic molecules in neurodegenerative diseases. Deletion of KFERQ in APP increased C-terminal fragments (CTFs) and secreted N-terminal fragments of APP and kept it away from lysosomes. KFERQ deletion did not abolish the interaction of APP or its cleaved products with heat shock cognate protein 70 (Hsc70), a protein necessary for CMA or microautophagy. These findings suggest that KFERQ motif is important for normal processing and degradation of APP to preclude the accumulation of APP-CTFs although it may not be important for CMA or microautophagy.

• BMB Reports
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• v.29 no.5
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• pp.411-416
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• 1996

In order to study the role of the protein shell in both iron uptake and iron core formation of ferritin, we constructed a deletion mutant of the ferritin gene and expressed the mutant gene in Escherichia coli, This mutant was obtained by introducing an amber mutation at position Pro-157 and a deletion of the 19 amino acid residues at the carboxy-terminus of the recombinant tadpole H-chain ferritin. The deleted amino acids correspond to E-helix forming the hydrophobic channel in the protein. E. coli harboring the plasmid pTHP157, which contains the deleted gene, was grown at $23^{\circ}C$ in the presence of 0.1 mM IPTG, and the induced protein appeared to be partly soluble. Nondenaturing polyacrylamide gel electrophoresis showed that the expressed mutant H-chains coassemble into holoprotein, suggesting that E-helix is not necessary for assembly of the subunits as reported for human H-chain ferritin. Its ability in iron core formation was proven in an Fe staining gel, the result disagreeing with the observation that the hydrophobic channel is necessary for iron core formation in human H-chain ferritin.

• BMB Reports
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• v.42 no.6
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• pp.350-355
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• 2009

Although previous studies on hexokinase (HK) II indicate both the N- and C-terminal halves are catalytically active, we show in this study the N-terminal half is significantly more catalytic than the C-terminal half in addition to having a significantly higher $K_m$ for ATP and Glu. Furthermore, truncated forms of intact HK II lacking its first N-terminal 18 amino acids ($\Delta$18) and a truncated N-terminal half lacking its first 18 amino acids ($\Delta$18N) have higher catalytic activity than other mutants tested. Similar results were obtained by PET-scan analysis using $^{18}F-FDG$. Our results collectively suggest that each domain of HK II possesses enzyme activity, unlike HK I, with the N-terminal half showing higher enzyme activity than the C-terminal half.

• BMB Reports
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• v.41 no.11
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• pp.820-825
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• 2008

Bacillus thuringiensis Cyt2Aa toxin is a mosquito-larvicidal and cytolytic $\delta$-endotoxin, which is synthesized as a protoxin and forms crystalline inclusions within the cell. These inclusions are solubilized under alkaline conditions and are activated by proteases within the larval gut. In order to assess the functions of the N-and C-terminal regions of the protoxin, several N- and C-terminal truncated forms of Cyt2Aa were constructed. It was determined that amino acid removal at the N-terminal, which disrupts the $\beta$1 structure, might critically influence toxin production and inclusion formation. The deletion of 22 amino acids from the C-terminus reduced the production and solubility of the toxin. However, the removal of more than 22 amino acids from the C-terminus or the addition of a bulky group to this region could result in the inability of the protein to adopt the proper folding. These findings directly demonstrated the critical roles of N- and C-terminal amino acids on the production and folding of the B. thuringiensis cytolytic $\delta$-endotoxin.

• Korean Journal of Microbiology
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• v.40 no.2
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• pp.87-93
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• 2004

Dynamic modification of cytoplasmic and nuclear proteins by O-linked N-acetylglucosamine (O-GlcNAc) on Ser and Thr residues is ubiquitous in higher eukaryotes. And this modification may serve as a signaling mod-ification analogous to protein phosphorylation. Addition and cleavage of O-GlcNAc are catalyzed by O-linked GlcNAc transferase (OGT) and O-linked N-acety1glucosaminidase (O-GlcNAcase), respectively. Two types of human O-GlcNAcase gene were cloned and expressed as three fusion proteins in Escherichia coli. O-GlcNA-case activity showed in the order of thioredoxin fusion> $6{\times}His$ tag> GST fusion. O-GlcNAcase had enzy-matic activity against only ${\rho}$NP-GlcNAc of seven tested substrate analogs. Blast search revealed that O-GlcNAcase has two conserved domains, amino terminal hyaluronidase-like domain and carboxy terminal N-acetyltransferase domain. Extensive deletion studies were done to define catalytically important domains. The deletions of hyaluronidase-like domain and N-acetyltransferase domain abolished enzyme activity. But, N-ter-minal 55 amino acid deletion and C-terminal truncation showed lower activity. Based on deletion analysis, we suggest that hyaluronidase-like domain is essential for enzyme activity and carboxy terminal N-acetyltrans-ferase domain may be modulatory function.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.11
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• pp.1572-1575
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• 2008

The xenotransplantation of pig organs and cells can be related with a risk of transmission of infectious diseases to human. Previous findings indicate that the regulatory region of PERV for retroviral transcription, replication and integration into the cellular DNA is located on the 5' Long Terminal Repeat (LTR). The objective of this study is the investigation of methylation and deletion status of the PERV 5' LTR region which can be used for regulating PERV expression. We compared the sequences of genomic DNA and bisulfite-treated genomic DNA from PK-15 cells expressing PERV to observe the methylation status of the 5' LTR. Our results showed that the CpG sites of U3 were methylated and methylation was inconsistent in the R and U5 regions. Also, variable numbers of 18 bp repeats and 21 bp repeats were detected on 5' LTR by sequencing analysis. The consistent U3 methylation might be indicative of host suppression of expression of the retroviruses.

• Korean Journal of Microbiology
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• v.42 no.4
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• pp.271-276
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• 2006

Previously we demonstrated that virus-inducible stress protein (VISP) is induced in fish cells by the infection of a fish rhabdovirus. In this paper, we investigated the subcellular localization of the VISP and determined the region of VISP responsible for the subcellular localization. The CHSE-214 cells were stained with monoclonal antibody raised against VISP and observed with confocal microscope to detect the endogenous VISP. The results showed that the VISP localizes to the perinuclear region as spots. A plasmid expressing VISP fused to enhanced green fluorescent protein (EGFP) was constructed. The transient expression of full-length VISP fused to EGFP in CHSE-214 cells confirmed the spot formation of the VISP at perinuclear region. To determine the region responsible for the perinuclear localization of the VISP, we constructed a series of deletion mutants and, by using these deletion mutants, we found that C-terminal region of the VISP (aa 612-710) is essential for the perinuclear distribution of VISP and that this region contained nuclear receptor binding motif (691-TLTSLLL-697). Our results suggest that VISP localizes to the perinuclear region and C-terminal regions are important for this localization. Further studies on the role of the perinuclear localization of VISP in IHNV growth mali reveal the novel mechanism of IHNV pathogenecity.

• Bulletin of the Korean Chemical Society
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• v.35 no.1
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• pp.83-86
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• 2014

Poly(A) polymerase (PAP) play an essential role for maturation of mRNA by adding the adenylate residues at the 3' end. PAP functions are regulated through protein-protein interaction at its C-terminal region. In this study, cyclophilin A (CypA), a member of the peptidyl-prolyl cis-trans isomerase family, was identified as a partner protein interacting with the C-terminal region PAP. The interaction between PAP and CypA was inhibited by the immunosuppressive drug cyclosporine A. Deletion analysis revealed that the N-terminal 56 residues of CypA are sufficient for the interaction with PAP. Interestingly, we observed that PAP and CypA colocalize in the nucleus during SDF-1-induced chemotaxis, implying that CypA could be involved in the regulation of polyadenylation by PAP in the chemotactic cells.

• Journal of Microbiology and Biotechnology
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• v.16 no.9
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• pp.1477-1480
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• 2006

The 63-amino-acid-encoding afsR2 is a global antibiotics-stimulating regulatory gene identified from the chromosome of Streptomyces lividans. To dissect a putative functional domain in afsR2, several afsR2-derivative deletion constructs were generated and screened for the loss of actinorhodin-stimulating capability. The afsR2-derivative construct missing a 50-bp C-terminal region significantly lost its actinorhodin-stimulating capability in S. lividans. In addition, site-directed mutagenesis on amino acid positions of #57-#61 in a 50-bp C-terminal region, some of which are conserved among known Sigma 70 family proteins, significantly changed the AfsR2's activity. These results imply that the C-terminal region of AfsR2 is functionally important for antibiotics-stimulating capability and the regulatory mechanism might be somehow related to the sigma-like domain present in the C-terminal of AfsR2.