• BMB Reports
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• 제33권2호
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• pp.155-161
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• 2000

The saccharomyces cerevisiae Rad27, a structure-specific endonuclease for the okazaski fragment maturation has been known to interact genetically and biochemically with Dna2, an essential enzyme for DNA replication. In an attempt to define the significance of the interaction between the two enzymes, we expressed and purified both Dna2 and Rad27 proteins. In this report, Rad27 could not form a complex with Dna2 in the three different analyses. The analyses included glycerol gradient sedimentation, protein-column chromatography, and coinfection of baculoviruses followed by affinity purification. This is in striking contrast to the previous results that used crude extracts. These results suggest that the interaction between the two proteins is not sufficiently stable or indirect, and thus requires an additional protein(s) in order for Rad27 and Dna2 to form a stable physical complex. This result is consistent with our genetic findings that Schizosaccharomyces pombe Dna2 is capable of interacting with several proteins that include two subunits of polymerase $\delta$, DNA ligase I, as well as Fen-1. In addition, we found that the N-terminal modification of Rad27 abolished its enzymatic activity. Thus, as suspected, we found that on the basis of the structure determination, N-terminal methionine indeed plays an important role in the nucleolytic cleavage reaction.

• BMB Reports
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• 제34권2호
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• pp.102-108
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• 2001

A time-dependent folding process was used to determine whether or not protein disulfide isomerase (PDI) plays an important role in the maturation of nascent lactoferrin polypeptides. Interaction between lactoferrin and PDI was analyzed according to the co-immunoprecipitation of the two proteins. The results indicate that lactoferrin folding requires a significant interaction with PDI and its binding is relatively brief compared to other nascent polypeptides. The amount of lactoferrin interacting with PDI increases up to half a minute and sharply decreases beyond this time point. During the refolding process that follows reduction by DTT, lactoferrin polypeptides heavily interact with PDI and the interaction period was extended compared to the normal folding process. In terms of the temperature effect on PDI-lactoferrin interaction, PDI binds to lactoferrin polypeptides longer at a lower temperature (here, $25^{\circ}C$) than $37^{\circ}C$. The lactoferrin-PDI interaction was also studied in vitro. According to the in vitro experiment data, PDI was still functional in cell lysates assisting lactoferrin folding into the mature form. PDI interacts with lactoferrin polypeptides for an extended period during the folding in vitro. During the refolding process in vitro, intermolecular aggregates and refolding oligomers matured into a functional form after PDI binds to the lactoferrin. These results suggest that PDI provides a prolonged chaperoning activity in the refolding processes and that there appears to be a greater requirement for PDI chaperone activity in the refolding of lactoferrin polypeptides.

• Molecules and Cells
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• 제20권2호
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• pp.157-166
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• 2005

The c-myc proto-oncogene encodes a nuclear protein that is deregulated and/or mutated in most human cancers. Acting primarily as an activator and sometimes as a repressor, MYC protein controls the synthesis of up to 10-15% of genes. The key MYC targets contributing to oncogenesis are incompletely enumerated and it is not known whether pathology arises from the expression of physiologic targets at abnormal levels or from the pathologic response of new target genes that are not normally regulated by MYC. Regardless of which, available evidence indicates that the level of MYC expression is an important determinant of MYC biology. The c-myc promoter has architectural and functional features that contribute to uniform expression and help to prevent or mitigate conditions that might otherwise create noisy expression. Those features include the use of an expanded proximal promoter, the averaging of input from dozens of transcription factors, and real-time feedback using the supercoil-deformable Far UpStream Element (FUSE) as physical sensor of ongoing transcriptional activity, and the FUSE binding protein (FBP) as well as the FBP interacting repressor (FIR) as effectors to enforce normal transcription from the c-myc promoter.

• Molecules and Cells
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• 제37권11호
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• pp.819-826
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• 2014

Protein modifications of recombinant pharmaceuticals have been observed both in vitro and in vivo. These modifications may result in lower efficacy, as well as bioavailability changes and antigenicity among the protein pharmaceuticals. Therefore, the contents of modification should be monitored for the quality and efficacy of protein pharmaceuticals. The interface of EPO and its receptor was visualized, and potential amino acids interacting on the interface were also listed. Two different types of modifications on the interface were identified in the preparation of rHu-EPO BRP. A UPLC/Q-TOF MS method was used to evaluate the modification at those variants. The modification of the oxidized variant was localized on the Met54 and the deamidated variants were localized on the Asn47 and Asn147. The extent of oxidation at Met54 was 3.0% and those of deamidation at Asn47 and Asn147 were 2.9% and 4.8%, respectively.

• Pediatric Gastroenterology, Hepatology & Nutrition
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• 제22권6호
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• pp.581-587
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• 2019

Arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome is a rare autosomal recessive multisystemic disease that is associated with the liver, kidney, skin, and central nervous and musculoskeletal systems. ARC occurs as a result of mutations in the VPS33B (Vacuolar protein sorting 33 homolog B) or VIPAR (VPS33B interacting protein, apical-basolateral polarity regulator) genes. A female infant presented with neonatal cholestasis with a severe clinical outcome. She was diagnosed with ARC syndrome using targeted exome sequencing (TES). Exome sequencing revealed compound heterozygous mutations, c.707A>T and c.239+5G>A, in VPS33B, where c.707A>T was a novel variant; the resultant functional protein defects were predicted via in silico analysis. c.239+5G>A, a pathogenic mutation that affects splicing, is found in less than 0.1% of the general population. Invasive techniques, such as liver biopsies, did not contribute to a differential diagnosis of ARC syndrome; thus, early TES together with clinical presentations constituted an apparently accurate diagnostic procedure.

• Biomolecules & Therapeutics
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• 제29권5호
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• pp.527-535
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• 2021

Sclerostin (SOST), a regulator of bone formation in osteocytes, inhibits the canonical Wnt signaling by interacting with low-density lipoprotein receptor-related protein 5/6 (LRP5/6) to prevent Wnt binding. Loss-of-function mutations of the SOST gene caused massive bone outgrowth and SOST-null mouse exhibited a high bone density phenotype. Therefore, SOST has been suggested as a promising therapeutic target for osteoporosis. A few previous studies with X-ray crystallography identified the binding interfaces between LRP6 and SOST, but there are limitations in these studies as they used truncated SOST protein or SOST peptide. Here, we analyzed the conformational dynamics of SOST-LRP6 E1E2 complex using hydrogen/deuterium exchange mass spectrometry (HDX-MS). We examined the effect of the C-terminal tail of SOST on LRP6 conformation upon complex formation. HDX-MS analysis suggested a new potential binding interface for the C-terminal region of SOST that was missing from the previous crystal structure of the SOST-LRP6 E1E2 complex.

• 한국자기공명학회논문지
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• 제25권2호
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• pp.17-23
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• 2021

High mobility group protein B1 (HMGB1) is a highly conserved, non-histone, chromatin associated nuclear protein encoded by HMGB1 gene. HMGB1 proteins may be general co-factors in Hox-mediated transcriptional activation that facilitate the access of Hox proteins to specific DNA targets. It is unclear that the exact binding interface of Hoxc9DBD and HMGB1. To identify the interface and binding affinity of Hoxc9DBD and HMGB1 A-box, the paramagnetic probe, MTSL was used in NMR titration experiment. It is attached to the N-terminal end of HMGB1 A-box by reaction with thiol groups. The backbone assignment of HMGB1 A-box was achieved with 3D NMR techinques. The ¹⁵N-labeled HMGB1 A-box was titrated with MTSL-labeled Hoxc9DBD respectively. Based on the chemical shift changes we can identify the interacting residues and further map out the binding sites on the protein structure. The NMR titration result showed that the binding interface of HMGB1 A-box is around loop-1 between helix-1 and helix-2. In addition, the additional contacts were found in N- and C-terminus. The N-terminal arm region of Hoxc9DBD is the major binding region and the loop between helix1 and helix2 is the minor binding region.

• Parasites, Hosts and Diseases
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• 제60권1호
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• pp.7-14
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• 2022

Acanthamoeba keratitis (AK) is a rare infectious disease and accurate diagnosis has remained arduous as clinical manifestations of AK were similar to keratitis of viral, bacterial, or fungal origins. In this study, we described the production of a polyclonal peptide antibody against the adenylyl cyclase-associated protein (ACAP) of A. castellanii, and evaluated its differential diagnostic potential. Enzyme-linked immunosorbent assay revealed high titers of A. castellanii-specific IgG and IgA antibodies being present in low dilutions of immunized rabbit serum. Western blot analysis revealed that the ACAP antibody specifically interacted with A. castellanii, while not interacting with human corneal epithelial (HCE) cells and other causes of keratitis such as Fusarium solani, Pseudomonas aeruginosa, and Staphylococcus aureus. Immunocytochemistry (ICC) results confirmed the specific detection of trophozoites and cysts of A. castellanii co-cultured with HCE cells. The ACAP antibody also specifically interacted with the trophozoites and cysts of 5 other Acanthamoeba species. These results indicate that the ACAP antibody of A. castellanii can specifically detect multiple AK-causing members belonging to the genus Acanthamoeba and may be useful for differentially diagnosing Acanthamoeba infections.

• Archives of Pharmacal Research
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• 제9권1호
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• pp.29-38
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• 1986

The effects of ginseng saponins on the sarcolemmal $Na^{+}$, $K^{+}$-ATPase were compared to gypsophila saponin, sodium dodecylsulfate (SDS), and Triton X-100 to elucidate whether the effects are due to the membrane distruption, using a highly enriched preparation of cardiac sarcolemma prepared from dog ventricular myocardium. About 26% and 29% of vesicles in the preparation, enriched in ouabain-sensitive $Na^{+}$, $K^{+}$-ATP ase, $\beta$-adrenergic and muscarinic receptors are rightside-out and inside-out orientation, respectively. Ginseng saponins (triol>total> diol) inhibited $Na^{+}$, $K^{+}$-ATP ase activity, $Na^{+}$, $K^{+}$-ATPase activity and [$^{3}$H]ouabain binding of sarcolemmal vesicles. However, gypsophila saponin, SDS (0.4$\mu$g/$\mu$g protein) and Triton X-100 (0.6 $\mu$g/$\mu$g protein) caused about 1.35 and 1.40-fold increase in $Na^{+}$, $K^{+}$-ATPase activity and [$^{3}$H] oubain binding, respectively. Especially, the activating effect of gypsophila saponin on membrane Na+, K+ ATPase was detected at gypsophila saponin to sarcolemmal protein ratios as high as 100. Low dose of ginseng saponin (3$\mu$g/$\mu$g protein) decreased the phosphorylation sites and the concentration of ouabain binding sites (Bmax) without affecting the turnover number and affinity for ouabain binding, while gypsophila saponin, SDS(0.4 ug/ug protein), ahd Triton X-100 (0.6$\mu$g/$\mu$g protein) increased the Bmax. The results suggest that ginseng saponins cause a decrease in the number of active sites by interacting directly with $Na^{+}$, $K^{+}$-ATPase before disruption of membrane barriers of sarcolemmal vesicles.

• 한국산학기술학회논문지
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• 제9권6호
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• pp.1795-1799
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• 2008

hnRNP K 단백질은 hnRNP 복합체를 구성하는 핵단백질들 중의 하나이며 시토신이 많은 RNA/DNA sequence에 잘 결합한다. 이 단백질은 핵 내에서만 머무르지 않고 핵과 세포질을 왕복하는 특징을 지니고 있다. hnRNP K의 기능을 조사하기 위하여 우선 hnRNP K와 상호 결합하는 세포내 단백질을 찾아내고자 하였다. 이를 위하여 본 연구에서는 이스트 two-hybrid 시스템을 사용하여 HeLa CDNA librar를 탐색하였다. 그 결과 얻은 클론들 중에는 사람의 hnRNP E1 (poly(rC) binding protein 1) cDNA (GenBank accession number XM_031585) 클론이 포함되어 있었다. 본 논문에서는 이스트 two-hybrid 시스템과 in vitro에서의 생화학적 실험을 통하여 hnRNP E1은 hnRNP K와 특이적으로 상호 결합한다는 것을 밝혔다.